The mucosal adjuvant cholera toxin B instructs non-mucosal dendritic cells to promote IgA production via retinoic acid and TGF-β

It is currently unknown how mucosal adjuvants cause induction of secretory immunoglobulin A (IgA), and how T cell-dependent (TD) or -independent (TI) pathways might be involved. Mucosal dendritic cells (DCs) are the primary antigen presenting cells driving TI IgA synthesis, by producing a proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), Retinoic Acid (RA), TGF-beta or nitric oxide (NO). We hypothesized that the mucosal adjuvant Cholera Toxin subunit B (CTB) could imprint non-mucosal DCs to induce IgA synthesis, and studied the mechanism of its induction. In vitro, CTB-treated bone marrow derived DCs primed for IgA production by B cells without the help of T cells, yet required co-signaling by different Toll-like receptor (TLR) ligands acting via the MyD88 pathway. CTB-DC induced IgA production was blocked in vitro or in vivo when RA receptor antagonist, TGF-beta signaling inhibitor or neutralizing anti-TGF-beta was added, demonstrating the involvement of RA and TGF-beta in promoting IgA responses. There was no major involvement for BAFF, APRIL or NO. This study highlights that synergism between CTB and MyD88-dependent TLR signals selectively imprints a TI IgA-inducing capacity in non-mucosal DCs, explaining how CTB acts as an IgA promoting adjuvant

Schistosomes Induce Regulatory Features in Human and Mouse CD1dhi B Cells: Inhibition of Allergic Inflammation by IL-10 and Regulatory T Cells

Chronic helminth infections, such as schistosomes, are negatively associated with allergic disorders. Here, using B cell IL-10-deficient mice, Schistosoma mansoni-mediated protection against experimental ovalbumin-induced allergic airway inflammation (AAI) was shown to be specifically dependent on IL-10-producing B cells. To study the organs involved, we transferred B cells from lungs, mesenteric lymph nodes or spleen of OVA-infected mice to recipient OVA-sensitized mice, and showed that both lung and splenic B cells reduced AAI, but only splenic B cells in an IL-10-dependent manner. Although splenic B cell protection was accompanied by elevated levels of pulmonary FoxP3+ regulatory T cells, in vivo ablation of FoxP3+ T cells only moderately restored AAI, indicating an important role for the direct suppressory effect of regulatory B cells. Splenic marginal zone CD1d+ B cells proved to be the responsible splenic B cell subset as they produced high levels of IL-10 and induced FoxP3+ T cells in vitro. Indeed, transfer of CD1d+ MZ-depleted splenic B cells from infected mice restored AAI. Markedly, we found a similarly elevated population of CD1dhi B cells in peripheral blood of Schistosoma haematobium-infected Gabonese children compared to uninfected children and these cells produced elevated levels of IL-10. Importantly, the number of IL-10-producing CD1dhi B cells was reduced after anti-schistosome treatment. This study points out that in both mice and men schistosomes have the capacity to drive the development of IL-10-producing regulatory CD1dhi B cells and furthermore, these are instrumental in reducing experimental allergic inflammation in mice

Anti-TNF therapy in IBD exerts its therapeutic effect through macrophage IL-10 signalling

Objective Macrophage interleukin (IL)-10 signalling plays a critical role in the maintenance of a regulatory phenotype that prevents the development of IBD. We have previously found that anti-tumour necrosis factor (TNF) monoclonal antibodies act through FcÎ 3-receptor (FcγR) signalling to promote repolarisation of proinflammatory intestinal macrophages to a CD206+ regulatory phenotype. The role of IL-10 in anti-TNF-induced macrophage repolarisation has not been examined. Design We used human peripheral blood monocytes and mouse bone marrow-derived macrophages to study IL-10 production and CD206+ regulatory macrophage differentiation. To determine whether the efficacy of anti-TNF was dependent on IL-10 signalling in vivo and in which cell type, we used the CD4+CD45Rb high T-cell transfer model in combination with several genetic mouse models. Results Anti-TNF therapy increased macrophage IL-10 production in an FcÎ 3R-dependent manner, which caused differentiation of macrophages to a more regulatory CD206+ phenotype in vitro. Pharmacological blockade of IL-10 signalling prevented the induction of these CD206+ regulatory macrophages and diminished the therapeutic efficacy of anti-TNF therapy in the CD4+CD45Rb high T-cell transfer model of IBD. Using cell type-specific IL-10 receptor mutant mice, we found that IL-10 signalling in macrophages but not T cells was critical for the induction of CD206+ regulatory macrophages and therapeutic response to anti-TNF. Conclusion The therapeutic efficacy of anti-TNF in resolving intestinal inflammation is critically dependent on IL-10 signalling in macrophages

CTB/LPS-primed BM-DCs promote IgA production in vitro.

<p>BM-derived DCs were cultured for 8 days with GMCSF, pulsed overnight with PBS, LPS (1 ng/ml) +/− CTB (10 µg/ml) or CTB alone, and then co-cultured with splenic B cells (ratio 1∶1) and anti-IgM Fab-fragments (10 µg/ml). After 7 days, IgA levels were determined by ELISA. (A) BM-DC and B cells of Balb/c background were used for the co-culture. In addition to the conditions described, a high concentration (100 ng/ml) of LPS was used to pulse the BM-DC (B) Besides BM-DC, PP-DC were used to put in co-coculture with B cells (all of C57/Bl6 background) (C) In addition to IgA, also other Immunoglobulin isotypes were measured in supernatant of cocultures (Balb/c) by ELISA. Mean+sem of at least 5 individual experiments are shown. * P<0.05, ** P<0.01, *** P<0.001.</p

Ex vivo CTB pulsed DCs promote IgA responses in the lungs via RA and TGF-β.

