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World Tuberculosis Day 2017 ARTICLES SUPPLEMENT

CONTENTSREVIEW: Understanding the pathophysiology of the human TB lung granuloma using in vitro granuloma techniques Future Microbiol. Vol. 11 Issue 8

COMMENTARY: Mechanisms of mycobacterial transmission: how does Mycobacterium tuberculosis enter and escape from the human host Future Microbiol. Vol. 11 Issue 12

EDITORIAL: New tests for detection of Myobacterium tuberculosis infection: sufficient to meet the WHO 2035 targets? Future Microbiol. Vol. 11 Issue 9

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1073ISSN 1746-0913Future Microbiol. (2016) 11(8), 1073–1089

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REVIEW

Understanding the pathophysiology of the human TB lung granuloma using in vitro granuloma models

Sudha Bhavanam1, Gina R Rayat2, Monika Keelan3, Dennis Kunimoto4 & Steven J Drews*,5

1Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, Alberta, Canada 2Department of Surgery, Surgical-Medical Research Institute, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta,

Canada 3Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, Alberta, Canada 4Department of Medicine, University of Alberta, Edmonton, Alberta, Canada 5Provincial Laboratory for Public Health, Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, Alberta,

Canada

*Author for correspondence: [email protected]

Tuberculosis remains a major human health threat that infects one in three individuals worldwide. Infection with Mycobacterium tuberculosis is a standoff between host and bacteria in the formation of a granuloma. This review will introduce a variety of bacterial and host factors that impact individual granuloma fates. The authors describe advances in the development of in vitro granuloma models, current evidence surrounding infection and granuloma development, and the applicability of existing in vitro models in the study of human disease. In vitro models of infection help improve our understanding of pathophysiology and allow for the discovery of other potential models of study.

First draft submitted: 6 January 2016; Accepted for publication: 17 May 2016; Published online: 9 August 2016

KEYWORDS • dormancy • granuloma • host factors • infection • in vitro models • microbial factors • Mycobacterium tuberculosis • new discoveries

Tuberculosis (TB) remains a major human health threat, with one in three individuals in the world infected and approximately 1.5 million deaths worldwide each year [1]. The Mycobacterium tuberculosis complex comprises several human and animal associated species and subspecies, with human TB primarily caused by M. tuberculosis (Mtb) and M. africanum [2,3]. The shift between latent and active infection requires an understanding of host and bacterial dynamics within the milieu of a granuloma [4], which is influenced by bacterial and host factors. Thus, it is no surprise that the disease is more prominent in populations of immunosuppressed individuals (e.g., AIDS patients) [5]. The need for immunotherapeutic vaccines and new antimycobacterial agents [6–8], highlights the importance of studying Mtb in the context of a rational infection model. Mtb uses various methods in its attempts to evade the host immune system, including the prevention of phagosome-lysosome fusion/maturation, avoidance of lysosomal acidic proteases, avoidance of exposure to bactericidal mechanisms within lysosomes, as well as prevention of both mycobacterial degradation and presentation of mycobacterial antigens to the immune system.

The question arises, why study in vitro models of Mtb? These models may provide an improved understanding of host–pathogen interactions and immune responses during mycobacterial infec-tion. These models might improve our understanding of correlation of immunity with infection, as well as the activity of new antimycobacterial agents against Mtb within a granuloma-like structure.

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Future Microbiol. (2016) 11(8)1074

REviEW Bhavanam, Rayat, Keelan, Kunimoto & Drews

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This review describes advances made in in vitro granuloma models, which have improved our understanding of how TB granulomas develop in the lungs of humans, as well as caveats to these models and their applicability to human disease. Current theories surround-ing infection, granuloma development and the progression of lesions are examined, along with a rationale for the need of in vitro granuloma models. Additionally, this review highlights the importance of in vitro granuloma models in understanding the host–mycobacteria interac-tions at stages of granuloma formation that may not be reasonably achieved with animal models.

Initiation of infection: the basicsThe steps during the initiation of infection are well described elsewhere [9] and will not be cov-ered in this review in detail. Briefly, Mtb is spread in aerosolized droplets from the sputum of an infected person. Alveolar macrophages (AMs) are thought to be the primary target for Mtb once it enters the lung. Figure 1A, illustrates how inhaled droplets enter the lung alveoli, where the bacteria are phagocytosed by AMs [9–11]. The interaction between Mtb and AMs decides the subsequent progression of infection (Figure 1B). If the host fails to eradicate the bacteria, the infection is preserved in a latent condition and the infection does not transform into active disease, but is instead contained in the form of granuloma (Figure 1C). Alternatively, if the host fails to eradicate the bacteria, the infection is transformed into active TB disease (Figure 1D). A granuloma is an active lesion that has the capac-ity to influence T cell migration both locally and distally. As the lung is the primary site of infec-tion, this review focuses on how in vitro models can be used to understand the development of pulmonary granulomas.

The infectious process described is specific for the lung. After inhalation of Mtb, the bacte-rium is deposited in the alveoli, but can infect human lung epithelial cells and activate mucosal associated invariant T cells [12]. Although lung epithelium can become infected, macrophages in the alveoli are the primary target for Mtb once it enters the lung. The interaction between Mtb and the alveolar macrophages determine the progression of infection. Lung hydrolases present in human surfactant alter the envelope of the invading Mtb, thereby reducing the abil-ity of the Mtb interaction to grow within alveo-lar macrophages [12]. Mtb pathogenicity in the

lung is linked to the capacity of the bacterium to express lipids on its cell surface. Specifically, the Mtb molecule, phthiocerol dimycoceroser-ate (PDIM), limits innate recognition of Mtb, thereby reducing recruitment of Toll-like recep-tor (TLR) activated macrophages [13]. In addi-tion, other lipids on the Mtb surface, such as the phenolic glycolipids, determine the recruitment of macrophages via chemokine CCL2 and the chemokine receptor CCR2 pathway [13].

The classic understanding of pulmonary granuloma formationAfter initial infection, dendritic cells and mono-cyte-derived macrophages phagocytose the bacil-lus [14–16]. Mtb can survive inside macrophages by inhibiting the fusion of phagosomes with the lysosomes thereby preventing the formation of a phagolysosome, the mature form of a phago-some [17]. During the initiation of the granuloma formation, there is an early recruitment and clustering event involving inflammatory mac-rophages. New macrophages and other immune cells are then recruited to the site of infection, and develop into granulomas, the characteristic lesions of tuberculosis [9–11,18]. After the initia-tion of the acquired immune responses, T cells migrate from the circulation into the paren-chyma of the lung and then into the infected site, composed largely of macrophages and den-dritic cells [19–21]. Mature granulomas form as multicellular structures composed of infected and uninfected macrophages, epithelioid cells, giant cells (multinucleated cells derived from fused macrophages), T cells and B cells [22–24] that can contain the bacilli and prevents spread of the infection. Inward migrating dendritic cells (DCs) express IL-12p40 [25], and the receptor IL-12Rβ1 [26]. Major histocompatibility com-plex (MHC) class II expressing DCs are required for T-cell activation but outward migrating DCs also end up transporting bacteria to local drain-ing lymph nodes [27]. Inhibition of growth or death of Mtb is in part due to enhanced mac-rophage activation and the creation of an oxygen and nutrient deprived environment [28,29].

As shown in Figure 2, a spectrum of granu-lomas is observed in humans [30]. Figure 2A describes solid granulomas, where mycobacteria are most likely dormant [31]. Figure 2B describes necrotic granulomas, which are present in early stages of active TB. Figure 2C describes caseous granulomas found in end-stage or severe TB. The solid noncaseating granuloma consists of CD4+

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Figure 1. Pathogenesis of tuberculosis. Mtb enters the host when inhaled droplets are transmitted to the lungs (A). The bacteria is phagocytized by alveolar macrophages and eliminated by different mechanisms, which include apoptosis and autophagy (B). When Mtb growth is contained inside granulomas, it is preserved in a latent condition seen in 90–95% of infected individuals and infection does not transform into disease (C). Mtb is replicated in the macrophages and transformed into active tuberculosis with symptoms of tuberculosis and can also be disseminated to other tissues and organs as seen in 5–10% of cases (D). Mtb: Mycobacterium tuberculosis; TB: Tuberculosis.

