Vibrio cholerae modulates the immune defense of human gut

mucosa

Aziz Bitar

Department of Clinical Microbiology, Section of Infection and Immunology Umeå 2018

This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD ISBN: 978-91-7601-881-1 ISSN: 0346-6612 New Series Number 1962 Front cover: Electron micrographs of small intestinal epithelium (upper left), V. cholerae bacteria (upper right and middle left), T84 tight monolayer (middle right and lower right) and outer membrane vesicles of V. cholerae (lower left). (With courtesy of Professors Marie-Louise Hammarström and Sun Nyunt Wai). Electronic version available at: http://umu.diva-portal.org/ Printed by: UmU-tryckservice, Umeå University Umeå, Sweden 2018

To my family

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Table of Contents

ABSTRACT ....................................................................................... iii Populärvetenskaplig sammanfattning .............................................. v Abbreviations .................................................................................. vii PAPERS IN THE THESIS .................................................................. ix INTRODUCTION ................................................................................ 1 1. BACKGROUND .............................................................................. 2

1.1 Overview of the immune system ................................................................................ 2 1.2 Adaptive immunity .................................................................................................... 2 1.3 Innate Immunity ........................................................................................................ 4

1.3.1 Cellular components of the innate immunity .................................................................. 4 1.4 Mucosal immunity and the intestine.......................................................................... 7

1.4.1 General structure and cellular composition of the intestine ........................................... 7 1.4.2 Tissues of the mucosal immune system and intestinal immune cells ........................... 11

1.6 Pattern recognition receptors ................................................................................... 13 1.6.1 Toll-like receptors ............................................................................................................ 13 1.6.2 Other families of pattern recognition receptors ............................................................ 14 1.6.3 Pattern recognition receptors in human intestine......................................................... 15

1.7 Cytokines ................................................................................................................... 17 1.7.1 Interleukin-1β and Interleukin-18 ................................................................................... 18 1.7.2 Tumor Necrosis Factor−α ............................................................................................... 19 1.7.3 Chemokines ...................................................................................................................... 19

1.9 MicroRNA.................................................................................................................. 21 1.9.1 Biogenesis, maturation and function of microRNA ...................................................... 22 1.9.2 MicroRNA in innate responses to pathogens ............................................................... 23

1.10 Bacterial interactions with the gastrointestinal tract ........................................... 25 1.11 Vibrio cholerae........................................................................................................ 25

1.11.1 O1 and O139-associated virulence factors..................................................................... 27 1.11.2 Non-O1 and non-O139 strains and associated virulence factors................................ 28

1.12 Bacterial outer membrane vesicles ........................................................................ 30 2. AIMS ........................................................................................... 33 3. METHODOLOGICAL CONSIDERATIONS ................................... 34

3.1 Clinical material ....................................................................................................... 34 3.2 Polarized tight monolayers of human intestinal epithelial cells ............................ 34 3.3 Bacterial work and effects on target cells ................................................................35 3.4 TER measurement techniques ................................................................................ 36 3.5 Gene expression analysis of total RNA using real-time qRT-PCR ......................... 37

3.5.1 Determination of expression levels of target-genes, cytokine and chemokine mRNAs by using real-time qRT-PCR ................................................................................................... 37 3.5.2 Determination of expression levels of microRNAs using real-time qRT-PCR ............ 38 3.5.3 Housekeeping genes for stable normalization ............................................................. 38 3.5.3 Gene expression analysis using genome-wide hybridization bead array .................. 39

3.6 In situ miRNA hybridization ................................................................................... 40 3.7 Determination of amounts of cytokine protein ...................................................... 40 3.8 Isolation and characterisation of outer membrane vesicles .................................. 40

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3.8.1 Nanotracking analysis ................................................................................................... 41 4. RESULTS AND DISCUSSION ...................................................... 42

4.1 Rationale of project .................................................................................................. 42 4.2 Innate epithelial responses to secreted Vibrio cholerae cytolysin and PrtV of Vibrio cholerae .............................................................................................................. 42

4.2.1 Effects of culture supernatant of V. cholerae on epithelial cells .................................. 42 4.2.2 Role of Vibrio cholerae cytolysin .................................................................................. 44 4.2.3 Post-translational modulation of inflammation by PrtV ............................................ 46 4.2.4 V. cholerae cytolysin induces interleukin-1 receptor activated kinase 2 ..................... 47

4.3 Cytokines and microRNA expression in small intestinal mucosa during acute Vibrio cholerae O1 infection .......................................................................................... 47

4.3.1 Analysis of cytokine mRNA expression .......................................................................... 47 4.3.2 Induction of regulatory microRNA in the intestinal epithelium ................................. 48 4.3.3 Analysis of targets for miR-146a in the intestinal mucosa .......................................... 50 4.3.4 In vitro analysis of V. cholerae and its secreted factors .............................................. 50

4.4 Effects of released outer membrane vesicles ........................................................... 51 4.4.1 Tightening of epithelia .................................................................................................... 51 4.4.2 Modulation of cytokine responses ................................................................................. 52 4.4.2 Induction of regulatory microRNA-146a ...................................................................... 53

4.5 Vibrio cholerae infection and the role of microRNA and released components – summary of results and hypothesis .............................................................................. 54

5. CONCLUSIONS ............................................................................ 56 6. ACKNOWLEDGEMENTS ............................................................. 58 7. REFERENCES .............................................................................. 60

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ABSTRACT

The key function of innate immunity is to sense danger signals and initiate effective responses as a defense mechanism against pathogens. Simultaneously, effector responses must be regulated to avoid excessive inflammation with resulting tissue damage. microRNAs (miRNAs), are small endogenous molecules, that has recently gained attention as important regulatory elements in the human inflammation cascade. The control over host miRNA expression may represent a previously uncharacterized molecular strategy exploited by pathogens to mitigate innate host cell responses.

Vibrio cholerae is a Gram-negative bacterium that colonizes the human small intestine and causes life-threatening secretory diarrhea, essentially mediated by cholera toxin (CT). It is considered a non-invasive pathogen and does not cause clinical inflammation. Still, cholera is associated with inflammatory changes of the small intestine. Furthermore, CT-negative strains of V. cholerae cause gastroenteritis and are associated with extra-intestinal manifestations, suggesting that other virulence factors than CT are also involved in the pathogenesis.

The innate immune response to V. cholerae is poorly investigated and the potential role of miRNA in cholera had not been studied before. Therefore, this thesis explores the role of intestinal epithelial cells in response to V. cholerae infection with a focus on regulatory miRNA as a potential contributor to the pathogenesis. The in vivo material was small intestinal biopsies from patients suffering from V. cholerae infection. As an in vitro model for V. cholerae attack on intestinal epithelium, we used tight monolayers of T84 cells infected with V. cholerae and their released factors. We analyzed changes in levels of cytokines, immunomodulatory microRNA and their target genes.

We showed that miRNA-146a and miRNA-155 reached significantly elevated levels in the intestinal mucosa at acute stages of disease in V. cholerae infected patients and declined to normal levels at the convalescent stage. Low-grade inflammation was identified at the acute stage of V. cholerae infection, which correlated with elevated levels of regulatory miRNA. Furthermore, outer membrane vesicles (OMVs) released by the bacteria were shown to induce miR-146a and live bacteria induced miR-155 in intestinal epithelial cells. In addition, OMVs decreased epithelial permeability and caused mRNA suppression of pro-inflammatory cytokines, including immune cell attractant IL-8 and CLL20, and the inflammasome markers IL-1β and IL-18. These results propose that V. cholerae regulates the host expression of miRNA during infection and may set the threshold for activation of the intestinal epithelium.

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Moreover, we showed that V. cholerae also harbors inflammatory-inducing capabilities, by secreting a pore-forming toxin, Vibrio cholerae cytolysin (VCC). By using genetically modified strains as well as soluble protein challenge experiments, VCC was found solely responsible for the increased epithelial permeability and induction of several pro-inflammatory cytokines in intestinal epithelial cells. In contrast to OMVs, VCC displayed strong upregulation of the pro-inflammatory cytokines IL-8, TNF-α, CCL20 and IL-1β and IRAK2, a key signaling molecule in the IL-1 inflammasome pathway. This suggest that VCC is an important virulence factor in the V. cholerae pathogenesis, particularly in CT-negative strains. Furthermore, we showed that the bacterium could control the inflammatory actions of VCC by secreting the PrtV protease, which degraded VCC and consequently abolished inflammation.

In summary, we showed that V. cholerae harbors immunomodulating capabilities, both at the gene level, through induction of host regulatory miRNA, and at the protein level, through secretion of VCC and PrtV. These strategies may be relevant for V. cholerae to promote survival in the gut and cause successful infections in the human host.

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Populärvetenskaplig sammanfattning

Många sjukdomsframkallande tarmbakterier använder sig av olika strategier för att reglera och undvika immunförsvaret i tarmen. För bakterien Vibrio cholerae, även kallad kolerabakterien, vilken utgör ett stort hot mot människors liv och hälsa världen över, är dock dessa immunreglerande mekanismer inte särskilt välstuderade. För att tillhandahålla bättre förebyggande och terapeutiska åtgärder är det viktigt att fördjupa förståelsen kring dessa mekanismer.

Tarmslemhinnan, eller tarmepitelet, som täcker tarmen insida, är en viktig del av kroppens immunförsvar och har en svår uppgift. Den måste tolerera den godartade tarmfloran och samtidigt försvara sig mot sjukdomsframkallande bakterier. Att reglera graden av inflammation, kroppens svar på infektion, är viktigt för att inte orsaka vävnadsskador i tarmen. På senare tid har mikroRNA (miRNA), små bitar av RNA som styr genuttryck, uppmärksammats som viktiga faktorer i regleringen av den inflammatoriska reaktionen. Det har också visats att vissa sjukdomsframkallande bakterier kan utnyttja miRNAs inflammations-hämmande egenskaper för att undkomma kroppens immunförsvar.

Bakterien V. cholerae infekterar tarmen och orsakar stora mängder vattnigt diarré som snabbt kan leda till livsfarlig uttorkning om inte behandling sätts in tidigt. Bakterien utsöndrar olika komponenter, till exempel toxiner och vesiklar, som deltar i sjukdomsförloppen. Toxiner är ett slags gift, och vesiklar är små membranomgivna bubblor som skickas iväg av bakterien och innehåller faktorer som kan påverka mottagarcellerna i tarmepitelet.

Den kolerastam som orsakar alla större epidemier och pandemier verkar främst genom det välstuderade så kallade koleratoxinet. Idag förekommer dock även andra kolerastammar som trots avsaknad av toxinet, ändå orsakar lokala utbrott av diarrésjukdom med inflammatoriska inslag. De sjukdomsframkallande mekanismerna hos dessa stammar är fortfarande inte kända. Det är heller inte kartlagt huruvida V. cholerae kan uttrycka miRNA i tunntarmsslemhinnan under infektionsförloppet. Det är heller inte känt hur vesiklar utsöndrade av V. cholerae påverkar immunförsvaret i tarmen.

Syftet med denna avhandling är att förstå reaktionerna i tarmsepitelet under V. cholerae-infektion, inklusive de utsöndrade bakteriella komponenternas påverkan på tarmepitelet.

I studie I har vi använt av oss av ett framodlat tunntarmsepitel, som har liknande egenskaper som det normala tarmepitelet. Genom denna modell har vi kunnat studera enskilda bakteriella komponenters påverkan på tarmepitelet. Vi

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visar att V. cholerae kan utsöndra en känd faktor kallad V. cholerae cytolysin (VCC), som i tunntarmen orsakar inflammation och ökad genomsläpplighet i epitelet. Denna reaktion innefattar en uppreglering av ett flertal inflammatoriska markörer, som kan signalera för immunceller att komma till infektionsområdet i tarmen. Vidare visar vi att bakterien kan utsöndra en annan faktor PrtV som bryter ner VCC och på så vis kan påverka mängden inflammation till sin fördel.

I studie II och III har vi, förutom det framodlade tunntarmsepitelet, också använt oss av tunntarmsbiopsier från patienter i olika stadier av kolerasjukdom. Genom dessa två metoder har vi studerat uttrycket av miRNA och hur immunförsvaret i tarmen påverkas under sjukdomsförloppet. I studie II visar vi att förhöjda nivåer av miRNA-146a och miRNA-155 återfinns i tarmepitelet hos patienter vid akut kolerainfektion, men sjunker sedan till normala nivåer vid tillfrisknandet. Fyndet av miRNA korrelerar väl med ett minskat inflammatoriskt påslag i tunntarmsslemhinnan. Vi fann vidare i studie III att inte bara bakterien, utan även utsöndrade vesiklar kan få värdcellerna att uttrycka miR-146a som därmed verkar hämmande för det inflammatoriska påslaget. Via vesiklar kan bakterien således dämpa det inflammatoriska svaret utan att själv behöva vara i direktkontakt med värdcellerna i tarmen.

Sammanfattningsvis visar våra resultat hur V. cholerae både på gennivå, via att uttrycka miRNA hos värdcellen, och på proteinnivå genom att utsöndra VCC och PrtV kan orsaka och modulera graden av inflammation i tarmslemhinnan och därmed skapa optimala förhållanden för sin överlevnad.

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Abbreviations

ADCC Antibody-dependent-cell mediated cytotoxicity AMP Antimicrobial peptide APC Antigen-presenting cell BCR B cell receptor CD Cluster of differentiation CTL Cytotoxic T-cell CTX Cholera toxin DAMP Danger-associated molecular pattern DC Dendritic cell FAE Follicle-associated epithelium GALT Gut-associated lymphatic tissue HBD Human β defensin HLA Human leukocyte antigen IBD Inflammatory bowel disease IEC Intestinal epithelial cell IFN Interferon Ig Immunoglobulin IL Interleukin ILC Innate lymphoid cell LI Large intestine LP Lamina propria LPL Lamina propria lymphocytes LPS Lipopolysaccharide LRR Leucin-rich repeat MHC Major histocompatibility complex MIC MHC class I chain related antigen mRNA Messenger RNA miRNA microRNA MUC2 Mucin 2 NF-κB Nuclear factor kappa B NLR NOD-like receptor NK Natural killer cell NKT Natural killer T cell NOD Nucleotide oligomerization domain PAMP Pathogen associated molecular pattern

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PCR Polymerase chain reaction PP Peyer’s patch PRR Pattern recognition receptor PrtV Protease of Vibrio cholerae qRT-PCR Real-time quantitative reverse transcriptase-PCR RLR Retinoic acid-inducible gene (RIG)-I-like receptor SI Small intestine sIgA Secretory IgA TCR T cell receptor TFF Trefoil factor TGF Transforming growth factor Th T helper cells TJ Tight junction TLR Toll-like receptor TNF Tumor necrosis factor Treg Regulatory T cell VCC Vibrio cholerae cytolysin

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PAPERS IN THE THESIS

This thesis is based on the following articles and manuscripts, which will be referred to in the text by Roman numerals (I-III).

I. Ou, G., Rompikuntal, P.K., Bitar, A., Lindmark, B., Vaitkevicius, K., Bhakdi, S., Wai, S. N., Hammarström, M-L. Vibrio cholerae cytolysin causes an inflammatory response in human intestinal epithelial cells that is modulated by the PrtV protease. PLoS One. 2009 Nov 12;4(11):e7806.

II. Bitar, A*, De, R*, Melgar, S., Aung, K.M., Rahman, A., Qadri, F., Wai, S.N., Shirin, T¤, Hammarström¤, M-L. Induction of Immunomodulatory miR-146a and miR-155 in Small Intestinal Epithelium of Vibrio cholerae Infected Patients at Acute Stage of Cholera. PLoS One. 2017 Mar 20;12(3): e0173817.

III. Bitar, A., Aung, K.M., Wai, S.N., Hammarström, M-L. Vibrio cholerae derived outer membrane vesicles modulate the inflammatory response of human intestinal epithelial cells by inducing microRNA-146a (Submitted)

*These authors contributed equally to this work

¤These authors shared senior authorship

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INTRODUCTION

The intestinal epithelium consists of a single layer of epithelial cells. It is constantly exposed to luminal antigens from food and the normal microflora and episodic infections by pathogens. The epithelium itself is an important and integrated compartment of the gut immune system. The powerful effector responses by the gut immune system must tolerate the commensal microflora and simultaneously avoid detrimental effects for the host.

Vibrio cholerae, a Gram-negative, small intestinal pathogen, infects the gut and causes severe life-threatening diarrhea. It was previously thought that cholera was solely a non-inflammatory and non-invasive disease. However, recent reports have provided evidence for inflammatory changes of the intestine during clinical infection. This was further illustrated by the fact that V. cholerae lacking the cholera toxin could still cause disease, such as gastroenteritis and extra-intestinal manifestations. These findings suggest that other virulence factors are involved in the pathogenesis. However, the nature of these inflammation-causing factor(s) in V. cholerae is largely unknown.

