ana carolina pagliaroni cláudia danella polli patrícia assis

Ana Carolina PagliaroniCláudia Danella PolliPatrícia Assis

Infected Cell in Trouble: Bystander Cells Ring the Bell

Immunity 33, November 24, 2010

IL-8 (CXCL2)TNF-αGM-CSF

Phagocytes

AP-1 H3

Proinflammatory cytokines

Shigella Listeria

Curr Opin Immunol. 2008; Aug 20(4):377-82

+

Entire animal

Tissues

Pool or population of cells

November 2010 | Volume 6 | Issue 11 | e1001194

Listeria monocytogenes model

Adhesion and invasion

LLOphospholipases

Actin polymerization

Intercellular transmission

Listeria monocytogenes

Infection mechanism

Shigella flexneri

OspF and OspG(virulence factors)

Potente inhibitor of JNK, ERK and p38 signaling

Shigella

J Biol Chem. 2003 Sep 5 278(36) 33878-86.

Intense inflammatory

response

Hypothesis : a horizontal intercellular

communication between intestinal epithelial

cells might help to amplify the innate immune

response?

Hypothesis : a horizontal intercellular

communication between intestinal epithelial

cells might help to amplify the innate immune

response?

Immunity 33, 804–816, November 24, 2010

Ana Carolina Ana Carolina PagliaronePagliarone

Aim: to investigate whether the intercellular comunication between infected and non-infected cells contributes to innate response against Listeria

monocytogenes infection .

The activation of intestinal epithelial cells depends on the L. monocytogenes intracellular localization?

actA mutant Listeria

(LLO-deficient)

m-ICcI2 cells + siRNA

actA mutant Listeria (no cell-to-cell spread)

ELISA

4h

m-ICcI2 cells

flowcytometry

hly mutant or WT Listeria expressing GFP

The activation of intestinal epithelial cells is dependent on bacterial presence in cytosol (endosomal lysis)

Non-infected epithelial cells are activated during infection challenge?

m-ICcI2 cells flow cytometryWT Listeria –GFP(PactA-gfp)

4h

Non-infected cells are activated during infection challenge?

m-ICcI2 cells

RT-PCR

PSOD–gfp Listeria

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?

Non-infected cells are activated even when there is no bacterial spread to neighboring cells.

m-ICcI2 cells flow cytometry or ELISA

actA PactA-gfp Listeria

Bacterial products are responsible for activating non-infected cells?

4h p.i

Bacterial products are not reponsible for activating non-infected cells.

WT Listeria

Recombinant LLO

m-ICcI2 cells

or

Cell culture medium +WT Listeria

filtrationBacteria free media

m-ICcI2 cells

4h p.i

http://www.clker.com/clipart-eppendorf-with-blood-zxc-1.html

Activation of non-infected cells occurs through gap junctions?

Gap junction inhibitors

actA PactA-gfp

4h

The non-infected cells can be activated even when gap junctions are inhibited.

actA PactA-gfp Listeria m-ICcI2 cells + gap junctions inhibitors

Flow cytometry

Secreted products of infected cells are responsible for inducting the activation of non-infected cells?

m-ICcI2 cells

Brefeldin A (BFA)

actA PSOD–gfp Listeria

30 min

OR

Brefeldin A (BFA)

actA PSOD–gfp Listeria

m-ICcI2 cells

60 min

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?


DPI: NADP oxidase inhibitor

L-NAME : nitric oxide sinthase (NOS) inhibitor

Oxygen radicals synthesis are involved in the activation of non-infected cells.

flow cytometry actA PactA-gfp Listeria m-ICcI2 cells + DPI orL-NAME

4h

ROIs induce ERK activation in non-infected cells during Listeria infection?

ROIs induce Erk activation in non-infected cells .

WT Listeria

ImmunoblottingWT Listeria m-ICcI2 cells + iRNA

50min

Nox4 is responsible for the Cxcl-2 production in non-infected cells?

Immunoblotting and ELISA assay


m-ICcI2 cells + siRNA

4h

Nox4 induces Cxcl-2 production in non-infected cells .

professional immune cells attraction

CONCLUSION

increased host Innate response

IL-8

AP-1 H3

OspF

Hypotesis

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

Hypotesis

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

HeLa1 h

p65 nuclear translocationImmunofluorescence microscopy

S. flexneri

Inflammation Mechanism of Epithelial Cells: characterization at the single-cell level

Uninfected cells surrounding infected cells shown NF-κB activation Uninfected cells surrounding infected cells shown NF-κB activation

Shigella spread to adjacent cells by actin-based motilityShigella spread to adjacent cells by actin-based motility

Bystander NF-κB activation is due bacterial intercellular motility?