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and CD8+ T cells, B cells and macrophages har-boring few tubercle bacilli (Figure 2A). A layer of fibroblasts surrounds this specific granuloma, with fibrosis occurring as the granuloma controls the infection and the inflammatory process is limited. In a latently infected individual, one or more granulomas controlling the infection can sometimes be observed; these granulomas can also be calcified [32,33]. As shown in Figure 2B, necrosis first occurs in the center of the structure and may further develop into a caseous necrosis of human lung tissue [34]. Figure 2C illustrates caseous granulomas can progress to form cavi-ties within the lung, leading to erosion of the

granuloma into a bronchus, and the subsequent release of bacteria into the airways [29]. They have a central necrotic area that contains extracellular bacteria surrounded by macrophages and phago-cytes, and contains lymphoid-like structures that are rich in T and B cells, as well as macrophages that contain tubercle bacilli.

Multiple components are involved in control-ling the development of a granuloma includ-ing immune cells, cytokines and chemokines (Figure 3). CD4+ Th1 cells [33,35–36], γ/δ T cells, CD4+ CD25+ FoxP3+ regulatory T cells [36,37], CD8+ T cells [35,38–39], as well as NKT cells [40] are involved in the control of TB infections.


Figure 2. Changing stability of the tuberculosis granuloma. The tuberculous granuloma is a compact, organized aggregate of epithelioid macrophages that tightly interlink cell membranes of epithelioid cells and adjacent cells. Depending on the immune response at the time of infection, the granulomas can either be solid, caseating or necrotic. In a solid granuloma, the infected macrophages are in the middle surrounded by other immune cells, such as CD4+ and CD8+ T cells, as well as macrophages that fuse to form multinucleated giant cells or have differentiated into foamy cells. A solid noncaseating granuloma (A) is seen during latent infection where the bacteria can be dormant and may have survived for decades. In a necrotic granuloma (B), the bacteria have multiplied and promoted the death of macrophages. In a caseous granuloma (C), the center of the granuloma is liquefied, which ultimately results in the dissemination of the bacteria and their spread to other parts of the body. The bacteria are also transmitted to other individuals due to their release via droplets. Mtb: Mycobacterium tuberculosis.

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The γ/δ T cells that secrete IL-17 and NK T cells that express both TCR and NK cell markers act as intermediaries between the innate and adaptive immune responses [40,41].

Proinflammatory cytokines, such as TNF, IL-1β [42–45] and IL-17 [46], and anti-inflam-matory cytokines, such as IL-4, IL-10 and TGF-β [45,47–48] maintain the granuloma. An appropriate equilibrium between pro- and anti-inf lammatory cytokines has been shown to be important in sterilizing the granulomas [45] (Figure 3). Individuals that have mutations in IL-12p40 or IL-12Rβ1 are deficient in both IL-12 and IL-23 signaling, and have reduced IFN-γ T-cell responses [49–51]. In addition,

signaling impairment, subsequent to IFN-γ deficiency and clinical phenotype was reported to vary between individuals [52]. These individu-als are difficult to treat with currently available anti-TB drugs and fail to develop granulomas in tissues infected with Mtb, demonstrating the requirement for IFN-γ signaling in granuloma formation [49].

Chemokines are produced by monocytes, macrophages and dendritic cells, and influence cellular migration, recruitment and activation of monocytes, macrophages and leukocytes. Alveolar epithelial cells and human bronchial epithelial cells produce chemokines CCL-2 and CXCL-8 in response to Mtb infection [53,54].

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Figure 3. Immune responses to tuberculosis infection. Following infection with Mycobacterium tuberculosis, the bacteria move to the lower respiratory tract where they are recognized by alveolar macrophages and trigger innate immune signaling pathways that lead to the production of various chemokines and cytokines, which promotes recruitment of other immune cells to the site of infection. Control of Mycobacterium tuberculosis is mainly dependent on the balance of pro- and anti-inflammatory cytokines. The CD4+ T-helper (Th) cells polarize into different subsets. Th1 cells produce IL-2 for T-cell activation, IFN-γ, or TNF for macrophage activation. Th17 cells, contribute to the early formation of protective immunity in the lung. Th2 cells and T regulatory cells (Treg) suppress Th1-mediated protection via IL4 and TGFβ or IL10, respectively. CD8+ T cells produce IFN-γ and TNF, which activate macrophages. The effector T cells (Teff) and memory T cells (TM) cells produce cytokines IL-12, IFN-γ and TNF.

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Many questions remain as to how host immune cells are recruited into sites of Mtb infec-tion in the lung. Although it is well known that T-cell migration to the parenchyma is important for the control of Mtb growth, it is still not fully understood how T cells migrate from the vas-culature into the parenchyma and whether they remain within the parenchyma. Expression of homeostatic chemokines CXCL13and and its receptor CXCR5 on T cells can act to recruit

T cells to the infected macrophage areas [55–57]. The role of neutrophils within the Mtb granu-loma is also currently not fully understood. In individuals with active TB infection, neutro-phils can be seen in airway samples [58] and in peripheral blood analysis [59]. Although neutro-phils might have a protective role immediately after exposure, it is evident that neutrophils are associated with the progression to active disease. One way in which neutrophils could interfere




with the protective response to Mtb is to limit the interaction between infected phagocytes and antigen-specific T cells [58,59].

The fate of granulomas are independent of each other in the same host: different developmental trajectories of lesions within an individual host tissueThere is growing evidence that within the lung, each granuloma may be on its own course of fate, and that the lung should not be consid-ered an environment where all granulomas are synchronized to a common state. The use of [18F]-fluorodeoxyglucose (FDG) positron emission tomography (PET)-CT scanning in human lungs during linezolid treatment stud-ies indicated diversity in the local inflamma-tory response leading to variability in the size of lesions and FDG avidity [60]. This has been cited as evidence that lesions within the lung are controlled not only by a systemic host immune response, but also by local factors that would determine the fate of individual lesions [61]. Furthermore, in nonhuman primate models, molecular tracking has been used to indicate the variability in the fate of multiple lesions within a host, possibly due to differences in host-mediated killing at the individual lesion level [62]. Finally, a recent mathematical model proposes that there may actually be no steady state for granulomas and that instead, there is a continuous progression of disease with each granuloma progressing at a different rate over time.

One hypothesis is that independent lesion fates would require locally acting agent(s) or factor(s) to modulate the pathophysiology of the granuloma environment in response to the pathogen. It is still not known what these local agents might be. It is possible that locally acting agents are the product of lipid metabo-lism at the site of an infection or other patho-physiologic process as described elsewhere in other organ systems [63]. These could include pro- and anti-inflammatory eicosanoids (lipid mediators derived from arachidonic acid, pros-taglandins, lipoxins and resolvins), those that modulate lesion resolution [63–65], or drive the lesion towards disease exacerbation and the development of necrotic foci [66]. It might be possible that these agents provide fine tuning to more systemic changes driven by cytokines and could possibly change the direction of lesion development within the host lung [61,67].

Lesion fate trajectories are controlled by host & bacterial factorsThe fate of an individual granuloma is most likely controlled by a variety of bacterial and host factors. Apart from the mediators described above and in Figure 3, these driving forces may also be impacted by the factors described in the following sections.

