The Phagocytic Synapse in Distinguishing Particulate and Soluble Stimuli David M. Underhill

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1 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli How does a cell know the difference between an inflammatory stimulus and a phagocytic stimulus? rof., h.d. Inflammatory Bowel & Immunobiology Research Institute Cedars-Sinai Medical Center, Los Angeles & UCLA Dept. of athology and Laboratory Medicine 1 The far-sighted macrophage Long distance surveillance Soluble microbial components Secretion of cytokines and chemokines to attract and activate other immune cells 2 The near-sighted macrophage hagocytosis Respiratory Burst Killing & Digestion Antigen rocessing 3 Secretion of cytokines and chemokines to attract and activate other immune cells 1

2 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli A far-sighted receptor mechanism Lipopeptide TLR2 TLR6 MyD88 Mal IRAKs Transcriptional regulation NF-κB, A-1, etc. 4 A near-sighted receptor mechanism Yeast Dectin-1 Dectin-1 Src - - Syk 5 hagocytosis Respiratory Burst NFAT, SRF NF-κB, But there s this inconvenient observation: For 3 years or more folks have been using soluble β-glucans to block yeast recognition by macrophages Laminarin a soluble glucan (storage polysaccharide) prepared from brown algae So, it must be too small, right? 6 2

3 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli β-glucan β-(1,3)-glucan β-(1,6)-glucan branch 7 MW = 15 Da Dectin-1 β-glucan 8 MMDB ID: 6461 Jones & Coworkers, rotein Science(27) MW = 15 Da 9 MW = 32 KDa 3

4 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli 1 MW = 13 KDa β-glucans have many structures, 11 there will be many binding sites available MW = 4 KDa So we got some β-glucans Laminarin Low MW Med. MW Med. High MW High MW Whole Glucan articles avg. 1 kda From Seaweed avg. 16 kda avg. 15 kda avg. 22 kda From S. cerevisiae avg. 4 kda >95% pure 12 4

5 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli Soluble β-glucans bind to Dectin-1 wild type bmdc Dectin-1 -/- 13 DTAF-MMW (22 kda) bright field A B bmm - ROS bmdc - ROS zymosan WG 8 particulate 4 particles particles MMW LMW 2 4 soluble soluble MHMW HMW soluble minutes minutes C bmm - TNF-α D bmdc - TNF-α particles soluble 2 *** F 16 *** particles 16 particles 12 soluble *** 12 8 soluble *** 8 n.s. n.s RLU ng/ml 4 Soluble glucans do not work regardless of their size or the assay unstim. zymosan E WG particles WG MMW LMW MHMW HMW RLU ng/ml 4 unstim. zymosan WG MMW LMW MHMW HMW soluble β-glucan MMW LMW MHMW HMW soluble unstim. G unstim. particles soluble phospho-p38 p38 14 Syk phosphorylation none β-glucan I-κBα GADH How does Dectin-1 discriminate between soluble and particulate β-glucans? 15 5

6 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli Immobilized β-glucans trigger Dectin-1 signaling 5 bmdc - TNF-α Immobilized ng/ml Soluble 16 unstim. MHMW HMW cmhmw chmw Soluble glucans coated onto bottoms wild type Dectin-1 -/- of tissue culture plates Immunological synapse TCR LFA1 * LCK Resting T cell lasma Membrane Abundant hosphatase () rimes Signaling Kinases, but Generally revents Spurious TCR Signaling 17 Immunological synapse (2) T Antigen resenting Cell MHC/peptide CD8/ CD86 ICAM1 AC LFA1 CD28/ CTLA4 Signaling 18 TCR 6

7 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli and CD148 Membrane tyrosine phosphatases Dual role in ITAM signaling 1. Activating remove inhibitory phosphorylation from Src-family 2. Inhibitory limit downstream Tyr kinase-based signaling 19 ABC is found on the surface of all nucleated hematopoietic cells On T and B cells it comprises up to 1% of the cell surface area 5 nm alternatively spliced The cytoplasmic tail contains two phosphatase domains Although there is low sequence identity between extracellular regions in different species, the cytoplasmic region is highly conserved RO 2-3 nm CD4 13 nm 2 * phosphatase domains ( * active) * CD4 MHC-eptide TCR Complex * Lck * Csk Zap7 Zap7 7