Micro(mi)RNAs are small endogenous RNAs that post-transcriptionally regulate eukaryotic gene expression. In addition to their involvement in a wide range of physiological conditions, miRNAs are increasingly implicated in the host cell response to bacterial pathogens. By gaining control over host miRNA, bacteria can modulate host responses and provide optimal conditions for successful infections.

In this thesis, in vitro and in vivo studies of V. cholerae infection are conducted and new insights are provided on how the bacterium can modulate host immune responses, at the post-transcriptional and post-translational level.

The following chapters give a brief background of the gut mucosal immune system, miRNA and V. cholerae before the discussion of the results obtained in this study.

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1. BACKGROUND

1.1 Overview of the immune system The word immunity, from the Latin, immunis, meaning “exempt” (or free of burden), originates from the juridical concept ‘exception’. Although, it was not until the late nineteenth century in Europe, that the word became a medical term referring to the state of protection from infectious disease.

The human immune system provides a vital defense mechanism and is defined by three main functions: Firstly, the ability to sense and deploy effective defense reactions against invading microorganisms, such as bacteria, virus, fungi, protozoa and parasites. Secondly, the constant immune surveillance that recognizes and removes damaged or transformed cells and thus contributes to the prevention of tumor development. Thirdly, promoting homeostasis, an attribute that is mainly important at constantly exposed surface areas, such as mucosal sites, where the external and sometimes hostile environment encounters bodily tissues. In addition, the immune system must also regulate and tolerate the beneficial commensal microbes that are localized along mucosal tissues.

The immune system is traditionally divided into two branches, the innate and the adaptive, which are interconnected and collaborate to protect the host against pathogens.

1.2 Adaptive immunity The adaptive immune system is traditionally defined by its three main characteristics: antigen specificity, immunological memory, and the ability to discriminate between self/non-self-antigens. In contrast to the rapid and unspecific innate immunity, adaptive responses take days to be activated following the first encounter with a specific pathogen. Adaptive immunity can be divided into a humoral and cell-mediated immune responses. The cell-mediated responses are mediated by specific T lymphocytes, T cells, and the humoral responses by antibodies derived from specific B lymphocytes, B cells. The specificity of the adaptive immune system is created by somatic rearrangements of the encoding genes that create enormous pools of T cells and B cells. Each have one specificity per cell that is mediated by T cell receptor (TCR) and B cell receptors (BCR), respectively. Antibodies are produced by plasma cells, which in turn are maturated from the specific B cells.

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The activation of adaptive immunity is based on recognition of exogenous and/or endogenous peptides presented by antigen-presenting cells (APCs), which harbor cell surface proteins of the major histocompatibility complex (MHC). MHC molecules can be divided into two classes: MHC class I and MHC class II. Most nucleated cells express MHC I class and therefore present peptides from endogenous antigens, such as viral and tumor antigens. MHC class II molecules present processed extracellular antigens to T cells and are normally expressed by professional APCs, like B cells, dendritic cells (DCs), and macrophages.

T cells originate from the bone marrow and mature in the thymes, where they learn to discriminate between self- and non-self-antigens through positive and negative selection processes. As the human thymus regress over time, other organs are also involved in T cells maturation, such as the intestinal mucosa1,2. T cells recognize processed antigens by their TCRs. Based on the composition of TCR polypeptide chains, T cells can be divided into αβ T cells and γδ T cells that differ in their biology and functions. In association with TCR, T cells express surface marker, cluster of differentiation 3 (CD3), which is involved in the activation of intracellular signaling upon antigen recognition.

αβT cells are further classified based on expression of co-receptors CD4 or CD8. The CD8+ T cells recognize antigens in association with MHC class I molecules, while CD4+ recognize antigens presented by MHC class II molecules. T cells require two signals for activation: recognition of antigens presented on MHC I or II by the TCR/CD3 complex, and interaction with the co-stimulatory molecules presented on APCs. The most important co-stimulatory signals are mediated through CD28 and CD40L on T cells, which bind to CD80 and CD40 on APCs. Naïve T cells refer to T cells that have not yet encountered an antigen.

T cells proliferate and differentiate into diverse types of effector T cells, depending on the stimuli and the cytokine milieu. CD8+ T cells can differentiate into cytotoxic T (CTLs) cells that kill infected or transformed cells. Most CD4+ T cells are T helper cells (Th) that assist other immune cells in immunologic processes and include different T cell subsets. For example, Th1, Th2 and Th17 are responsible for pathogen clearance and produce cytokines and other soluble mediators involved in efficient removal of their respective target pathogens. In addition, a regulatory T cell subset (Treg) is responsible for maintaining intestinal homeostasis by down-regulating the production of pro-inflammatory cytokines, and thus dampening inflammation responses.

The B cells differentiate in the bone marrow and they express the BCR, which is a membrane-bound immunoglobulin (Ig) with a unique antigen binding site.

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Each B cell has only one antigen-binding site i.e. specificity. The BCR is composed of two identical light and two identical heavy chains with variable V(D)J and constant (C) regions. The V(D)J region determines the antigen specificity and the C region determines the effector functions and antibody class. Naive B cells express BCR of IgM and IgD classes on their cell surfaces. Upon activation, which generally occur with the help of T cells, B cells differentiate into plasma cells, a differentiation process that induces changes in the constant region to IgG, IgA or IgE, a process called isotope switching. Antibodies have several important functions, such as toxin neutralization, prevention of adhesion of pathogens to mucosal surfaces, activation of the complement system, opsonization of microorganism for phagocytosis and involvement in antibody-dependent-cell-mediated-cytotoxicity (ADCC). Antibody producing plasma cells are usually short lived, but a fraction of these cells become memory cells3.

1.3 Innate Immunity Innate, derived from the Latin, innātus, meaning inborn, refers to the immune response system that we are born with. It is an evolutionary ancient defense mechanism, found in plants, fungi, insects and other multicellular organism. Innate immunity constitutes the “first” line of host defense and controls the first step of immune response. Despite constant exposure to microorganisms, the innate immune systems keep the incidence of infectious diseases relatively low in the healthy human body. It senses and provides immediate protection against potential threats, without requiring previous exposure to pathogens.

Essential elements in innate immunity includes: provision of physical and biochemical barriers, sensing the presence of microbes and tissue damage, production of antimicrobial compounds, e.g. antimicrobial peptides (AMPs), lysosome, lactoferrin, reactive oxygen and nitrogen species, activation of the complement cascade, cytokines for activation and recruitment of immune cells to the site of an infection, and direct or indirect killing of microbes by immune cells that compromise the innate immunity.

The basis of recognition in innate immunity, resides in the ability to sense unique patterns that are common to most microbes, referred to as MAMP (microbe-associated molecular pattern), which are recognized by host sensors known as pattern recognition receptors (PRRs).

1.3.1 Cellular components of the innate immunity The leucocytes involved in innate immunity include macrophages/monocytes, dendritic cells (DCs) granulocytes, unconventional T cells and innate lymphoid cells (ILCs) and natural killer (NK) cells, which together are responsible for the rapid recognition and elimination of pathogens. In addition to pathogen

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removal, most cellular components of the innate immune system also serve as links to activate the adaptive immune system.

1.3.1.1 Monocytes are the largest cell of the leukocytes in term of size and originate from a precursor in the bone marrow. They enter the peripheral blood and migrate by the help of specific chemokine and adhesion receptors to sites of inflammation or infection and then mature to macrophages. Macrophages display important functions in both innate and adaptive immunity. They reside in almost all tissues and are believed to maintain tissue integrity. Further, they are usually the first immune cells to encounter foreign material, such as bacteria and soluble proteins. They can engulf and kill microbes and promote inflammation by producing cytokines and chemokines that activate and recruit other immune cells to the site of infections.

1.3.1.2 Dendritic cells (DCs) are potent APCs cells and serve as messengers between the innate and adaptive immune responses. Morphologically, long cytoplasmic processes that allow intimate contact with several immune cells, characterize the DCs. These cells can present antigens in the context of MHC class II molecules and express the surface molecules CD80 and CD40, which are accessory molecules required for T cell activation. DCs are found in all lymphoid organs and mucosal sites.

1.3.1.3 Granulocytes Granulocytes are characterized by their cytoplasmic granules, which are secretory vesicles containing various antimicrobial factors. They are also called polymorphonuclear leukocytes (PMN) because of the varying shape of the nucleus, which is usually lobed into three segments. PMNs often refer to neutrophils, since this group is the most abundant of all granulocytes. Other groups of granulocytes are eosinophils, mast cells, and basophils.

Neutrophils comprise more than half of the circulating leukocytes and are usually the first immune cells to arrive at the site of infection or inflammation. They are recruited by a process called chemotaxis, meaning that they migrate towards a chemical gradient to substances such as interleukin-8 secreted by macrophages and epithelial cells at the site of infections. They eliminate pathogens by phagocytosis or by degranulation that releases soluble antimicrobial compounds such as defensins. Furthermore, they can form massive amounts of reactive oxygen and nitrogen species and other toxic molecules that destroy pathogens. They also generate neutrophil extracellular traps (NET), which are a network of composed neutrophil DNA that binds pathogens4.

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Eosinophils comprise a small part of circulating leucocytes. They are not phagocytic, but contain antimicrobial granules that are released upon activation and usually involved in defense against parasites5.

Mast cells are characterized by their content of numerous secretory granules, containing proteases, histamine, serotonin, heparin and chemokines. They express IgE receptors (FcεR), which bind antibodies of the IgE class. If appropriate antigen (often called allergen in this context) is bound to the receptor-bound IgE, cross-linking occurs which in turn leads to degranulation. Mast cells are associated with allergy and anaphylaxis, but are also involved in defense against pathogens, including parasitic infections.

Basophils are the smallest population of blood leukocytes and may functionally be considered as circulating form of mast cells. Basophils release effector molecules and vasodilating substances, (e.g. histamine), usually involved in allergen responses.

1.3.1.2 Unconventional T cells Unconventional T cells share features of cells belonging to both innate and adaptive immunity. They primary recognize lipids and small-molecule metabolites. Further, they express a TCR, which generally has low diversity and respond rapidly upon activation. Among the most studied unconventional T cells are mucosal associated invariant T cells (MAIT), γδ T cells and NKT cells.

MAIT cells are evolutionarily conserved T cells expressing CD8 and recognizing bacterial metabolites via the non-classical MHC class I- related molecule (MR1). They are primarily found in the circulation, but can also be found in the gut6.

γδ T cells constitute only a small proportion of the lymphocytes in human blood, although they are widespread within the intestinal epithelium7. They can recognize a variety of antigens, such as small non-peptide ligands and larger protein antigens, such as MHC class I chain-related antigens A and B (MICA/MICB) expressed on stressed cells. γδ T cells attack target cells directly through degranulation of cytotoxic granules or indirectly through the activation of other immune cells.

Natural killer T cells (NKT cells) are a subset of lymphocytes that express TCR, but also other molecules that are characteristic of NK cells. They recognize glycolipid antigens presented by the non-classical MHC I molecule CD1d and are found with the highest frequency in the lamina propria (LP) of the small intestine8. Upon activation, they are involved in defense mechanisms against tumours and infectious microbes, but can also suppress cell-mediated immune responses9.

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1.3.1.4 Innate lymphocyte cells Like unconventional T cells, the innate lymphocyte cells (ILCs) share features with cells of both the adaptive and innate immune system. One of their characteristics is the absence of TCR. ILCs are divided into three groups (ILC1, ILC2 and ILC3) based on cytokine-production profiles10. ILC1 consist of IFN-γ-producing cells that play a major role in defense against viruses and intracellular bacteria. ILC2 produce cytokines associated with parasitic infections and allergy, while ILC3 are involved in lymphoid tissues development as well as maintaining the intestinal barrier integrity11.

1.3.1.5 NK cells are cytotoxic lymphocytes that are essential in innate immunity and kill target cells with low expression of MHC class I molecules (i.e. virus-infected cells or tumour cells). NK cells lack antigen-specific receptors and are not dependent on MHC for their activation. Instead, their cytotoxic activity is tightly regulated by stimulatory and inhibitory receptors on the cell surface12. Antibodies attached to infected cells can be recognized by NK cells and trigger them to kill the infected cell through antibody-dependent cell-mediated cytotoxicity (ADCC).

1.4 Mucosal immunity and the intestine The mucosal immune system consists of diffusely distributed immune cells and cells organized into lymphoid tissues as well as within epithelial cells. It is present in the gastrointestinal (GI), respiratory, and urogenital tracts. Together it constitutes the largest areas within the body that is in contact with the external environment. These surface areas separate the bodily tissues from pathogenic microbes and commensal flora, often by a single layer of epithelial cells. Functions associated with the mucosal immune system is mucus production, providing a physical barrier, ability to tolerate the normal microbiota and in the GI also food constituents. Constant peristalsis in the intestine may also be considered functions of this system.

1.4.1 General structure and cellular composition of the intestine The intestine is the largest mucosal surface in the body, and therefore houses the largest reservoir of immune cells. It is divided into the small intestine (SI) and the large intestine (LI). The SI compromises about 80 % of the entire intestine and is about 8.5 m long in adult humans 13. The small intestine (SI) can be further divided into three segments: the duodenum, which begins at the pylorus, and is followed by the jejunum, and the ileum. Ileum ends in the ileocecal valve, which is the entry point into the LI. The LI begins at the caecum, followed by the ascending colon, the transverse colon, the descending colon and the rectum.

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The SI plays a role in nutrient absorption and consists of villi, finger-like projections extending into the lumen, which increases the surface area for nutrition absorption. The base of each villus is surrounded by numerous epithelial invaginations, known as crypts of Lieberkühn. The LI, which is primarily involved in water absorption and elimination of undigested dietary components, lacks villous structures and has deeper crypts.

Histologically, four distinct layers can be distinguished along the whole intestine. These are: the mucosa, the submucosa, the muscularis externa and the serosa. The mucosa faces the gut lumen and can be further divided into the intestinal epithelium, the lamina propria (LP), and the muscular mucosa. Under the intestinal epithelium, is a layer of connective tissue, the LP. The LP supports the epithelium and is a compartment that houses different types of cell populations, most of them belonging to the immune system, such as T and B cells, plasma cells, macrophages, DCs and occasionally eosinophils. Additionally, connective tissue cells, such as fibroblast and smooth muscle cells are present in LP. A network of blood capillaries and lymphatic capillaries are also present in LP. The muscularis mucosae is a thin muscle layer separating the LP from the submucosa. The submucosa in turn is a layer of connective tissue harboring fibroblast, mast cells, blood- and lymphatic vessels and nerve fiber plexus. Under the submucosa reside layers of smooth muscle tissue, the muscularis externa, which play a role in the peristalsis. The fourth layer of the intestine wall is the adventitia or serosa, which consists of connective tissues that separate the intestine from the surrounding peritoneal cavity.

1.4.1.1 Cell junctions Intestinal epithelial cells (IECs) are tightly packed together in a single layer that prevents passage of bacteria to the underlying tissues. At the same time, the epithelium remains semi-permeable to allow passage of nutrients and ions from the lumen to the IECs and underlying tissues. Transport can take place both through (intracellular) and between (paracellular) IECs. The IECs are therefore connected to one another with the help of protein complexes, which provide layers of cell dynamic junction structures, referred to as the epithelial junction complex.

The junction complex comprises tight junctions (TJ; located closest to the lumen), followed by adherence junctions (AJs), and desmosomes. TJs are intercellular complexes made up of transmembrane proteins, such as claudins and occludins that prevent paracellular leakage14. AJs consist of transmembrane proteins, such as E-cadherin and catenin proteins15. Both AJs and desmosomes are located beneath the TJs and are responsible for strengthening cell-to-cell adhesion. Together, these junction structures act as a selective physical barrier. In addition, several components of luminal contents, including dietary

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components and stimuli from the microbiota can act beneficially on IECs by affecting tight junction structures16,17.

1.4.1.2 Intestinal epithelial cell types The IECs includes several cell types. These are derived from stem cells, which are located at the bottom of the intestinal crypts. Daughter cells to the stem cells proliferate before they differentiate into highly specialized and differentiated IECs18. IECs consist of two classes: absorptive and secretory enterocytes. The secretory type can be further subdivided into four populations: mucus-producing goblet cells, antimicrobial peptide-producing Paneth cells, hormone-secreting enteroendocrine cells, and chemical-sensing Tuft cells.

Enterocytes are the predominant absorptive cell type in the intestine villi. One of their primary functions is to absorb digested nutrients. They have typical characteristics, such as dense microvilli, which further increase the absorptive area towards the lumen. The cell membrane of the microvilli contains enzymes needed to digest dietary components, and membrane-bound glycoproteins, like transmembrane mucins, which constitute the glycocalyx. Enterocytes are also capable of producing chemokines in the event of infection or epithelial damage19. Furthermore, enterocytes in both SI and LI constantly express antimicrobial peptides, such as β−defensins 120,21.