●● Is the RD1 region of Mtb required for the creation of a granuloma?Recent literature suggests that the genomic and proteomic nature of an Mtb strain could have a direct impact on its ability to create a granuloma or other pathophysiological events. A variety of genes have been proposed to play a role in the persistence of Mtb in hosts [68]. Complete genome sequencing has now been carried out on a variety of Mtb complex strains including H37Rv, CDC1551, H37Ra and M. bovis BCG. It is clear that there are differences in both the presence and the expression of specific gene products amongst members of the Mtb com-plex [69,70]. DNA sequence analysis identified the region of difference-1 (RD1) as an approximately 9.5-kb region of Mtb [70] responsible for virulence that is absent in M. bovis BCG [71]. The RD1 region contains genes that encode two secretory proteins, EsxA coding for ESAT-6) and EsxB coding for CFP-10, as well as the rv3877 gene, a putative translocation pore in the cytoplasmic membrane; all are located within the ESX-1 locus that encodes a secretory system [71,72]. RD1 is associated with pathogenesis of TB and con-tributes to secretion of specific proinflammatory cytokines (e.g., IL-1) [24,72]. RD1 may also be driving nonspecific cell damage (e.g., mitochon-drial damage), as RAW264 cell infection models showed that strains of H37Rv lacking RD1 have less measurable mitochondrial damage and did not have depleted ATP levels when compared with cells infected with wild-type H37Rv [24]. The soluble RD1 component ESAT-6 may also play a role in tissue remodeling and cell recruit-ment to support granuloma formation. ESAT-6 has been shown to induce MMP-9 production in epithelial cells and the recruitment of mac-rophages to the site of infection [73]. MMP-9 might drive tissue remodeling that would allow for intact solid granuloma production [74–76]. However, sequence analysis alone is not enough to determine the differences between strains as seen with Mtb. H37Ra, which contains the RD1 region but has noticeable downregulation

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of the RD1 gene products CFP-10 and ESAT-6 proteins [77].

●● Host genetic polymorphisms may impact the balance of granuloma stability-instability & the progression to active diseasePrevious work has suggested that some host genetic polymorphisms, such as the polymor-phism in the promoter (-403G/A and -28C/G) and intron (In1.1T/C) regions of the Ccl5 gene, may play a role in host resistance to Mtb infec-tion [78,79]. However, once infection has been established, it is also possible that there are several human polymorphisms that may play a role in increasing patient risk for a destabilized granuloma and active disease. A functional pro-moter polymorphism in the -2518A>G of the MCP-1 gene Ccl2 (chromosome region 17q11.2) has been associated with increased susceptibility to Mtb infection in some non-BCG vaccinated populations [80]. The proposed model is that the polymorphism is associated with increased levels of MCP-1, which in turn is associated with decreased levels of IL-12p40 and greater likelihood of progression to active disease [80]. Another functional polymorphism is found in the promoter region of Mmp1 (chromosome region 11q22.2), an insertion of a guanine at position – 1607 (-1607_1608insG), creates an Ets-1 transcription factor binding site [81] and enhances gene expression [82]. It is thought that polymorphisms in Ccl2 and Mmp1 may jointly act to increase the chance of active disease in the hosts [82].

The rationale for supporting studies using in vitro granuloma modelsThere are varieties of approaches for understand-ing the pathophysiology of Mtb, as well as bacte-rial responses to antimycobacterial agents. The rationale for using in vitro models to study Mtb infections are presented below.

●● In vitro studies outside of the granuloma milieu only give a partial picture of bacterial–host interactionsSo why not avoid an in vitro granuloma model and just study the impact of specific environ-mental or host components on Mtb? There is already a significant history of research using different aspects of the granuloma environment (e.g., starvation, changes ion oxygen concen-tration) to study Mtb alone in culture without

host cells [83–86]. These perturbed environmen-tal conditions can then be used to understand mycobacterial growth and the efficacy of anti-mycobacterial agents/growth inhibitors [85,87–88]. Although host cells are not present in these models, this work should not be discounted. For example, starvation studies of Mtb in the absence of host cells have identified possible roles for toxin-antitoxin systems in the response to nutrient starvation conditions [89]. Other models of phosphate depletion and nutrient starvation have been used to understand environmental impacts on mycobacterial two-component cell signaling systems [90]. These models may be ideal for the initial analysis of varying culture con-ditions on Mtb growth and survival responses but they are limited because that they do not represent the dynamic environment where cells are dying, changing or being recruited. The per-turbation of mycobacteria in a liquid or pellicle environment does not represent the true physi-ological environment that exists within the lung granuloma.

●● The granuloma provides a distinctly different environment for drug-induced bacterial killing than within normal lung or bloodGrowing evidence suggests that bacteria within the granuloma are not subject to the same host and environmental pressures as bacteria within the normal lung or within blood. It has been proposed that the concentration of antimycobac-terial drugs in the blood represents the drug con-centration in the lesions of the lung. However, initial work in rabbits indicates that drug pen-etration into lesions varies among individuals, and among drugs, and differs from the penetra-tion into the lung in general as estimated by pen-etration coefficients. Work by Kjellsson et al. has indicated that there may be no numerical differ-ences for drug penetration across lesion type for rifampin (RIF) and isoniazid (INH), while for pyrazinamide (PZA) and moxifloxacin (MXF) there were modest numerical differences in the penetration for the suppurative and coalescing lesions compared with caseous and solid types of lesions [91]. Within lesions, other work has reported a lower concentration of MXF in the caseous versus the cellular fraction of the gran-uloma [92,93]. The lesions themselves are com-pletely different environments than the normal lung. Recent modeling suggests that; antibiot-ics are frequently below effective concentrations




inside granulomas leading to bacterial growth between doses and prolonged treatment times, and antibiotic concentration gradients form within granulomas, with lower concentrations toward their centers [92].

●● In vivo experimental models to study the TB granulomaThere are several nonhuman models that can be used to study the pathophysiology of the TB granuloma, (1) the humanized murine model, (2) the guinea pig model, (3) the rabbit model; and (4) the primate model. The mouse model has been very useful in obtaining the information on the cytokine and immune cell responses for granuloma formation. Benefits of the mouse model include the inexpensive nature of the assay system, the ease in handling mice, the different variant strains available and reagents available for the perturbation of the system [94,95]. However, significant differences exist between the granulomatous response to Mtb infection in the lung between mice and humans [96]. Necrosis is lacking or difficult to achieve in murine models (Figure 4B) and may not represent the necrosis seen in human granulo-mas (Figure 4A) [97–101]. Finally, although latency can be studied, there is no standardized model of latency in the murine model [102]. In guinea pig and rabbit models, the benefits include ease of handling and the ability to produce necrosis within the granuloma [103,104]. There are now latent models of infection that have been created in the guinea pig host but these have not been widely tested [105]. The rabbit model is the only animal model that can represent the progres-sive disease as seen in human infection [106,107]. However, drawbacks to both the guinea pig and rabbit models include the limited variety of rea-gents to study pathophysiology when compared with human or murine models. Finally, primate models of infection allow for granuloma produc-tion that is similar to humans and latency can be established [45,108], with necrosis also produced in granulomas [109]. Drawbacks to using pri-mates for this type of work include the expense, difficulty in handling and ethical concerns in primate research.

●● Mathematical & computational models of TB infection are not sufficientMathematical and computational models have been used to predict the granulomatous response in TB infection based on experimental

observations and the available information about the disease [110–117]. Mathematical models are inexpensive and allow investigators to test a vari-ety of new hypotheses and incorporate a num-ber of complex parameters without the cost and time issues encountered in wet laboratory experi-ments. These models have been useful to address questions in TB that are difficult to approach experimentally. For example, differential equa-tion (DE) based models describe a relationship between numbers of cells and concentrations of molecules and their rates of change in the gran-uloma and time [113–114,117]. In contrast to DE based models, individual based models, or agent-based models (ABMs), are rule-based models that capture the events occurring in the immune systems (e.g., immune cells, bacteria, environ-mental factors) using a 2D grid representing a section of lung tissue [110,115–116]. Although these models have been useful in answering complex questions, they are highly dependent on the parameters chosen, and require previous observations in different systems to extrapolate the results. As a result, they can miss unknown factors.

