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“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?
Tissue Distribution
Liver
Bone
Kupfer Cells
Osteoclasts
Lung
Lymph
Node
Brain
Alveolar Macrophages
Subcapsular Macrophages
Glia
Spleen
Marginal Zone Macrophages
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
•
Emigration from the BM is CCR2 dependent
•
Differentiation
TIP Dendritic cells (TNFa, iNOS)
Inflammatory DCs
Inflammatory Macrophages
Patrolling Monocytes
•
Crawling on vascular endothelium (CX3CR1/CXCL1)
•
Enter Non-inflamed Tissue
•
Role is not clear During Tissue injury (M2-Macrophages)
Macrophage Phenotypes and Function
Classically
Activated
Macrophages
Host Defense + Antitumor
Activity
MDCS
Myeloid derived
Suppressor Cells
TAM
Tumor –
Associated
Macrophages
Precursors of TAM
Suppression of Anti Tumor
Immunity
Regulatory
Macrophages
Anti-Inflammatory - secretion of
large amounts of IL-10
Wound Healing
Macrophages
Anti-Inflammatory
Wound Healing
Activation States in Macrophages
Under Steady State Conditions Macrophages
are Intrinsic Anti-Inflammatory
Intrinsic Anti-Inflammatory Macrophages
Spleen
Colonic Macrophages
•
Bathed in Interleukin-10 (regulatory T-cells)
•
Mice genetic deleted for IL-10 develop
severe Colitis
Marginal Zone Macrophages
•
suppress Adaptive Immune Responses
against arriving apoptotic Cells
•
Deletion of MZM in mice lead to auto
reactive T- and B-cells with AB Production
against ds DNA and Lupus Erythematodes
Symptoms
Macrophage Activation during Infection and
Inflammation
Classically
Activated
Macrophages
Wound Healing
Macrophages
Regulatory
Macrophages
Classical Activation Pathway
Classical Activation Pathway
Bacteria, DNA, RNA
Via PPR
Cytokines
IFNg and TNF-a
Common Host Response
• Micro Arrays of Monocyte-derived
Macrophages stimulated with diverse
bacteria revealed - 100 genes
MHC-2
Cytokines
TNF- a, IL-1a, IL-1b, IL-6, IL-12
Costimulatory Molecules
CD80, CD86
Chemokines
CXCL-9 CXL10, CCL2, CCL5, CXCL8.
Enzymes
iNOS, ROS, Oxygen Radicals
Classical Activation Pathway
Induction of The Classical Activation Pathway by
Pathogens
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
Infection
Classical Activation Pathway
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
Classical Activation Pathway
RIG-1
• the 50-triphosphate of ssRNA from many positivestrand ssRNA viruses
• Both recognition pathways trigger robust type I
pathways trigger robust type IIFN productions
Classical Activation Pathway
Classical Activation Pathway induced by Innate and
Adaptive Immune System
Classical Activation Pathway
Early IFN-g Response
Cell Stress / Tissue damage
NK-Cells
Transient Production
Maintenance of IFN-g secretion
Adaptive Immune System
TH1 Cells
Classical Activation Pathway
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
Host Defense
How Do Classically Activated Macrophages Fight
Invading Pathogens such as Bacteria ?
• Reactive Oxygen Species
• Proteases
• Proinflammatory Lipid Mediators
• Proinflammatory Cytokines
• Intracellular Killing
Classical Activation Pathway
Host Defense
Reactive Oxygen Species ROS/NOS
• Superoxide Anion, Hydrogen Peroxide, NO• At low levels Important function in tissue homeostasis
by regulating cell signaling molecules
Excessive•Quantities
of ROS/NOS
canof
affect
Proteins
and DNA are
During infection
– secretion
large Host
quantities
By Macrophages
inducing Necrosis
Apoptosis
and amplify
the inflammatory
the key to and
destruction
of invading
Pathogens
Response by activating NFKB and AP-1
NO-
L-Arginine
NOS-2
Superoxide Anion
Peroxinitrite
Classical Activation Pathway
Host Defense
Proteases
Neutral Proteases
Elastase, Matrix, Metalloproteinases
Collagenase
Acid Hydrolases
Lipase, Ribonuclease, Cathepsin
Glycosidase and Lysozyme
Extent of Tissue Injury Depends on the Amount of
Antiproteases Generated in the Tissue
Classical Activation Pathway
Host Defense
Pro-inflammatory Lipid Mediators
LTB4 and PGE2
Neutrophil chemoattractant and stimulation of
TNF-a and IL-1 in Macrophages
PGD2
LTB4
Classical Activation Pathway
Host Defense
Proinflammatory Cytokines
Upregulation of Adhesion Molecules and
Chemokine expression
Chemokine Expression
By the Endothelium to Recruit
Leukocytes
CXCR2, CCL-2 and CCL-5
IL-1
TNF-a
Induction of Apoptosis
and Cytotoxic Effects
Classical Activation Pathway
Host Defense
Intracellular Killing
Role of IFN-g / TNFa on IC- Killing during L.