Paneth cells mainly resides in the SI and unlike enterocytes, they are long-lived cells (about two months). They are located at the bottom of the crypts, juxtaposed with the stem cells and function as nursing cells by providing growth factors to the stem cells22. Paneth cells are characterized by their apically-oriented secretory granules in the cytoplasm, containing antimicrobial proteins, including defensins and lysosome. Defensins are bactericidal and anti-viral components, as they can disrupt bacterial membranes or target the viral envelopes23,24. The high amounts of various antimicrobial proteins produced by Paneth cells help to guard the intestinal crypt and create an almost sterile local environment in the crypt lumen25. Altered expression of defensins is associated with inflammatory bowel diseases (IBDs), like Crohn’s disease21,26

Goblet cells are large mucus-producing cells, interspersed between enterocytes, located both in the crypts and at the villus/luminal compartments. They are named goblet, because they contain mucin-containing vacuoles that shape the cytoplasm, which is reminiscent of the structure of a goblet. In contrast to Paneth cells, the frequency of mucus-producing goblet cells progressively increases along the GI being highest in the most distal part of the intestine.

The entire intestinal surface is covered by mucus with the gel-forming mucin (MUC) 2 as its main constituent. The small intestine has one layer of mucus that

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is relatively loose, while the colon has a thicker two-layered system with an inner and an outer mucus layer27. Goblet cells also express the protease resistance trefoil factor 3 (TFF3), which interacts with MUC2 and influences the mucus viscosity28. The intestinal mucus layer has several important functions, which include: lowering antigen exposure to the immune system, protecting the epithelium from self-digestion by endogenous enzymes, and providing matrix for secreted components of both the innate immune system, such as AMPs and adaptive immune system, such as secretory IgA (sIgA)27. Goblet cells may also be induced to secrete mucin in response to inflammatory conditions29,30.

Enteroendocrine cells, are hormone-producing cells located throughout the whole intestinal tract31. They produce immunologically active factors, such as substance P, a mediator of inflammation32-34. They are classified into several types, depending on their hormone production. Their main function is to sense changes in the luminal content and secrete neuropeptides and hormones into adjacent capillaries and regulate various digestive functions35,36.

Tuft cells are a rare type of cells, characterized by long microvilli projections and deep surface invaginations into the intestinal epithelium with as yet unknown functions. They are proposed to act as chemical sensors for the luminal contents. Recently, it was shown that tuft cells are involved in immune responses in parasitic infections37.

In addition to enterocytes, the intestinal epithelium contains microfold cells (M cells), found in the follicle-associated epithelium (FAE) of subepithelial lymphoid aggregates that lie in the mucosa and submucosa. M cells have a flat apical surface and are specialized for the uptake and transport of antigens from the lumen into the underlying tissue for further presentation to the adaptive immune system.

1.4.1.4 Microbiota The biogeography of the microbiota is still being investigated. However, the bacterial communities seem to differ dramatically between intestinal compartments from the stomach to the rectum. In the SI, the bacterial load is several order of magnitude lower, compared to the colon, probably due differences in the nutritional and biochemical milieu. Shorter transit time, as well as antimicrobials are factors that limit bacterial growth in the SI38. In contrast, conditions in the colon, promote a dense and diverse community of bacteria, such as anaerobes with the ability to utilize undigested carbohydrates. The intestinal microbiota of an individual consists of tens of thousands of species, dominated mainly by the phyla Bacteroidetes and Firmicutes, Proteobacteria and Actinobacteria39. Studies have shown that the mucosa-associated microbiota in the SI is different from that which is found in the colon

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and is dominated by two bacterial genera, namely Streptococcus and Neisseria39. Microbiota can be affected by diet and environmental factors, although despite fluctuations, the microbiota has been shown to be relatively stable within the individual40.

In addition to production of important nutrients, such as vitamins and short-chain fatty acids (SCFAs), the microbiota is essential for the function of the immune system41. Studies in germ-free animals have demonstrated the importance of gut microbiota in the development and regulation of innate and adoptive immune responses. For example, germ-free mice have less developed gut-associated lymphoid tissue structures and reduced levels of sIgA compared to conventionally raised mice42. By occupying biological niches within mucosal areas, the microbiota also take part in the prevention of invasion of pathogenic bacteria43.

1.4.2 Tissues of the mucosal immune system and intestinal immune cells

1.4.2.1 Gut-associated lymphoid tissue (GALT) Organized lymphoid tissue in the gut is referred to as the GALT. It constitutes the largest immunological organ in the body. The GALT includes Peyer’s patches (PPs), the appendix and solitary intestinal lymphoid tissues (SILTs). SILTs consist of dynamic microscopic lymphoid aggregates ranging from small cryptopatch-like structures to large, mature isolated lymphoid follicles (ILFs)44. PPs are localized in the SI and have developed before birth, while SILTs are located along the GI tract and develop early after birth, which is considered to be due to stimulation by the microbiota45.

The best studied components of GALT are the macroscopically visible PPs. The size and density of PPs increase from the jejunum to the ileum and they are particularly concentrated in the distal ileum46. Each PP consists of at least five aggregated lymphoid follicles. A follicle can be divided into three main domains. These domains include the follicular area, the interfollicular area, and the FAE47. The follicular area has a germinal center (GC), and contains primarily proliferating B lymphocytes, which when matured to plasma cells, are the major source of intestinal IgA48,49. The follicle is flanked by the interfollicular domain, which is rich in T cells. The FAE is separated from the follicle by the subepithelial dome (SED) region, which lies directly beneath the FAE and is rich in APCs and lymphocytes. DCs in the SED region have been shown to extend their dendrites into the gut lumen and sample antigens50.

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1.4.2.2 Lamina propria immune cells The LP and the epithelium are the effector sites of the intestinal immune system. The LP contains T cells, B cells and plasma cells together with numerous types of innate immune cell populations, including ILCs, DCs, macrophages, and occasional eosinophils and mast cells.

Both CD4+ T cells and CD8+ T cells with effector memory phenotype are abundant in LP, while γδ T cells are rarely found. The CD4+ T cell compartment of LP is highly diverse and contains several subsets, such as Th1, Th17 and Tregs51. Furthermore, LP contains plasma cells, which constitute the basis for the secretory sIgA52. IgA are recognized by polymeric immunoglobulin receptors on the basolateral surface of IECs, which shuttle IgA dimers across the cells through a process called transcytosis and release sIgA53. Plasma cells also secrete β-defensins and thereby contribute to the protection from invasion by bacteria into the LP54. ILCs are found in LP with important roles in intestinal immunity, inflammation and GALT development11. Intestinal DCs have a key role in maintaining homeostasis in the gut55. As such, they produce factors that favor tolerogenic responses, which promotes differentiation of Treg cells56. Macrophages in the gastrointestinal mucosa represent the largest pool of tissue macrophages in the body57. During non-pathological conditions, macrophages have inflammation-suppressive phenotypes and thus contribute to homeostasis in the gut58. Eosinophil and mast cells are also present in normal intestinal mucosa and have important physiological roles, such as tissue repair and epithelial integrity as well as influencing peristalsis51.

1.4.2.3 Intraepithelial lymphocytes The intestinal epithelium contains numerous T cells that are located between enterocytes. The T cells are of different phenotypes and are distributed differently in the SI and the LI1,59. As such, IELs are more frequent in SI where they comprise 15-20% of the cells in the epithelium1. The various cell populations of IELs, include conventional αβ T cells with coreceptors CD4 or CD8 and unconventional T cells including TCRαβ coreceptor-negative (or double-negative, DN) T cells and γδ T cells1,60. Conventional CD8+ T cells dominate in the SI, while in the LI there is an almost equal proportion of CD8+ and CD4+ T cells and DN T cells1. γδ T cells are about 10% of IEL in both SI and LI1. Most IELs are antigen experienced T cells with effector memory phenotype. The majority of IELs, regardless of other cell surface molecules, express CD103, also known as αE integrin, which anchors IELs to the epithelial cells61. Many IELs have cytolytic capabilities characterized by the presence of cytotoxic granulae in the cytoplasm62. Additionally, they have other functions, like induction and maintenance of oral tolerance and surveillance of the IECs’ functions60.

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1.6 Pattern recognition receptors The basis for innate immunity relies on pattern recognition receptors (PRRs). PRR are proteins specialized in recognizing evolutionarily conserved structures of components in microbes, known as microbial associated molecular patterns (MAMPs). Most PRRs can also be activated by non-microbial and endogenous signals, which are generated during tissue damage, known as damage-associated molecular patterns (DAMPs). PRRs can be classified into several families based on protein domain homology. Examples are Toll-like receptors (TLRs), nucleotide binding domain, leucine-rich repeat (LRR)-containing receptors (NLRs), RIG-I like receptors (RLRs) and C-type lectin receptors (CLRs). Activation of PRRs leads to production of innate responses that help the host to remove the threat and restore tissue homeostasis63. This includes induction of pro-inflammatory cytokines and regulatory miRNA (described more in Chapter 1.9). Further, PRR activation can lead to cellular responses, such as induction of phagocytosis, autophagy and/or cell death64,65.

PRRs are strategically located in different subcellular compartments. PPRs can be either membrane-bound at the cell surface and at the endosomal membranes or occur free in the cytoplasm. For instance, TLRs and CLRs are found at cell surfaces or in association with endocytic compartments, while NLRs and RLRs are mainly located in the cytoplasm, where they survey for the presence of intracellular pathogens. This distribution of PRR is meant to effectively detect different types of threats and to prevent inappropriate activation. The multiplicity and diversity MAMPs present on microbes and the recognition by numerous PRRs, trigger different signaling pathways and contribute to the complexity and magnitude of innate responses. In addition to sensing danger, PRR also play an important role in the maintenance of epithelial barrier integrity, production of antimicrobial proteins (such as defensins) and transcytosis of IgA to the gut lumen63.

1.6.1 Toll-like receptors The best-known PRRs are members of the TLR family. They are transmembrane glycoproteins characterized by an extracellular N-terminal LRR motif, which recognize ligands, and a cytoplasmic Toll/IL-R homology (TIR) domain that initiates the intracellular signaling pathways in response to receptor activation66. TLRs can recognize a broad range of microbes, such as bacteria, virus, fungi and parasites. Depending of the type and location of TLRs, different regulatory signaling pathways are activated.

There are 10 identified TLRs in humans. TLRs located to within cell membranes (TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10) mainly recognize bacterial products, while TLR, usually located in intracellular membranes (TLR3, TLR7,

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TLR8 and TLR9), appears to be suited for viral detection and recognize nucleic acids66.

Each receptor shows specificity by recognizing distinct bacterial MAMPs. For instance, TLR4 recognizes lipopolysaccharide (LPS), a molecule found in the outer membranes of Gram-negative bacteria67. Activation of TLR4 further involves the accessory molecules myeloid differentiation protein 2 (MD-2) and LPS-binding-protein (LBP) as well as CD14, which bind LPS and act as a co-receptor68,69. TLR5 senses bacterial flagellin, which is the structural component of the flagellum, a locomotor organ that is mostly associated with Gram-negative bacteria70. TLR2 plays a role in recognition of peptidoglycan (PGN) and lipoproteins71. TLRs occur as homo- or heterodimer in different combinations of TLRs, which further increase the variety of PAMPs that can be recognized. For instance, TLR2 can form a heterodimer with either TLR1 or TLR6 and recognize lipoprotein with different lipid moieties72. TLR3 is a receptor for viral dsRNA73, while TLR7 and TLR8 recognize ssRNA from RNA virus74-76. TLR9 primarily recognize unmethylated CpG DNA, which is uncommon in the mammalian genome77.

1.6.1.1 Toll-like receptor signaling pathways There are two well-studied signaling pathways in TLR activation, which depend on the type of TIR-adaptor protein being recruited after receptor activation. The myeloid differentiation primary response gene 88 (MyD88) is the commonly used TLR adaptor for initiating signal cascades, also referred to as the MyD88-dependent signaling pathway. Following activation, MyD88 oligomerizes to form a large signaling platform, which includes several MyD88 molecules, TIR domain–containing adapter protein (TIRAP) and members of the IRAK family proteins. This complex assembles with TNF receptor–associated factor 6 (TRAF6)78 and through further intracellular molecular reactions, NF-κB is activated, which drives the transcription of several genes involved in innate immune responses79. For TLR7, TLR8 and TLR9, the MyD88-dependent signaling can also result in the expression of type I interferons (IFNs), which are important in controlling viral infections80. The other signaling pathway is referred to as the MyD88-independent/TRIF-dependent pathway, which is used by TLR3 or TLR481 and leads to induction of members of the interferon regulatory transcription factor (IRF) family, responsible for the induction of IFNs82.

1.6.2 Other families of pattern recognition receptors NLRs (Nucleotide-binding oligomerization domain-like receptors) are cytosolic sensors with more than 30 members. They are characterized by the presence of a C-terminal LRR domain, a central NLR, and an N-terminal domain

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responsible for signal transduction. These receptors are expressed in many cell types, including immune cells, epithelial cells and endothelial cells. NLRs are divided in subfamilies based on the N-terminal-containing domain, which can mediate signaling, either with the caspase recruitment domain (CARD) or the putative protein-protein interaction (PYRIN) domain. NOD1 and NOD2 are the most studied receptors within this family and can sense components of bacterial outer membranes or cell walls. NOD1, specifically recognizes the peptidoglycan moiety of Gram-negative bacteria, named γD-glutamyl-meso-diaminopimelic acid (iE-DAP)83. NOD2 recognizes the peptidoglycan fragment muramyl-dipeptide (MDP), that is present in the peptidoglycan of both Gram-negative and Gram-positive bacteria84.

NLRPs (NACHT, LRR and PYD domains-containing proteins) are one subfamily of NRLs that contain a PYRIN, instead of a CARD domain. They are involved in the formation of a multiprotein complex, referred to as the inflammasome, which initiate innate immune responses characterized by the secretion of pro-inflammatory cytokines IL-1β and IL-1885. Inflammasome formation is considered to be an important host response to a wide range of diseases, such as inflammatory diseases, cancer, metabolic and autoimmune disorders86.

RLRs (Retinoic acid-inducible gene-I-like receptors, or RIG-I-like receptors) are DEXD/H box RNA helicases that detect the presence of foreign RNA within the cytosol of the cell. They are situated at specific locations within the cytosol where viral- entry or replication can occur and recognize parts of the viral genome components formed as replicative intermediates during viral growth. The RLRs generate activation signals that drive IFNs production and elicit an intracellular immune response to control virus infection87. There are currently three members of RLRs: retinol acid-inducible gene I protein (RIG-I), melanoma differentiation associated gene-5 protein (MD5) and laboratory of genetics and physiology protein (LGP2)88-90.

CRLs (C-type lectin receptors) are a family of membrane-associated receptors, which recognize the carbohydrate structures present on pathogens. There are currently, more than 60 identified CLRs in humans. The extracellular portion of CLR consists of a calcium-dependent carbohydrate recognition domain (CRD). These receptors are widely recognized to play an essential role in antifungal immunity91.

1.6.3 Pattern recognition receptors in human intestine IECs are normally the first to detect the presence of pathogens or secreted bacterial factors in the gut lumen and respond immediately by producing a

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variety of mediators, depending on the type of stimuli. Therefore, IECs represent an important site of crosstalk between the immune system and the microbiota. Members of the following families, TLRs, NLRs, RLRs and CRLs have been detected in the intestinal mucosa92. To avoid unappropriated stimulation, PRR activation is tightly regulated in several ways.

PRRs are generally present at low levels in IECs. For instance, TLR4 as well as its co-receptor MD2 are expressed at low levels in IECs, which is thought to explain the relative hypo-responsiveness to PAMPs, such as LPS93. However, during pathological conditions, for instance in IBD, TLR4 expression is up-regulated94. Similarly, NLRP2, NLRP6 and NLRP8 are up-regulated in autoimmune conditions such as celiac disease95.

Expression of TLRs are generally restricted to the basolateral surface of IECs, such as for TLR5, where it can trigger production of cytokines in response to flagellin that has reached the internal tissue side of the epithelium96. The spatial regulation of this TLR, is thought to ensure that an immune response is only mounted when bacteria have breached the host epithelial layer63. Thus, the consequences of PRRs signaling depend on the site of the cell where the ligand is encountered. It has been shown that, TLR activation through apical or basolateral surface domains produces distinct transcriptional responses. For example, basolateral exposure of IECs to TLR9 ligands results in activation of NFκB, while apical stimulation results in an opposite, inhibitory signaling cascades and further provides intracellular tolerance to subsequent TLRs challenges97. Similar mechanisms may also explain why challenges with bacterial components can differ depending on whether challenge with bacteria are done using cells in ordinary tissue culture compared to differentiated polarized tight monolayers98

The microbial ligands recognized by PRRs are not unique to pathogens but are also expressed by commensal microorganisms. The commensal bacteria are under normal conditions recognized by PRR, such as TLRs and NODs and this interaction plays a crucial role in the maintenance of intestinal epithelial homeostasis99,100. Several protective features following PRR activation have been demonstrated, which include epithelial cell proliferation, maintenance of tight junctions and induction of antimicrobial peptides100-103. By which mechanisms commensal bacteria modulate inflammatory responses in the gut is unclear. Meanwhile, the regulation of PRR expression and how these receptors are spatially distributed within the polarized IECs has been suggested to play a major role. Negative regulators of TLR signaling, such as expression of microRNA, is another mechanism employed by IECs that probably contributes to creating a tolerant environment within the intestine104.