In vitro granuloma models●● The experimental human lung tissue

modelRecently, an early granuloma model was estab-lished using experimental human lung tis-sue [118], which used a previously described 3D tissue model of the human lung mucosa [119]. Macrophages were infected with Mtb strains prior to infection of the model and then cointro-duced with PKH26 red fluorescent dye-labeled monocytes into the lung model. Using confo-cal microscopy, only virulent strain infections (e.g., H37Rv) and not avirulent strains (H37Ra, BCG, ΔRD1 and ΔESAT-6) were associated with monocyte/macrophage clustering at the infection sites [118]. As an indicator of necrosis, another group stained for HMGB-1 protein, a marker released from cells undergoing necro-sis [120]. They observed a significantly higher level of HMGB-1 staining in tissues infected with H37Ra than in uninfected areas or in tissues infected with avirulent or ΔRD1 or ΔESAT-6 strains [118]. However, it should be noted that this is a very preliminary model which uses indirect indicators (e.g., macrophage, monocytes clus-tering and HMGB-1 protein production) and only involves macrophages, monocytes, fibro-blasts and lung-specific epithelial cells [118]. A

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Figure 4. Differences in granuloma structure between mouse and human granulomas. (A) In human granulomas, the infected macrophages are in the middle surrounded by other immune cells such as CD4+ and CD8+ T cells, as well as macrophages that fuse to form multinucleated giant cells or differentiated into foamy cells. This type of granuloma is seen during latent infection where the bacteria can be dormant and survive for decades. (B) Granuloma in mice are comprised of loose nonnecrotic aggregates, surrounded by lymphocytes and macrophages that have fused and differentiated into foamy cells. Mtb: Mycobacterium tuberculosis.

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key message in this study is that mycobacterial factors may be important in initiating early phases of granuloma production, as well as the development of necrosis.

●● Peripheral blood mononuclear cell modelsPeripheral blood mononuclear cell (PBMC) granuloma models allow for the systematic determination of bacterial and host factors that drive granuloma formation and pathophysiol-ogy [121]. Over the last 10 years, a variety of approaches have been used.

In early models, proof of principle studies used Mtb antigens attached to agarose beads instead of viable mycobacteria [122]. These

antigen-bead complexes, when incubated with PBMCs, induced the production of granuloma-like structure. The composition of immune cells within these structures was similar to that found in natural Mtb granulomas. Later studies used cyanogen bromide (CNBr)-activated Sepharose beads coated with purified protein derivative (PPD) [123]. In a study by Puissegur et al., blood samples were collected from healthy BCG-vaccinated, PPD-reactive nontuberculous con-trol individuals [124]. Monocyte-like cells were recruited to the bead surface on day one. By day four, lymphocyte-like cells were recruited and were seen to bind to the attached monocyte-like cells. By day 5, bead surfaces were completely




covered by recruited cells and multilayers of monocytes and lymphocytes were formed with pseudopodia (indicating cell differentiation) identified [124]. Staining at day 9 identified CD68-positive staining cells that might repre-sent multinucleated giant cells (MGC). These MGC-like cells were also surrounded by CD3-stained lymphocytes. Potential macrophages stained strongly positive for CD168, and possible epithelioid cells were weakly stained for CD168.

Other PBMC models have avoided antigen-coated beads and utilized viable BCG to create granuloma-like structures. This approach also showed that PBMCs from healthy donors were able to form granuloma like structures around BCG within 9 days [125]. These granulomas included activated lymphocytes in tight contact with macrophages, as well as multinucleated giant cells. BCG-containing phagosomes were also present in what appeared to be epithelioid cells.

However, it is not clear how BCG gener-ates a granuloma in the absence of RD1 and how these models would vary from other Mtb models. BCG granulomas are possible as BCG pulmonary granulomas have been described in highly immunocompromised patients [125]. The growing number of models using human donor PBMCs suggest that the immune status, previous PPD or BCG exposure, previous Mtb infection status and health (including acute infections) of the PBMC donor must be accounted for when in vitro granuloma models are studied [124].

Concerns remain about these in vitro PBMC granuloma models. Are these models in fact just cell aggregates with similar cellular characteris-tics to human granulomas? Perhaps they have some key characteristics that may improve our knowledge about Mtb infections. They allow us to study Mtb infections of cells in a mixed multi-cellular environment. They may also account for PBMC donor factors that might have a down-stream impact on how Mtb infections occur, and how granulomas are formed. These models can be quite dynamic and can be used to study the proliferation of specific cell types during a mock infection.

●● Dormancy models of TB infectionAs described earlier, host cell free Mtb culture experiments have often been used to mimic the conditions encountered by the bacteria within host granulomas [126–128]. These have then been applied to simple infection models with some

success. For example, using a hypoxic induced environment model, it was shown that Mtb accu-mulates triacylglycerides and goes into a dor-mant state but can regrow after reexposure to oxygen [127]. When this information was applied to a lipid loading THP-1 infection model, the Mtb was found to be dormant [127,128]. We believe that this work has been useful in improving our understanding of the metabolic adaptation of Mtb during processes resembling dormancy. However, these models have been unable to demonstrate resuscitation under conditions that mimic immune suppression, which is a key ele-ment in Mtb pathophysiology leading to active TB. To achieve this goal, 3D in vitro models of granulomas have been studied.

●● 3D in vitro modelsSeitzer and Gerdes were the first to report the 3D granuloma model using PBMCs infected with Mtb [129]. In their studies, PBMCs were infected with Mtb strain H37Rv at different MOI (number of bacteria/cell) and seeded into agarose-coated wells. After 4 days of incubation, the lowest MOI in their study (1:150) produced host cell aggregates. However, infection at a higher MOI did not result in a large aggregate formation but rather numerous very small aggre-gates. An increase in the number of dead cells was also observed. By histological analysis the aggregates were confirmed to have similar phe-notypical characteristics (e.g., cell population type) to natural Mtb granulomas. Another study combined human PBMCs, autologous mac-rophages and Mtb in ultralow attachment tis-sue culture plates and the nonadherent PBMCs were added on day 2 and 5 after infection to mimic the natural infection process in which additional lymphocytes are recruited to the infection site [130]. The formation of granuloma was observed and acid-fast bacilli were observed within the host cells composing the granuloma. This study also showed that addition of IL2, IFN-γ and/or TNF-α, enhanced the formation of the granuloma and host cell recruitment. Although these models have provided research-ers with important information about granuloma cell differentiation, these models were unable to establish dormancy and resuscitation inside the 3D-generated granulomas.

Kapoor et al. developed a 3D in vitro granu-loma model in which Mtb progresses to dor-mancy and subsequently resuscitates under con-ditions that mimic weakening of the immune

1083



system [131]. PBMC were mixed at room tem-perature with an extracellular matrix (ECM). Mtb H37Rv was added to the ECM at an MOI of 1:10. After 8 days of incubation, micro gran-ulomas, and an aggregation of lymphocytes around infected macrophages were observed, as well as the presence of multinucleated giant cells. Cytokines IFN-γ, TNF-α, IL-12p40 and IL-10 were detected in the culture supernatants. Dormancy was demonstrated by the loss of acid fastness, accumulation of lipid bodies, develop-ment of rifampicin tolerance, and gene expres-sion changes. Treating the granulomas with immunosuppressant anti-TNF-monoclonal anti-body reactivated the dormant Mtb. Several other studies used the 3D granuloma model to study other mycobacterial diseases, such as M. lep-rae [132], M. bovis [129] and M. avium [130]. These studies had the advantage of showing Mtb going into dormancy and subsequently resuscitating under conditions that mimic weakening of the immune system, these studies were not able to mimic the different cell types in a lung tissue exposed to Mtb infection [124,130–132].

