Neuronal guidance protein semaphorin 7A influences neutrophil arrest and aggravates inflammatory lung injury. Dr. Tiago Granja
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1 Neuronal guidance protein semaphorin 7A influences neutrophil arrest and aggravates inflammatory lung injury Dr. Tiago Granja
2 Neuronal guidance proteins (NGPs) force cells to move NPGs give axons and dendrites the ability to locate and recognize their appropriate synaptic partners 1 Filopodia filaments are oriented toward the filopodium tip 2 Microtubules stabilize the axon shaft 3 A growth cone drives the filipodia to extend and retract 4 Strong directions stabilise and dilate a single filopodium and following microtubuli
3 Neuronal guidance proteins (NGPs) force cells to move NPGs families and receptors n Semaphorin7A (CDw18) is ligand of: PlexinC1 (CD232) Β1 integrin (CD29) β 1
4 ring The neutrophil recruitment cascade Rolling Adhesion Crawling Transmigration Slow rolling Neutrophil drive: Full arrest Indirect the endothelial leukocyte recruitment signals cascade from tissue involves destruction the fol- (Cytokines, leukotrienes, histamine) Intraluminal crawling Firm Neutrophil adhesion extravasation detection of pathogens that increase expression of adhesi on molecules; within minutes, they upregulate pre- The neutrophil recruitment cascade. In most tissues, stored P-selectin from Weibel Palade bodies. E-selectin, lowing commonly recognized steps: tethering, rolling, which is synthesized de novo, is upregulated within Tethering adhesion, crawling and, finally, carried transmigration by PSGL1,CD62L (FIG. 1). 9 (L-selectin), minutes 2,45. These two selectins have partially overlapping functions and maximize neutrophil recruitment. Neutrophil recruitment is initiated by changes on the Slow surface rolling of endothelium that CD11a/CD18 result from stimulation (LFA1) by (CD62P Once present - P-selectin on the endothelial induced) surface, P-selectin and a Paracellular, inflammatory mediators (including between histamine, cysteinylleukotrienes and cytokines) CD11b/CD18 that endothelial are released cells from (MAC1) tissue- -P-selectin β2 integrins, glycoprotein ligand 1 (PSGL1) 2,46, leading E-selectin bind to their glycosylated ligands, including Adhesion resident sentinel leukocytes when they come into contact Extravasated to the tethering (capturing) of free-flowing neutrophils to Crawling with pathogens 1,2,4. Endothelial MAC1 OR cells can also be activated neutrophil the surface of endothelium and their subsequent rolling directly by pattern-recognition receptor (PRR)-mediated along the vessel in the direction of blood flow (TABLE 1). Direct endothelial signals derived from PPR B oodf o (E-selectin) Transmigration LFA1, MAC1 REVIEWS Rolling Adhesion Crawling Transmigration Extravasated neutrophil Free circulating neutrophil neutrophil Adhering neutrophil Tethering Slow rolling Intraluminal perpendicular crawling Selectin Full arrest b Transcellular via individual cells (potential dome formation a b) Chemokine receptor Firm adhesion Inactive integrin Intraluminal crawling Extravasated neutrophil Integrin ligand Selectin Active Neutrophil recruitment and function in health and inflammation Chemokine Rolling neutrophil ligand integrin Kolaczkowska, E. Kubes, P. Nat Rev Immunol; 213 with a tether 1.138/nri3399 Figure 1 The updated classical neutrophil recruitment cascade. Shown are the sequential steps of neutrophil recruitment from the vasculature to the tissue. Two possible methods of transmigration are acknowledged: Nature Reviews Immunology a Paracellular, between endothelial cells OR B oodf o Extravasated neutrophil
