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![Page 1: “The Dance with the Wolf - Interplay of Gamma Delta T-cells and Macrophages In Acute Lung Injury” Fabian Wehrmann, MD Instructor School of Medicine Division.](https://reader034.fdocuments.us/reader034/viewer/2022051113/56649dbf5503460f94ab27ee/html5/thumbnails/1.jpg)
“The Dance with the Wolf - Interplay of Gamma Delta T-cells and Macrophages In
Acute Lung Injury”
Fabian Wehrmann, MD Instructor
School of Medicine Division of Allergy & Clinical Immunology
![Page 2: “The Dance with the Wolf - Interplay of Gamma Delta T-cells and Macrophages In Acute Lung Injury” Fabian Wehrmann, MD Instructor School of Medicine Division.](https://reader034.fdocuments.us/reader034/viewer/2022051113/56649dbf5503460f94ab27ee/html5/thumbnails/2.jpg)
Where are Macrophages localized and how do they control infection?
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Osteoclasts Kupfer Cells
Subcapsular Macrophages Alveolar Macrophages
Marginal Zone Macrophages
Glia
Spleen Liver Bone
Lung Lymph Node Brain
Tissue Distribution
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During Tissue Injury the Bone Marrow Function as a Reservoir for Macrophage Precursors
Inflammatory Monocytes• 2-5% of WBC
• Rapid Recruitment to the Site of Injury
• Differentiation
• Emigration from the BM is CCR2 dependent
• Crawling on vascular endothelium (CX3CR1/CXCL1)
• Role is not clear During Tissue injury (M2-Macrophages)
TIP Dendritic cells (TNFa, iNOS)
Inflammatory Macrophages
• Enter Non-inflamed Tissue
Inflammatory DCs
Patrolling Monocytes
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Macrophage Phenotypes and Function
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Classically Activated
Macrophages
TAM
Tumor –Associated
Macrophages
Wound Healing Macrophages
Regulatory
Macrophages
MDCS
Myeloid derived
Suppressor Cells
Host Defense + Antitumor Activity
Anti-InflammatoryWound Healing
Suppression of Anti Tumor Immunity
Anti-Inflammatory - secretion of large amounts of IL-10
Precursors of TAM
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in vivo
in vitro
Activation States in Macrophages
Under Steady State Conditions Macrophages are Intrinsic Anti-Inflammatory
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• suppress Adaptive Immune Responses against arriving apoptotic Cells
Marginal Zone Macrophages
• Deletion of MZM in mice lead to auto reactive T- and B-cells with AB Production against ds DNA and Lupus Erythematodes Symptoms
• Bathed in Interleukin-10 (regulatory T-cells)
Colonic Macrophages
• Mice genetic deleted for IL-10 develop severe Colitis
Spleen
Intrinsic Anti-Inflammatory Macrophages
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Wound Healing Macrophages
Regulatory
Macrophages
Classically Activated
Macrophages
Macrophage Activation during Infection and Inflammation
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Classical Activation Pathway
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Common Host Response
• Micro Arrays of Monocyte-derived Macrophages stimulated with diverse bacteria revealed - 100 genes
Cytokines TNF- a, IL-1a, IL-1b, IL-6, IL-12
Chemokines CXCL-9 CXL10, CCL2, CCL5, CXCL8.
Costimulatory Molecules CD80, CD86
Enzymes iNOS, ROS, Oxygen Radicals
Bacteria, DNA, RNA Via PPR
Classical Activation Pathway
MHC-2
Cytokines IFNg and TNF-a
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Signaling Pathogen Recognition Receptors (PPR)
• Toll Like Receptors TLR-1,-2,-3,-4,-5 , -7,-8 and -9
• RLR - Retinoic Acid Inducible Gene (RIG) Like Receptors
• NLR – NOD Like Receptors
Induction of The Classical Activation Pathway byPathogens
Classical Activation Pathway
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Classical Activation Pathway
Infection
Cpg-motifs
specific sequences in the DNA with a length of 6 nucleotides
• Center: 1) Cytosine 2) Phosphate 3) Guanine
• Human: every 60 nucleotides you have a cpg sequence (90% methylated)• Bacteria: Every 16 nucleotides (un-methylated)
Intracellular TLRs
• TLR-3: ds RNA • TLR-7 and TLR-8: ssRNA• TLR-9: cpg motifs / ds DNA
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Classical Activation Pathway Flagellin
• Surface Recognition via TLR-5• Cytosolic Recognition of via NAIP5 and IPAF
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Classical Activation Pathway
RIG-1
• the 50-triphosphate of ssRNA from many positive-strand ssRNA viruses
• Both recognition pathways trigger robust type I pathways trigger robust type IIFN productions
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Classical Activation Pathway induced by Innate and Adaptive Immune System
Classical Activation Pathway
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Classical Activation Pathway
Early IFN-g Response
NK-CellsTransient Production
Cell Stress / Tissue damage
Maintenance of IFN-g secretion
Adaptive Immune System
TH1 Cells
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Self Regulatory Functions of Classically Activated Macrophages via IL-12 and IL-27
• Interleukin-12 induce further Proliferation of TH1 Cells
• Interleukin-27 block further Proliferation of TH1 Cells
Classical Activation Pathway
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Classical Activation Pathway
Host Defense
How Do Classically Activated Macrophages Fight Invading Pathogens such as Bacteria ?