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1.7 Cytokines Cytokines are small soluble signalling proteins that function in cellular communications through ligand and receptor interactions. They are messengers in immune responses and can be produced by many cell types, such as immune cells and epithelial cells. Cytokines can act in an autocrine, paracrine or endocrine manner and therefore can produce both local and systemic effects. Binding to the corresponding receptor(s), they induce intracellular signaling, leading to cellular responses, which include activation, proliferation, and differentiation. Cytokines are pleiotropic and redundant molecules, meaning that the same cytokine can affect multiple cell-types and many cytokines can exert similar actions in the same target cell. They further function in both innate and adaptive immune responses, which further illustrates their complex biological actions.

Cytokines can be divided into families, based on target cells and proposed functions. Interleukins (ILs; cytokines that signal between immune cells), lymphokines (cytokines produced by lymphocytes), monokines (cytokines produced by monocytes/macrophages/DCs), colony stimulating factors (CSFs; cytokines involved in stem cell proliferation and differentiation), chemokines (cytokines with the capacity to attract immune cells), and interferons (IFNs; cytokines that interfere with virus infection) are well-described, partly over-lapping families of cytokines. Further type of classification is based on the outcome immune responses, i.e. whether the cytokine promotes or inhibits inflammation or acts on adaptive immune cells. Representative examples of pro-inflammatory cytokines are TNF-α, IL-1β, IL-6, IL-8 and IFN-γ. IFN-γ is a member of the IFN family and is a typical pleotropic cytokine promoting cell-mediated immune responses, increase in antigen presentation and expression of other pro-inflammatory cytokines105. Expression of pro-inflammatory cytokines results in hallmarks of the inflammation phenotype, which include vasodilation, increased vascular permeability, and influx of immune cells, e.g. monocytes and neutrophils to the site of infection. In contrast, cytokines that suppress the activity of inflammation signaling are called anti-inflammatory cytokines. IL-10 and transforming growth factor-β (TGF-β) prevent host cell damage from excessive inflammation by counteracting the functions of pro-inflammatory cytokines106.

IECs are one of the important cellular sources for cytokine production. During infection several different cytokines are produced in order to mobilize the immune system and eliminate the infectious agents. After the elimination and resolution of diseases, the expression of the cytokines is down-regulated and usually returned to normal baseline levels. Therefore, aberrant expression of cytokines, often long-standing production, can give signals that can sustain

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pathophysiological processes. For example, increased production of IL-1β, IL-6, IL-8, TNF-α and CCL20 are associated with several inflammatory disorders, such as IBD and ongoing infiltration of immune cells to the site of inflammation107. Therefore, an understanding of how pathogens can affect cytokine production at sites of infection may explain some of the pathological outcomes in many infections or inflammatory diseases.

1.7.1 Interleukin-1β and Interleukin-18 IL-1β and IL-18 are members of the IL-1 family, currently comprising 11 cytokine members107. IL-1β and IL-18 are considered pro-inflammatory cytokines and are produced by various cell types, including macrophages, neutrophils and epithelial cells108. IL-1β is mainly induced in response to microbial recognition, mediated by activated PRR, like TLRs and NODs109. IL-18, on the other hand is constitutively expressed in nearly all cells in healthy tissues110. Members of the IL-1 family, like IL-1β and IL-18 are typically synthesized as precursors and must be cleaved by enzymes, for example caspase-1 (CASP1), to become active secreted cytokines111. The secretion of IL-1β and IL-18 are part of the inflammasome forming response, which is activated by pathogens and are responsible for the activation of crucial inflammatory responses85.

There are two types of receptors for IL-1: IL-1 receptor type 1 (IL-R1) and IL-1 receptor type 2 (IL-1R2). The heterodimeric complex of IL-1R1 is associated with an IL-1R accessory protein (IL-1RAcP), which together constitutes the functional receptor for IL-1112. IL-1R has a long and conserved cytoplasmic Toll/IL-1R (TIR) domain, while IL-R2 has a short intracellular domain and acts as a suppressor of IL-1 activity, supposedly by competing for IL-1 binding109.

The receptor for IL-18 (IL18R) consists of IL-18 receptor α−chain (IL-18Rα), and the IL-18 co-receptor, termed IL-18 receptor β−chain (IL-18Rβ), which together are required for effective signaling113. Following binding, the complex of IL-18 with the IL-18Rα and IL-18Rβ is similar to that formed by IL-1β and the accessory protein IL-1RAcP107. The activity of IL-18 is balanced by the presence of an inhibitor, called IL-18 binding protein (IL-18BP), which is secreted mainly by immune cells upon pro-inflammatory response to inactivate IL-18. Similar to IL-R2, IL-18BP is considered a decoy receptor that ensures a balance between the amplification of innate immunity and the regulation of cytokine signaling114.

The signal transduction for IL-1β and IL-18 share common features and are mediated through IRAK family members and TRAF6, which activate mitogen-

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activated protein kinases (MAPKs) and the transcription factor NF-κB, resulting in expression of pro-inflammatory cytokines107.

1.7.2 Tumor Necrosis Factor−α TNF-α was initially identified as a factor responsible for necrosis of certain tumor cells115. Now, it is known that TNF-α is a potent inflammatory mediator and plays a central role for induction of inflammation, including cytokine production, expression of adhesion molecules and growth stimulation 116. The multitude of functions elicited by TNF-α makes it one of the most pleiotropic mediators in immune responses107. TNF-α is a classical member of the TNF superfamily of proteins, which includes 30 receptors and 19 associated ligands with diverse functions in cell differentiation, inflammation, and apoptosis117. TNF-α is mainly secreted by activated macrophages, however other cell types, including, T cells, mast cells and epithelial cells can also produce TNF-α118.

TNF-α is synthesized as a transmembrane precursor, arranged in homodimers at the cell membrane. To generate the soluble form, the cytokine is released by proteolytical cleavage by proteases, like the TNF-α converting enzyme (TACE)118. Both the soluble and the cell membrane-bound form of TNF-α are active and have distinct effects in innate responses119. The protease TACE is further involved in the processing of several other cell membrane-associated receptors and can execute the release of soluble forms of receptors, such as TNF-α receptors, which can neutralize the action of TNF-α and thus limit cytokine availability to other cells120.

The receptors for TNF-α are TNF receptor type 1 (TNFR1) and TNF receptor type 2 (TNFR2). The two TNFRs have been reported to mediate distinct biological effects. As such, TNFR2 mediate signals promoting tissue repair and angiogenesis121, while TNFR1 are suggested to support the pro-inflammatory effects of TNF-α107.

1.7.3 Chemokines Chemokines are mainly produced to recruit leukocytes of both innate and adaptive immunity to inflammatory sites. There are 44 chemokines and 23 chemokine receptors in the human genome122. Chemokines are a group of small (8–12 kDa) peptide mediators, classified into four families, based on the positioning of the N-terminal cysteine residue. The four families are: C-, CC-, CXC- and CX3C- families, where C represent the number of N-terminal region cysteine residues and X represents the number of intervening amino acids123. Chemokine signals mainly through binding to members of the G protein–coupled receptors (GPCR) superfamily124.

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The CC family is characterized by cysteine residue located adjacent to one another. The CC chemokine ligand 20 (CCL20) is an example of one such chemokine. CCL20 is a small chemokine of about 8 kDa that is constantly expressed at low levels by epithelial cells in various peripheral tissues125. Immune cells such as activated neutrophils and lymphocytes can also produce this chemokine. Only one receptor, CC receptor 6 (CCR6) for CCL20 is known. The CCR6 receptor is found prominently on immune cells, such as DCs, B cells and T cells, including T cell subsets Th17 and Treg126. In the literature, this interaction is referred to as the CCR6–CCL20 axis, and is implicated in several autoimmune diseases, including IBD127,128.

CXC cytokines can be further subdivided based upon the presence or absence of the amino acid sequence, glutamic acid-leucine-arginine (the ELR motif), prior to the first cysteine. The ELR+ CXC chemokines are: CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8/IL-8). They act mainly through CXCR2 and attracts neutrophils and possess angiogenic properties. Some of the ELR− CXC chemokines are: CXCL4, CXCL9, CXCL10 and CXCL11, which act mainly through CXCR3 and attract lymphocytes, like Th and NK cells and harbors angiostatic properties129.

Interleukin-8 (IL-8) or CXCL8 is one of the first chemokines to be isolated (1987) and is the most studied chemokine130. Originally, IL-8 was named neutrophil chemotactic factor (NCF), because this chemokine mainly attracted neutrophils to sites of infection131. IL-8 can also attract several types of immune cells and is considered a key mediator of the inflammation response132. There are several receptors for IL-8. The most studied are the two GPCRs; CXCR1 and CXCR2133. Following chemokine binding to the receptor, intracellular signaling cascades are activated within target cells, such as neutrophils and induce several cell responses, including chemotaxis and/or degranulation of neutrophil contents134. In addition, IL-8 signaling induces expression of adhesion molecules in target cells, such as the lymphocyte function-associated antigen 1 (LFA-1) and the macrophage-1 antigen (Mac-1), which are required for chemotaxis135.

CX3CL1 is a member of the CX3C chemokine family. This cytokine is mainly produced as a cell membrane protein. Once cleaved from the cell surface it functions as a soluble chemoattractant. CX3CL1 attracts predominantly T and B cells, NK cells, and monocytes via chemokine receptor CX3CR1136.

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1.9 MicroRNA It is now widely accepted that non-coding(nc)RNAs, both small and long, participate in important regulatory functions in cells137. The class of ncRNA, that has gained most attention so far are miRNAs.

miRNAs are a family of small (18-24 nucleotide long) RNAs that regulate gene expression at the post-transcriptional level by targeting mRNA. The first miRNA was discovered in a study of genes involved in the development of the nematode worm, Caenorhabditis elegans in 1993138. So far, 38,589 miRNAs have been identified in humans139. Data analysis predicts that miRNA can regulate more than 60% of all proteins encoded in the human genome140. This enormous impact of miRNA in regulating gene expression is based on the ability of miRNA to target multiple mRNAs. Each miRNA can have hundreds of mRNA targets, while the same target can also be regulated by multiple miRNAs141. It is proposed that each single miRNA has a relatively modest repressive effect on the target mRNA by altering gene expression in an estimated range of 1.2- to 4-fold, suggesting that these regulatory elements do not function as on-off switches, but instead modulate and fine-tune the expression levels of proteins142.

A variety of cellular processes, such as growth, proliferation and differentiation can be influenced by miRNA regulation143. miRNA has been shown to control intestinal epithelial differentiation and barrier function144. Moreover, it is proposed that miRNA contribute to the tissue specificity, as these molecules are expressed in a tissue-dependent manner145. For example, miR-375 is important in the development and differentiation of goblet cells and enteroendocrine cells in the SI146,147. miRNAs are recognized to function as important regulators of immune responses since they seem to target critical intermediates in PRR signaling pathways. Consequently, deregulation of miRNA is linked to various pathological conditions, such as cancer and immunological disorders143,148,149. The tissue-specific expression of miRNAs and their differential expression in a variety of diseases, have prompted investigation into their potential use as diagnostic biomarkers150.

More recently, numerous miRNAs were reported to be present in the extracellular environment, in association with protein complexes, or within exosomes, small vesicles that bud off the cell surface into the extracellular space 151,152. Exosomal miRNAs from one cell can be taken up and processed by other cells at both local and distant sites. This may represent a novel mode of communication between cells151.

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1.9.1 Biogenesis, maturation and function of microRNA In the genome, genes for miRNAs can be located in introns of both coding and non-coding transcripts and occasionally in exons153. There are two well-characterized steps in the process of forming mature and functional miRNA. In the first step, miRNA genes are transcribed by RNA Polymerase II in the nucleolus into long primary sequence miRNA (pri-miRNA) and are processed by a microprocessor complex, which is composed of Droscha, (a ribonuclease protein) and an RNA-binding protein, named DiGerorge syndrome critical region 8, (DGCR8)154,155. The pri-miRNA is processed into about 70-100 nucleotide hairpin-shaped precursor miRNA, referred to as pre-miRNA. Next, pre-miRNA is exported to the cytosol, through the nuclear pore complex by Exportin-5 (XPO5), for further processing156. In the second step, pre-miRNA is cleaved by a ribonuclease, named Dicer, into about 20-25 nucleotide miRNA-duplex (ds-miRNA)156. A single strand of this duplex is subsequently incorporated into the RNA-induced silencing complex (RISC), which is a large multicomplex with AGO protein as its primary components157. Once incorporated, the miRNA can guide the RISC-complex to a target mRNA and modulate its activity. The target recognition is mediated through the pairing of a 6-8-nucleotide-long sequence in the miRNA (known as the seed sequence) to the complementary sequences within the 3’ untranslated region (3’ UTR) of a target mRNA158. The outcome of binding target mRNA leads to gene silencing, either by inhibition of translation or degradation of the target gene mRNA159.

Figure 2. Biogenesis of miRNAs. Reproduced with permission from Sonkoly & Pivarcsi 160.

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1.9.2 MicroRNA in innate responses to pathogens A role for miRNAs in the response of human cells to bacterial infections was originally deduced from experiments involving the sensing of purified PAMPs by TLRs in immune cells. A study investigating the expression of a panel of hundreds of miRNAs in human monocytes following stimulation of TLR4 by LPS, revealed induction of miR-146a and miR-155 as the foremost significantly up-regulated miRNA161.

The first studies that a live bacterial pathogen could induce changes in the miRNA profile of host infected cells was obtained using the gastrointestinal Helicobacter pylori. Elevated levels of miR-146a and miR-155 were significantly up-regulated in gastric epithelial cells following H. pylori infection which resulted in negative modulation of the bacterial induced inflammatory response162,163. Studies have later demonstrated miR-146a and miR-155 to be important regulatory miRNAs that modulate the inflammatory responses in immune cells in response to bacterial stimuli159,164-168.

Given the importance of miR-146a and miR-155, it is not surprising that aberrant expression of these two miRNAs are also linked to diseases characterized by abnormal immune response, such as autoimmune disorders169,170

microRNA-146a Two transcripts of miR-146 exist, miR-146a and miR-146b, differing only by 2 nucleotides. miR-146a is the most studied transcript. It was shown to be induced by TLR4 in an NF-κB-dependent manner161. Later studies have confirmed the significance of miR-146a in response to other families of TLRs and in other cell-types than immune cells. In epithelial cells, such as keratinocytes, overexpression of miR-146a significantly suppressed the production of IL-8, CCL20, and TNF-α and also functionally repressed the chemotactic attraction of neutrophils171. The primary effect of miR-146a is mediated by targeting key mediators in the MyD88-dependent pathway. So far validated targets of miR-146 include key signaling molecules, IRAK1, IRAK2, and TRAF6172. More recently, CARD10, involved in the GPCR-mediated signal pathways, was also added to the list of miR-146 target genes173. The negative regulatory effects of miR-146a seems to be essential, as mice deficient in miR-146 spontaneously developed autoimmune disorders and early mortality168. Furthermore, miR-146a was found to be important in promoting endotoxin tolerance and promoted protection from bacteria-induced epithelial damage in neonatal mice, via targeting IRAK1 that repressed TLR-induced NFκB activity 174,175.

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Figure 3. A model for the role of miR-146a in TLR-mediated signal transduction. Reproduced with permission from Nahid et al 175.

microRNA-155 miR-155 is recognized as a multifunctional miRNA, because of its numerous targets of signaling molecules involving several types of signaling pathways. For example, miR-155 is induced by cytosolic sensing NODs, which through a TLR-independent pathway regulate activation signaling176. Furthermore, miR-155 was shown to negatively regulate inflammation by targeting MyD88 and consequently reduces IL-8 production in H. pylori infection163. However, miR-155 can target both inducers and suppressors of signaling, suggesting a more complex role of miR-155 in regulating immune responses177. The functions of miR-155 are proposed to be counterbalanced by other miRNAs, such as miR-146178. Even though, miR-146a and miR-155 are co-expressed during TLR activation, differences in expression pattern have been observed, depending on the type and strength of TLR ligand-activation. It is proposed that miR-146 responds to sub-inflammatory stimuli that do not trigger inflammatory responses, while miR-155 is associated with pro-inflammatory transcriptional responses, once the miR-146-dependent barrier has been breached176. Furthermore, miR-155 have been demonstrated to be induced in T cells, B cells and DCs, thus implicating its regulatory functions in adaptive immunity as well179,180.