5 Lung injury as a model to study Neutrophil recruitment Events during Acute Lung Injury (ALI) Platelet aggregation Endothelial gap formation Neutrophil migration Tissue destruction Acute lung injury and the acute respiratory distress syndrome in the injured patient; Magdalena Bakowitz , Brandon Bruns and Maureen McCunn; Scandinavian Journal of Trauma; Resuscitation and Emergency Medicine 212 Release of pro-inflammatory mediators: Fibrin Thrombin MPO TNFα IL6 IL8 IL1β
6 Semaphorin7a is pro-inflammatory NaCl DAPI vwf Sema7a merge NaCl ALI induces the release of proinflammatory cytokines in the lung ALI up-regulates Semaphorin7a in the lung mice have less Platelet-neutrophil complex (PNCs) infiltration
7 Block Sema7a, PlexinC1 and β1 integrin inhibits aggregation Aggregation (Norm. a.u.) Aggregation (AU) Multiplate analysis Whole blood incubated TRAP6 - Thrombin Receptor Activator Peptide 6 - Amino Acid Sequence Ser-Phe-Leu-Leu-Arg-Asn Control CD18 antibody CD29 Antibody PlexinC1 Antibody Semaphorin 7A Antibody - PAR 1 hexapeptide agonist Time (min) TRAP6 Aggregation Inhibition of thrombin derived aggregation by antibody block of: β1 integrin (CD29) Sema7a (CDw18) PlexinC1 (CD232).2 Acquisition of electrical impedance over time. Ctrl CD29 ab CD18 ab SEMA7a ab PLXNC1 ab
8 Neutrophil transmigration (cells/ml) Semaphorin7a slows neutrophils and induce transmigration Primary adhesion with E-Selectin Sema7a ICAM1 ICAM1Sema7a Neutrophils rolling and attach Whole Blood Blood Flow E-selectin E-selectin Semaphorin7a Whole blood from n=5 healthy donors. Capillaries are coated with adhesion molecules PNC transmigration HMEC culture Platelets 1/5 Neutrophils 37 C PNCs E-selectin ICAM-1 E-selectin ICAM-1 Semaphorin7a Semaphorin7a slows Neutrophils in flow Neutrophils isolated from n=5 healthy donors. Platelets isolated and diluted 1/5 Membrane pores of 3µm fmlp as chemoattractant for 9min fmlp 3µm NaCl SEMA7A Semaphorin7a induces Neutrophils to transmigrate
9 s CD11b function is controlled by Semaphorin7a CD66b - FITC CD66b - FITC ICAM1 PerCp (Norm. Freq. of parent) TNFα TNFα Rolling Neutrophils Inactive Neutrophil integrin CD18/CD11b Active Neutrophil integrin CD18/CD11b 85% PMNs Alive % PMNs Dead CD11b block Semaphorin7a ICAM-1 DRAQ7-APC FSC ICAM1 - PerCp Neutrophils were isolated from n=5 healthy donors. 1 Proper controls set for CD11b block Reactions take min TNFα induces ICAM1 adhesion in neutrophils 4 2. Semaphorin7a Alone induces max. ICAM-1 adhesion through CD11b 2 3. Semaphorin7a TNFα inhibits CD11b function Semaphorin7a has different functions during haemostasis and inflammation CD11b Block TNFa Sema7a Sema7a TNFa