• Reactive Oxygen Species
• Proteases
• Proinflammatory Lipid Mediators
• Proinflammatory Cytokines
• Intracellular Killing
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Classical Activation Pathway
Host Defense
Reactive Oxygen Species ROS/NOS
• At low levels Important function in tissue homeostasis by regulating cell signaling molecules
• Superoxide Anion, Hydrogen Peroxide, NO-
• During infection – secretion of large quantities By Macrophages are the key to destruction of invading Pathogens
L-Arginine NO-
Superoxide Anion
Peroxinitrite
NOS-2
Excessive Quantities of ROS/NOS can affect Host Proteins and DNA inducing Necrosis and Apoptosis and amplify the inflammatory
Response by activating NFKB and AP-1
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Classical Activation Pathway
Extent of Tissue Injury Depends on the Amount of Antiproteases Generated in the Tissue
Neutral Proteases Elastase, Matrix, Metalloproteinases
Collagenase
Acid Hydrolases Lipase, Ribonuclease, Cathepsin
Glycosidase and Lysozyme
Host Defense
Proteases
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Classical Activation Pathway
Host Defense
Pro-inflammatory Lipid Mediators
LTB4 and PGE2Neutrophil chemoattractant and stimulation of
TNF-a and IL-1 in Macrophages
PGD2 LTB4
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Classical Activation Pathway
Host Defense Proinflammatory Cytokines
Upregulation of Adhesion Molecules and Chemokine expression
Induction of Apoptosis and Cytotoxic Effects
Chemokine ExpressionBy the Endothelium to Recruit
LeukocytesCXCR2, CCL-2 and CCL-5
IL-1 TNF-a
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Classical Activation Pathway
Host Defense Intracellular Killing
Lysteria Monocytogenes
Role of IFN-g / TNFa on IC- Killing during L. Monocytogenes Infections
IFN-g / TNF-a deficient
IFN-g and TNF deficient miceMice die due to impaired bacterial
killing
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Classical Activation Pathway
Host Defense
Evolved Mechanism to Escape Intracellular Killing
Mycobacterium Tuberculosis
ESAT-6
ESAT-6 ESAT-6
Impaired M1-Polarization
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Classical Activation Pathway
What Situations Lead to an Excessive or Prolonged M1-Program?
Host Defense
Escherichia Coli • Neonatal Meningitis• Gastroenteritis • Uterine Tract Infection
Sepsis
Immune Dysregulation
Severity Correlates with M1 Typical cytokines levels
Model of Peritoneal Sepsis Survival is associated with a more
balanced M1/M2 Phenotype
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Macrophages and their Role in the Resolution of Inflammation and Initiation of Tissue Repair
Alternative Activation Pathway
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Alternative Activation Pathway
Interleukin-4 Source: Basophils / Mast Cells
Cytokines that Activate the Alternative Pathway in Macrophages
One of the first Innate Signals during Tissue Injury
IL-4 / IL-13 and IL-10
Resident Macrophages
Wound Healing Program
Wound Healing Program
• Mannose Receptor – Mrc1
• Resistin- like a (Retnla, Fizz1)
• Chitinase 3–like 3 (Chi3l3, Ym1)
• Arginase
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Alternative Activation Pathway
Arginase
• uses L-Arginine to synthesize Proline via Ornithine
• Proline is used for Production of Extracelluar Matrix Protein such as Collagen
CAP - Cytokines
CAP – Classical Activation Pathway / AAP – Alternative Activation Pathway
Wound Healing Program Arginase Induction
AAP - Cytokines
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Alternative Activation Pathway
Wound Healing Program Arginase Induction
Competition for Limited Intracellular L-Arginine
L-Arginine
L-Ornithine iNOS/Citrullin
Arginase iNOS Sythetase
Arginase -/- Arginase overexpressing Accelerated inflammation/Increased T-cell
proliferation
Impaired CD4 Proliferation Decreased IFN-g
Schistosoma Mansonii
Leishmania Donovani
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Alternative Activation Pathway
IL-4 IL-13 and IL-10
Inhibitory Effects on NFKB via STAT6 Activation
CAP
AAP
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Alternative Activation Pathway
Wound Healing Program
Mannose Receptor CD206
Carbohydrate recognition domain
CD206 is Expressed on The Surface and Intracellular
• Mediating Endocytosis • May play a role in antigen presentation