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It is known that various stimuli can influence miRNA expression either rapidly or in a delayed manner. These findings have led to classification of miRNA as early- or late-response miRNAs181. For instance, miR-155 is rapidly expressed upon LPS-stimulation, while expression levels of miR-146a are accumulated in a delayed manner and therefore most likely function at later stages to regulate inflammatory responses182.

Despite the large number of studies on miRNA so far, not all targets and functions of miRNAs, including miR-146a and miR-155, have been found. Furthermore, less is known about miRNAs in human tissues other than immune cells.

1.10 Bacterial interactions with the gastrointestinal tract The vast majority of bacteria in the gut do not cause disease under normal circumstances. Orally introduced bacteria including pathogens must overcome numerous physical, chemical and biological factors produced by the host in order to establish colonization or successful infection in the gut. The acidic environment of the stomach and other soluble factors like bile salts and antimicrobial peptides provide an initial barrier to prevent bacteria from reaching the intestine. Moreover, the bacteria must overcome interactions with mucins and mucosal secretory IgA in the mucous layer. In addition, a pathogen must compete for nutrients and physical space with resident microorganisms in the gut. Some enteric pathogens have developed virulence mechanisms that allow them to breach the intestinal epithelial barrier. These bacterial strategies can be either non-invasive, like causing toxin-induced diarrhea or invasive with abilities to manipulate the host cells from within and damage the intestinal mucosal tissues. One important part of the bacterial pathogenesis is the ability to modulate innate host cell responses, either by the bacteria itself or by means of secreted factors.

1.11 Vibrio cholerae One important human intestinal pathogen is the Gram-negative bacterium Vibrio cholerae causing the severe diarrhea disease, cholera. The physician Pacini Filippo first described the disease-causing bacterium in 1854 when the Asiatic Cholera Pandemic in 1846-63 came to Florence. Through his autopsy and histological examinations of cholera victims, he discovered a highly motile and flagellated comma-shaped bacillus, which he described as a Vibrio, derived from the Latin word vibrāre, meaning to ‘to vibrate’.

The clinical presentation of V. cholerae infection ranges from asymptomatic colonization to cholera gravis, which is the most severe form of the disease.

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Figure 4. Electron microscopy of highly motile Vibrio cholerae which possesses a single polar flagellum as a locomotion organelle. (courtesy of Prof. S.N. Wai).

An infectious dose ranges from 106 to 108 bacterial cells that is transmitted through contaminated food and water183. Bacteria that were recently shed from infected individuals appear to be transiently more infectious than organisms isolated from the aquatic environment183. Symptomatic patients may shed V. cholerae before the onset of illness and usually continue to shed organisms in the feces for 1 to 2 weeks184. The bacterium exists in the SI after a half day to three days of incubation, before the onset of symptoms, which include stomach cramps and vomiting and is followed by diarrhea. Symptoms may progress and lead to death if acute rehydration therapy is not administered185. The total volume loss over the course of the disease may be up to 100 percent of the body weight186. Epidemiological data estimates that 2.9 million cases and 95,000 deaths occur annually in countries with endemic cholera187.

Killed whole-cell oral cholera vaccines (kOCVs) are becoming an important tool in cholera prevention, although they provide limited protection. A recently published systematic review and meta-analysis of kOCVs showed overall efficacy to be 56% during the first year with reduction to 39% protection in the third year, and even less protectiveness in children under 5 years of age188. Natural infection with V. cholerae is known to provide protection against subsequent disease, although the mechanism of this protective immunity is not well understood185,189.

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V. cholerae strains may be identified serologically based on the O antigen of its LPS. Only bacteria of serogroups O1 and O139 have caused pandemic cholera, due to their ability to produce cholera toxin (CT) and the toxin-coregulated pilus (TCP). The O1 serogroup is subdivided into two phenotypically distinct biotypes, classical and El Tor biotype, the latter remains the dominant disease-causing strain globally184. Remaining serogroups of V. cholerae are collectively called non-O1 non-O139 V. cholerae (NOVC).

The bacterium exists as a normal component of some marine ecosystems, surviving without a human host. V. cholerae has been found in association with zooplankton and other non-mammalian hosts, which serve as reservoirs during periods between epidemics190.

1.11.1 O1 and O139-associated virulence factors V. cholerae possesses a number of factors, which assist the bacterium in several processes, such as motility, colonization of the host and other processes inside the host. CT and TCP represent the primary virulence factors in V. cholerae191. The genes for CT are encoded by a filamentous bacteriophage (CTXΦ), which is integrated in the genome of the bacterium192.

Since it was first discovered in 1959193, CT has been extensively studied. CT is an A-B toxin, consisting of a central enzymatic A-subunit (CT-A) and a binding B-subunit (CT-B)194. After secretion, the CT-B subunit binds to GM1 ganglioside receptors located in lipid rafts on the plasma membrane of the intestinal epithelium. In the cytosol, through a series of intracellular reactions, CT-A activates adenylate cyclase, which increases cAMP levels within the host cell, leading to an efflux of chloride ions, through activation of cAMP-dependent chloride channels. This results in an imbalance of electrolyte transport across the cell membrane and enhances the osmotic movement of water into the intestinal lumen, causing severe diarrhea and dehydration195. The diarrhea flashes the V. cholerae bacteria out of the host body, where it can colonize other human hosts183.

Although CT is the primary toxin responsible for the watery diarrhea, toxigenic strains also carry other factors that may contribute to disease. This notion is supported by the fact that volunteers inoculated with a CT deletion mutant (Δctx) strain of V. cholerae still developed diarrhea196. Further, despite being considered a non-inflammatory disease, cholera is associated with inflammatory changes of the human small intestine, suggesting that inflammatory components are released during the early stages of acute infection197-201. Other virulence factors than CT are therefore likely to be involved. The role of innate immune responses to infection and potential activation of immune regulatory

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mechanisms have so far been subjected to only a limited number of investigations in clinical patient material.

1.11.2 Non-O1 and non-O139 strains and associated virulence factors Serogroups other than O1 and O139, collectively named NOVC, lack the ability to produce CT and TCP202-204. Despite the absence of these major toxins, NOVC strains can still cause cholera symptoms. Although these strains are not associated with pandemic disease, they represent an emerging threat and are of increasing concern in endemic areas and worldwide205. More than 25% of documented V. cholerae infections were attributed to NOVC strains in Kolkata, India, in 2003202. Molecular analysis from hospitalized V. cholerae patients in China, revealed NOVC to be an important contributor to incidence of diarrhoeal infections203. Therefore, infections by these strains are more clinically relevant than previously thought. Furthermore, the Haitian cholera outbreak in 2010, estimated to have caused more than 7,000 deaths and have sickened over half a million people, revealed that an NOVC strain was solely responsible for 21% of the clinical cases206. Importantly, there is a lack of understanding about the causes of NOVC infection and the subsequent host immune response. Molecular analysis from hospitalized patients with confirmed NOVC-induced-diarrhea, detected a gene named hemolysin or V. cholerae cytolysin gene (hly/vcc), as a common gene of potential virulence202,203,207.

In contrast to O1 and O139 serogroups, NOVC strains can cause extra-intestinal infections, including wound-infection, otitis, meningitis, bacteremia and even septicemia207-210. Invasive infections have also been reported to affect the Northern part of Europe. Some NOVC-strains like V:5/O4, a clinical isolate that caused a severe sporadic outbreak in Sweden in 2004, expresses factors, such as VCC. In Sweden and Finland, the highest incidence of NOVC infections were observed in 2014, causing ear infections, wound-infection and bacteremia, presumably because the victims had been bathing in sea water211.

Vibrio cholera cytolysin One subset of toxins are called pore-forming toxins (PTFs), as they have the ability to form transmembrane pores on biological membranes, and consequently disrupt the epithelial integrity212. Bacteria can utilize PTFs as tools to harvest vital nutrients, promote growth and dissemination within the host213,214.

V. cholerae Cytolysin (VCC), a PFT of V. cholerae, encoded by the hly/vcc gene is widespread across both pathogenic and environmental strains of V. cholerae, suggesting that VCC provide an advantage to the pathogenesis of the organism215. Although the precise role of VCC in clinical infection is unknown,

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the toxin is considered a major virulence factor in V. cholerae infection with NOVC strains216,217.

VCC is synthesized and secreted in a precursor form, as a 79 kDa monomer (pro-VCC) and requires proteolytic cleavage, which is achieved by proteases (both bacterial or mammalian) to a 65-kDa mature form218,219. In the presence of cholesterol-rich membranes, mature VCC forms heptameric oligomers, which penetrate the membrane and form pores with an internal diameter of 1-2 nm, in a highly intricate process220,221.

In response to PFTs, host eukaryotic cells have developed multiple highly conserved processes to sense and mount a defense or even repair functions of damaged cells 222. VCC causes lysis of erythrocytes and therefore, it has been traditionally designated as V. cholerae hemolysin221. However, red blood cells are not necessarily the physiological host target cells of VCC and probably the action towards other cell types are more important in their function as virulence factors. VCC have been shown to cause vacuolization, necrosis or autophagy in numerous cell lines depending on the dosage221,223,224.

The understanding of the structures and mechanisms of action of PFTs, like VCC, can lead to new opportunities for development of therapeutic measures. For example, small drug molecules or engineered antibodies can be designed to target specific sites in the structures of PFTs213. Prior to this study, innate immune response to VCC in a polarized and differentiated intestinal cell monolayer had not been investigated before.

Protease of V. cholerae (PrtV) Bacterial proteases have important roles in microbial physiology, metabolism, and pathogenesis 225. In V. cholerae, the protease production is under the control of the transcriptional regulator HapR226. PrtV protease, as described by Vaitkevicius et al, 227 was found to be encoded by the prtV gene, which is located downstream of the hemolysin (hly) gene loci. Despite its proximity to the hly gene, PrtV, it did not show any cytotoxic effect in an infant mouse model228. Although in a study using a non-mammalian host model, PrtV had an essential role in killing the nematode Caenorhabditis elegans and was shown to be necessary for survival from grazing by flagellates and ciliates229. In a human intestinal model, PrtV was not cytotoxic, but instead reduced the production of the inflammatory cytokine IL-8, induced by as yet unidentified secreted component(s) of V. cholerae229.

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1.12 Bacterial outer membrane vesicles In all domains of life, i.e., Eukaryote, Archaea and Bacteria, cells produce and release membrane-bound material, often referred to as exosomes, microvesicles or membrane vesicles230. Bacterial membrane vesicles (MVs) are spherical membrane-enclosed particles released from Gram-positive and Gram-negative bacterial species231. All types of bacteria produce MVs, including both pathogenic and commensal bacteria as part of their normal behavior232. Outer-membrane vesicles (OMVs) from Gram-negative bacteria have been mostly studied.

Outer membrane vesicles (OMVs) are spherical, 20 nm to 250 nm in size, membranous structures, consisting of a lipid bilayer, that is released from the outer membrane of Gram-negative bacteria233. The vesicle membrane is usually composed of lipopolysaccharide (LPS), glycerophospholipids and outer membrane proteins (OMPs). The regulation of OMVs biogenesis is not completely understood, although several models exist234. Moreover, the OMVs production can increase under environmental stress235,236. Since their discovery, almost 50 years ago237, OMVs have emerged as factors causing damage or eliciting protective effects in infectious diseases. The utility of OMVs in the development of effective diagnostic tools and as vaccines against diverse bacterial species has gained recent interest238.

OMVs contain PAMPs, which are recognized by PRR in the host cells. A recent proteomic and biochemical analysis revealed that V. cholerae OMVs contain numerous bacterial components, including DNA, RNA, LPS, various proteins, including enzymes, and peptidoglycan239.

Secreted OMVs function as a transportation system for delivery of important factors into host cells at both local and distal sites, without the requirement of close contact between the bacterium and the host240. The effects of OMVs rely in their capacity to mediate transport of bacterial components to the interior of eukaryotic host cells236. The mechanism for such intracellular delivery usually suggests adherence of OMVs to the host cell followed by internalization. The exact mechanism of the uptake of OMVs is still not completely understood. Several different pathways for OMVs to enter host cells have been described. These include clathrin-dependent and clathrin-independent endocytosis, caveolin mediated and caveolin-non-mediated endocytosis as well as fusion with eukaryotic plasma membranes241. Host uptake mechanisms seem to depend on the composition and size of the OMVs and the type of bacteria they are derived from234.

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In the case of V. cholerae, isolated OMVs have been shown to be internalized by lipid raft-dependent endocytosis242. H. pylori OMVs also enter host cells via lipid rafts and intact vesicles were detected in the intracellular compartment of gastric epithelial cells243. Furthermore, OMVs can fuse with cell membranes in human airway epithelium and thereby deliver their contents into the host cell cytosol, as was observed for Pseudomonas aeruginosa OMVs244.

Many gastrointestinal pathogens, such as Enterotoxigenic E. coli (ETEC), Campylobacter jejuni and H. pylori use OMVs as cargo for the delivery of virulence factors245-248. More recently, secreted V. cholerae OMVs were shown to mediate transport of biologically active VCC protein249. However, it is known that challenge with bacterial components can differ depending on whether stimulation is performed with cells in ordinary tissue culture compared to cells in differentiated polarized tight monolayer98.

Some studies show that OMVs can dampen the production of inflammatory mediators in host cells. OMVs derived from Porphfyromonas gingivalis instantly degrade the IL-8 protein250 and therefore modulate the neutrophil recruitment activity of IL-8. Further, OMVs have been described to limit cytokine response to secondary stimuli. Pre-treatment with OMVs from Brucella abortus limited the production of the pro-inflammatory cytokines TNF-α and IL-8 in response to TLR ligands in a monocytic cell line251. In human gastric epithelial cells, H. pylori, OMVs showed different cellular responses, depending on the dosage of OMVs following stimulation, i.e. lower doses increased cell proliferation, while higher amounts led to growth arrest and increased IL-8 production252. Stimulation by OMV derived from Neisseria meningitidis resulted in production of anti-inflammatory IL-10 in a whole blood experimental model253. To date, only one study has investigated the role of OMVs and the expression of miR-146a in infection. According to that study, OMVs from Legionella pneumophila facilitated bacterial replication in a monocytic cell line in an miR-146a-dependent manner, leading to dissemination of the bacteria within the host254.

In summary, OMVs play important roles in delivering bacterial factors into the host cells and shape their cellular responses. An important element is their ability to modulate the immune system in an activating or inhibitory direction. To investigate the role of OMVs from V. cholerae as immune modulators is one of the aims of this thesis.

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2. AIMS

The over-all aim of this thesis is to understand the epithelial immune response in the human intestine upon infection with the small intestinal pathogen Vibrio cholerae. The specific aims were:

• To gain knowledge of the local immune reaction in the small intestinalmucosa of patients with V. cholerae infection by determining levels ofinflammasome cytokines, pro-inflammatory cytokines, chemokines andimmunomodulatory miRNAs and their target genes in biopsies ofcholera patients at acute and convalescent stages of disease.

• To get an understanding of how V. cholerae bacteria and their releasedproducts influence the epithelium and contribute to the immunereaction of the intestinal mucosa of cholera patients by performingchallenge experiments in the tight monolayer in vitro model of humanintestinal epithelium.

• To investigate the effects of V. cholerae OMVs on the function ofintestinal epithelium, including permeability, expression ofimmunomodulatory miRNAs and their target genes in the inflammatoryresponse, and expression of inflammation mediating cytokines usingthe tight monolayer model for intestinal epithelium.

• Performing challenge experiments in the tight monolayer model inorder to get an understanding of how the pore-forming, cytolytic toxinVibrio cholerae cytolysin (VCC) influences the function of intestinalepithelium when secreted in its soluble form and when released inassociation with OMVs. Further, to explore whether protease of V.cholerae (PrtV), which can degrade VCC, can also moderateinflammatory effects by VCC.

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3. METHODOLOGICAL CONSIDERATIONS

This section gives a brief overview of the material and methods applied in this thesis, together with rationales and comments. For a more detailed description of the material and methods used, please see papers I-III.

3.1 Clinical material To explore the immune status and role of miRNA in V. cholerae infection, we used duodenal biopsy specimens from patients at the acute and at the convalescent stage of infection. The specimens were obtained through our collaboration with the International Centre for Diarrheal Disease Research in Daccha, Bangladesh (icddr,b).

The clinical material consisted of duodenal biopsies from six adult male patients, aged 22-46 years, with culture-confirmed V. cholerae O1 infection. They all suffered from a moderate-to-severe degree of dehydration. Paired duodenal biopsies were collected from each patient at the acute stage (day 2 post-onset of disease) and at day 21 post-onset of disease. Biopsies were taken from the second part of the duodenum using a standard endoscope. Duodenal biopsies were also collected from five healthy adults, defined as individuals who did not have a history of diarrheal illness in the preceding three months. Biopsies were directly stored in Trizol for RNA extraction and further qRT-PCR analysis or embedded in Optimal-cutting-temperature compound (OCT-Tissue-Tek) for in situ hybridization.