Recently, Braian et al. developed a cell-based in vitro human lung tissue model [133]. In this model, the human lung-specific fibroblasts were cultured in a matrix of collagen. A transwell insert matrix was layered over the top of the mem-brane, monocytes or macrophages isolated from the PBMC were infected with Mtb and added to the matrix layer, human lung-specific epithelial cells were added to the culture. The culture was then exposed to air to initiate the production of extracellular matrix proteins, mucus secretion and stratification by the epithelium. The advan-tage of this 3D tissue model is that it displays the characteristic features of human lung tissue, including epithelia integrated with macrophages, formation of extracellular matrix, stratified epi-thelia and mucus secretion [133]. Although this model has several advantages over other in vitro models, it has some limitations. This model consists of only monocytes and macrophages, besides lung-specific cells. It lacks neutrophils and lymphocytes that are also known to be pre-sent in a granuloma. This model can be used to study both active infection and latent states. Continued work is needed to determine if this model is representative of true dormancy.

In summary, 3D in vitro models are poten-tially comparable to granulomas observed in clinical specimens. Potential applications of the in vitro models include the study of functional

characterization of individual cell types, pat-terns of cell surface expression, cytokine and chemokine secretion, development of mycobac-terial dormancy, and resuscitation under condi-tions that suppress the host immune response. These models could potentially provide insights into mechanistic aspects of host defences, such as phagosomal maturation, autophagy and antimi-crobial peptides. Interestingly, 3D in vitro models allow manipulation of one or more cells types and provide a relevant tissue microenvironment, a platform for testing vaccine and drug candidates against dormant, as well as active, mycobacte-ria. These models could also potentially provide insights between mycobacteria and host–cell interactions within granuloma structures, which have limitations to address in the animal models.

●● In vitro granuloma models are amenable to microdissection approachesNew developments in microdissection [134] have raised the possibility that individual granulomas within an infection model could be interro-gated. The previous PBMC granuloma models described above have all been approached as a single system with no attempt to dissect indi-vidual granulomas. Given the previous discussion that each granuloma may be on its own devel-opmental trajectory, it is possible that dissection approaches could provide more information on how individual granulomas develop within a larger system. Microdissection studies have previously been used to study the expression of host cell genes in lung tissue from Mtb infected mice [135]. Tissue from microdissection has also been used for qPCR, and immunohistochemistry from M. bovis induced granulomas in cattle.

DiscussionThis review has described a variety of in vitro experimental models available that can help in understanding the complex host–pathogen rela-tionship that takes place within the TB granu-loma. An understanding of the pathophysiology of granulomas is critical for the design of new TB drugs and vaccines. A major caveat in the in vitro granuloma model is whether in vitro models of Mtb complex granulomas are truly representative of human pulmonary granulomas or just the cell aggregates. We propose that these PBMC models are representative of human pulmonary granulo-mas in many ways. Both natural and in vitro Mtb granulomas contain basic elements, such as mac-rophages, T cells and bacilli, and are multilayered




with multicellular structures [123–124,131]. In vitro granuloma models can help us understand the early stages of cell recruitment and immune responses over multiple environmental condi-tions in a manner that is not feasible with animal models or in clinical studies. Human infections and in vitro models also share common immune responses. In humans, patients show increased mRNA and protein expression of IL-8, IL-12, IFN-γ and TNF-α in granulomatous lymph nodes [136], IL-8 and TNF-α in PBMCs [137], and IL-6 and TNF-α in bronchealveolar lavage fluid [138–140]. The production of these cytokines, as well as time-dependent variability in cytokine production has also been described within in vitro granuloma models. Some in vitro granu-loma models may also mimic the surrounding lung tissue environment where the granuloma is anchored by extracellular matrix proteins, including osteopontin and fibronectin [140].

However, some questions about the exact nature of in vitro granulomas still remain unan-swered. For example, the exact role of Mtb factors in granuloma formation is unclear. In addition, questions still exist about the role of the Mtb RD1 region in the creation of granulomas in vitro and in vivo [118]. We still do not completely under-stand how host responses drive granuloma forma-tion. Hopefully some of these questions can be further elucidated by a variety of experimental models, each with their own peculiar strengths and weaknesses [118,130].

Conclusion & future perspectiveThis review identifies the reasons why in vitro granuloma models have a practical purpose for

the study of TB pathophysiology, host immune responses and mycobacterial responses to anti-mycobacterial agents. In vitro granuloma mod-els allow for the study of defined cellular and soluble host factors, as well as specific natural strains, acellular mycobacterial components and other species. This ability to work with simpler systems involved in granuloma forma-tion, while still maintaining a sense of com-plexity, allows for the creation of well-focused scientific hypotheses in a model that is more representative of the natural host. It is clear that the health status of the donor can impact the formation and recruitment of the cells to the granuloma. In vitro granuloma models allow the exploration of genetic and immunologic variables contributing to TB immunopatho-genesis, including the metabolic state of myco-bacteria contained inside human granulomas. In doing so, our understanding of the patho-physiology of the human Mtb granulomatous response will be greatly improved, thus ena-bling new ideas for biomarkers, therapy and development of effective vaccines against TB infection.

Financial & competing interests disclosureFinancial support for S Bhavanam comes from the University of Alberta, Department of Laboratory Medicine and Pathology. The authors have no other rel-evant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

EXECUTivE SUMMARY ● Tuberculosis remains a major human health threat, with one in three indidivuals in the world infected and

approximately 1.5 million deaths worldwide each year.

● Treatment and prevention options are limited and there is an urgent need for new immunotherapeutic vaccine and new antimycobacterial agents. These will require an improved understanding of infections within a granuloma environment, as well as host correlates of effective immune responses to infection.

● The outcome of Mycobacterium tuberculosis infection in the lung depends on the initial environment encountered by the bacterium and the genetics of the host.

● Multiple models have been created that allow us to understand the interaction of Mycobacterium tuberculosis and the host within the granuloma. Each has its strengths and weaknesses.

● In vitro granuloma models allow for the systematic determination of bacterial and host factors that drive granuloma formation and pathophysiology in ways that are not possible with culture-based methods or animal infections.

● Newer 3D in vitro granuloma models may allow for new studies as these models can be used to mimic events, such as dormancy and resuscitation.

1085future science group www.futuremedicine.com


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Commentary

Mechanisms of mycobacterial transmission: how does Mycobacterium tuberculosis enter and escape from the human host

Michael U Shiloh*,1,2

1Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA 2Department of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA


In 1882, while lecturing on his discovery of the cause of tuberculosis, Robert Koch said “If the importance of a disease for mankind is measured by the number of fatalities it causes, then tuber-culosis must be considered much more important than those most feared infectious diseases, plague, cholera and the like.” Although Koch’s identifica-tion of Mycobacterium tuberculosis (Mtb) as the cause of tuberculosis heralded many future dis-coveries and treatments, Mtb remains responsible for 1.5 million deaths annually. Therefore, more research is needed on all stages of Mtb infec-tion. This commentary provides a framework for understanding two central questions in Mtb biol-ogy at the extremes of the infectious life cycle: how does Mtb enter the body and how does it escape?

