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Ana Carolina Pagliaroni Cláudia Danella Polli Patrícia Assis

Infected Cell in Trouble: Bystander Cells Ring the Bell. Ana Carolina Pagliaroni Cláudia Danella Polli Patrícia Assis. Immunity 33, November 24, 2010. Initially …. epithelial intestinal cells Innate immunity. Over the past decade …. IEC: Immune response. IL-8 (CXCL2) TNF- α GM-CSF.

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Ana Carolina Pagliaroni Cláudia Danella Polli Patrícia Assis

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  1. Infected Cell in Trouble: Bystander Cells Ring the Bell Ana Carolina Pagliaroni Cláudia Danella Polli Patrícia Assis Immunity 33, November 24, 2010

  2. Initially… epithelial intestinal cells Innateimmunity

  3. Over the pastdecade…. IEC: Immune response IL-8 (CXCL2) TNF-α GM-CSF Phagocytes Epithelia do play an active role in coordinatingdefense

  4. Proinflammatory cytokines are produced by the infected cell itself… Shigella Listeria P Proinflammatory cytokines H3 AP-1 P Curr Opin Immunol. 2008; Aug 20(4):377-82

  5. Most studies of immunyresponseinduced by pathogens have employed… Entire animal Tissues + Pool or population of cells Purifiedpathogen-derivedmolecules

  6. What’s new? Focus on single cellresponse Pathogen mutants deficient in cell-to cellspread +

  7. November 2010 | Volume 6 | Issue 11 | e1001194 Listeria monocytogenes model

  8. Listeria monocytogenes Infection mechanism Adhesion and invasion LLO phospholipases Intercellular transmission Actin polymerization

  9. Shigella flexneri

  10. Immunosuppressive activity of bacterial effector proteins Shigella OspF and OspG (virulence factors) Potente inhibitor of JNK, ERK and p38 signaling

  11. 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.

  12. Hypothesis : a horizontal intercellular communication between intestinal epithelial cells might help to amplify the innate immune response?

  13. Immunity 33, 804–816, November 24, 2010

  14. Aim: to investigate whether the intercellular comunication between infected and non-infected cells contributes to innate response against Listeriamonocytogenes infection . Ana Carolina Pagliarone

  15. The activationof intestinal epithelialcellsdependsontheL. monocytogenesintracellularlocalization? 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 activationof intestinal epithelialcellsisdependentonbacterialpresence in cytosol (endosomallysis) flow cytometry

  16. Non-infectedepithelialcells are activatedduringinfectionchallenge? 4h WT Listeria –GFP (PactA-gfp) flow cytometry m-ICcI2 cells

  17. Non-infectedcells are activatedduringinfectionchallenge? PSOD–gfp Listeria m-ICcI2 cells RT-PCR Non-infectedepithelialcellsare ativatedto a greaterextendthaninfectedcellsduringinfection .

  18. The activationof non-infectedcellsdependsonthebacterialcell-to-cell spread? actA PactA-gfp Listeria flow cytometry or ELISA m-ICcI2 cells Non-infectedcells are activatedevenwhenthereis no bacterial spread toneighboringcells.

  19. Bacterialproducts are responsible for activating non-infectedcells? WT Listeria Cell culture medium + WT Listeria filtration Bacteria free media or Recombinant LLO m-ICcI2 cells m-ICcI2 cells 4h p.i 4h p.i Bacterialproducts are notreponsible for activating non-infectedcells.

  20. Activationof non-infectedcellsoccursthrough gap junctions? m-ICcI2 cells + gap junctions inhibitors 4h actA PactA-gfp Listeria Flow cytometry actA PactA-gfp Gap junctioninhibitors The non-infectedcellscanbeactivatedevenwhen gap junctions are inhibited.

  21. Secretedproductsofinfectedcells are responsible for inductingtheactivationof non-infectedcells? Brefeldin A (BFA) actA PSOD–gfp Listeria 30 min m-ICcI2 cells m-ICcI2 cells OR 60 min Brefeldin A (BFA) actA PSOD–gfp Listeria Flow cytometry Secretedproductsofinfectedcells are notresponsible for inductingnon-infectedcellsactivation.

  22. ....but unstable and highly reactive host-derived factors cannot be excluded by the previous results!!!

