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Delineation of Innate Immune Response of Vibrio Cholerae and its Outer Membrane Vesicles in an Epithelial Cell-Dendritic Co-Culture Model

IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata


Field	Value

Title	Delineation of Innate Immune Response of Vibrio Cholerae and its Outer Membrane Vesicles in an Epithelial Cell-Dendritic Co-Culture Model

Creator	Chatterjee, Debashree

Subject	Molecular & Human Genetics

Description	Cholera remains a devastating bacterial cause of human morbidity and mortality in India and other developing countries. The disease is produced by a Gram-negative, motile organism Vibrio cholerae that colonizes in the human intestine and secretes a potent cholera toxin, which ultimately stimulates cellular adenylate cyclase to cause massive intestinal fluid loss leading to profuse watery diarrhea. To combat the disease from newly emerging threats, besides the study of organisms V. cholerae, a thorough understanding of the host response following V. cholerae infection and the response generated by bacterial components such as lipopolysaccharide, flagellar proteins, CT as well as outer membrane vesicles (OMVs) is indispensable. Such findings will eventually improve the present vaccine strains as well as design drugs for appropriate targets. Vibrio cholerae induces acute inflammatory response at intestinal epithelial surface; the underlying cellular immune mechanisms for such effects are largely unexplored. Mucosal immune response is controlled by the crosstalk between the intestinal epithelial cells (ECs) and dendritic cells (DCs). ECs act as a sensor for current environmental conditions and release a variety of mediators which instruct nearby DCs accordingly. The present study has established for the first time an ECDC co-culture model for V. cholerae infection. Our studies revealed that an ECderived cytokine thymic stromal lymphopoietin (TSLP) is highly elevated in ECs stimulated with V. cholerae and its recombinant flagellin (rFlaA). V. cholerae treated human ECs produce DC-attracting chemokine MIP-3α (CCL20). Flagellin, a potent V. cholerae factor was responsible for maximum stimulation of epithelial CCL20 production and subsequent DC activation. Activated DCs express high levels of costimulatory molecules and secrete inflammatory cytokines TNF-α, IL-6 and IL-1β. Bacteria stimulated ECs conditioned DCs to produce Th2 cell-attracting chemokines CCL17 and CCL22. TSLP and other mediators present in the V. cholerae stimulated EC-culture filtrate potently activated DCs, which subsequently primed CD4+T cells to differentiate into T helper type 2 (Th2) cells that produce high amounts of IL-4, IL-13 and TNF-α and low IFN-γ. TSLP-induced proinflammatory response in DCs involved the transcriptional mechanisms, MAPKs (ERK1/2, p38 and JNK) and STAT3 activation. This study suggests TSLP and other mediators released from ECs in response to V. cholerae colonization actively influence DCs in initiating inflammatory response. Another important component of V. cholerae is outer membrane vesicles or OMVs which are naturally produced bacterial vesicles. They are discrete, closed outer membrane blebs having an average diameter of 10-300 nm with a bilayer membrane and electron-dense luminal content and consist only of the protein and lipids of OM and periplasm. The existence of OMVs has been demonstrated in a variety of Gramnegative bacteria. Our study for the first time demonstrates that a fraction of cholera toxin (CT), the major virulence factor of pathogenic V. cholerae is associated with the outer membrane vesicles (OMVs). Atomic force microscopy (AFM) and also transmission electron microscopy (TEM) of purified OMVs from toxigenic V. cholerae O395 strain revealed spherical shaped vesicles of size range 20–200 nm. Immunoblotting of purified OMVs with polyclonal anti-CT antibody and GM1 ganglioside dependent ELISA suggests that CT is associated with OMVs. CHO cell assay indicated that OMV associated CT is physiologically active. OMVs labelled with fluorescent dye interacted with intestinal epithelial cells via the CT-receptor and were internalized increasing the cAMP level. Thus OMVs may represent an important vehicle in delivering CT to epithelial cells. In pathogenic bacteria, as OMVs are associated with a mixture of different pathogen associated molecular patterns (PAMPs) like LPS, peptidoglycan and with toxins as in V. cholerae, these OMVs are expected to elicit host immune response.The present study evaluated the interaction of OMVs with intestinal epithelial cells and with EC-DC co-culture. It was observed that, cytosolic pattern recognition receptor (PRR) NOD1 significantly upregulated in epithelial cells during OMV stimulation. In contrast, another PRR NOD2 expression was constitutive in nature and not up regulated upon OMV treatment. Therefore we sought to investigate the production of proinflammatory cytokines in response to V. cholerae O395 OMV stimulation in NOD1 dependent manner. We observed that vesicles induced expression of proinflammatory cytokines such as IL-8 and GM-CSF, chemokines like CCL2, CCL20 and also TSLP in ECs through activation of ERK1/2 and p38 MAPK and NF-kB pathways in NOD1 dependent manner. ECs stimulated with OMVs activate DCs in a direct co-culture system. DC activation was indicated by an increased number of cells expressing high levels of the activation markers HLA-DR, Synopsis CD80 and CD83. Activated DCs express high levels of costimulatory molecules and released inflammatory cytokines IL-1β, IL-6, TNF-α, IL-23 and chemokines CCL22, CCL17 and subsequently primed CD4+T cells leading to IL-4, IL-13 and IL-17 expression. It was observed that neutralizing of IL-23p19 caused a significant decrease in IL-17 production. These results suggest that V. cholerae O395 OMVs modulate the epithelial pro-inflammatory response and activate DCs which promoted the T cells polarization towards an inflammatory Th2/Th17 response. Thus the overall study suggest that V. cholerae and its component outer membrane vesicle modulate epithelial function and induce dendritic cell mediated Th2/Th17 proinflammatory response and OMV act as a potent vehicle in delivering a percentage of cholera toxin to intestinal epithelial cells.

Date	2013

Type	Thesis NonPeerReviewed

Format	application/pdf

Identifier	http://www.eprints.iicb.res.in/1887/1/Thesis_DC_25062013.pdf Chatterjee, Debashree (2013) Delineation of Innate Immune Response of Vibrio Cholerae and its Outer Membrane Vesicles in an Epithelial Cell-Dendritic Co-Culture Model. PhD thesis, Jadavpur University.

Relation	http://www.eprints.iicb.res.in/1887/