How is tuberculosis spread?The tubercle bacillus is spread person-to-per-son almost exclusively by aerosolized particles. The size of infectious droplets from Mtb in infected patients ranges from 0.65 (small) to >7.0 μm (medium–large) [1]. While small Mtb aerosol particles are expected to transit past the nasopharyngeal or tracheobronchial region to be deposited in the distal airways, larger

particles can be trapped in the upper airway or oropharynx where they can potentially lead to tuberculosis of the oropharynx or cervical lymph nodes [2]. Despite the apparent ease with which Mtb is spread – how else could nearly 2 billion humans be infected currently? – and the strong epidemiologic evidence linking airborne transmission to close personal con-tacts, directly demonstrating aerosol transmis-sion of Mtb has been exceedingly difficult [3]. Indeed, Riley et al. needed to expose hundreds of Mtb-susceptible guinea pigs to air from a ward of Mtb-infected patients continuously for months to demonstrate airborne transmission of Mtb [4], indicating that aerosolization and transmission of Mtb is a rare event. This obser-vation is corroborated by epidemiologic and modeling data indicating that the likelihood of transmission of Mtb is proportional to the duration of exposure to an infectious person and inversely proportional to the volume of space within which exposures occur [5].

What causes active disease?The simplest model of human interaction with Mtb is represented by the binary outcome ensuing

“Novel approaches are needed to further elucidate these critical events in the pathogenesis of

tuberculosis.”

First draft submitted: 22 September 2016; Accepted for publication: 30 September 2016; Published online: 10 November 2016



Commentary Shiloh


from exposure of a naive individual to Mtb: devel-opment of either asymptomatic latent infection or active disease characterized by fever, weight loss and a bloody cough. Latently infected individuals can either remain asymptomatic throughout their lifetime or develop active disease at a remote time from the primary infection in a process known as reactivation. Epidemiologic studies have shown that only 5% of otherwise healthy individuals will develop active disease when first exposed to Mtb, thus leading to the fundamental question in Mtb biology of why so few acute infections prove symptomatic? One straightforward expla-nation relates to inoculum dose: multiple studies have demonstrated a direct correlation between the number of bacilli in an infected person’s spu-tum and the likelihood that contacts will develop symptomatic active tuberculosis. Likewise, ani-mal studies show that a lower initial Mtb inocu-lum in wild-type animals results in a quiescent but persistent infection, whereas a higher inoc-ulum leads to active disease, pneumonia and death. Since a coughing, actively infected person typically produces only a few aerosolized bacte-ria [3], his or her contacts likely will inhale a low number of bacteria, skewing the probability to development of latent infection rather than active disease. As well as the importance of inoculum size, environmental factors such as proximity and duration of the contact as well as micronutrient or relative vitamin D deficiency have been associated with susceptibility to tuberculosis. Furthermore, being immunocompromised at the time of infec-tion due to either host genetics or acquired defi-ciencies can also increase a person’s likelihood of developing active disease from an initial infec-tion. In addition, it has been recently proposed [2] that trapping of larger particles in the upper air-way may induce a protective immune response. Finally, as described below, direct translocation of bacteria across the epithelium overlying mucosa-associated lymphatic tissue (MALT) might also dictate whether acute or latent infection occurs.

How does tuberculosis cross the airway mucosa?The current paradigm of primary tuberculosis infection is that small airborne particles distrib-ute to the terminal alveoli, where resident alveolar macrophages or tissue dendritic cells ingest the airway bacteria. Subsequently, infected mac-rophages or dendritic cells migrate to draining lymph nodes, activate adaptive immunity and then return to the initial site of infection where a

granuloma forms [6]. However, prior to reaching the terminal alveoli, some small and large par-ticles likely become trapped along the mucosa, where they can interact with noninnate immune cells between and above MALT. Oropharyngeal MALT, widespread in childhood but regressed in adults, includes nasal-associated lymphatic tissue, the tonsils and adenoids of Waldeyer’s ring, and bronchus-associated lymphatic tissue. Interestingly, MALT is prevalent during a vulner-able period of early childhood and adolescence when tuberculosis manifests in more severe and disseminated forms. Overlying MALT are both primary epithelial cells and specialized cells called microfold cells (M cells), the latter whose function in both the airway and GI tract is to ingest and transcytose foreign antigens [7,8]. It has previously been demonstrated that airway epithelial cells can mediate dissemination of Mtb from the airway [9], and a functional role for M cells in Mtb entry was recently established [10], extending observations made over 15 years ago [11]. M cell depletion prior to airway infection results in fewer Mtb recov-ered from draining lymph nodes during nasal or airway infection and protection from long-term Mtb-mediated mortality in mice [10]. Not tested in these experiments is the impact of Mtb trans-cytosis by M cells on adaptive immunity, nor how inoculum dose affects the immune response. Since M cells targeting has recently been proposed as a method for inducing vaccine responses in MALT as well as systemically [12], M cells are prominent in nasal-associated lymphatic tissue [13] and intra-nasal vaccination with BCG provides enhanced protection from Mtb infection [14], it is also pos-sible that low level or repeated infection (i.e., with <10 bacteria) in the upper airway results in direct delivery of Mtb to APCs in MALT in a pathway that stimulates a more robust protective immune response than when Mtb are ingested by alveolar macrophages directly. Thus, both inoculum dose and particle size could influence the outcome of disease such that individuals whose M cells tran-scytose either paucibacillary or larger Mtb parti-cles develop latent disease or even remain com-pletely uninfected. However, if the initial dose of Mtb transcytosed by M cells in the upper airway mucosa overwhelms the primary local response, then drainage of Mtb to local lymph nodes could result in lymphatic disease, as is seen in scrofula. Likewise, if the particles are very small, they might bypass epithelial cells altogether and reach the t erminal alveoli to be ingested by airway phagocytes.

“The success of any bacterial pathogen

ultimately depends on its ability to multiply and

infect new hosts.”

1505

Entry and escape mechanisms of Mycobacterium tuberculosis Commentary


How does tuberculosis escape from the lung?The success of any bacterial pathogen ulti-mately depends on its ability to multiply and infect new hosts. Many pathogenic bacteria interact with hosts by secreting virulence mol-ecules like proteins and lipids. A classic exam-ple is the production of cholera toxin by Vibrio cholerae, which enhances the pathogen’s spread from person-to-person by promoting profuse watery diarrhea. Mtb also employs sophisti-cated means of dissemination, including medi-ating caseation, access to the bronchial tree and cough-mediated airborne transmission.

role of tissue destruction & caseationRecently, it was proposed that the transition of airway tuberculosis from the initial, asympto-matic infection to symptomatic, necrotic and cavitary disease occurs in three stages [15]. In this model, the first stage reflects early infec-tion of alveolar macrophages by Mtb and acti-vation of cell-mediated immunity, followed by immune control with granuloma formation. In some individuals, a second stage ensues, repre-sented by accumulation of Mtb antigens and host lipids in the airway, leading to bronchial obstruction and obstructive lipid pneumonia. Finally, depending on the host response, the third stage is characterized by either cavitary or fibrocaseous disease with communication to the outside world. The mechanisms accounting for each stage, and in particular, the formation of obstructive, lipid pneumonia are not well known, though some studies have identified critical roles for host matrix metalloproteinases in tissue destruction and dissemination [16].

Cough in tuberculosis transmissionAlthough cough is a well-known hallmark feature of active pulmonary tuberculosis, very little is known about the pathogenesis of infectious cough [17]. Cough may be a natural consequence of lung inflammation and host

production of prostaglandins, bradykinin and other inflammatory mediators that acti-vate afferent neuronal C-fibers in the lung mucosa [18]. In addition, cavity or cyst for-mation itself might induce mechanical acti-vation of either rapidly adapting receptors or slowly adapting stretch receptors that can sensitize the lungs to cough triggers [19,20]. Conversely, cough itself, perhaps triggered by secreted mycobacterial factors, could lead to Mtb aerosolization and/or cavity formation. In this model, a granuloma or region of case-ous necrotic pneumonia could be induced to form a cavity by very high mechanical forces (intrathoracic pressures as high as 300 mm Hg and expiratory velocities as high as 800 km/h) generated by a strenuous cough [18], forcing weakened extracellular matrix and elastic tis-sue [16] to stretch into a cavity. Thus, while cough may be a major route of aerosolization and spread, it may precede and/or overlap with the cavitary disease stage.