  23. Listeria infection induces reactive oxygen intermediates (ROIs) production in non-infected cells Act A mutant Listeria

  24. Oxygenandnitrogenradicals are involvedtheactivationof non-infectedcells? 4h actA PactA-gfp Listeria m-ICcI2 cells + DPI or L-NAME flow cytometry actA PactA-gfp Listeria DPI: NADP oxidase inhibitor L-NAME : nitric oxide sinthase (NOS) inhibitor Oxygenradicalssynthesis are involved in theactivationof non-infectedcells.

  25. ROIsinduce ERK activation in non-infectedcellsduringListeriainfection? 50min WT Listeria m-ICcI2 cells + iRNA Immunoblotting WT Listeria ROIsinduceErkactivation in non-infectedcells .

  26. Nox4 isresponsible for the Cxcl-2 production in non-infectedcells? actA PactA-gfp Listeria m-ICcI2 cells + siRNA 4h Immunoblotting and ELISA assay Nox4 induces Cxcl-2 production in non-infectedcells .

  27. CONCLUSION professional immune cells attraction increased host Innate response

  28. OspF X P X IL-8 H3 AP-1 P

  29. Hypotesis A host cell-cell communication mechanism are circumvents the bacterial effector proteins amplifying IL-8 expression

  30. Inflammation Mechanism of Epithelial Cells: characterization at the single-cell level p65 nuclear translocation Immunofluorescence microscopy S. flexneri 1 h HeLa Uninfected cells surrounding infected cells shown NF-κB activation

  31. Shigella spread to adjacent cells by actin-based motility

  32. 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

  33. JNK, ERK e p38 are also activated in bystander cells in S. flexneri infection? p-JNK, p-ERK and p-p38 Immunofluorescence microscopy S. flexneri ∆virG 90 min HeLa p-ERK p-p38 p-JNK JNK, ERK and p38 also propagates from infected to bystander cells during S. flexneriinfection

  34. Bystander cells are actively producing IL-8? S. flexneri ELISA IL-8 ∆virG 6 h HeLa Bystander cells are the main producers of IL-8 ?

  35. Bystander cells are actively producing IL-8? monesin S. flexneri IL-8 Immunofluorescence microscopy ∆virG 3 h HeLa Green: S. flexneri Red: IL-8 Blue: Hoechst Gray: F-actin Bystander cells are the main source of IL-8 during S. flexneriinfection

  36. Bacterial virulence factors could impair bystander cell activation? p38 desphosphorylation by OspF S. flexneri p-p38 Immunofluorescence microscopy ∆virG ∆ospF 90 min HeLa

  37. Bacterial virulence factors could impair bystander cell activation? IL-8 Immunofluorescence microscopy S. flexneri ∆virG ∆ospF 3 h HeLa OspF failed to impair the ability of the host to spread p38 activation to neighboring cells and induce IL-8 expression

  38. Pathogen sensing via Nod-1 is sufficient to induce bystander IL-8 expression? TriDAP monesin IL-8 Immunofluorescence microscopy TriDAP 3 h IgG Alexa 488 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

  39. What is the mechanism of cell-cell communication between infected and bystander cells?

  40. Bystander cells activation is due to factors secreted by the infected cell? S. flexneri IL-8 Immunofluorescence microscopy ∆virG BFA IL-8 Immunofluorescence microscopy S. flexneri Flow Chamber 60 min 90 min ∆virG 10 min HeLa Culture Flow Staining Imaging Cell-cell propagation of proinflammatory signals was not mediated by secreted proteins or soluble factors

  41. Bystander cells activation is due to cell-cell contact with the infected cell? 18β-GA S. flexneri S. flexneri ∆virG Immunofluorescence microscopy ∆virG Immunofluorescence microscopy 90 min 90 min Subconfluent density 1 2 X 3 IL-8 expression by bystande cells was mediated by comminication through gap junctions 18β-glycyrrhetinic acid

  42. Bystander activation via cell-cell contact is dependent of gap-junction? S. flexneri ∆virG Immunofluorescence microscopy S. flexneri ∆virG 90 min A431 A431-Cx43

  43. Bystander activation via cell-cell contact is dependent of gap-junction? IL-8 Immunofluorescence microscopy Infected Cell 90 min 4 1 3 2 A431-Cx43

  44. Bystander activation via cell-cell contact is dependent of gap-junction? S. flexneri ∆virG 70/30 + Immunofluorescence microscopy A431 A431-Cx43 10/90 IL-8 Cx-43 The propagation of inflammation during bacterial infection of epithelial cells depends on conexin gap junctions

  45. CONCLUSION

  46. Ca2+ IP3 cAMP

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