10 25min Neutrophil interaction (stationary cells over 1µm) Platelet sedimentation (Norm. a.u.) 2min min Tramsmigration distance (mm) cell transmigration (cell/mm 2 ) 1min 5min Cell speed (mm/sec) Stationary cells (cells/mm 2 ) min i.v. Neutrophils Platelets neutrophils do not adhere Sham Sham Neutrophil rolling Stationary cells Time (min) Time (min) 4 3 Transmigration distance PNC transmigration Time (min) Time (min) 4 3 Neutrophil contact Platelet sedimentation Time (min) Time (min)
11 Neutrophil survey time (sec) Rolling cells after min i.v. PNC buildup aftermin (Platelet fluo. a.u.) PNC buildup after min (Platelet fluo. norm. a.u.) neutrophils do not crosstalk min i.v. min PNC cross section (µm) 2 1 and Semaphorin7a -/- mice are equally able to build PNCs after i.v. Semaphorin7a -/- neutrophils in flow do not crosstalk
12 CD42b -FITC CD42b FITC Platelet Activity Ly6G-/ CD42b High PLTs Ly6G-/ CD42b/ CD62P SSC SSC Platelet Activity Ly6G-/ CD42b High PLTs Ly6G-/ CD42b/ CD62P Ab cocktail incubation (min) Semaphorin7a inhibits Platelet CD62P Scanning for Platelet function Par-4 Whole blood 1 withdrawn from n=5 healthy 25 8 mice per group Platelets were 2 focused by invert gate of 5 FSC 1 CD42 HIgh - FITC FSC CD42b /Ly6G Ly6G - BV421 Ly6G - BV421 CD62P - APC and both hold their platelet reactivity (CD42b-GPIbα) CD62P is not active in mice after thrombin stimulation CD42b - FITC CD42b -FITC
13 CD42b -FITC CD42b FITC SSC SSC Ab cocktail incubation (min) PNC adhesion is inhibited in Understand PNC function Par-4 Whole blood withdrawn from n=5 healthy mice per group PNCs were focused as CD42b /Ly6G FSC FSC Ly6G - PC7 Ly6G - PC7 Ly6G - PC7 Ly6G - PC7
14 PNCs Ly6G/ CD42b/ CD11b PNCs Ly6G/ CD42b/ CD11b PNCs Ly6G/ CD42b/ CD11b PNCs PNCs Ly6G/ CD42b/ CD11b (Med. (Med. Fluo. Fluo. Units) Units) PNCs Ly6G/ CD42b/ LFA1 PNCs Ly6G/ CD42b/ LFA1 PNCs Ly6G/ CD42b/ LFA1 PNCs Ly6G/ CD42b/ LFA1 (Med. Fluo. Fluo. Units) PNCs Ly6G/ CD42b/ CD62P PNCs PNCs Ly6G/ CD42b/ CD62P (Med. (Med. Fluo. Fluo. Units) Units) PNCs Ly6G/ CD42b/ CD62P PNCs Ly6G/ CD42b/ CD62P (Med. Fluo. Fluo. Units) PNCs Ly6G/ CD42b/CD62L PNCs Ly6G/ CD42b/CD62L PNCs Ly6G/ CD42b/CD62L PNCs Ly6G/ CD42b/CD62L (Med. Fluo. Fluo. Units) PNCs Ly6G/ CD42b Freq. of Parent PNCs Ly6G/ CD42b Freq. of Parent PNCs Ly6G/ CD42b Freq. of Parent PNCs Ly6G/ CD42b Freq. of Parent PNCs Ly6G/ CD42b Freq. of Parent Ab cocktail incubation (min) PNC adhesion is inhibited in Understand PNC function CD62P - APC PNCs Ly6G/ CD42b High PNCs Ly6G/ CD42b High CD42B - FITC CD62L - PerCp PNCs Ly6G/ CD42b High PNCs Ly6G/ CD42b High PNCs Ly6G/ CD42b High Sema7a -/- - mice form less PNCs after stimulation - PNCs from mice have impaired adhesion through: CD62P CD11b LFA1 (CD11a/CD18) CD11b - BV LFA1 -PE
15 SSC CD42b -FITC SSC PNCs Ly6G/ CD42b Freq. of Parent PlexinC1 and β1 integrin control aggregation in vivo Understand PNC function Inhalation (45min) Ab cocktail incubation (min) 8 6 CD18 block CD232 block CD29 block After inhalation blood was withdrawn Samples were separately blocked and stained PNCs were focused as Ly6G /CD42 FSC 4 2 inhalation - PlexinC1 (CD232) blocks - β1 integrin (CD29) blocks - β2 integrin (CD18) Ly6G - BV421 Ly6G - BV421
