“The Dance with the Wolf - Interplay of Gamma Delta T-cells and Macrophages In Acute Lung...

“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

Where are Macrophages localized and how do they control infection?

Osteoclasts Kupfer Cells

Subcapsular Macrophages Alveolar Macrophages

Marginal Zone Macrophages

Glia

Spleen Liver Bone

Lung Lymph Node Brain

Tissue Distribution

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

Macrophage Phenotypes and Function

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

in vivo

in vitro

Activation States in Macrophages

Under Steady State Conditions Macrophages are Intrinsic Anti-Inflammatory

• 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

Wound Healing Macrophages

Regulatory

Macrophages

Classically Activated

Macrophages

Macrophage Activation during Infection and Inflammation

Classical Activation Pathway

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


MHC-2

Cytokines IFNg and TNF-a

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



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

Classical Activation Pathway Flagellin

• Surface Recognition via TLR-5• Cytosolic Recognition of via NAIP5 and IPAF


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

Classical Activation Pathway induced by Innate and Adaptive Immune System



Early IFN-g Response

NK-CellsTransient Production

Cell Stress / Tissue damage

Maintenance of IFN-g secretion

Adaptive Immune System

TH1 Cells

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



Host Defense

How Do Classically Activated Macrophages Fight Invading Pathogens such as Bacteria ?

• Reactive Oxygen Species

• Proteases

• Proinflammatory Lipid Mediators

• Proinflammatory Cytokines

• Intracellular Killing


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


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


Host Defense

Pro-inflammatory Lipid Mediators

LTB4 and PGE2Neutrophil chemoattractant and stimulation of

TNF-a and IL-1 in Macrophages

PGD2 LTB4


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


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


Host Defense

Evolved Mechanism to Escape Intracellular Killing

Mycobacterium Tuberculosis

ESAT-6

ESAT-6 ESAT-6

Impaired M1-Polarization


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

Macrophages and their Role in the Resolution of Inflammation and Initiation of Tissue Repair

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


• Mannose Receptor – Mrc1

• Resistin- like a (Retnla, Fizz1)

• Chitinase 3–like 3 (Chi3l3, Ym1)

• Arginase


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


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


IL-4 IL-13 and IL-10

Inhibitory Effects on NFKB via STAT6 Activation

CAP

AAP



Mannose Receptor CD206

Carbohydrate recognition domain

CD206 is Expressed on The Surface and Intracellular

• Mediating Endocytosis • May play a role in antigen presentation


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



Impaired IL-4/IL-13 Pathway lead to Pathogenesis



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



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


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


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


Balance between Inflammation (M1) and Tissue Repair/Resolution (M2)

Acute Lung Injury

Role of the Innate Immune System

Pathomechanism

Clinical Relevance

Predisposing Factors

Symptoms in Acute Lung Injury

• Dyspnea - Trouble Breathing

• Tachypnea – faster breathing

• Tachycardia – increased heart rate

• Central Cyanosis (Hypoxia)

Symptoms Acute Lung Injury

Pulmonary

• Pneumonia• Aspiration of Gastric Content

Non-Pulmonary

• Sepsis (most common) • Multi-Trauma Injuries• Burn• Chronic Alcohol Abuse• Genetic Predisposition

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

Pathomechanism

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)

To Investigate The Role of Gamma Delta T-Cells During Acute Lung Injury, We Used a Mouse

Model for Acute Lung Injury

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

How can we determine the Extent of Lung Injury?

Barrier Function in the Pulmonary Vascular System

“Vascular Leak”

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

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

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

Figure 3: Increased Numbers of Lung Cell Counts in the Absence of Gamma Delta T-cells

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

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

Figure 3: Absence of γδ T-cells lead to significant higher numbers of classically activated Macrophages

within 4 days

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

.

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

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

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

WTB6 LPS Day 1

Vγ4Vγ1 Vγ5 Vγ6 Vγ7

WTB6 LPS Day 4


WTB6 LPS Day 7


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

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

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

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

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

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

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

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.

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

“The Dance with the Wolf - Interplay of Gamma Delta T-cells and Macrophages In Acute Lung...

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