8 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli Do and CD148 regulate Dectin-1 signaling? Reactive Oxygen roduction TNF-α RLU 4 3 CD148 -/- wild type 2 -/- 1 -/- CD148 -/ β-glucan particles (minutes) pg/ml none β-glucan particles 22 BMMO wild type -/- CD148 -/- -/- CD148 -/- Do Dectin-1 & form a synapse-like structure during phagocytosis? Red = Green = Dectin-1 Blue = zymosan core 23 Dectin-1 phagocytic synapse -3 sec sec 3 sec 6 sec 9 sec WG Dectin-1 24 Dectin-1/-expressing RAW264.7 macrophages 8

9 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli Dectin-1 phagocytic synapse (2) Dectin-1 (Green) (Red) Zymosan (Blue) 25 RAW264.7 macrophages Dectin-1 phagocytic synapse (3) 26 Dectin-1-expressing RAW264.7 macrophages Dectin-1 phagocytic synapse Aspergillus fumigatus Dectin-1 Merge Bright field 27 Dectin-1/-expressing RAW264.7 macrophages 9

10 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli CD148 Glucan article Active Signaling Complex Dectin-1 X X Inactive src Active src Ca ++ NFAT Calmodulin Calcineurin NFAT p38 LCγ2 KCδ ERK RAC GT CARD9 Signalosome CARD9 ASC BCL1 MALT1 CASASE 8 hagocytic Synapse hagocytosis Respiratory Burst IL-1β rocessing & Secretion 28 NFAT Jun A-1 Fos SRF NF-κB p5 p65 Transcriptional Response IL-12, IL-23, IL-6, TNF-α, IL-1 CD148 Soluble Glucans X X Inactive src Active src No Formation of a hagocytic Synapse Soluble glucans bind & cluster Dectin-1, but do not sufficiently exclude the membrane phosphatases to allow signaling to progress 29 Soluble glucans immobilized on a plastic surface activate signaling Active src Ca ++ NFAT Calmodulin Calcineurin NFAT p38 GT RAC LCγ2 KCδ CARD9 Signalosome CARD9 ASC BCL1 MALT1 ERK CASASE 8 hagocytic Synapse hagocytosis Respiratory Burst IL-1β rocessing & Secretion 3 NFAT Jun A-1 Fos SRF NF-κB p5 p65 Transcriptional Response IL-12, IL-23, IL-6, TNF-α, IL-1 1

11 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli Dectin-1 phagocytic synapse plate-coated β-glucan Uncoated HMW β-glucan-coated sec 3 sec sec 3 sec Dectin-1 31 Dectin-1/-expressing RAW264.7 macrophages What happens if you also coat the plastic surface with anti- antibodies as well to prevent movement of the proteins? Anti- CD148 X 32 X Inactive src Active src hagocytic Synapse? co-immobilization bmdc - TNF-α ng/ml.8.6 ***.4.2 unstim. chmw cls WG 33 none IgG anti- 11

12 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli Summary hagocytic synapse formation allows a cell to distinguish between microbes in the immediate vicinity and components shed from microbes at a distance revents inappropriate initiation of phagocytosis and direct anti-microbial responses 34 On the term phagocytic synapse The term phagocytic synapse or engulfment synapse has previously been proposed in the literature to describe the re-organization of macrophage cell surface proteins upon engagement of apoptotic cells See: Fadok et al., 21 J Clin Invest 18: 957, Grimsley and Ravichandran 23 Trends Cell Biol 13: 648) In this context, the term has been applied simply to denote the points of contact between phagocytic cells and the receptors & ligands found there It is not clear yet whether this contact site bears any functional similarity to the Immunological Synapse that forms between an AC and a T cell It is this similarity that has lead to the use of the term hagocytic Synapse to denote the mechanism by which cells discriminate soluble from particulate ligands 35 Do other receptors require a phagocytic synapse? Myosin II-dependent exclusion of from the site of Fcγ receptor activation during phagocytosis Yamauchyi et al., FEBS Letters, 19: (212) Myosin II contributes to FcγR activation and subsequent F-actin assembly at the nascent phagocytic cup; While FcγRs cluster beneath an IgG-opsonized particle without myosin II activity, myosin II is required to exclude and permit full activation of signaling and phagocytosis 36 12

13 The hagocytic Synapse in Distinguishing articulate and Soluble Stimuli Further reading: Goodridge et al., Activation of the innate immune receptor Dectin-1 upon formation of a hagocytic Synapse, Nature, 472: (211) Goodridge & Underhill, Information processing during phagocytosis, Nature Reviews Immunology 12: (212) Goodridge et al., Mechanisms of Fc receptors and dectin-1 activation for phagocytosis, Traffic 13: (212) Kerrigan & Brown, hagocytes: Fussy about Carbs, Current Biology, 21: R5-52 (211)