1 Department of Immunology The Weizmann Institute of Science Rehovot, Israel The mononuclear phagocyte system Van Furth and Cohn (1968) Ilya (Eli) Metchnikoff (1845-1916) Nobel prize 1908 Macrophages Ralph Steinman (1943-2011) Nobel prize 2011 Dendritic cells 2 Role in immunity Homeostasis (physiological inflammation) Unrivalled migratory Naïve T cell stimulators The mononuclear phagocyte system (2) Van Furth and Cohn (1968) Macrophages Dendritic cells 3 Outline of presentation: Mononuclear phagocyte development Mononuclear phagocyte functions in the intestine 1
The hematopoetic system Myeloid lineage Common Myeloid Precursor GRANULOCYTES (PMN) MONOCYTES MONONUCLEAR PHAGOCYTES MACROPHAGES Hematopoetic Stem Cell Common Lymphoid Precursor DENDRITIC CELLS, DC T CELLS 4 Lymphoid lineage LYMPHOCYTES B CELLS NK CELLS Mononuclear phagocyte origins CX 3CR1 CX 3CR1 2005 HSC CMP MP MΦ DC Progenitor 5 CLP MEP Granulocytes Lin - CD34 + ckit + CD16 + CX 3CR1 + Varol et al., 2007 Monocyte subsets Mouse CD115 Ly6C/G CX 3CR1/ CX 3CR1/ blood CX 3CR1 6 Geissmann, Jung & Littman, 2003 2
Monocyte subsets (2) CD115+ blood cells Human monocyte subsets Ziegler-Heitbrock et al., 1996 Human Mouse CD14 ++ CD16 +/- CD14 dim CD16 ++ 7 Cros et al., 2010 Monocyte subsets (3) CCR2 + Inflammatory monocytes Patrolling monocytes blood 8 Geissmann, Jung & Littman, 2003 Auffray et al., 2009 Monocyte origins Which pathway dominates in steady state? MΦ DC Precursor? CCR2 + CCR2 + 2 Ly6C alternative + but not routes to Ly6C monocytes - Sunderkotter et al., 2007 express CCR2 Yrlid et al., 2006 Varol et al., 2007 CCR2 is required for exit of monocytes from BM Serbina et al., 2006; Shi et al.. 2011 Bone marrow Blood 9 Fogg et al., 2006 Varol et al., 2007 Geissmann, Jung & Littman, 2003 3
Monocyte origins (2) Mixed BM chimeras Blood Lymphocytes equally represented CCR2 -/- (CD45.2) BM from both genotypes WT () BM B cells 52.2 1.54 CD115 Gr1 BM B220 0.12 46.2 CD45.2 Monocyte precursors (s) equally represented from genotypes 10 CD135 FSC CD117 CD115 % of Max CD45.2 Ki-Wook Kim Yochai Wolf Mixed BM chimeras CCR2 -/- (CD45.2) BM WT () BM Monocyte origins (3) Ly6C 84.5 0.375 87.9 0.325 CX3CR1 0.0536 CD45.2 15.1 0.0464 CD45.2 11.7 21 Both monocyte subsets equally affected by CCR2 deficiency Wt / CCR2 -/- ratio 18 15 12 9 6 3 1.5 1.0 0.5 11 0.0 MP Ly6C+ BM blood spleen monocytes Conclusion I Mononuclear phagocyte origins Steady state monocytes form a developmental series MΦ DC Precursor 12 Bone marrow Blood 4
Monocyte fates Blood 13 Conditional mononuclear phagocyte ablation -DTR mice DTx Receptor (DTR) Diphtheria Toxin (DTx) Mice are naturally DTx resistant because they lack high affinity DTx receptor (hbegfr) Primate DTR transgene confers DTx sensitivity Saito et al., Nat. Biotech. 2001 Jung et al., Immunity 2002 DTA Promoter DTR + DTx 14 Ablation of : expressing cells All high cdcs Defined macrophage populations Mononuclear phagocyte origins -DTR mice Intestine without graft CX 3CR1 monocytes + DTx Intestine with graft reconstitute macrophages in vivo differentiation 15 Varol et al., JExMed 2007 5
Mononuclear phagocyte origins (2) -DTR mice Donor * gated on + cells DC-depleted mouse + DTx hi cdcs recipient Monocyte recipient s, but not monocytes yield classical splenic cdcs in vivo differentiation 16 Varol et al., JExMed 2007 Mononuclear phagocyte origins (3) are absent from circulation Identification of circulating dedicated cdc precursors, pre-cdc Liu et al., Science 2009 Adoptive transfers 17 Pre cdc B220 CD11b % Donor-derived cells mono Pre-cDC cdc Conclusion II: Mononuclear phagocyte origins YOLK SAC Primitive macrophages E7.5-8.5 Self renewal HSC BM Through Adulthood FETAL LIVER Prenatal MP Granulocytes monocytes CDP Pre-DC PDC Self renewal Macrophages monocyte-derived Macrophages CD4 + DN CD8α + CD103 + 18 MoDCs FLT3-dependent classical DCs 6