The ethical review committee of icddr,b approved the study and all participants gave written informed consent to participate in the study.

3.2 Polarized tight monolayers of human intestinal epithelial cells To explore the role of bacteria and secreted factors, we used human intestinal epithelial cell polarized tight monolayers of T84 cells. V. cholerae is an intestinal pathogen that infects the small intestine and it would therefore have been preferable with a polarized tight monolayer composed of epithelial cells of the small intestine as an in vitro model. However, there are only a few intestinal cell lines and they cannot form polarized tight monolayers. Thus, they do not enable measurement of trans-epithelial resistance (TER) as an indication of cell permeability255. We performed some attempts with the small intestine-like colon carcinoma cell line, (HT29) and human fetal small intestine cell line (FHs74int). However, even when grown as a confluent monolayer, they did not

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become tight and adequate TER values that indicate formation of tight junctions were not obtained.

Hence, tight monolayers of T84 cells are presently one of the best, established in vitro models for investigating human intestinal epithelium interactions with a variety of different stimuli, such as bacteria, isolated bacterial factors or cytokines. The monolayers show great resemblance to normal intestinal epithelium since they are polarized, connected by tight junctions, form microvilli, express a dominating glycocalyx component and carcinoembryonic antigen at the apical side, and even harbor cells that have differentiated into goblet-like cells, yielding the possibility of production of secreted mucins98.

Figure 5. T84 human colon carcinoma cell-line shows spontaneous differentiation that leads to the formation of a tight monolayer on the porous membrane (left). Electron micrograph of T84 monolayer cells (right) with microvilli of normal height (thin arrows) at the apical side and tight junctions connect the cells (fat arrows). Adopted with permission from Ou et al 98.

3.3 Bacterial work and effects on target cells To explore the effects of the V. cholerae pathogen and secreted factors on intestinal epithelium, we choose to work with V. cholerae El Tor C6706 in Paper I and Paper II, since it remains the dominant disease-causing strain globally185 The El Tor C6706 strain is a well-established laboratory strain of V. cholerae and is extensively used in the research field. In Paper II, we further obtained clinical isolates of V. cholerae El Tor strain, which allowed us to compare possible differences between the laboratory strain and relative newly isolated clinical samples. To study host-microbe interactions in the absence of CT and TCP, we used V:5/04 isolates (in Paper III), which caused a severe sporadic outbreak of Vibrio infection in Sweden in 2004.

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To investigate the effects of V. cholerae bacteria and its secreted virulence factors on the host cells, bacteria and culture supernatants were isolated from strains grown in different culture media and at different growth phases. It is known that different culture conditions can affect the production of virulence factors in V. cholerae256. We studied the effects of different kind of media compositions, such as Luria-Bertani (LB) and brain-heart-infusion (BHI) broth. It is known that high titers of VCC are observed in bacteria grown in more nutrition-rich media, such as BHI broth257. We therefore used conditions that could promote VCC production in order to study its effects on target host cells. All bacteria strains and clinical isolates were grown under the same cultural conditions prior to challenge experiments, as optimized by previous work in our lab98. Furthermore, our aim was to use an optimized number of bacteria or released bacterial factors to provoke an epithelial innate response, without causing signs of cell death. A cell cytotoxicity assay was used measuring lactate dehydrogenase (LDH) release from T84 monolayers. Innate responses are normally activated within minutes or hours, and therefore optimal time-points were set to induce detectable mRNA and cytokine production, without causing signs of cell damage or death in the T84 in vitro model. LB, BHI or tissue culture mediums were used as control media in sham-treated monolayers, depending on the media-containing stimuli used in the experiments, respectively.

3.4 TER measurement techniques To estimate the permeability of tight monolayers, we used transepithelial electrical resistance (TEER or TER) measurements. TER is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of single-layer epithelia, such as T84 monolayer cells. TER values are robust indicators of the integrity of the cellular barriers255,258. One advantage of this method is that measurements can be performed in real time without inflicting cell damage.

There are several factors that can affect TER values, such as temperature and medium composition. Therefore, to minimize variations, all our measurements were conducted under similar conditions. To avoid any TER changes due to temperature fluctuation, Transwell-plates were equilibrated from 37°C to room temperature at least 20 minutes before and after challenge experiments prior to an initial measurement. TER values at >1000 Ohm were used in challenge experiments as they are correlated with a polarized and differentiated intestinal epithelium98.

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Figure 6. The transepithelial electrical resistance (TER) increases gradually as tight monolayers are established by T84 cells grown on semipermeable polycarbonate membranes in a two-chamber cell culture system. Graphs shows a summary of 10 independent experiments. Data are represented as mean ± 1 SD. Adapted with permission from Ou et al 98.

3.5 Gene expression analysis of total RNA using real-time qRT-PCR

3.5.1 Determination of expression levels of target-genes, cytokine and chemokine mRNAs by using real-time qRT-PCR To analyse the gene expression levels of selected mRNAs and for validation of mRNA of interest from genome-wide hybridization bead array, we used real-time quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) based on Taqman EZ-technology. In the Taqman EZ-technology, sequence-specific primers bind to a specific mRNA and transcribes it to specific cDNA and are thereafter directly amplified by real-time quantitative PCR. Sequence-specific primers are useful for determination of small amounts of mRNA in an excess of unrelated mRNA259. The qRT-PCR analyses were run in triplicate with 45 cycles in the qPCR step. As a cut of level, a CT-value of 35 was applied. The mRNA concentrations were normalized to the 18S rRNA concentration in the sample by calculating mRNA copies/18S rRNA unit for mRNAs analyzed by assays with an RNA copy standard and by calculating the ΔCT between the CT-value for the mRNA species and the CT-value for 18S rRNA (CTsample−CT18S rRNA) for mRNAs analyzed by commercial Taqman gene Expression Assays.

This method was chosen for several reasons: It is a one-step procedure, and thus reduces possible errors and risks for contamination. By using sequence-

Days

Ohm

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specific primer, the mRNA of interest is specifically transcribed to cDNA from the total RNA sample in the reaction mixture, which brings about very high sensitivity and good chances of detecting mRNAs present in small amounts.

3.5.2 Determination of expression levels of microRNAs using real-time qRT-PCR Expression levels of miRNAs in T84 monolayer cells and patient biopsies were determined by real-time qRT-PCR using the TaqMan MicroRNA Reverse Transcription (RT) Kit and TaqMan MicroRNA (TM). There are other methodologies that can be applied to examine miRNA expression. However, qRT-PCR is considered the gold standard for studying specific detection of selected miRNAs260-262. The samples were analysed in duplicates using the ABI prism 7900 HT sequence detection system. To ensure accurate ∆CT values, expression analysis of the housekeeping gene RNU48 was continuously assessed in the same PCR plate together with the selected miRNA gene of interests. Both the RT step and the real-time quantitative PCR were consequently analysed in the same day of analysis. These procedures were applied to minimize the risk that differences were due to technical alterations between the runs. Expression levels were estimated by calculating the relative quantity (RQ) by using the 2(-∆∆ct)-method263.

3.5.3 Housekeeping genes for stable normalization The qRT-PCR signal of an mRNA or miRNA depends on the amount of starting material, i.e. total RNA, which can differ between samples and experimental conditions. This is especially relevant in clinical material where the samples have been obtained from different individuals and will result in misinterpretation of the results, unless adequate normalization analysis is applied. Therefore, it is of great importance to have a stable housekeeping gene, where the amounts of mRNAs and miRNAs in the samples can be related to and obtain normalized expression levels. Features of an ideal housekeeping gene should be: 1) expressed at a constant level in cells regardless of activity state, 2) similar levels in different cell-types and tissues, 3) unaffected by experimental conditions.

18S rRNA is a reliable normalization RNA for mRNA expression There are three commonly used housekeeping genes that are used for normalizing qRT-PCR data: β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S rRNA. However, several studies have reported that levels of ACTB and GAPDH are highly variable among cell types, at different activation states of cells, during cell differentiation and in inflammation. 18S RNA, on the other hand, was shown to be more stably

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expressed265,266. We used 18S rRNA for normalization of mRNA expression levels.

RNU48 rRNA is a reliable normalization RNA for miRNA expression Normalization with a small nucleolar RNA, RNU48 (also referred to SNORD48), which is a well-studied and a suitable reference gene for normalization for miRNA expression analysis262,267. A recent study comparing the most commonly used reference genes for miRNA analysis demonstrated that RNU48 was the most stable RNA for normalization in qRT-PCR analysis260.

3.5.3 Gene expression analysis using genome-wide hybridization bead array To address the ability of VCC to induce additional markers of inflammation in the polarized T84 cell monolayer, we performed genome-wide hybridization bead array analysis of VCC challenged and sham-treated monolayer by Illumina technology.

A brief description of the procedure follows: Total RNA is reverse transcribed into cDNA by using an oligo-dT primer. The oligo-dT primer binds to the poly-A tail that is only present on mRNA, hence, only coding RNA is transcribed into cDNA. This is followed by cDNA purification, in vitro transcription to cRNA, amplification of cRNA, and finally cRNA purification. The in vitro transcription step incorporates biotin-labeled nucleotides. Thus, the output of this step is a biotin-labeled cRNA. The labeled cRNA is then hybridized to the oligo probes on the beadchips and stained with streptavidin-Cy3. Finally, fluoroscence emission from Cy3 is quantitatively detected by Illumina Beadstation. Sentrix HumanRef-8_v3 expression BeadChip (more than 18,000 annotated genes with more than 25,000 probes) were used to analyze T84 tight monolayer samples. The purity of total RNA and cRNA were determined by measuring optical density at 260 nm (OD260) and OD280 in a NanoDrop. The integrity of cRNA was determined by agarose gel analysis (Bioanalyser). The raw data was analyzed using Illumina Beadstudio software (version 3.1). Background was first subtracted from the data and then normalized using Beadstudios cubic spline algorithm. Significant differential expression was calculated using Beadstudio software by applying the Illumina Custom algorithm. Raw and analyzed data files from a hybridization bead array were submitted to Gene Expression Omnibus (GEO) DataSets (www.ncbi.nlm.nih.gov/geo).

The analysis was focused on up-regulated genes. Therefore, potentially down-regulated genes were excluded. A signal≥ 60 arbitrary units was used as the cut-off. Only genes with an RQ ≥ 1.8 between signals in VCC challenged and sham-treated samples were considered as candidate genes up-regulated by VCC95.

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Data presented from genome-wide screening in this thesis were further validated using specific qRT-PCR assays for the selected mRNA.

3.6 In situ miRNA hybridization To detect miRNA in small intestinal biopsies, we used in situ miRNA hybridization (ISH) in Paper II. ISH is a useful tool to visualize and detect the presence of miRNAs in both tissues and isolated cells. This method can provide evidence on the cellular source of miRNAs present in tissue. For detection of miRNA in the duodenal epithelial tissues, Digoxigenin (Dig)-labeled miRCURY locked nucleic acid (LNA) was used. LNA probes are designed to increase the binding strength of the probe making it possible to hybridize to single-stranded RNA as short miRNAs and thereby allow detection of miRNAs in tissue sections with high specificity268. The probes in our study were, the miR-146a complementary probe hsa-miR-146, the miR-155 complementary probe hsa-miR-155, the RNU6 complementary probe hsammurno (positive control) and Scramble-miR (negative control). ISH has its advantages and limitations. One limitation is that miRNAs may occur in low copy numbers in tissues, which make them difficult to detect by ISH. However, ISH is useful for addressing the cellular presence of miRNA and therefore was suitable for the aims of Paper II.

3.7 Determination of amounts of cytokine protein To measure amounts of cytokine protein produced in T84 monolayer cells in response to challenges with the bacterial culture supernatant or VCC protein, we performed enzyme-linked immunosorbent assays (ELISA). ELISA is a fast, simple and accurate assay that allows detection of proteins with high sensitivity. Concentrations of IL-8 and CCL20 proteins were determined in tissue culture medium collected from the lower chamber of challenged monolayer cultures or from cell-lysates of challenged monolayers cells. The total cell-lysate of monolayer cells contains both soluble cytoplasmic proteins and membrane-bound proteins. Analyses of increases were done by comparing concentrations in culture medium collected from the lower chamber of challenged monolayer to concentrations in the lower chamber of sham-treated monolayers and in cell lysates of challenged monolayers compared to control monolayers.

3.8 Isolation and characterisation of outer membrane vesicles OMVs were isolated as described in earlier studies249. Briefly, culture supernatants were isolated by using low-speed centrifugation to remove bacterial cells. The supernatants obtained was the filtered through a 0.22 µm filter followed by ultracentrifugation yielding a crude OMV preparation. The ultracentrifugation step efficiently sediments OMVs, but also some bacterial cell

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membrane fragments, flagella and non-vesicle-associated secreted proteins. These contaminants may affect host immune response and should be removed from the crude preparations in order to study “true” host responses to OMVs. For instance, the flagellum plays a well-documented role in innate immunity and as a dominant antigen of the adaptive immune responses269. In our studies, isolated vesicle samples were further subjected to density gradient centrifugation249 in order to obtain purified OMVs.

3.8.1 Nanotracking analysis Differences in size and concentration of OMV preparations are a factors that might affect target host cells responses241. Therefore, analysis of these factors provides further understanding of the function of OMVs. Analysis of OMVs particle concentrations is suggested to be important, since it has become evident that quantification of OMVs based on measurement of total protein concentration can be problematic. Thus, the number of particles per amount of protein can vary between OMV preparations derived from different bacterial strains and also OMVs isolated from bacteria subjected to different growth conditions. To obtain better control of these factors, OMV-preparations were characterized by Nanotracking analysis (NTA) was performed. NTA was performed using the ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and its corresponding software (ZetaView 8.02.28). Size and concentration of OMVs were assessed by ZetaView according to the manufacturer’s instructions270. Average values were calculated based on five measurements for each OMV sample.

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4. RESULTS AND DISCUSSION

4.1 Rationale of project Disease caused by V. cholerae remains a major threat to human health worldwide and there is still a necessity to understand mechanisms of virulence to develop preventive or therapeutic strategies. V. cholerae NOVC strains lacking V. cholerae toxins CT and TCP are emerging as more clinically relevant than previously thought. Most importantly, there is a lack of understanding of the mechanism behind these inflammatory-attributed infections, as well as the subsequent host immune response.

A previously uncharacterized molecular strategy that is exploited by bacteria to control host cell defense might be to regulate host miRNA expression. Therefore, we choose to study the role of regulatory miRNAs in intestinal epithelial cells during V. cholerae infection and monitor the immune responses. Using clinical duodenal biopsies, from patients during acute infection of V. cholerae and an in vitro model for intestinal epithelium to study consequences of infection at the epithelial lining, we could delineate the impact of released components by the bacterium. Further, we chose to study the effects of secreted VCC and the contributing role of PrtV in immunomodulation. Our objective was to elucidate the role of regulatory miR-146a and miR-155 in V. cholerae infection and the role of the secreted factors, PrtV, VCC and OMVs in intestinal epithelial responses.

4.2 Innate epithelial responses to secreted Vibrio cholerae cytolysin and PrtV of Vibrio cholerae Bacteria can communicate with their host or other organism by secreting macromolecules into the environment271. PrtV was previously identified as a secreted protease involved in protecting V. cholerae from non-mammalian hosts, while in the T84 intestinal cell model, the protease seemed to degrade the ability of secreted factor(s) to induce an IL-8 cytokine response229. Inspired by the contrasting effects of PrtV in different host models, we aimed to identify the secreted component(s) of V. cholerae that induced the epithelial inflammatory response and investigate whether VCC could be a substrate for the PrtV protease.

4.2.1 Effects of culture supernatant of V. cholerae on epithelial cells To address the question of whether PrtV directly affects the intestinal epithelium, cell-free supernatants of overnight cultures in LB broth of wild-type and PrtV deletion mutant (∆prtV) were added to the apical side of T84 tight

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monolayers. Outcomes in permeability, as measured by TER, and cytokine expression levels, measured by qRT-PCR, and secretion of IL-8 measured as concentration in the lower chamber of tight monolayer culture medium by ELISA, were compared to that of the wild-type El Tor strain (C6706wt) and unstimulated (control) cells. The results showed that culture supernatant isolated from the ∆prtV mutant caused a significant drop (about 80%) in TER compared to tight monolayers challenged with supernatant of C6706wt or control monolayer cells challenged by LB broth only, indicating a clear increase in permeability (Figure 1, Paper I). Furthermore, supernatant of the ∆prtV mutant bacteria caused a more than 20- fold increase in mRNA expression levels of neutrophil attractant IL-8 and pro-inflammatory TNF-α, while supernatant of C6706wt only caused a marginal increase in expression levels of these cytokines. Increases in IL-8 mRNA levels were accompanied by increased levels of IL-8 protein in the culture medium of the tight monolayers indicating that the epithelial cells indeed secrete this chemokine. To exclude the possibility that the observed pro-inflammatory responses were a consequence of CT, we performed experiments with culture supernatant derived from a deletion mutant of the cholera toxin gene (∆ctx) and showed that it did not cause any changes compared to the supernatant of the isogenic C6706wt. Thus, we speculated that PrtV was involved in decreasing the stability of secreted factor(s) to cause a pro-inflammatory response in T84 monolayer cells.

