docHYPOXIA AND ITS IMPACT ON THE IMMUNE SYSTEM
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HYPOXIA AND ITS IMPACT ON THE IMMUNE SYSTEM
Plenary Symposium: New insights into the Basics of Tumor Immunology Luigi Varesio (Italy) HYPOXIA AND ITS IMPACT ON THE IMMUNE SYSTEM Hypoxic Tissue Microenvironment Local Receptor Independent Signals pH Exosomes Cell migration HIF Oxygen tension Cell interaction Hypoxia Genes HIF Systemic Signals HYPOXIA Cytokines Hormones NORMOXIA Tissue hypoxia: a condition of low oxygen tension O2 O2 Tumor hypoxia Tumor Hypoxia is patchy VEGF Immunohystocheminstry of neuroblastoma sections ANA-1 Mouse tumor stained by hypoxyprobe Experimental hypoxia Luigi Varesio Pimo HIF-1α Hypoxic workstation Normoxia (20%O2) Hypoxia (1%O2) VEGF Mn Infiltrate •Phenotype •Geneexpressionprofile •Functionalresponse CD68 Hypoxia signatures NB-hop (hypoxia outcome prediction) Luigi Varesio Hypoxia inducible genes Hypoxia Inducible Genes Muscle Cancer cells Core hypoxia genes Leukocytes Epithelium Liver Hypoxia signatures Neurons Tissue/tumor specific Hypoxia risk factors Prior knowledge Other known risk factors Cancer Prognosis NB-hop PGK1 ALDOC PDK1 EGLN1 AK4 FAM162A MTFP1 Neuroblastoma patients stratification by NB-hop Luigi Varesio NB-hop splits the 636 patients dataset into two groups of 174 and 462 patients low hypoxia Survival curves high hypoxia Multivariate Cox regression Factor P HR NB-hop (high vs low) 9.11E-05 2 (1.4-3.3) MYCN (amplified vs normal) 0.0002 2.1 (1.4-3.1) Age group (<1 year vs >=1 year) 0.001 3 (1.5-6.0) INSS stage (st4 vs not st4) 6.53E-14 6.3 The NB-hop signature is an independent risk factor for neuroblastoma Tumor hypoxia promotes tumor aggressiveness 95%CI (3.9-10.3) NB-hop signature measures tumor hypoxia and predicts poor outcome Luigi Varesio CA IX Stage 2 Good prognosis NB-hop: ”low” Stage 2 Poor prognosis NB-hop: “high” HIF‐1 alpha Tumor hypoxia is associated to poor outcome VEGF Hypoxia and cancer • How does hypoxia affect tumor progression? – Understanding tumor microenvironment • • • • Tumor cells Vasculature Stroma Leukocyte HYPOXIA INFLAMMATION CANCER Life, hypoxia and immune system Years ago: 3.8 billion first procariotic fossil 2.0 billion photosynthetic organisms 1.8 billion first eucariotic organism Hypoxic Atmosphere (1% oxygen) 0.6 billion animal evolution Normoxic Atmosphere ( 20% oxygen) Adaptive immunity Innate % O2 immunity 2000 900 700 550 Mil. years ago Effects of hypoxia on innate versus adaptive immunity Monocyte Dendritic Langherans Lymphocyte Macrophage NK cell Neutrophil INNATE Immunity O2 (inflammation) ADAPTIVE Immunity (effector) Hypoxia-induces more than 1000 genes in human monocytes Costimulatory/adhesion molecules Scavenger/Pattern Recognition Receptors Transcription Factors Immunoregulatory Molecules Chemokines/Receptors Regulators of Cholesterol Biosynthesis/Transport ECM Components/Regulators Cytokines/Growth Factors/Receptors osco et al. 2006 Juvenile Idiopathic Arthritis Clinical manifestations: - Persistent synovial joint inflammation - Leukocyte infiltration - Synovial fibroblast proliferation - Synovial tissue hyperplasia - Decreased capillary density and vascular system disorganization - Progressive cartilage destruction, bone erosion Synovial pO2 = 0.5-2% HIF-1 HIF-1 HIF-2 JIA synovial tissue is hypoxic HIF-2 CD68 Monocytes are trapped in the tissue Bosco et al., A.R., 2008 O2 Hypoxic tissue lymphnode Monocyte Chemokine receptors ↓ Gro-chemokine ↑ Circulation Monocyte trapping DENDRITIC CELL GENERATION GM-CSF IL-4 MONOCYTES GM-CSF IL-4 IMMATURE DC TNF- IL-1 IL-6 PGE2 MATURE DC •Phenotype •Gene expression profile •Functional responses O2 levels Hypoxia modulates a chemokine/receptor gene cluster in H-DCs Hypoxia CCL18 CCR2 CCL23 •Inhibition of NK, Mn and naïve/resting T cell influx and Th2 polarization CCR3 CCL24 CCL26 CXCR4 CCL13 CX3CR1 CCL14 CCL2 •Increased responsiveness to chemoattractants CCR5 H-iDC Migratory Phenotype Hypoxia CCL20 CCL3 CCL5 •Inhibition of naïve T cell influx CCL18 CXCL2 CCL23 CXCL3 CXCL5 H-mDC Proinflammatory Phenotype CXCL6 CXCL8 •Recruitment of Mn, iDC, and activated/memory Th1/Th17 cells •Mn trapping •Neutrophil