Monocytogenes Infections
IFN-g / TNF-a deficient
IFN-g and TNF deficient mice
Mice die due to
impaired
bacterial killing
Lysteria
Monocytogenes
Classical Activation Pathway
Host Defense
ESAT-6
ESAT-6
Evolved Mechanism to Escape Intracellular Killing
Mycobacterium Tuberculosis
Impaired M1-Polarization
ESAT-6
Classical Activation Pathway
Host Defense
What Situations Lead to an Excessive or Prolonged M1Program?
Escherichia Coli
•
Neonatal Meningitis
•
•
Gastroenteritis
Uterine Tract Infection
Immune Dysregulation
Sepsis
Severity Correlates with M1 Typical
cytokines levels
Model of Peritoneal Sepsis
Survival is associated with a more
balanced M1/M2 Phenotype
Alternative Activation Pathway
Macrophages and their Role in the Resolution of
Inflammation and Initiation of Tissue Repair
Alternative Activation Pathway
Cytokines that Activate the Alternative
Pathway in Macrophages
IL-4 / IL-13 and IL-10
One of the first Innate Signals during Tissue Injury
Interleukin-4
Source: Basophils / Mast Cells
Resident Macrophages
Wound Healing Program
Wound Healing Program
•
Arginase
•
Mannose Receptor – Mrc1
•
Resistin- like a (Retnla, Fizz1)
•
Chitinase 3–like 3 (Chi3l3, Ym1)
Alternative Activation Pathway
Wound Healing Program
Arginase Induction
Arginase
CAP - Cytokines
AAP - Cytokines
CAP – Classical Activation Pathway / AAP – Alternative Activation Pathway
•
uses L-Arginine to synthesize
Proline via Ornithine
•
Proline is used for Production
of Extracelluar Matrix Protein
such as Collagen
Alternative Activation Pathway
Wound Healing Program
Arginase Induction
Competition for Limited Intracellular L-Arginine
L-Arginine
L-Ornithine
Arginase
Schistosoma
Mansonii
Arginase -/-
iNOS/Citrullin
iNOS Sythetase
Accelerated inflammation/
Increased T-cell
proliferation
Impaired CD4 Proliferation
Decreased IFN-g
Arginase overexpressing
Leishmania
Donovani
Alternative Activation Pathway
IL-4
IL-13 and IL-10
Inhibitory Effects on NFKB
via STAT6 Activation
CAP
AAP
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
Alternative Activation Pathway
Wound Healing Program
Resistin Like a (RELM-a)
Schistosoma
Mansonii
Relm-a -/-
Accelerated inflammation/
Increased Fibrosis and Granuloma
Formation
Increased TH2 Cytokine
Mechanism is unclear how Relm-a protect
against accelerated Inflammation
Alternative Activation Pathway
Wound Healing Program
Impaired IL-4/IL-13 Pathway lead
to Pathogenesis
Alternative Activation Pathway
Wound Healing Program
Impaired IL-4/IL-13 Pathway lead to Pathogenesis
Intracellular Killing of
Cryptococcus
Neoformans
Overexpression of IL-13
mice have increased intracellular growth rate of
Cryptococcus Neoformans
Lack of IL-13 Expression
mice are more Resistent to infection with
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
Alternative Activation Pathway
Wound Healing Program
Resolution of Inflammation
TGF beta
Vascular Endothelial Growth Factor
Endothelial Growth Factor
M2 – derived TGF-beta and the Role for Fibrosis/Cancer
Interleukin-10
IL-10 -/-
Decreased
Pulmonary Fibrosis
TGF beta
IL-10 overexpressing
Increased
Pulmonary Fibrosis
Alternative Activation Pathway
Other Mediators that Activate the Alternative
Pathway in Macrophages
Bioactive Lipids
Lipoxins and 15d PGJ2
Phenotypic Switch of Cyclooxygenase-2 at
a Later Stage of Inflammation
Prostaglandin D2
Prostaglandin-E2
COX-2
Prostaglandin d PG J2
Alternative Activation Pathway
Other Mediators that Activate the Alternative
Pathway in Macrophages
Lipoxins and 15d PGJ2
Prostaglandin d PG J2
Neutrophil Clearance
Anti-oxidant enzymes
Heat shock Proteins
Apoptosis
Alternative Activation Pathway
Balance between Inflammation (M1) and
Tissue Repair/Resolution (M2)
Acute Lung Injury
Symptoms in Acute Lung Injury
Predisposing Factors
Clinical Relevance
Pathomechanism
Role of the Innate Immune System
Symptoms Acute Lung Injury
• Dyspnea - Trouble Breathing
• Tachypnea – faster breathing