Next, we investigated the presence and amount of PrtV and VCC in the culture supernatants used in the challenge experiments by immunoblot. We detected both VCC and PrtV in the culture supernatants. However, supernatants of ∆prtV mutant contained more VCC than supernatants of the C6706wt, when bacteria were grown in LB broth. Thus, we hypothesized that supernatants lacking PrtV protease allowed more stable VCC protein to target host cells and induce an inflammatory response when the bacteria were grown in LB broth.

We next assessed whether culture media composition could influence the activity and amount of PrtV and VCC secreted by the bacteria. Therefore, the same challenge experiment of T84 tight monolayer as previously described was conducted using a rich media for growth of the bacteria, i.e. BHI broth. The C6706wt culture supernatant induced significantly higher levels of IL-8 and TNF-α compared to when the same bacteria were cultured in LB broth, indicating that cytokine repression in wild-type strain was over-ridden in BHI broth. Immunoblot analysis of supernatants of bacteria cultures in BHI revealed decreased amounts of PrtV in C6706wt compared to cultures in LB and in parallel the exact opposite results were found for VCC. The amount of PrtV seemed therefore reduced when the bacteria were cultured in BHI, suggesting

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that the absence of PrtV allowed more un-degraded VCC or other factors(s) to induce an inflammatory response in the monolayer cells.

To investigate whether VCC was the only factor contributing to the induction of pro-inflammatory cytokines, we performed a challenge experiment with BHI culture supernatant of a deletion mutant of VCC (∆vcc) strain from the same genetic background as the wild type. In contrast to C6706wt, supernatant of the ∆vcc mutant strain had no effect on TER or expression of IL-8 and TNF-α as these cytokines remained at the same levels as in control monolayer cells. This data suggested that VCC is essential for the induction of the pro-inflammatory responses and the increased membrane permeability observed in this in vitro system. This notion was further supported by the fact that expression levels of IL-8 and TNF-α as well as alternation in TER could be reproduced using wildtype and genetically modified strains from other serogroups of V. cholerae, such as NOVC strain V:5/O4 (Paper 1 and unpublished results), suggesting that the capacity to cause VCC-induced inflammatory responses in epithelial cells could be a universal feature in Vibrio strains.

4.2.2 Role of Vibrio cholerae cytolysin To investigate whether stimulation with VCC alone could induce inflammatory responses, we incubated tight monolayers with serial dilutions of the purified VCC protein at concentrations ranging from 0.26 ng/ml to 20 mg/ml. Changes in permeability, cytokine production as well as cytotoxicity, measured as LDH release, was assayed. The results showed a concentration-dependent response following VCC-stimulation in T84 monolayer cells. At concentrations as low as 6.4 ng/ml, a significant drop in TER (more the 50%) was observed, without signs of cell death or cytokine production. Upregulation of IL-8 and TNF-α was observed at 160 ng VCC/ml, where expression values reached their highest levels. The increased IL-8 and TNF-α mRNA levels were inversely correlated with TER up to this point. At higher VCC concentrations, a decrease in cytokine mRNA levels was observed and, in parallel, an increase in cell death.

The cytotoxic and damaging effect of VCC is in line with previous studies demonstrating the role of VCC in inducing toxicity in the gut and causing lethality in mouse and rabbit models217,272. Therefore, at higher concentrations the activity of VCC may generate epithelial destruction that can allow other V. cholerae factors to penetrate the mucosa197,198.

To identify additional markers of inflammation in response to VCC, we performed genome-wide expression analysis to detect up-regulated cytokines in T84 monolayer cells.

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Figure 7. Dose-dependent effects of VCC on permeability, cell viability and cytokine levels in intestinal epithelium tight monolayers. TER (a, left y-axis), cytolysis (a, right y-axis), and IL-8 (b) and TNF-a (c) mRNA levels in polarized tight monolayer cells challenged with serial dilutions of pure VCC protein in LB broth at the apical side for 5 hours at 37 °C. Results are from three independent experiments and presented as mean +/- 1 SD for each concentration. Rectangles indicate results at 160 ng VCC/ml (From Figure 5, Paper I).

Data obtained from genome-wide screening were further validated using specific qRT-PCR assays. The results showed that, in addition to IL-8 and TNF-α, expression levels of CCL20, CXCL1 CX3CL1, and IL-1β were significantly induced in intestinal monolayer cells in response to non-toxic concentrations of VCC (Paper I, Paper III and unpublished results). Although, it cannot be certain that all these mRNAs are translated into proteins, ELISA analysis showed that this indeed was the case for IL-8 and CCL20. Increased levels of IL-8 protein was secreted into the basolateral compartment. CCL20 was not highly secreted but instead was detected in lysates of challenged monolayer cells, suggesting elevated levels of cell bound CCL20 compared to unstimulated cells (Paper I and unpublished results).

Upregulation of IL-1β may suggest inflammasome activation in IECs. Toxin-induced inflammasome activation was previously reported for other PFTs, like aerolysin in Gram-negative Aeromonas hydrophilia273. The upregulation of the chemokines CCL20, CXCL1 and CX3CL1 suggested strong signaling for recruiting of lymphocytes to the site of inflammation. Therefore, these results highlight the possible involvement of IECs in supporting induction of adaptive immune response following VCC exposure. In a recent clinical screening, among several hundred immune reactive antigens in V. cholerae, VCC was indeed one of the top candidates eliciting high adaptive immune reactivity274. Our results suggest that, VCC is an important virulence factor with potential high immunogenicity in V. cholerae disease.

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4.2.3 Post-translational modulation of inflammation by PrtV In order to investigate the ability of PrtV to degrade the inflammation-inducing VCC, we co-incubated these two proteins before challenge experiments in the intestinal epithelium in vitro model. We found that the VCC-induced inflammatory responses were completely abolished in the presence of PrtV. Within 5 minutes PrtV degraded the majority of VCC protein without any remaining adverse inflammatory effects on the monolayer cells (Figure 6, Paper I).

To investigate the possibility that PrtV influences the haemolytic activity of VCC, we tested LB broth culture supernatants of ∆prtV and the deletion mutant of hapR (ΔhapR), the regulator of secreted proteases, for their abilities to lyse red blood cells relative to that of the wild-type strain. Both ∆prtV and ΔhapR mutant strains demonstrated increased hemolytic activity compared to the wild-type, indicating that hemolytic activity is in fact regulated by the transcription regulator HapR, and also directly by PrtV protease (Figure 3, Paper I). Taken together, these results indicate that HapR represses hemolytic activity by activating the transcription of PrtV, which degrades the hemolysin VCC.

The bacteria seem thus able to modulate the level of inflammation caused by VCC. The clinical significance of this modulation remains to be clarified. It would however be of interest for the V. cholerae bacterium to modulate and fine-tune the effects of VCC and thereby evade very strong immune responses during infection. The difference in pro-inflammatory activity in cultures supernatant of bacteria grown in different media, suggest that this can be an adaptation for diverse environments. Since V. cholerae resides in harsh aquatic environments, that differ radically from the environments in the human small intestine, it must be important for the bacteria to evolve different persistence strategies. The dual role of PrtV in V. cholerae could therefore be a key adaptation for survival in the different host organisms.

The amount of VCC and PrtV that are produced during V. cholerae infection in humans is unknown because the human gut is too complex and therefore difficult to recreate in vitro. It is possible that at a certain time point during infection, the content in the gut favors a milieu with decreased protease activity, and thereby allowing VCC to alter the epithelial integrity and induce inflammatory responses. It is further possible that once the bacterium and/or its released factors have penetrated the epithelial barrier into the tissues, additional virulence factors and mechanisms may come into play. Since an inflammatory component in V. cholerae infections have been confirmed in clinical cases198,275, it is likely that VCC plays an important role in human infections.

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4.2.4 V. cholerae cytolysin induces interleukin-1 receptor activated kinase 2 To study expression of regulatory miRNA in response to VCC, we analyzed expression of miR-146a and known target genes of miR-146a, which included signaling molecules IRAK1 and IRAK2. The results showed that inflammation-inducing VCC was not correlated with induction of miR-146a or miR-155 in T84 monolayer challenge experiments (Paper III). Interestingly, IRAK2 but not IRAK1, was significantly elevated in response to VCC, suggesting a role of IRAK2 in the intracellular signaling pathway upon stimulation by VCC (Paper III). The fact that VCC caused increased levels of IRAK2 suggests that it can indeed cause severe inflammation, since studies in mice showed that IRAK2 is associated with sustained inflammation and LPS-induced shock276. Taken together, it is possible that VCC may play a role in overcoming regulatory mediators of the innate immune system and promote a more severe and invasive infection, as observed in the clinical manifestation of NOVC strains.

4.3 Cytokines and microRNA expression in small intestinal mucosa during acute Vibrio cholerae O1 infection

4.3.1 Analysis of cytokine mRNA expression To better understand the immune status to V. cholerae O1 infection, expression levels of selected cytokines in duodenal mucosa of six cholera patients was analyzed using qRT-PCR. Paired biopsies were obtained at two consecutive time points for each patient during infection, i.e. at the acute stage and the convalescent stage. We included analysis of cytokines and chemokines known to be induced in epithelial cells during intestinal inflammation based on previous cytokine profile in response to VCC (Paper I) and in vivo studies of immune responses in inflamed human mucosa277. These included pro-inflammatory cytokines (TNF-α and IL-6), inflammasome markers (IL-1β and IL-18) and chemokines (IL-8/CXCL8, CXCL9 CXCL10 CXCL11 and CX3CL1). In addition, pro-inflammatory cytokines expressed by lymphocytes (IL-17A and IFN-γ) were also analyzed, since these two cytokines are known to be produced by intraepithelial lymphocytes in the inflamed small intestinal mucosa of patients with active celiac disease277,278. Furthermore, these cytokines were analyzed, since T helper cells of individuals who had gained immunity through natural V. cholerae infection produced these cytokines in vitro following challenge with V. cholerae antigens199.

The results showed increased levels of IL-8, CXCL9 and IL-1β at the acute stage of infection compared to the convalescent stage of cholera patients, i.e. expression levels of these cytokines changed with disease activity. Of these, the increase of IL-8 was statistically significant. The results from the investigation

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of cytokine expression in biopsies are summarized in Table 1 of Paper II. The observation of elevated IL-8 levels and its role in neutrophil recruitment is supported by previous findings on elevated IL-8 in lamina propria lymphocytes, derived from duodenal biopsies at acute stage of V. cholerae O1 infection199. Furthermore, histological observations have detected the presence of neutrophils during the acute stage of V. cholerae infection198. Elevated levels of IL-8 were also observed in response to VCC in in vitro challenge experiment in monolayer cells (Paper I).

Collectively, our results indicated inflammation of low-grade when estimated as cytokine mRNA levels in the acute stage. Expression levels for TNF-α and IL-6 was not altered during infection and no changes related to disease activity were seen for the expression levels of T cell cytokines. These data did not corroborate previous work with small intestinal biopsies from V. cholerae infected patients and in vitro analysis, where upregulation of TNF-α, IL-6, IL-17A and IFN-γ was reported at the protein level199. Regardless, we could still detect a transient and weak inflammatory response in the gut at the acute stage of infection, which supported previous work demonstrating the presence of inflammatory components in acute V. cholerae infection198,200,201,275.

4.3.2 Induction of regulatory microRNA in the intestinal epithelium As levels of pro-inflammatory cytokines were only marginally elevated in acute infection of V. cholerae and further declined in the convalescent stage, we hypothesized that induction of miRNAs may be involved as negative regulators in the early stages of inflammation. To address the question whether miRNAs are induced during clinical infection, the same samples that were analyzed for cytokine expression were also analyzed for expression levels of miR-146a and miR-155. In addition to paired biopsies of cholera patients, we also analyzed expression of miRNA compared to biopsies from healthy controls.

We showed that expression of both miR-146a and miR-155 in the duodenal mucosa were significantly up-regulated at the acute stage of V. cholerae infection, compared to healthy controls. To minimize the effects of biological variations, we further analyzed the levels in each patient in relation to disease activity, i.e. paired samples from acute and convalescent stages. This analysis further confirmed that miR-146a and miR-155 were significantly higher at the acute stage of infection compared to the convalescent stage for each patient in the study (Figure 2, Paper II).

Next, we aimed to determine the cell type(s) responsible for the up-regulation of miR-146a and miR-155 in the duodenal mucosa at acute stage of V. cholerae infection. Therefore, we visualized the expression of these miRNAs using in situ hybridization of duodenal biopsies of a subgroup of patients in the study.

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Figure 8. The relative quantities (RQ) of miR-146a and miR-155 decrease at the convalescent stage in patients with V. cholerae O1 infection. Lines connect the RQ-values at acute and convalescent stage of the same patient. P-values for statistically significant differences are given. Adapted from (Figure 2, Paper II)

We found that both miR-146 and miR-155 were easily detected in the mucosa of patients with V. cholerae infection at the acute stage, while no expression was detected in the convalescent stage. Interestingly, miR-146a and miR-155 were detected mainly in the intestinal epithelium and only a few positive scattered cells were seen in the lamina propria (Figure 3, Paper II). It has previously been reported that activated immune cells express miR-146a and miR-155142,161, making it likely that lamina propria immune cells and lymphocytes within the epithelium may also provide a source of miRNA in the patient biopsies. However, the strong detection of miR-146a and miR-155 in the epithelium demonstrates that the intestinal epithelium probably is the most important compartment of production miR-146a and miR-155.

That miR-146 and miR-155 may function as regulators of inflammation in the cholera patients was suggested by the finding that significantly up-regulated levels of miRNA in each patient correlated with a low grade of inflammation in the acute stage of infection. This proposes that V. cholerae may alter host regulatory RNA and modulate cytokine responses. To our knowledge, this is the first study demonstrating the upregulation of miRNA during clinical V. cholerae infection. The regulatory role of miRNA in bacterial infections has been studied before. In H. pylori infection, elevated expression levels of miR-146a and miR-155 negatively regulated production of pro-inflammatory cytokines and chemokines162,163.

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4.3.3 Analysis of targets for miR-146a in the intestinal mucosa As we identified significant increase of miR-146 and miR-155 in all duodenal biopsies collected at the acute stage of infection, we next analyzed expression levels of known mRNA targets for miR-146a. The results showed that the levels of miR-146a targets such as IRAK1, TRAF6 and CARD10 were lower in the acute stage as compared to the convalescent stage in only two, three and four patients, respectively. There was a clear trend towards downregulation, although, it did not reach statistically significant levels.

However, as duodenal biopsies were obtained at only two consecutive time-points during infection (day 2 and day 21), we do not know the expression levels of mRNA targets prior to day 2 and in the time span between the two time points. It is possible that pro-inflammatory responses were more rapidly produced at the onset of infection, as many innate responses are initiated within hours. Furthermore, other studies have shown that there is a delayed onset of miR-146a target gene regulation after miR-146 induction182. The long lasting and sustained expression of miR-146a and transient expression of pro-inflammatory cytokines has been demonstrated before in other epithelial cells169. The kinetics expression of miR-146a could therefore explain the unchanged expression levels of miR-146a target genes for some of the patients in our study.

The absence of significant down-regulation of targets in elevated miR-146a tissues, has also been observed in other studies. For example, increased levels of miR-146 in synovial fibroblasts of rheumatoid arthritis patients, did not correlate with decreased levels of IRAK1 and TRAF6170. Similarly, miR-146a did not cause significant suppression of IRAK1 or TRAF6 in lung alveolar epithelial cells279. Another explanation is that the mRNA target genes for miR-146a are differently expressed depending on cell types and tissues161. Furthermore, whether alterations at the protein level of the target genes occurred in the duodenal biopsies is not known because it was not investigated.

Taken together, our results suggest that the immune response at the acute stage of cholera is mainly innate and to a large extent originates from epithelial cells. The moderate cytokine expression levels and the induction of miR-146a and miR-155 indicated that the V. cholerae have the capacity to induce regulatory miRNAs that limit the inflammatory response.

4.3.4 In vitro analysis of V. cholerae and its secreted factors To address the question of whether live bacteria per se or factors released to culture broth was the causative agent for the elevated levels of miR-146a and miR-155, as observed in our in vivo analysis, we performed challenge

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experiments in vitro in polarized T84 monolayers. We included live bacteria isolates from two of the patients in the study and the laboratory wild-type V. cholerae O1 El Tor strain C6706.