recruitment •Angiogenesis Hypoxiamodulatesimmune‐relatedgeneclustersinDCs TREM‐1 AngiogenicFactors •AbsentonDC(expressed onPMN,Mn,Mf) Scavenger/Pattern RecognitionReceptors TREM-1 •AssociatedwithITAM‐bearingadaptorproteinDAP12forsignaling Immunoregulatory TREM-1 signalingreceptors CD37 CD180 •Amplifierofinflammatoryresponses,implicatedintheperpetuationof CD53 CLEC-2D/2B bothchronicinfectiousandnon‐infectiousinflammatorydiseases CD9 TLR1,2 Chemokine/Receptors CD32 ECMComponents/ CD31 •Potential ligands:DAMPmoleculesHMGB1andHSP70 Regulators CD69 iDCs Cytokines/Receptors • H-DCsC mDCs Costimulatory/adhesion molecules Members of the Ig and the C-type lectin superfamilies of inhibitory /stimulatory cell surface receptors, broadly expressed on myeloid cells Damage Associated Molecular Pattern (DAMPs) Necrosis Chronic Inflammation HYPOXIA (pO2 0-20mm Hg) Reducing environment DAMPs extended persistance DAMPs (Redox sensitive) Extracellular oxidation HypoxiainducesTREM‐1inDCs Hypoxia Fold Change 50 -- + - + - + TREM-1 40 actin 30 Donor 1 20 Donor 3 Donor 5 10 0 H-DC mDC H-iDC 9.5 80 0 2 0 98 H-mDC 83% 1% TREM-1 0.5 10 CD83 CD1a Normo events TREM-1 i-DC DC 0% Hypo 76% (33) Reox (24h) 52% (11) Synovial fluid PO2 measurement (0.8-5.5%) 43 37 15 5 6% Patient 1 SFMC purification SSC-H 4.6% HIF-1 -actin TREM-1 R1 60 15 15 10 Patient 3 R1 PBMC SFMC PBMC SFMC PBMC SFMC Patient 1 Patient 3 Patient 6 64 16 16 4 6.6% Patient 6 R1 CD1a CD83 TREM‐1ismarkerofhypoxicDCsinvivo Langherhans cells in the hypoxic environment of Scar and Ulcer are TREM-1+ LANGERIN HIF-1α TREM-1 HEALTHY SKIN HEALTHY SKIN HYPERTROPHIC SCAR HYPERTROPHIC SCAR DECUBITUS ULCER LANGERIN TREM-1 DECUBITUS ULCER 0 * 5 10 15 20 25 *** *** HYPERTROPHIC SCAR DECUBITUS ULCER ** HEALTHY SKIN * * 0 5 10 15 n° of positive cells (0.05 20 mm2 25 0 5 10 tissue section) 15 20 25 MERGE TREM‐1activation amplifiesofDCpro‐inflammatoryfunctions Agonist mAb TREM-1 IL-12p70 TNF DAP12 p-Akt IL-6 CCL4 P-Erk p-IB CCL17 CCL5 TREM-1 cross-linking triggers DAP12 signaling pathways D o no r 6 T0 Activation of Th1 cells Inflammation I g T RE M D o no r 7 Ig TR EM p -E rk E rk -t ot p -A kt A kt -t o t CXCL8 OPN IL1β Mn, activated T cell recruitment Neutrophil recruitment Angiogenesis Inflammation Hypoxia amplifies chronic inflammation through chemokine production and TREM1 expression Angiogenesis HIF-1 CC-chemokines Hyperplasia Hypoxia HIF-2 CXC-chemokines iDC/mDC TREM-1 •Recruitment of Mn, iDC, Th1/Th17 cells •Mn trapping •Neutrophil recruitment •Angiogenesis Polarization toward a Th1/Th17proinflammatory direction Inflammation Hypoxic tissue lymphnode O2 Monocyte trapping CC-chemokine receptors ↓ Gro-chemokines ↑ Circulation iDC migratory phenotype Chemokine receptors ↑ IRS receptors ↑ mDC Inflammatory phenotype Proinflamatory, Th1-priming cytokines/chemokines ↑ TREM-1 ↑ Hypoxia (and TREM-1 stimulation) promote inflammatory macrophages OO2 2 Hypoxia Normoxia TREM-1 Increase of pro-TREM-1 CD 80 Pro-inflammatory Inhibition of Th1 cells Activation of Th1 cells inflammatory activity activity IL6 IL6 IL8 IL8 OPN OPN CD 80 M1 M1 TNFα IL12 TNFα IL12 TNF M0 Mn CD80 IL8 TREM-1 ACTIVATION H-iDC MCSF ( 0-6d) TREM-1 Anti –inflammatory TREM-1 CD 206 activity Anti –inflammatory activity TGFβ IL10 TGFβ IL10 OPN CD 206 Inhibition of Th2 Activation of Th2 cells cells IL6 IL12 M2 M2 CCL24 CCL18 CCL24 CCL18 Inflammatory phenotype (M1 switch) Hypoxia PROMOTES innate immunity Hypoxia INHIBITS adaptive immunity Monocyte Proliferation Survival Effector functions Dendritic Langherans Lymphocyte Macrophage Activating receptors Killing Extravasation Survival Anti-microbial activity Inflammatory responses NK cell Neutrophil INNATE Immunity O2 (inflammation) ADAPTIVE Immunity (effector) Thanks to… Lab of Molecular Biology Department of Pediatrics Federica Raggi Marco Gattorno Maria Carla Bosco Martina Morini Cristina Vanni Clinical Bioinformatics Davide Cangelosi Massimiliano Izzo Department of Pathology Claudio Gambini Clinical Oncology Alberto Garaventa Amsterdam Medical Center Lab of Tumor Immunology Jan Koster Mirella Giovarelli Rogier Versteeg Daniele Pierobon Torino Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
