OmpA stimulates phosphorylation of protein tyrosine kinases, ERK, JNK and p38 and induces NF-κB activation.

<p>B cells were incubated with 5 µg/ml of OmpA for the indicated times, and cell lysates were probed for phosphotyrosine (pY) (<b>A</b>), and phosphorylated and total ERK, JNK and p38 (<b>B</b>). Representative blots from three independent experiments are shown. Arrows in the pY blot show bands with higher tyrosine phosphorylation. (<b>C</b>) B cells were purified from the spleen of BALB'/c mice, stimulated with OmpA (5 µg/ml) for the indicated times, and lysates were probed with phosphorylated and total IκBα. β-Actin was used as an internal control. The data is representative of three independent experiments. (<b>D</b>) B cells were cultured in absence and presence of OmpA for 30 min. The cytoplasmic (Cyto) and nuclear (Nuc) extracts were analyzed for p65, Lamin B1 (nuclear marker) and α-tubulin (Cytoplasmic marker) by Western blot analysis. The data shown are representative of three independent experiments.</p

Effect of NF-κB-, p38-, JNK- and ERK-specific inhibitors on OmpA-stimulated production of IL-6 and IL-10.

<p>(<b>A-D</b>) B cells were purified from spleen, cultured for 1 h with vehicle or the inhibitors of NF-κB (SN50; 100 µg/ml), <b><i>A</i></b>, p38 (SB203580; 5 µM), <b><i>B</i></b>, JNK (SP600125; 5 µM), <b><i>C</i></b>, MEK1/2 (U0126; 5 µM) <b><i>D</i></b>, followed by incubation with OmpA (5 µg/ml). After 72 h of culture, the IL-6 and IL-10 levels in the cell supernatants were determined via sandwich ELISA. The data are the mean ± S.E.M of three independent experiments. <i>**, p</i><0.01, relative to OmpA treated group in the absence of the inhibitor.</p

TLR2 is essential for OmpA-induced nuclear translocation of NF-κB, activation of ERK and expression of MHCII and CD86 on B cells.

<p>(<b>A</b>) B cells were incubated with anti-mouse TLR2 blocking antibody (100 ng/ml) for 1 h at 37°C prior to treatment with OmpA (5 µg/ml) for 30 min. The cytoplasmic (Cyto) and nuclear (Nuc) extracts were analyzed for p65, Lamin B1 (nuclear marker) and α-tubulin (Cytoplasmic marker) by Western blot analysis. The data shown are representative of three independent experiments. (<b>B</b>) B cells were pretreated with mouse anti-TLR2 blocking mAb (100 ng/ml) for 1 h. Cells were then stimulated with OmpA (5 µg/ml) for 5 min and cell lysates were probed for phosphorylated and total ERK. Representative blots from three independent experiments are shown. (<b>C, D</b>) B cells were incubated with anti-mouse TLR2 blocking antibody (100 ng/ml) for 1 h at 37°C (red line). Cells were then cultured in the absence (blue line) and presence (green line) of OmpA (5 µg/ml) for 24 h. Cells were harvested and assayed for cell surface expression of MHCII (<b>C</b>) and CD86 (<b>D</b>). The shaded histograms denote the isotype control antibodies. Representative data from three independent experiments are shown.</p

Involvement of TLR2 and protein tyrosine kinase in the OmpA-induced B cell differentiation and cytokine secretion.

<p>(<b>A, B</b>) B cells were incubated with anti-mouse TLR2 blocking antibody (100 ng/ml) or isotype (Mouse IgG1) control antibody (100 ng/ml) for 1 h at 37°C, followed by addition of OmpA (5 µg/ml) for a further 72 h. (<b>A</b>) The total number of ASCs (IgM + IgG-secreting cells) was analyzed by ELISPOT. Number of ASCs/10⁵ B cells. The results are the mean ± S.E.M of three independent experiments. <i>***, p</i><0.001, relative to OmpA treated group in the absence of TLR2 mAb. (<b>B</b>) The cell-free supernatants were assayed for IL-6 and IL-10 production by ELISA. The data are the mean ± S.E.M of three independent experiments. <i>**, p</i><0.01, relative to OmpA treated group in the absence of TLR2 mAb. (<b>C, D</b>) B cells were incubated with anti-mouse TLR4/MD2 blocking antibody (100 ng/ml) or isotype (Rat IgG2a) control antibody (100 ng/ml) for 1 h at 37°C, followed by addition of OmpA (5 µg/ml) for a further 72 h. (<b>C</b>) The total number of ASCs (IgM + IgG-secreting cells) was analyzed by ELISPOT. Number of ASCs/10⁵ B cells. The results are the mean ± S.E.M of three independent experiments. (<b>D</b>) Production of IL-6 and IL-10 was examined by ELISA of the cell-free supernatants. The data represent the mean ± S.E.M of three independent experiments. (<b>E, F</b>) Purified B cells were cultured for 1 h with vehicle (DMSO) or the PTK inhibitor (Herbimycin A; 1 µM), followed by incubation with OmpA (5 µg/ml) for 72 h. (<b>E</b>) The total number of ASCs (IgM + IgG-secreting cells) was analyzed by ELISPOT. Number of ASCs/10⁵ B cells. The results are the mean ± S.E.M of three independent experiments. <i>***, p</i><0.001, relative to OmpA treated group in the absence of the inhibitor (DMSO). (<b>F</b>) Production of IL-6 and IL-10 was examined by ELISA in the cell-free supernatants. The data represent the mean ± S.E.M of three independent experiments. <i>**, p</i><0.01, relative to OmpA treated group in the absence of the inhibitor.</p

