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Infected Cell in Trouble: Bystander Cells Ring the Bell Immunity 33, November 24, 2010 Ana Carolina Pagliaroni Cláudia Danella Polli Patrícia Assis epithelial intestinal cells Innate immunity IEC: Immune response IL-8 (CXCL2) TNF-α GM-CSF Phagocytes Epithelia do play an active role in coordinating defense Proinflammatory cytokines are produced by the infected cell itself… Shigella AP-1 H3 Listeria Proinflammatory cytokines Curr Opin Immunol. 2008; Aug 20(4):377-82 Most studies of immuny response induced by pathogens have employed… Entire animal Tissues + Purified pathogen-derived molecules Pool or population of cells Focus on single cell response + Pathogen mutants deficient in cell-to cell spread November 2010 | Volume 6 | Issue 11 | e1001194 Listeria monocytogenes model Listeria monocytogenes Infection mechanism Adhesion and invasion LLO phospholipases Intercellular transmission Actin polymerization Shigella flexneri Immunosuppressive activity of bacterial effector proteins Shigella OspF and OspG (virulence factors) Potente inhibitor of JNK, ERK and p38 signaling During intestinal infection challenge ..... Despite the immunosuppressive activity of bacterial effector proteins…Massive IL-8 expression is observed in IECs during infection… Intense inflammatory response J Biol Chem. 2003 Sep 5 278(36) 33878-86. Hypothesis : a horizontal intercellular communication between intestinal epithelial cells might help to amplify the innate immune response? Immunity 33, 804–816, November 24, 2010 Aim: to investigate whether the intercellular comunication between infected and non-infected cells contributes to innate response against Listeria monocytogenes infection . Ana Carolina Pagliarone The activation of intestinal epithelial cells depends on the L. monocytogenes intracellular localization? actA mutant Listeria actA mutant Listeria (no cell-to-cell spread) m-ICcI2 cells + siRNA 4h ELISA (LLO-deficient) hly mutant or WT Listeria expressing GFP m-ICcI2 cells The activation of intestinal epithelial cells is dependent on bacterial presence in flow cytometry cytosol (endosomal lysis) Non-infected epithelial cells are activated during infection challenge? WT Listeria –GFP (PactA-gfp) 4h m-ICcI2 cells flow cytometry Non-infected cells are activated during infection challenge? PSOD–gfp Listeria m-ICcI2 cells RT-PCR Non-infected epithelial cells are ativated to a greater extend than infected cells during infection . The activation of non-infected cells depends on the bacterial cell-to-cell spread? actA PactA-gfp Listeria m-ICcI2 cells flow cytometry or ELISA Non-infected cells are activated even when there is no bacterial spread to neighboring cells. Bacterial products are responsible for activating non-infected cells? WT Listeria Cell culture medium + WT Listeria or Recombinant LLO m-ICcI2 cells filtration Bacteria free media m-ICcI2 cells 4h p.i 4h p.i Bacterial products are not reponsible for activating non-infected cells. Activation of non-infected cells occurs through gap junctions? actA PactA-gfp Listeria m-ICcI2 cells 4h + gap junctions inhibitors Flow cytometry actA PactAgfp Gap junction inhibitors The non-infected cells can be activated even when gap junctions are inhibited. Secreted products of infected cells are responsible for inducting the activation of non-infected cells? actA PSOD–gfp Listeria Brefeldin A (BFA) 30 min m-ICcI2 cells m-ICcI2 cells OR actA PSOD–gfp Listeria 60 min Brefeldin A (BFA) Flow cytometry Secreted products of infected cells are not responsible for inducting non-infected cells activation. ....but unstable and highly reactive host-derived factors cannot be excluded by the previous results!!! Listeria infection induces reactive oxygen intermediates (ROIs) production in non-infected cells Act A mutant Listeria Oxygen and nitrogen radicals are involved the activation of non-infected cells? actA PactA-gfp Listeria m-ICcI2 cells + DPI or L-NAME 4h flow cytometry actA PactA-gfp Listeria DPI: NADP oxidase inhibitor L-NAME : nitric oxide sinthase (NOS) inhibitor Oxygen radicals synthesis are involved in the activation of non-infected cells. ROIs induce ERK activation in non-infected cells during Listeria infection? WT Listeria m-ICcI2 cells + iRNA 50min Immunoblotting WT Listeria ROIs induce Erk activation in non-infected cells . Nox4 is responsible for the Cxcl-2 production in non-infected cells? actA PactA-gfp Listeria m-ICcI2 cells + siRNA 4h Immunoblotting and ELISA assay Nox4 induces Cxcl-2 production in non-infected cells . CONCLUSION professional immune cells attraction increased host Innate response OspF IL-8 AP-1 H3 Hypotesis A host cell-cell communication mechanism are circumvents the bacterial effector proteins amplifying IL-8 expression Inflammation Mechanism of Epithelial Cells: characterization at the single-cell level S. flexneri HeLa 1h p65 nuclear translocation Immunofluorescence microscopy Uninfected cells surrounding infected cells shown NF-κB activation Shigella spread to adjacent cells by actin-based motility Bystander NF-κB activation is due bacterial intercellular motility? S. flexneri Wt ∆virG HeLa 1 hora F-actin (FITC-phalloidin) p65 nuclear translocation Immunofluorescence microscopy NF-κB activation was not caused by intercellular motility, but reflected instead a novel host response to bacterial infection JNK, ERK e p38 are also activated in bystander cells in S. flexneri infection? S. flexneri ∆virG HeLa p-JNK 90 min p-JNK, p-ERK and p-p38 Immunofluorescence microscopy p-ERK p-p38 JNK, ERK and p38 also propagates from infected to bystander cells during S. flexneri infection Bystander cells are actively producing IL-8? S. flexneri ∆virG HeLa 6h ELISA IL-8 Bystander cells are the main producers of IL-8 ? Bystander cells are actively producing IL-8? monesin S. flexneri ∆virG HeLa 3h IL-8 Immunofluorescence microscopy Green: S. flexneri Red: IL-8 Blue: Hoechst Gray: F-actin Bystander cells are the main source of IL-8 during S. flexneri infection Bacterial virulence factors could impair bystander cell activation? S. flexneri ∆virG ∆ospF p-p38 p38 desphosphorylation by OspF HeLa 90 min Immunofluorescence microscopy Bacterial virulence factors could impair bystander cell activation? S. flexneri ∆virG ∆ospF HeLa 3h IL-8 Immunofluorescence microscopy OspF failed to impair the ability of the host to spread p38 activation to neighboring cells and induce IL-8 expression Pathogen sensing via Nod-1 is sufficient to induce bystander IL-8 expression? TriDAP monesin TriDAP IgG Alexa 488 3h IL-8 Immunofluorescence microscopy IgG Green: TriDAP Red: IL-8 Blue: Hoechst Gray: F-actin Nod-1-mediated recognition was necessary and sufficient to induce IL-8 expression L-Ala-D--Glu-Meso-diaminopimelic acid What is the mechanism of cell-cell communication between infected and bystander cells? Bystander cells activation is due to factors secreted by the infected cell? S. flexneri S. flexneri ∆virG BFA ∆virG HeLa 60 min 10 min Culture Flow Staining Imaging IL-8 IL-8 Immunofluorescence Flow Immunofluorescence 90 minChamber microscopy microscopy Cell-cell propagation of proinflammatory signals was not mediated by secreted proteins or soluble factors Bystander cells activation is due to cell-cell contact with the infected cell? 18β-GA S. flexneri ∆virG Immunofluorescence microscopy 90 min Subconfluent density S. flexneri ∆virG Immunofluorescence microscopy 90 min 1 2 3 IL-8 expression by bystande cells was mediated by comminication through gap junctions 18β-glycyrrhetinic acid Bystander activation via cell-cell contact is dependent of gap-junction? S. flexneri ∆virG S. flexneri ∆virG A431 90 min A431-Cx43 Immunofluorescence microscopy Bystander activation via cell-cell contact is dependent of gap-junction? Infected Cell 1 2 3 4 90 min IL-8 Immunofluorescence microscopy A431-Cx43 Bystander activation via cell-cell contact is dependent of gap-junction? S. flexneri ∆virG 70/30 A431 A431-Cx43 Cx-43 10/90 Immunofluorescence microscopy IL-8 The propagation of inflammation during bacterial infection of epithelial cells depends on conexin gap junctions CONCLUSION Ca2+ IP3 cAMP