ConclusionThough significant progress has been made to understand the innate and adaptive responses to Mtb infection since Koch’s discovery of tuber-culosis, both how Mtb penetrates the mucosa to initiate infection and how it reverses the process to escape remain poorly understood. Novel approaches are needed to further eluci-date these critical events in the pathogenesis of tuberculosis.

Financial & competing interests disclosureM Shiloh is supported by NIH funding, grant numbers AI099439, AI109725, AI125939, AI111023. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.


“...direct translocation of bacteria across the

epithelium overlying mucosa-associated

lymphatic tissue might also dictate whether acute or latent infection occurs.”

references1 Fennelly KP, Martyny JW, Fulton KE, Orme

IM, Cave DM, Heifets LB. Cough-generated aerosols of Mycobacterium tuberculosis: a new method to study infectiousness. Am. J. Respir. Crit. Care Med. 169(5), 604–609 (2004).

2 Fennelly KP, Jones-Lopez EC. Quantity and quality of inhaled dose predicts

immunopathology in tuberculosis. Front. Immunol. 6, 313 (2015).

3 Roy CJ, Milton DK. Airborne transmission of communicable infection – the elusive pathway. N. Engl. J. Med. 350(17), 1710–1712 (2004).

4 Riley RL, Mills CC, Nyka W et al. Aerial dissemination of pulmonary tuberculosis. A

two-year study of contagion in a tuberculosis ward. 1959. Am. J. Epidemiol. 142(1), 3–14 (1995).

5 Pienaar E, Fluitt AM, Whitney SE, Freifeld AG, Viljoen HJ. A model of tuberculosis transmission and intervention strategies in an urban residential area. Comput. Biol. Chem. 34(2), 86–96 (2010).


Commentary Shiloh


6 Russell DG, Barry CE 3rd, Flynn JL. Tuberculosis: what we don’t know can, and does, hurt us. Science 328(5980), 852–856 (2010).

7 Wang J, Gusti V, Saraswati A, Lo DD. Convergent and divergent development among M cell lineages in mouse mucosal epithelium. J. Immunol. 187(10), 5277–5285 (2011).

8 Mabbott NA, Donaldson DS, Ohno H, Williams IR, Mahajan A. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium. Mucosal Immunol. 6(4), 666–677 (2013).

9 Bermudez LE, Goodman J. Mycobacterium tuberculosis invades and replicates within type II alveolar cells. Infect. Immun. 64(4), 1400–1406 (1996).

10 Nair VR, Franco LH, Zacharia VM et al. Microfold cells actively translocate Mycobacterium tuberculosis to initiate infection. Cell Rep. 16(5), 1253–1258 (2016).

11 Teitelbaum R, Schubert W, Gunther L et al. The M cell as a portal of entry to the lung for the bacterial pathogen Mycobacterium tuberculosis. Immunity 10(6), 641–650 (1999).

12 Lo DD, Ling J, Eckelhoefer AH. M cell targeting by a Claudin 4 targeting peptide can enhance mucosal IgA responses. BMC Biotechnol. 12, 7 (2012).

13 Mutoh M, Kimura S, Takahashi-Iwanaga H, Hisamoto M, Iwanaga T, Iida J. RANKL regulates differentiation of microfold cells in mouse nasopharynx-associated lymphoid tissue (NALT). Cell Tissue Res. 364(1), 175–184 (2015).

14 Derrick SC, Kolibab K, Yang A, Morris SL. Intranasal administration of Mycobacterium bovis BCG induces superior protection against aerosol infection with Mycobacterium tuberculosis in mice. Clin. Vaccine Immunol. 21(10), 1443–1451 (2014).

15 Hunter RL. Tuberculosis as a three-act play: a new paradigm for the pathogenesis of

pulmonary tuberculosis. Tuberculosis (Edinb.) 97, 8–17 (2016).

16 Elkington PT, D’armiento JM, Friedland JS. Tuberculosis immunopathology: the neglected role of extracellular matrix destruction. Sci. Transl. Med. 3(71), 71–76 (2011).

17 Turner RD, Bothamley GH. Cough and the transmission of tuberculosis. J. Infect. Dis. 211(9), 1367–1372 (2015).

18 Polverino M, Polverino F, Fasolino M, Ando F, Alfieri A, De Blasio F. Anatomy and neuro-pathophysiology of the cough reflex arc. Multidiscip. Respir. Med. 7(1), 5 (2012).

19 Mazzone SB. An overview of the sensory receptors regulating cough. Cough 1, 2 (2005).

20 Mazzone SB, Undem BJ. Vagal afferent innervation of the airways in health and disease. Physiol. Rev. 96(3), 975–1024 (2016).


part of


EDITORIAL

New tests for detection of Mycobacterium tuberculosis infection: sufficient to meet the WHO 2035 targets?

Morten Ruhwald*,1 & Peter L Andersen1

KEYWORDS • correlate of risk • IGRA • IP-10 • prediction of progression • specific skin test • transcriptomics

The new WHO End TB Strategy sets ambitious 2035 targets and emphasizes targeted treatment of the Mycobacterium tuberculsis infected at risk of developing tuberculosis (TB). New short-course treat-ment regimens have shown promise for better compliance; however, the available diagnostic tests are insufficient to guide treatment.

This editorial discusses current state of art and future developments in the field of latent TB diagnostics, with emphasis on Quantiferon-TB Gold® Plus (Qiagen), IP-10 release assays, specific skin tests and transcriptomic signatures that sug-gests a brighter future with more accurate predictive tools.

With 7.3 million annual cases and 1.3 million deaths, TB remains among the most significant infectious killers [1]. Each year, close to half a million new cases of multidrug-resistant cases emerge and HIV-associated TB affects more than a million people [1]. Before diagnosis and

treatment, every TB patient will have passed on the bacteria to on average 11 contacts [2], hereby feeding the huge res-ervoir of M. tuberculosis (Mtb) infected at risk of developing active TB disease. The implementation of direct observed therapy and roll out of PCR-based diagnostics and drug susceptible testing have prevented millions of lives; however, it is clear that radical means are required to curb the epidemic [1].

A central pillar in the new WHO End TB Strategy is a recommendation of target treatment of Mtb infection among at-risk populations in upper-middle and high-income countries with an incidence <100 per 100,000 population [1,3]. This is an extension of the global recommendations for targeted treatment of children [4] and people living with HIV [5], and is among the core activities expected to facilitate the achievement of the ambitious targets of 90% reduction in TB incidence and 95% reduction in TB deaths by 2035 [1].

1Statens Serum Institut, Artillerivej 5, 2300 Copenhagen, Denmark


First draft submitted: 29 June 2016; Accepted for publication: 14 July; Published online: 22 August 2016

“The new WHO End TB Strategy sets ambitious 2035

targets and emphasizes targeted treatment of the Mycobacterium tuberculsis

infected at risk of developing tuberculosis.”

“...to meet these ambitious goals there is a need for shorter treatment regimens and better diagnostics to

guide the treatment.”



EDitORial Ruhwald & Andersen


The recommendations are supported by mod-els suggesting a significant impact of targeted treatment [6]; however, to meet these ambitious goals there is a need for shorter treatment regi-mens and better diagnostics to guide the treat-ment. The introduction of the 3-month weekly isoniazid and rifapentine ensures better compli-ance and higher treatment success [7,8], however, the currently available toolbox of tests to guide preventive treatment is a bottleneck.