16 Ly6G/ CD42b/ LFA1 (Mean Fluo. PE) Ly6G/ CD42b/ LFA1 (Mean Fluo. PE) Ly6G/ CD42b/ CD11b (Mean Fluo. Pac. Blue) Ly6G/ CD42b/ CD11b (Mean Fluo. Pac. Blue) Ly6G/ CD42b/ CD62P (Mean Fluo. APC) Ly6G/ CD42b/ CD62L (Mean Fluo. PerCp) SSC Ly6G/ CD42b/ CD62P (Mean Fluo. APC) SSC Ly6G/ CD42b/ CD62P (Mean Fluo. APC) Ly6G/ CD42b/ CD62L (Mean Fluo. PerCp) Ly6G/ CD42b/ CD62L (Mean Fluo. PerCp) Ly6G/ CD42b (Freq. of Parent) Ly6G/ CD42b/ JON/A (Mean Fluo. PE) Ly6G/ CD42b/ CD42b (Mean Fluo. FITC) Ly6G/ CD42b (Freq. of Parent) Ly6G/ CD42b (Freq. of Parent) Ly6G/ CD42b/ CD42b (Mean Fluo. FITC) Ly6G/ CD42b/ CD42b (Mean Fluo. FITC) Platelets from Sema7a-/- mice have impaired function Scanning for Platelet function Inhalation (45min) Ab cocktail incubation (min) untreated FSC CD42b - FITC 1 5 JON/A - PE /- - and are able to build PNCs 2 - PNCs from 1 mice have 5 impaired function of Platelet: JON/A (active GPIIb/IIIa) CD62P d 25 2 Ly6G - BV d CD62P - APC d d Reconstitution 1 with induces injury (gain 5 of function)
17 Ly6G/ CD42b/ LFA1 (Mean Fluo. PE) Ly6G/ CD42b/ CD11b (Mean Fluo. Pac. Blue) SSC SSC Ly6G/ CD42b/ LFA1 (Mean Fluo. PE) Ly6G/ CD42b/ LFA1 (Mean Fluo. PE) Ly6G/ CD42b/ CD11b (Mean Fluo. Pac. Blue) Ly6G/ CD42b/ CD62P (Mean Fluo. APC) Ly6G/ CD42b/ CD11b (Mean Fluo. Pac. Blue) Ly6G/ CD42b/ CD62L (Mean Fluo. PerCp) Ly6G/ CD42b/ CD62P (Mean Fluo. APC) Ly6G/ CD42b/ CD62P (Mean Fluo. APC) Ly6G/ CD42b/ CD62L (Mean Fluo. PerCp) Ly6G/ CD42b/ CD62L Ly6G/ (Mean CD42b Fluo. (Freq. PerCp) of Parent) Ly6G/ CD42b/ CD42b (Mean Fluo. FITC) Ly6G/ CD42b (Freq. of Parent) Ly6G/ CD42b (Freq. of Parent) Ly6G/ CD42b/ CD42b (Mean Fluo. FITC) Ly6G/ CD42b/ CD42b (Mean Fluo. FITC) Sema7a induces PNC adhesion -/- 2 1 Understand PNC function Inhalation (45min) CD62L - PerCp Ab cocktail incubation (min) 2 untreated 1 5 CD62L 1 CD11b - PNCs from mice have impaired adhesion through neutrophil: 5 LFA1 (CD11a/CD18) FSC CD11b - BV Reconstitution with induces injury (gain of function) Ly6G - BV421 LFA-1 - PE 5 1
18 Ly6G/ CD42b/ CD29 (Mean Fluo. PE) PlexinC1 is chemorepulsive, β1 integrin is chemoattactive Understand PNC function Inhalation (45min) Ab cocktail incubation (min) untreated 1 Integrin ß1 (CD29) Relative transcript expression Ly6G/ CD42b/ CD232 (Mean Fluo. PE) Plexin C1 (CD232) Relative transcript expression CD232 - PE Whole Blood Lung CD29 - PE Whole Blood Lung PlexinC1 is not regulated during inhalation in and mice β1 integrin (CD29) expression increases in the blood and is maximum in the lung Reconstitution with in induces PlexinC1 expression after 45min inhalation
19 Summary Semaphorin7a controls cell communication during haemostasis and inflammation Neutrophil Platelet Blood Flow Neutrophils from Semaphorin7a -/- mice ( i.v.) Keep Rolling speed Have no stationary cells Do not trans-migrate Do not cross-talk PlexinC1 Semaphorin7a α1β1 Inactive α1β1 active Neutrophil Free Cells Platelet Activated PNC formation Semaphorin7a-/- mice under inflammation have impaired - Platelet adhesion, CD62P, JON/A (active GPIIb/IIIa), - neutrophil adhesion, CD62L, CD11b, CD11a, CD18 PNC Arrest Sema7a PlexinC1 is chemorepulsive between PMNs and PLTs (haemostasis) Sema7a β1 integrin is a chemoattractant between PMNs and PLTs (inflammation)
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