The results showed that challenges with live wtC6706 bacteria induced significant levels of miR-146a and miR-155 in intestinal monolayer cells, while the clinical strains VC08 and VC09, induced significant expression of miR-155 only (Figure 6, paper II). Live bacteria did not cause inflammatory responses, as expression levels of IL-1β, CXCL9 and IL-8 were comparable to unstimulated control cells. In contrast, VCC-containing culture supernatants from clinical isolates induced an inflammatory response and did not induce expression of regulatory miRNAs. The absence of regulatory miR-146a and miR155 was also observed after challenges with VCC (Paper I and III).

It seems that the intestinal epithelium receives contradictory signals during infection, i.e., pro-inflammatory signaling from secreted factors, including VCC and inflammation-limiting signaling from the bacterial cells. The variation in magnitude of inflammatory responses between patients may therefore reflect differences in the amount of secreted VCC and PrtV as well as the milieu in the gut. The ability of the bacteria to induce regulatory miR-146a and miR-155 in surface epithelium of host cells may favour survival of the bacteria at the epithelial lining which subsequently may lead to tissue penetration.

4.4 Effects of released outer membrane vesicles To study the response of OMVs, we challenged T84 monolayers cells with V. cholerae OMVs derived from the NOVC strain V:5/04. Since crude preparations of OMVs contain non-vesicular components such as flagella filaments, which can affect immune host responses, we further purified OMVs by density gradient centrifugation. To investigate the role of OMV-associated VCC, we analyzed OMVs with (wt-OMVs) and without VCC-cargo (∆vcc-OMVs). Nanoparticle tracking analysis (NTA) and Transmission microscopy (TEM) was used to estimate the size and particle concentrations in each OMV preparation. The ability of OMVs to alter epithelial permeability, cytokine production and levels of regulatory miRNA was assessed.

4.4.1 Tightening of epithelia Interestingly and rather unexpectedly, challenge with purified wt-OMVs caused a significant increase in TER (20 %) compared to sham-treated controls and TER remained unchanged in monolayers challenged with purified Δvcc-OMVs (Figure 2, Paper III), suggesting that OMVs have a positive, tightening effect on the intestinal epithelium. It is interesting to note that OMVs carrying VCC caused significant increase in TER, while soluble VCC (as shown in Paper I and

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Paper II) caused a marked decrease, suggesting that the two forms affect tight junction structures differently. The mechanism by which OMVs can reduce gut permeability, i.e. increasing TER, is not known. However, it was recently demonstrated that OMVs from a probiotic E. coli strain caused similar increase in TER by reinforcing tight junction structures in T84 monolayer cells280. Activation of TLR2 has been reported to positively alter barrier function in IECs and it was previously shown that TLR2 is present in T84 cells281,282. Therefore, one might speculate that NOVC-derived OMVs mediate a protective-barrier phenotype through the involvement of TLR2. However, V cholerae OMVs are normally internalized into host cells, therefore it is also possible that intracellular PRRs are involved in the observed phenotype.

4.4.2 Modulation of cytokine responses Since purified V. cholerae OMVs were shown to positively alter epithelial integrity by increasing TER, we next investigated whether OMVs could modulate cytokine responses in the T84 tight monolayer model. We have previously shown that culture supernatants of V. cholerae bacteria and purified VCC cause increased expression levels of IL-8, TNF-α and IL-1β mRNAs in monolayer cells. Furthermore, it was also shown that T84 monolayers constitutively express detectable levels of mRNA for CCL20 and high levels of the inflammasome cytokine IL-1895. Therefore, cytokines IL-8, TNF-α, CCL20, IL-1β and IL-18 were selected for investigation in T84 monolayer cells in response to OMVs.

The results showed that challenge with OMVs did not cause induction of the investigated cytokines (Figure 2, Paper III). mRNA levels for TNFα, IL-8 and IL-1β remained unchanged after challenges with wt-OMVs. Notably, challenges with the ∆vcc-OMVs caused a significant 1.4-2.0-fold decrease in the level of CCL20 and IL-18 mRNAs. The absence of pro-inflammatory cytokines and the down-regulation of IL-18 and CCL20 suggest that OMVs may prevent the ability of IECs to recruit T cells to the site of infection and evade inflammasome activation. The innate responses of OMVs derived from both wild-type and ∆vcc mutant bacteria are different in a way that could not be directly explained by the presence or absence of VCC. One explanation for the differences may be due to an alteration in composition and size of the OMVs. OMVs of the VCC deletion mutant had both higher numbers of vesicles per mg protein and a smaller, more uniform size than OMVs of the wild-type. It is known that entry of OMVs into host cells can dependent on several factors such as composition and size because different up-take mechanism exist242. It can be speculated that the size and/or composition of the OMVs may use different entry- and/or sub-cellular trafficking pathways within the host cell leading to different immune modulatory responses.

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4.4.2 Induction of regulatory microRNA-146a Intrigued by the ability of OMVs to cause a decrease in cytokine expression levels in monolayer IECs, we investigated whether OMVs could induce up-regulation of immunomodulatory miR-146 and miR-155. The results showed that both wt-OMVs and Δvcc-OMVs induced significant expression levels of miR-146a (Figure 3, Paper III). The induction of miR-146a seemed not depended on VCC-cargo since it was induced by OMVs of both the wild-type and the VCC deletion mutant. It is worth mentioning that miR-146a can target multiple key mediators in the NFκB pathway, therefore a significant increase of miR-146a may have major effects on the overall NFκB-dependent cellular signaling responses. However, no decrease in the expression levels of the miR-146a target genes IRAK1, IRAK2, and TRAF6 were observed after OMV-challenge. In Paper II and Paper III, we show that levels of IRAK1, IRAK2 and TRAF6 are increased in the T84 monolayer cells upon challenge with soluble components released by V. cholerae. Thus, it seems that miR-146a prevents up-regulation rather than cause down-regulation of these signaling molecules in IECs and thereby limit or suppress cytokine responses.

That OMVs induced elevated expression levels miR-146a, suggest that they are anti-inflammatory. This notion is in line with the finding that challenge with OMVs, either with or without VCC-cargo, did not cause increases in levels of any of the inflammatory cytokines analyzed, i.e. IL-8, TNF-α, CCL20, IL-1β, and IL-18. The finding that OMVs without VCC-cargo even suppressed the baseline levels of mRNAs for CCL20 and the inflammasome cytokine IL-18 further strengthen the notion that OMVs are anti-inflammatory. Down-regulation of CCL20 together with IL-18 suggests reduced capacity to recruit and activate intraepithelial lymphocytes (IELs) that normally execute immune protection at the epithelial lining95,277. Thus, in vivo exposure of the epithelium to OMVs might reduce the defense capacity of the two cell-types of the intestinal epithelium that together build a protective barrier towards the gut lumen, i.e. the epithelial cells and the IELs. In line with this hypothesis, we observed that patients with V. cholerae O1 infection at the acute stage of disease expressed miR-146a and miR-155 in the SI-epithelium and had only a weak cytokine response in their intestinal mucosa with no increases in IFN-γ or IL-17A, that are typically produced by activated IELs (Paper II).

By releasing OMVs the bacteria can thus manipulate the host immune responses without requiring direct contact with the target cells. To our knowledge, this is the first observation that V. cholerae OMVs may regulate cytokine responses via modulation of host miRNAs. By evading the immune system, the bacteria can thrive within the host and may even cause severe disease, as observed in the case of NOVC strains of V. cholerae.

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4.5 Vibrio cholerae infection and the role of microRNA and released components – summary of results and hypothesis In this thesis, the role of IECs in response to V. cholerae infection and secreted factors of this bacterium were explored by using in vivo analysis of duodenal biopsies during clinical infection and by in vitro analysis of T84 cells in polarized monolayer cells.

In Papers I and III we investigated the role of secreted VCC and PrtV in interactions with target host cells. By using culture supernatants of genetically modified strains of V. cholerae and soluble, pure VCC in challenge experiments in vitro we showed that VCC elicits a sustained and full-blown immune response. Our results suggest participation of the epithelium by recruitment of granulocytes through secretion of IL-8, recruitment of T lymphocytes by secretion of CCL20, general acute inflammation by TNF-α and inflammasome activation through IL-1β and IRAK2. VCC should be regarded as an important virulence factor in V. cholerae pathogenesis, especially in non-CT-producing V. cholerae strains. Further, we showed that PrtV protease, secreted by the bacterium itself, efficiently degraded VCC and thus provides the ability to modulate the inflammatory response in intestinal epithelium.

In Paper II, we investigated the role of the epithelium during clinical infection of V. cholerae in patients. By analyzing expression of cytokines and miRNA in the small intestinal biopsies of patients during V. cholerae O1 infection, we showed that miR-146a and miR-155 were significantly up-regulated and correlated inversely with cytokine levels at the acute stage of infection. More importantly, the epithelium was shown to be the major source of the immunomodulatory miRNAs as demonstrated by in situ hybridization. Further, in vitro complementary analysis showed that live bacteria but not released factors could induce miR-155 in T84 tight monolayer epithelial cells.

In Paper III, we investigated the impact of isolated OMVs from a V. cholerae NOVC strain on IECs in the tight monolayer in vitro model. We show that OMVs: induced regulatory miR-146a, were found not inflammatory and could even suppress base-line levels of CCL20 and IL-18. OMVs, either with or without VCC-cargo, seemed to have opposite effect to the strong inflammatory soluble VCC protein. These results give important clues about the immunomodulatory role of OMVs in V. cholerae infection, as induction of miR-146a may set the threshold of activation in intestinal cells and thus facilitates infection of the bacteria.

In summary, we showed that V. cholerae harbor capacities to dampen inflammatory responses, by themselves, by releasing OMVs and secreting

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soluble factors such as PrtV and at the same time raise inflammatory responses by soluble VCC. These results can be further added to the overall complexity of immunomodulation strategies deployed by V. cholerae. The capabilities of V. cholerae to induce and regulate inflammation in the intestinal epithelium may represent a relevant strategy for the bacteria to promote survival and cause successful infections.

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5. CONCLUSIONS

• Vibrio cholerae cytolysin (VCC) was shown to be solely responsible for induction of inflammatory response in the tight monolayer in vitro model of human intestinal epithelium. Thus, challenge with VCC caused increased epithelial permeability and production of the pro-inflammatory cytokines IL-8, TNF-α, CCL20 and IL-1β and IRAK2, a key signaling molecule in the IL-1 inflammasome pathway. IL-8 mRNA levels were up-regulated in the duodenal mucosa of patients at acute stages of V. cholerae infection and followed disease activity with decreased levels at the convalescent stage. IL-1β mRNA levels showed a similar trend. Thus, tight monolayers appear to be a reasonably good in vitro model for the intestinal epithelium of the patients. Taken together these findings suggest that the patient can raise a strong inflammatory response against VCC during clinical infection and that VCC could be the cause to the gastroenteritis symptoms seen in patients with intestinal infection by NOVC strains.

• Protease of V. cholerae (PrtV) degrades the VCC protein and PrtV treatment of VCC could abolish the inflammatory effects of soluble VCC on tight monolayers, suggesting that the bacterium might modulate the immune response of the host by production of PrtV.

• Both miR-146a and miR-155 were significantly up-regulated in the epithelium of the duodenal mucosa of patients at the acute stage of V. cholerae and the epithelium itself was the main site where these miRNAs were expressed. Live V. cholerae bacteria themselves caused up-regulation of miR-155 in the tight monolayer model, while OMVs of both wild-type and the VCC deletion mutant of V. cholerae, caused up-regulation of miR-146a. Taken together, these data suggest that the expression of miR-146a and miR-155 observed in the patients are caused by bacteria and OMVs reaching the epithelial lining with consequent down-regulation of an innate defense reaction thereby paving the way for manifest infection.

• The mechanisms by which miR-146a and miR-155 down-regulate the inflammatory response in intestinal epithelial cells is still not known. However, mRNA levels of the miR-146a target genes IRAK1, TRAF6 and CARD10 increased during the convalescent stage in three out of six cholera patients, supporting the notion that the MyD88/NFκB pathway is involved. Challenge of tight monolayers with culture supernatants of

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V. cholerae and/or soluble VCC caused significant increases in the levels of IRAK1, IRAK2 and CARD10 mRNAs. This suggests that inflammatory responses in intestinal epithelial cells is normally associated with increased levels of these target genes and that the miRNAs actually prevent this increase leading to less inflammatory responses. The relatively weak inflammatory response in the duodenal mucosa of cholera patients at active stage of disease most likely reflects that it is dampened by regulatory miRNAs.

• V. cholerae bacteria seem to be immunomodulatory during infection by affecting the epithelial lining of the small intestine in several ways, 1) the bacteria themselves induce immunomodulatory miRNAs in the epithelial cells, 2) the bacteria release OMVs that induce immunomodulatory miRNAs and tighten the epithelial junctions, and 3) the bacteria secrete PrtV, which degrades the pore-forming, inflammation-inducing toxin VCC. These features may all contribute to the capacity of V. cholerae O1- and O139 strains with CT as well as NOVC V. cholerae strains that lack CT to cause severe intestinal infection.

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6. ACKNOWLEDGEMENTS

There are several people that I would like to explain my gratitude to:

First, I would like to express my gratitude to my supervisor, Professor Marie-Louise Hammarström for giving me this great opportunity to do my PhD studies. Thank you for support thorough all these years, for your patience and encouragement and for believing in me and sharing your knowledge of Immunology.

Professor Sun Nyunt Wai, my co-supervisor. Thank you for your interest and support with all the projects through all these years, as well as for introducing me to the exciting world of bacterial OMVs.

Professor Sten Hammarström. Thank you for your interest and thoughtful and valuable suggestions into my thesis and projects through the years.

Gangwei Ou, one of the co-authors. Thank you for your support and for teaching me to grow monolayer.

Anne Israelsson, tack för din ovärderliga hjälp, med alla metoder som jag fått lära mig av dig, med alla problem som du löst, och alltid haft tid för mig när jag behövt hjälp, under och utanför arbetstid. Tack för att du lyssnat på mig och att du alltid varit vänlig mot mig.

Tahmin Shirin, another of the co-authors. Thank you for a good cooperation with the biopsy project and Dr Fidausi Qadri at icddr,b for providing us with the clinical material used in this study.

Kristina Lejon, min examinator. Tack för att du fått mig att hålla mina immunologiska kunskaper på topp.

Lena Hallström-Nyhlén. Tack för din hjälp under alla dessa år och för din vänliga inställning varje gång när jag behövt hjälp.

Mariann Sjöstedt. Tack för din hjälp att du lärde mig om cell-odling och det laborativa arbetet.

Kyaw, Pramod and Rituparna, three of the co-authors. Thank you for your collaboration.

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Jag vill tacka alla medarbetare och doktorander på Immunologen som gjort det trivsamt genom åren. Till Greg, för våra trevliga pratstunder både på jobbet och utanför. Radha och Viqar, som jag delat rum med, tack för er ständigt trevliga närvaro på jobbet. Tack till Maria, David, Lina, Mia, Therese, Eva, Sofia, Torgny, Basel, Constantin, Mattias, Shrikant, Priscilla och Andy. Tack Mari för ditt intresse för min doktorandutbildning. Alla kollegor på Klinisk immunolog. Tack Eva för din tekniska hjälp med att mäta vesiklarna. Tack till Lucia och Vladimir för trevliga diskussioner och för att ni delat med er eran kunskap om immunologi.

Mårten Strand, tack för alla dessa trevliga luncher och vetenskapliga diskussioner genom åren. Och för att du tog tiden att läsa min avhandling.

Till alla Vänner, för alla kvalitativa och mycket trevliga stunder tillsammans både på jobbet och utanför.

Jag vill tacka min stora familj. Till mamma Eva, och pappa Josef, för er kärlek och omsorg och för att ni stöttat mig att flytta till Umeå för att studera och fullfölja mina mål. Mickhael, min broder, tack för att du är här i Umeå och förgyller helgerna med dina kulinariska förmågor. Till Abir och Maria-Therese, jag är så tacksam att ni är mina underbara systrar. Till alla mina farbröder och fastrar. Till Jeanette och Zahra, jag vet allt ni gjort för mig.

Emelie – Tack för ditt värdefulla stöd, för att du gjort det möjligt för mig att ta mig igenom många hinder längs vägen. För att du tog dig tiden att läsa hela min avhandling och gav mig värdefulla kommentarer. Jag hoppas jag kan vara ett lika bra stöd för dig, när du snart ska göra samma sak.

This work was financially supported by grants from the Swedish Research Council-Natural Sciences and Engineering Sciences, the TORNADO-project within the 7th EU framework program theme, the Kempe Foundations, the Found of Donations to the Medical Faculty, Umeå University and a fellowship for combined medical practice- and Ph.D. studies from the County of Västerbotten.

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