Gastrointestinal tract Bioreactor for food breakdown (>10 13 bacteria) Large surface & thin border (single cell epithelial layer) Challenge to the immune system Tolerance > Commensals Defense > Pathogenic enterobacteria Colon 10 11-12 CFU/ml Breakdown of balance Inflammatory Bowel Disease (IBD) 19 Intestinal macrophages CX 3CR1 CX 3CR1 20 The CX 3 CR1/L1 reporter animals CX 3CR1 CX 3CL1 Fractalkine CX 3CR1 45 kb CX 3CR1CX 3CR1 :CX 3CL1 cherry Targeted replacement of chemokine receptor with reporter prominent expression in mononuclear phagocytes BAC transgene encoding reporter under CX 3 CL1 promoter control ATG CX 3C mucin stalk T M 21 CX 3 CR1 : CX 3 CL1 Cherry mice Jung et al., MCB 2000 Kim et al., BLOOD, 2011 7
CX 3CR1 :CX 3CL1 cherry Macrophages Epithelial/ Goblet cells 22 Ki-Wook Kim Blood 2011 Intestinal lamina propria mononuclear phagocyte compartment Analysis of colon of CX 3 CR1 gfp mouse Gated on living CD45 + cells 87 % R1 + CD11b - R1 R2 MHC II (I-A b ) R3 2.5 % R2 + CD11b + FSC CD11b + CD103 + CD11b - CD103 + CD11b + DCs 82 % R3 - CD11b - 23 CX 3 CR1 + CD11b + CX 3 CR1 - CD11b + MΦ s CD103 CX 3CR1 / 82 % Zigmond Intestinal lamina propria mononuclear phagocyte compartment (2) CX 33 CR1 + + Macrophages macrophages monocyte-derived CD103 + Dendritic cells predc-derived Varol, C., et al. Bogunovic, M. et al., Immunity 2009 24 Varol, Zigmond & Jung, Nature Reviews Immunology 2010 8
Task division between migratory DCs and resident MFs LAMINA PROPRIA CD103 + dendritic cells CCR7 Th2 CX 3 CR1 + macrophages Th2 Th17 Th1 Treg Treg CCR9, a4b7 Effector site (Maintenance selective expansion) NHC gd LTi Innate lymphoid cells steady state MsnLN 25 naïve CD4 T cell priming site Th1 Th17 Scheme compiled from data of Pabst, Agace, Kelsall, Mowat, Powrie and Pulendran groups Steady state Resident MFs Inflammation Exposure to dextran sodium sulfate (DSS) in drinking water for 7 days Lesions in epithel CD11b Cx3cr1/ 26 Inflammation recruits a novel CX 3 CR1 int cell subset Steady state Inflammation Resident MFs day 4 day 7 CD11b 27 Cx3cr1/ See also: Weber et al., EJI 2010, Platt et al., JI 2010 Rivollier et al., JExMed 2012 % out of CD45 + living cells CD11b 20 10 Cx3cr1/ 0 0 5 10 15 20 CX3CR1 high F4/80 high CX3CR1 dim F4/80 low Neutrophils 9
CX 3 CR1 int infiltrate is monocyte-derived w/o graft DSS 2% in drinking water day 0 2 4 5 6 7 Monocyte transfer Analysis Ly6C hi monocytes seed tissue, give rise to Ly6C lo cells Monocyte recipient Ly6C day 1 CX 3CR1/ F4/80 MHC II 28 CX 3CR1/ Ly6C F4/80 MHC II day 3 Cells gain CCR7 expression CX 3 CR1 int infiltrate is monocyte-derived, requires CCR2 DSS 2% in drinking water day 0 2 4 5 6 7 Mixed monocyte graft CCR2 KO WT Monocyte transfer Analysis Mixed monocyte recipient CX 3CR1/ 29 Only WT cells but not CCR2 deficient cells seed inflamed tissue CX 3 CR1 int monocyte-derived infiltrate promotes inflammation DSS 1% in drinking water day 0 2 4 5 6 7 Anti-CCR2 ab Monocyte depletion DSS only DSS + MC21 30 CD11b Ly6C 10
Expression analysis of monocyte progeny during steady state and inflammation monocytes Steady state CX 3 CR1 hi resident macrophages CX 3 CR1 int infiltrating macrophages DSS Colitis Resident MFs Infiltrating MFs Steady state DSS Colitis 31 CX 3 CR1 hi resident MFs express IL-10 (Pabst, Kelsall, Pulendran) CX 3 CR1 int MFs express pro-inflammatory cytokines (IL6, IL23) fold change CX3CR1 int over CX3CR1 hi 16 12 8 4 0-4 -8-12 -16 IL6 IL23 QRT-PCR IL10 TNFa CX 3 CR1 int MFs respond to bacterial products Expression of recruited CX 3 CR1 int cells over resident CX 3 CR1 hi cells TLR2 & NOD-like receptor signaling pathway PDG TLR-2 ie- DAP NOD1 RIP2 CARD9 IKKa IKKg IKKb IkB NFkB IL-6 NOD2 32 CX 3 CR1 int MFs respond to bacterial products (2) Infiltrating MFs, but not resident MFs secrete IL-6 in response to TLR2 / NOD2 ligands Pam3 in vitro assay Muramyldi-peptide 33 11
Conclusion CD103 + DCs CX 3 CR1 + MF s give rise Education to CX 3 CR1 + macrophages Homeostasis in lamina propria Are in steady state educated by environment Under inflammation monocyte infiltrate fails LN to be tamed respond to bacterial product and exacerbate disease 34 IL10 IL10 IL6 IL6 Inflammation LN CCR 7 Dan Littman Frederic Geissmann Skirball Institute King s College, London NYU Medical Center Simon Yona Ki-wook Kim Diana Rashkovan Yochai Wolf Tegest Aychek Alexander Mildner Rita Krautgammer Chen Varol Tel Aviv-Sourasky Medical Center 35 36 12