Current diagnostic toolbox to guide targeted treatmentThe tuberculin skin test (TST) has been the standard method of determining whether a person is infected with Mtb [6] for over a cen-tury. The test is performed by intradermal injec-tion of purified protein derivative tuberculin, a precipitate of species-nonspecific antigens [7]. A major limitation to the use of TST, is false-positive reactions occurring in persons infected with nontuberculous mycobacteria and in per-sons vaccinated with Bacille Calmette–Guérin (BCG) resulting in lower test specificity and compromised potential to guide targeted treatment [9].

The IFN-γ release assays (IGRAs) is an in vitro diagnostic alternative to TST [8]. These tests are based on the Mtb-specific antigens ESAT-6, CFP10 and TB7.7, and provide an objective read-out and solve the problem of false-positive TST results. Two IGRA tests are commercially avail-able, the whole blood-based Quantiferon Gold® In-Tube test (QFT, Qiagen, Germany) and the purified peripheral blood mononuclear cells-based test T-SPOT.TB® test (Oxford Immunotec, UK). Compared with the TST, IGRAs are more complex and labor intensive, requiring labora-tory infrastructure and skilled staff [9]. Although IGRAs have solved the problem of false-positive reactions in BCG vaccinated, both IGRA and TST are unable to differentiate latent infection from active disease [9,10]. Thus, current tests have low-positive predictive value for the development of active TB and require treatment of >30 contacts to prevent one case of TB [11–13].

Imminent futureIn 2015, Qiagen launched the fourth genera-tion Quantiferon-TB Gold® Plus (QFT 4G), as a test with improved diagnostic sensitivity for infection in immunosuppressed [14]. QFT 4G builds on the QFT 3G in-tube design and comprises two blood collection tubes, TB1 and

TB2, instead of the single TB-Ag tube known from QFT 3G. TB1 contains a cocktail of pep-tides from ESAT-6 and CFP10 identical to the predecessor [15] but without the TB7.7 anti-gen [9,14]. TB2 comprise the same ESAT-6 and CFP10 peptides as TB1, and – in addition – an unknown number of shorter peptides intended to activate the CD8-positive T cells. The ration-ale for the TB2 tube is to boost diagnostic sen-sitivity for infection, for example, in people liv-ing with HIV (PLHIV) with low CD4 T cells. QFT 4G is interpreted positive if either TB1 or TB2 induces an antigen-specific IFN-γ release ≥0.35 IU/ml, a modification of the algorithm that in itself will increase the positivity rate by doubling the chance for samples with low IFN-γ levels (and very high analytical variability [16]) of becoming positive.

Registration studies and the first independ-ent evaluations found that the QFT 4G pro-vides a marginal and nonsignificant increase in the detection rates in TB patients and con-tacts [14,17–18]. The million-dollar question yet to be answered experimentally is whether the extra detected individuals are found among those that progress to TB – hereby truly improving the test; or whether it picks up more in the group who never progress to TB and, therefore, will be a detriment to the predictive value. Nevertheless, the fourth tube adds cost and complexity to an already laborious test, wherefore it is difficult to appreciate QFT 4G as the panacea enabling the End TB Strategy targets.

Further aheadMany activities are underway aiming to improve the IGRA. Several groups have explored the use of alternative readout markers expressed in higher levels compared with IFN-γ. It is now established that cell-mediated immune responses can be detected with other cytokines and chemokines, among which IP-10 is the leading alternative [19]. IP-10 is a chemokine expressed in 100-fold higher levels than IFN-γ, allowing for simpler detection technology, such as the lateral flow quick test format or even extrac-tion from dried blood spots [19,20]. IP-10-based IGRAs are highly concordant with IFN-γ-based IGRA, but the high magnitude of response allows for improved sensitivity in children and PLHIV with confirmed Mtb infection [19]. Of particular relevance for the implementation of the End TB strategy, the lateral flow-based IP-10 IGRA would enable implementation of a rapid

“...the lateral flow-based IP-10 IFN-γ release assay

would enable implementation of a rapid and simple IFN-γ release assay-like diagnostic with minimal equipment and

training.”

1103

New tests for LTBI: sufficient for WHO targets? EDitORial


and simple IGRA like diagnostic with minimal equipment and training.

Another approach to improving the predic-tive potential of the IGRA is alternative antigens associated with controlled infection. One exam-ple is heparin-binding hemagglutinin, a protein expressed on the surface of several mycobacterial species. Heparin-binding hemagglutinin-specific cellular immune responses are associated with Mtb containment suggesting a not yet fully explored predictive potential when combined with specific antigens, such as ESAT-6 and CFP10 [21], in a two-tube test vis-a-vis QFT 4G.

The era of specific skin tests?Specific skin tests are another promising and field friendly alternative to IGRA. C-Tb developed by Statens Serum Institut (Copenhagen, Denmark) is a skin test based on a recombinant double ESAT-6 protein and single CFP10 monomer expressed in Lactococcus lactis. Phase II data suggest that C-Tb drives induration sizes on level with TST and deliv-ers positivity rates in TB cases and unexposed con-trols on par with QFT [22,23]. As C-Tb is unaffected by previous BCG vaccination, the responses seen in infected are more clearly separated from unin-fected, and the cutoff can be lower and universal (at ≥5 mm) irrespective of HIV infection or age [18,19]. Phase III trial data recently shown at conferences suggest that C-Tb is safe and delivers positivity rates highly concordant with QFT 3G in recent contacts, but also that C-Tb appears more robust than QFT in PLHIV with active TB and low number of circulat-ing CD4 T cells [24] [Ruhwald, Aggerbaek, Pers. Comm.]. Diaskintest developed by Pharmstandard (Moscow, Russia) using recombinant ESAT-6 and CFP10 dimer expressed in Escherichia coli is another specific skin test. Despite having been on the market in several former soviet republics since 2005, few results of this test has emerged in the international scientific literature. Diaskintest seems to deliver IGRA like diagnostic performance in BCG vacci-nated, but reports suggest a relatively high number of adverse events associated with this test [25].

Prediction beyond the IGRAOutside the IGRA concept, a recent landmark paper from Zak et al. suggested that whole-blood

transcriptomic mRNA expression signatures accu-rately predict risk of progression in latently infected individuals [26]. A correlate of risk was identified by mining RNA-sequencing data from large pro-spective cohorts of Mtb-infected adolescents from South Africa, and a 63 mRNA transcript from 16 genes was migrated to a chip-based PCR assay for high-throughput analysis. Validation in independ-ent prospective cohorts from South Africa and Gambia demonstrated sensitivity of 54% and speci-ficity of 83% for progression to active disease within a year [26]. The mRNA signature was not in itself specific for infection, but indicates a future two-step approach with a doorstep IGRA followed by a PCR-based risk prediction in IGRA-positive cases.

In conclusion, the field of immunodiagnos-tics is in transition. Innovative new specific skin tests and field-friendly IGRA like test formats will enable specific and likely cheaper diagnos-tics. Transcriptomic signatures could be a game changer, guiding rational use of targeted treat-ment. Encouraging, the predictive potential of transcriptomic signatures could also provide a revolution in TB vaccine development, enabling much smaller and cost-effective efficacy trials in high-risk populations to triage vaccine candi-dates [27]. Advances on all fronts are required to meet the ambitious 2035 targets of the End TB strategy, but without a new and efficacious TB vaccine and drastic improvements in living condi-tions and global access to healthcare, the targets still seem a high bar to meet.

Financial & competing interests disclosureM Ruhwald and PL Andersen are employed by SSI, a gov-ernmental not-for-profit research organization that holds intellectual property rights on several antigens used for immunodiagnostic tests and vaccines for tuberculosis. M Ruhwald is registered as inventor on patents disclosing the use of IP-10 as immunodiagnostic marker for Mycobacterium tuberculosis infection. All rights have been assigned to SSI. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.


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