Activation of ERK is critical for OmpA-induced differentiation of B cells into ASCs.

<p>B cells were purified from spleen, cultured for 1 h with vehicle or the inhibitors of MEK1/2 (U0126; 5 µM) <b><i>A</i></b>, NF-κB (SN50; 100 µg/ml), <b><i>B</i></b>, p38 (SB203580; 5 µM), <b><i>C</i></b>, JNK (SP600125; 5 µM), <b><i>D</i></b>, followed by incubation with OmpA (5 µg/ml) for 72 h. The total number of ASCs (IgM + IgG-secreting cells) was analyzed by ELISPOT. Number of ASCs/10⁵ B cells. The results are the mean ± S.E.M of three independent experiments. <i>***, p</i><0.001, relative to OmpA treated group in the absence of the inhibitor.</p

Outer Membrane Protein A (OmpA) of <i>Shigella flexneri</i> 2a Induces TLR2-Mediated Activation of B Cells: Involvement of Protein Tyrosine Kinase, ERK and NF-κB

<div><p>B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA) of <i>Shigella flexneri</i> 2a has been demonstrated to induce the production of IgG and IgA <i>in vivo</i> following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response <i>in vitro</i>. We show that OmpA of <i>S. flexneri</i> 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs). The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs), ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of <i>S. flexneri</i> 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an optimal vaccine antigen.</p></div

A hypothetical model for <i>S. flexneri</i> 2a OmpA-mediated activation of B cells and its role in regulation of host immune response.

<p>A hypothetical model for <i>S. flexneri</i> 2a OmpA-mediated activation of B cells and its role in regulation of host immune response.</p

OmpA evokes proliferation and differentiation of splenic B cells as well as induces secretion of IL-10 and IL-6.

<p>(<b>A</b>) CFSE-labeled B cells were cultured in medium alone or with an increasing concentration of OmpA for 96 h followed by flow cytometry. A representative of three independent experiments is shown and each experiment was done in triplicate. (<b>B</b>) The total number of ASCs (IgM + IgG-secreting cells) was analyzed by ELISPOT at the end of a 72 h culture period. Results are expressed as numbers of ASCs/10⁵ cells seeded and correspond to the mean ± S.E.M of quadruplicate determinations. <i>**, p</i><0.01, relative to the untreated (media alone) group. (<b>C</b>) Purified splenic B cells were cultured in the absence or presence of indicated concentrations of OmpA for 72 h. The culture supernatants were assayed for IL-6 and IL-10 by enzyme-linked immunosorbent assay. The values are derived from IL-6 and IL-10 standard curves and represent the mean ± S.E.M of three independent experiments performed. <i>**, p</i><0.01, relative to the untreated (media alone) group. (<b>D</b>) Time kinetics of IL-6 and IL-10 production by B cells incubated without or with OmpA (5 µg/ml). The values are derived from IL-6 and IL-10 standard curves and represent the mean ± S.E.M of three independent experiments performed. <i>**, p</i><0.01, relative to the untreated (media alone) group.</p

OmpA triggers expression of MHCII and CD86 on B cell.

<p>Splenic B cells were incubated without (red) or with (black) OmpA (5 µg/ml) for 24 h. Cells were harvested and assayed for cell surface expression of MHCII, CD80 and CD86. The shaded histograms indicate the isotype control antibodies. Representative data from three independent experiments are shown.</p

OmpA of <i>S. flexneri</i> 2a induces expression of TLR2 and TLR6 in B cell.

<p>(<b>A</b>) Splenic B cells were stimulated with OmpA (5 µg/ml), total RNA was extracted after 3 h and subjected to RT-PCR using the appropriate primer. PCR products were quantified and expressed as the ratio of each product to GAPDH band density. The data are representative of three independent experiments. (<b>B</b>) Purified splenic B cells were incubated in the absence (red line) or presence (green line) of OmpA (5 µg/ml) for 6 h. Cells were harvested and assayed for cell surface expression of TLR1, TLR2, TLR4, and TLR6. The blue lines represent the isotype control antibodies. Representative data from three independent experiments are shown.</p