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Alternative Activation Pathway
Wound Healing Program Resistin Like a (RELM-a)
Relm-a -/- Accelerated inflammation/Increased Fibrosis and Granuloma
Formation
Schistosoma Mansonii
Increased TH2 Cytokine
Mechanism is unclear how Relm-a protect against accelerated Inflammation
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Alternative Activation Pathway
Wound Healing Program
Impaired IL-4/IL-13 Pathway lead to Pathogenesis
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Alternative Activation Pathway
Wound Healing Program
Impaired IL-4/IL-13 Pathway lead to Pathogenesis
Overexpression of IL-13 mice have increased intracellular growth rate of
Cryptococcus Neoformans
IL-4 Treatment
During Infection with Mycobacterium Tuberculosis, IL-4 Treatment lead to increased susceptibility due
to auto-mediated Killing.
• Treatment During Infection with Leishmania Major –induces polyamine biosynthesis and can contribute to the intracellular growth of the parasite
Intracellular Killing of Cryptococcus Neoformans
Lack of IL-13 Expression mice are more Resistent to infection with
Cryptococcus Neoformans
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Alternative Activation Pathway
Wound Healing Program
Resolution of Inflammation
TGF betaVascular Endothelial Growth Factor
Endothelial Growth Factor
M2 – derived TGF-beta and the Role for Fibrosis/Cancer
Interleukin-10
TGF beta
IL-10 -/-
IL-10 overexpressing
Increased Pulmonary Fibrosis
Decreased Pulmonary Fibrosis
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Alternative Activation Pathway
Phenotypic Switch of Cyclooxygenase-2 at a Later Stage of Inflammation
Prostaglandin-E2
Other Mediators that Activate the Alternative Pathway in Macrophages
Bioactive LipidsLipoxins and 15d PGJ2
COX-2
Prostaglandin D2
Prostaglandin d PG J2
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Alternative Activation Pathway
Prostaglandin d PG J2
Heat shock Proteins
Apoptosis
Anti-oxidant enzymes
Neutrophil Clearance
Other Mediators that Activate the Alternative Pathway in Macrophages
Lipoxins and 15d PGJ2
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Alternative Activation Pathway
Balance between Inflammation (M1) and Tissue Repair/Resolution (M2)
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Acute Lung Injury
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Role of the Innate Immune System
Pathomechanism
Clinical Relevance
Predisposing Factors
Symptoms in Acute Lung Injury
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• Dyspnea - Trouble Breathing
• Tachypnea – faster breathing
• Tachycardia – increased heart rate
• Central Cyanosis (Hypoxia)
Symptoms Acute Lung Injury
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Pulmonary
• Pneumonia• Aspiration of Gastric Content
Non-Pulmonary
• Sepsis (most common) • Multi-Trauma Injuries• Burn• Chronic Alcohol Abuse• Genetic Predisposition
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Clinical and Non-Clinical Relevance
• 58 new cases per 100 000
• Total of 141 500 new cases
• Annual Death Rate 59 000 per year
• Health Care Costs
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Pathomechanism
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Pathomechanism
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Some Facts about Gamma Delta T-cells
• Small Subset of T-cells
First Line Defense
Able to Present Antigen to T-cells
• TCR consist of one Gamma and one Delta Chain unlike CD4/CD8 T-cells which Bear an Alpha and Beta Chain
• Complex Behavior Which is Still Elusive
• Highest Abundance in the Gut Mucosa
Bridge Innate and Adaptive Immunity
Able to Mature Dendritic Cells
Can Induce Phenotype Isotype Switch from IgG to IgE
And suppress (Vg-1) and enhance IgE Responses (Vg-4)
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To Investigate The Role of Gamma Delta T-Cells During Acute Lung Injury, We Used a Mouse
Model for Acute Lung Injury
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Assesment of Acute Lung Injury during LPS induced Lung injury
LPS (O55B5-5ug/g body weight in 60 ul PBS
0 1 4 7 10 14
Days
Model of Sterile Inflammation
Allows To Elucidate Self Regulatory Function of The Innate Immune System Without Potential Regulatory
Interactions By Pathogens
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How can we determine the Extent of Lung Injury?