• Tachycardia – increased heart rate
• Central Cyanosis (Hypoxia)
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
Pathomechanism
Some Facts about Gamma Delta T-cells
•
Small Subset of T-cells
•
TCR consist of one Gamma and one Delta Chain
unlike CD4/CD8 T-cells which Bear an Alpha and Beta Chain
•
Highest Abundance in the Gut Mucosa
•
Complex Behavior Which is Still Elusive
First Line Defense
Bridge Innate and Adaptive Immunity
Able to Present Antigen to T-cells
Able to Mature Dendritic Cells
Can Induce Phenotype Isotype Switch from IgG to IgE
And suppress (Vg-1) and enhance IgE Responses (Vg4)
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
Days
0
1
4
Model of Sterile Inflammation
Allows To Elucidate Self Regulatory Function of The
Innate Immune System Without Potential Regulatory
Interactions By Pathogens
7
10
14
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 Samples in Order to Compare
Fluorescence of BAL samples
Normalized to 60 x 106
Normalization of Serum/BAL Samples
Fluorescence Intensity in Sera of Mice 5080 x106
60 x 106 Units - set as 1
Serum sample: 69x106 Units FI (100%) - 60x106 (86%)
BAL:
36597 Units Fi (100%) - 31603 (86%)
Figure 1: Mice deficient for γδ T-cells develop significant worse Lung Injury determined by
Fluorescence Intensity
Fluorescence Intensity
the Pulmonary Vascular Barrier Function after intravenous application of FITC Dextran
Wildtype (WT)
no γδ T-cells (γδ-/-)
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
F4/80
γδ -/- LPS Day 4 TLC
CD206
MHC-2
CD206
MHC-2
CD11b
F4/80-1 / Ly6C high MHC-2 pos
F4/80-2 / Ly6C high MHC-2 pos
CD206
MHC-2
Ly6C
WT LPS D4 TLC
Ly6C
F4/80high /Ly6C high / MHC-2 pos / CD11b pos / CD64pos CD80 pos / CD86pos IL-4R pos
CD11c
CD206
CD64
CD80
CD86
MHC-2
IL-4R
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 TNFa 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
CD3 pos /IL-4 pos
γδ TCR
IL-4 GFP
γδ TCR
CD3 neg /IL-4 pos
CD3
18s rRNA
Interleukin-4
.
Identification of Vg-1, Vg-4 and Vg-7 expanding γδ T-cells during LPSinduced Lung Injury
Day 0
Day 1
Day 4
γδ
A
100000
80000
60000
40000
20000
7
D
ay
4
D
ay
1
ay
D
ay
0
0
D
B
Number of γδ T cells
CD3+
Day 7
WTB6 LPS Day 1
Vγ1
Vγ4
Vγ5
Vγ6
Vγ7
Vγ6
Vγ7
WTB6 LPS Day 4
Vγ1
Vγ4
Vγ5
WTB6 LPS Day 7
Vγ1
Vγ4
Vγ5
Vγ6
Vγ7
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
B
18srRNA
CD3
Vg-1 Vg-4 Vg-7
Interleukin-4
Vg-1
Vg-4
Vg-7
IL-4 amplification: chromosomal DNA = 1504 bp
mRNA = 204bp
IL-4 amplification: chromosomal DNA = 1504 bp
mRNA = 204bp
Gating Strategy is used to identify the Alternative Activation Pathway in Macrophages (Anti-inflammatory)
during LPS-induced Lung Injury:
CD206
CD11b
F4/80-1 / Ly6C low / CD206 pos
F4/80-2 / Ly6C low / CD206 pos
CD206
F4/80
Ly6C
Isotype
Ly6C
F4/80
Ly6C
CD206
γδ -/- LPS Day 4 TLC
Ly6C
Ly6C
WT LPS D4 TLC
Ly6C
F4/80 high /Ly6Clow /CD206 pos / MHC-2 pos / CD11b pos / CD11c pos / IL-4R pos/ MAC-3 pos
CD206
Isotype
MHC-2
CD64
CD11c
IL-4R
MAC-3
γδ -/-
Ly6C
Ly6C
C57B6
LPS Day 1
Ly6C
F4/80
CD206
Ly6C
CD206
CD206
LPS Day 4
γδ -/-
Ly6C
C57B6
Ly6C
CD206
CD206
CD206
Ly6
C
F4/80
Ly6C
F4/80
CD206
CD206
Normalization of the Inflammatory Response at 7 Days after LPS-induced Lung Injury
γδ -/- LPS Day 7
Ly6C
C57B6 LPS Day 7
Ly6C
LPS Day 7
F4/80
Ly6C
F4/80
CD206
CD206
Ly6C
CD206
CD206
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
no γδ T-cells (γδ-/-)
Wildtype (WT)
“Treatment with recombinant IL-4 reduces TNF-a expression in resident Alveolar
Macrophages”
Treatment with
recombinant IL-4
Ly-6C
Isotype PE
F4/80
CD206
Isotype
TNF-a PE
TNF-a PE
Hypothesis:
Gamma Delta T-cells protect against LPS-Induced Lung Injury via Interleukin-4
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
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.
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.
Acknowledgments
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