HeLa

1 hora

F-actin (FITC-phalloidin)

p65 nuclear translocationImmunofluorescence microscopy

S. flexneriWt

∆virG

NF-κB activation was not caused by intercellular motility, but reflected instead a novel host response to bacterial infection

NF-κB activation was not caused by intercellular motility, but reflected instead a novel host response to bacterial infection

HeLa90 min

p-JNK, p-ERK and p-p38Immunofluorescence microscopy

S. flexneri∆virG

JNK, ERK e p38 are also activated in bystander cells in S. flexneri infection?

p-JNK p-ERK p-p38

JNK, ERK and p38 also propagates from infected to bystander cells during S. flexneri infection

JNK, ERK and p38 also propagates from infected to bystander cells during S. flexneri infection

Bystander cells are actively producing IL-8?

HeLa6 h

ELISA IL-8S. flexneri

∆virG

Bystander cells are the main producers

of IL-8 ?

HeLa3 h

IL-8Immunofluorescence microscopy

S. flexneri

monesin

∆virG

Green: S. flexneri

Red: IL-8

Blue: Hoechst

Gray: F-actin

Bystander cells are actively producing IL-8?

Bystander cells are the main source of IL-8 during S. flexneri infectionBystander cells are the main source of IL-8 during S. flexneri infection

HeLa90 min

S. flexneri∆virG∆ospF

p-p38Immunofluorescence microscopy

Bacterial virulence factors could impair bystander cell activation?

p38 desphosphorylation by OspFp38 desphosphorylation by OspF

HeLa3 h


S. flexneri∆virG∆ospF

OspF failed to impair the ability of the host to spread p38 activation to neighboring cells and induce IL-8 expression

OspF failed to impair the ability of the host to spread p38 activation to neighboring cells and induce IL-8 expression

Bacterial virulence factors could impair bystander cell activation?

3 h

TriDAP

IgG Alexa488

monesin

Pathogen sensing via Nod-1 is sufficient to induce bystander IL-8 expression?


Green: TriDAP

Red: IL-8

Blue: Hoechst

Gray: F-actin

IgG

TriDAP

Nod-1-mediated recognition was necessary and sufficient to induce IL-8 expressionNod-1-mediated recognition was necessary and sufficient to induce IL-8 expressionL-Ala-D--Glu-Meso-diaminopimelic acid

What is the mechanism of cell-cell communication

between infected and bystander cells?

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?

90 min

BFAS. flexneri

∆virG

60 min

IL-8Immunofluorescence

microscopy Flow Chamber

HeLa


microscopy

S. flexneri

∆virG 10 min

Culture Flow

Staining Imaging

Cell-cell propagation of proinflammatory signals was not mediated by secreted proteins or soluble factors

Cell-cell propagation of proinflammatory signals was not mediated by secreted proteins or soluble factors

Bystander cells activation is due to cell-cell contactwith the infected cell?

90 minImmunofluorescence

microscopy

S. flexneri∆virG


microscopy

S. flexneri∆virG

18β-GA

Subconfluent density

1

2

3

18β-glycyrrhetinic acidIL-8 expression by bystande cells was mediated by comminication through gap junctionsIL-8 expression by bystande cells was mediated by comminication through gap junctions

Bystander activation via cell-cell contact is dependent of gap-junction?

A431-Cx43

S. flexneri∆virG

A431

S. flexneri∆virG


microscopy

90 min1 2 3 4

Infected Cell



microscopy

A431-Cx43

S. flexneri∆virG

A431-Cx43A431

70/30

10/90

Cx-43 IL-8


Immunofluorescence microscopy

The propagation of inflammation during bacterial infection of epithelial cells depends on conexin gap junctions

The propagation of inflammation during bacterial infection of epithelial cells depends on conexin gap junctions

CONCLUSION

Ca2+ IP3 cAMP

Ca2+ IP3 cAMP

ana carolina pagliaroni cláudia danella polli patrícia assis

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