Barrier Function in the Pulmonary Vascular System
“Vascular Leak”
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D
Assessment of Endothelial/Epithelial Barrier Function in the Lung
• 100ul FITC Dextran (70KD / Murine Albumin 69 kD)
• (30 mg/ml) iv injection into the retro-orbital sinus
• circulation for 2h
• Collection of Blood and Broncho-Alveolar lavage (BAL)
• 2 x 500ul PBS
Fluorescence Intensity of samples measured at 520 nm
Fluorometer
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k
Normalization of Serum/BAL Samples
Fluorescence Intensity in Sera of Mice 50-80 x106
60 x 106 Units - set as 1
Serum sample: 69x106 Units FI (100%) - 60x106 (86%)
BAL: 36597 Units Fi (100%) - 31603 (86%)
Normalized to 60 x 106
Normalization of Serum Samples in Order to Compare Fluorescence of BAL samples
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Flu
ores
cenc
e In
tens
ity
Wildtype (WT) no γδ T-cells (γδ-/-)
Figure 1: Mice deficient for γδ T-cells develop significant worse Lung Injury determined by
the Pulmonary Vascular Barrier Function after intravenous application of FITC Dextran
Flu
ores
cenc
e In
tens
ity
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Figure 3: Increased Numbers of Lung Cell Counts in the Absence of Gamma Delta T-cells
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Figure: Screening for Innate Cytokines in the Lung after LPS-induced Lung Injury revealed
significant higher levels of M1-typical Cytokines/Chemokines in γδ T-cell deficient mice at day 4
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Gating Strategy to identify Classically Activated Macrophages in LPS-induced Lung Injury
F4/80high /Ly6C high / MHC-2 pos / CD11b pos / CD64pos CD80 pos / CD86pos IL-4R pos
CD11b CD11c CD64 CD80 CD86 IL-4R
F4/80-1 / Ly6C high MHC-2 pos
F4/80-2 / Ly6C high MHC-2 pos
γδ -/- LPS Day 4 TLC
CD206
CD206
MHC-2
F4/80
Ly6
C
Ly6
C
WT LPS D4 TLC
MHC-2 CD206
CD206 MHC-2
MHC-2
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Figure 3: Absence of γδ T-cells lead to significant higher numbers of classically activated Macrophages
within 4 days
![Page 58: “The Dance with the Wolf - Interplay of Gamma Delta T-cells and Macrophages In Acute Lung Injury” Fabian Wehrmann, MD Instructor School of Medicine Division.](https://reader034.fdocuments.us/reader034/viewer/2022051113/56649dbf5503460f94ab27ee/html5/thumbnails/58.jpg)
Cyotkines, that drive the polarization of Classically Activated Macrophages (M1/Pro-inflammatory) are TNF-a and IFN-g. Figure: “The Early TNF-a response at day 1 is significantly higher in γδ T-cell-deficient
mice suggesting that these Gamma Delta T-cells regulate TNF-a expression.
That would explain why the Knockout mice develop much more inflammation after day 2 (when TNF-a kicks in) – So one mechanism
would be that the γδ T-cells regulate TNF-a expression in Alveolar Macrophages
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.
Mice deficient for γδ T-cells display significant lower levels of Interleukin 4 Expressed in the Lung at 1 day after LPS-induced Lung Injury
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Induction of LPS-induced Lung Injury in IL-4 GFP-Reporter Mice revealed that γδ T-cells express Interleukin-4 at day 1
18s rRNA Interleukin-4
γδ
TC
R
IL-4
GF
P
CD3 pos /IL-4 posCD3 neg /IL-4 pos
γδ
TC
R
CD3
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Day 4 Day 7Day 1
CD3+
γδ
Day 0A
Day 0
Day 1
Day 4
Day 7
0
20000
40000
60000
80000
100000
Nu
mb
er
of
γδ
T c
ell
sB
.
Identification of Vg-1, Vg-4 and Vg-7 expanding γδ T-cells during LPS-induced Lung Injury
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WTB6 LPS Day 1
Vγ4Vγ1 Vγ5 Vγ6 Vγ7
WTB6 LPS Day 4
Vγ4Vγ1 Vγ5 Vγ6 Vγ7
WTB6 LPS Day 7
Vγ4Vγ1 Vγ5 Vγ6 Vγ7
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CD3 18srRNA Interleukin-4
IL-4 amplification: chromosomal DNA = 1504 bp mRNA = 204bp
Vg-1 Vg-4 Vg-7
B
Vg-1 Vg-4 Vg-7
Sorting on expanded γδ-T-cell populations revealed γδ-1, γδ-7 T-cells, but not γδ-4, as Interleukin-4 expressing populations at Day 1
IL-4 amplification: chromosomal DNA = 1504 bp mRNA = 204bp
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F4/80-1 / Ly6C low / CD206 pos
F4/80-2 / Ly6C low / CD206 pos
CD206
CD206
Isotype
Isotype
F4/80
Ly6
C
Ly6
C
Ly6
C
Ly6
C
CD11b MHC-2 CD64 CD11c IL-4R MAC-3
Gating Strategy is used to identify the Alternative Activation Pathway in Macrophages (Anti-inflammatory)
during LPS-induced Lung Injury:
γδ -/- LPS Day 4 TLC WT LPS D4 TLC
CD206
Ly6
C
CD206
F4/80
Ly6
C
F4/80 high /Ly6Clow /CD206 pos / MHC-2 pos / CD11b pos / CD11c pos / IL-4R pos/ MAC-3 pos
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LPS Day 1
CD206
CD206
F4/80
Ly
6C
L
y6
C
Ly
6C
L
y6
C
C57B6 γδ -/-
CD206
CD206
CD206
CD206
F4/80
Ly
6C
L
y6
C
CD206
CD206
F4/80
Ly
6C
L
y6
C
C57B6 γδ -/-
LPS Day 4
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CD206
CD206
F4/80
Ly6
C
Ly6
C
CD206
CD206
F4/80
Ly6
C
Ly6
C
C57B6 LPS Day 7 γδ -/- LPS Day 7
LPS Day 7
Normalization of the Inflammatory Response at 7 Days after LPS-induced Lung Injury
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Wildtype (WT)no γδ T-cells (γδ-/-)
Figure: At 4 days of LPS-induced Lung Injury Mice deficient for Gamma Delta T-cells display An Imbalance of Pro-inflammatory M1 and Anti-inflammatory M2-Macrophages
![Page 68: “The Dance with the Wolf - Interplay of Gamma Delta T-cells and Macrophages In Acute Lung Injury” Fabian Wehrmann, MD Instructor School of Medicine Division.](https://reader034.fdocuments.us/reader034/viewer/2022051113/56649dbf5503460f94ab27ee/html5/thumbnails/68.jpg)
F4/80 CD206 Isotype
Ly-6
C
“Treatment with recombinant IL-4 reduces TNF-a expression in resident Alveolar Macrophages”
Isotype PE TNF-a PE TNF-a PE
Treatment with recombinant IL-4
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Hypothesis:
Gamma Delta T-cells protect against LPS-Induced Lung Injury via Interleukin-4
Finding No-2:
The Significant Worse Barrier Function in γδ -/- Mice is Accompanied By Increased Levels of M1-typical Cytokines at Day 4 such as IL-1a, IL-1beta, CXCL9 and CXCL-10.
Finding No-3:γδ -/- Mice Display a Dysbalance of Cells that have a Phenotype of Classical Activated and Alternatively Activated Macrophages with significant higher numbers of F4/80 high Ly6C high And MHC-2 high compared to Wildtype Mice.
Finding No-1:
Mice Deficient for Gamma Delta T-cells Develop Significant Worse Lung Injury within 4 days after LPS-instillation, determined by the Pulmonary Vascular Barrier Function
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Summary
Finding No-4: At Day 1 after LPS-induced Lung Injury γδ -/- Mice display higher levels of TNF-a and lower levels of IL-4 at day 1. Two Cytokines that regulate CAP and AAP.
Finding No-5:
3 Expanding Populations of γδ T-cells during LPS-induced Lung Injury: Vg-1, Vg-4 and Vg7. We found Vg-1 and Vg-7, but not Vg-4 as Interleukin-4 Expressing Populations.
Current Experiments focusing on the Treatment with Recombinant IL-4 During LPS-induced Lung Injury with to show
• Improvement of Barrier Function in γδ -/- Mice at Day 4
• Restore a more Balanced M1/M2 Phenotype
• a Reduction in Early TNF-a Expression in Resident Macrophages and a Restorage of the Diminished Number of Alternatively Activated Macrophages at Day 1.
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Simonian Lab
• Jim Lavelle, MD• Fabian Wehrmann, MD
Fontenot Lab
• Amy McKee, PhD• Alex Tinega• Michael Falta PhD• Natalie Bowerman, PhD• Douglas Mack• Allison Laham
Cancer Core Center
• Karen Helm
Acknowledgments