<p>PBS, OVA (800 µg/80 µl/mouse), OVA+CTB (0.1 or 0.001 µg/80 µl) or CTB alone were instilled in lungs of naïve mice. (A) After OVA challenge, lung B cells were isolated, cultured and restimulated by LPS (10 µg/ml) for 7 days and IgA levels were determined (B) AF488-labeled CTB (and as a control Fitc-labeled OVA) was used. After 36 hrs, MedLN and lung tissue were studied for the presence of AF488-positive cell populations: Dead cells are excluded based on FSc and SSc; AF488 positive cells selected; migratory cDCs (MHCII^hi) subdivided into CD11b+ and CD11b- subsets; resident DCs (MHCII^int); CD11c^loMHCII^lo cells, subdivided into pDC (B220+) and alveolar macrophages (CD11b+) (C) As in B. Percentage of AF488-positive DC subsets total AF488+ cells in MedLN (D) After 36 hrs, MedLN were isolated. ALDH enzyme activity of different DC subsets was determined using ALDEFLUOR assay, by flowcytometry. (E) BM-DC were generated as described and pulsed with PBS, OVA (100 µg/ml), OVA+CTB (10 µg/ml) or CTB only (10 µg/ml) and the next day intratracheally injected in naïve mice. One day before until 3 days after DC instillation, mice were treated i.p with LE135 and anti-TGFβ antibody (or isotype Ab and DMSO). After one week, the mice were challenged by OVA for 3 consecutive days. One day after challenge, the number of IgA+ cells in digested lungs was determined by flowcytometry. Data are mean +/− s.e.m, of at least 2 individual experiments with 4 mice per group (for D: 12 mice per group, pooled per 3 mice, resulting in 4 datapoints per group). ^# p = 0.10 * P<0.05, ** P<0.01, *** P<0.001.</p

MyD88 driven pathways synergize with CTB priming to drive IgA promoting.

<p><i>DCs.</i> (A) BM-DCs from WT or MyD88^−/− mice (C57/Bl6 background) were pulsed with PBS, LPShi (100 ng/ml), LPShi+ CTB (10 µg/ml) or CTB alone and co-cultured as described at <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059822#pone-0059822-g001" target="_blank">Figure 1</a>. (B) BM-DCs were pulsed with PBS, or different TLR ligands (Poly I:C 25 µg/ml, LPS 100 ng/ml, Flagellin 1 µg/ml, FLS1 10 µg/ml, Cl97 1 µg/ml, CpG 2.5 µg/ml) or CTLs (Zymozan 10 µg/ml, Curdlan 150 µg/ml) with and without CTB, followed by co-cultures with splenic B cells. Data of one representative experiment out of three is shown. * P<0.05, ** P<0.01, *** P<0.001.</p

In vitro, RA and TGF-β are responsible for IgA induction by CTB-primed DCs.

<p>BM-DCs were pulsed with PBS, LPS (100 ng/ml), LPS+ CTB (10 µg/ml) or CTB alone and co-cultured as described at <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059822#pone-0059822-g001" target="_blank">Figure 1</a>. (A) During the co-culture the RA receptor was blocked by LE135 (1 µM). After 7 days, IgA levels were measured in supernatant. (B) Similar as in A, but here in the presence of SB-431542 (5 µM) (or vehicle) alone to inhibit TGFβ-specific signaling or together with LE135. After 7 days, IgA levels were measured in supernatant. (C) BM-DC and B cells were co-cultured as described, however the DC were WT or deficient for iNOS (C57/Bl6 background). Data shown are mean +/− s.e.m from at least 3 pooled experiments * P<0.05, ** P<0.01, *** P<0.001.</p

Expression of candidate IgA inducing molecules of BM-DCs.

<p>(A) BM-DCs were cultured as described previously and pulsed with PBS, LPS (1 ng/ml) +/− CTB (10 µg/ml) or CTB alone. After 24 hrs the following gene expression was analyzed (A) RALDH1, RALDH2, iNOS, BAFF, APRIL and TGF-β. Expression is normalized against the house keeping gene GAPDH and displayed as relative to conventional unpulsed BM-DCs. Mean+sem of 4 indvidual BM-DC cultures are shown. (B) BM-DCs were generated by culture in GM-CSF or Flt3-L, and pulsed as described. After 24 hours, cells were washed and stained for ALDEFLUOR activity (according to the instructions of the manufacturer), CD11c, CD11b and MHCII expression and analyzed by flowcytometry. The results are a mean+sem from 4 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.</p

Treg cell induction by IL-10-producing Breg cells.

<p>(A) Irradiated splenic B cells (1×10⁵) were cultured with CD4⁺CD25⁻ T cells (1×10⁵) for 5 days in the presence of anti-CD3 and anti-CD28. Induction of CD4⁺CD25⁺FoxP3⁺ Treg cells (in %) by PBS-uninfected B cells was set at one. Fold change in Treg cell percentage for OVA-uninfected and OVA-infected B cells was calculated. Graph expresses results from three independent experiments. (B) OVA-sensitized DEREG mice were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030883#pone-0030883-g002" target="_blank">Fig. 2B</a> in addition to a DT or PBS injection. This graph expresses two experiments, consisting of five mice per group. (C) WT and IL-10^−/− B cell chimeras were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030883#pone-0030883-g001" target="_blank">Fig. 1A</a>. The MedLNs were collected and the percentage of CD4⁺CD25⁺FoxP3⁺ Treg cells was determined. Figure contains two independent experiments and each group consists of 6 to 8 mice. (D) <i>In vitro</i> co-culture were performed as described in (A) in the presence of blocking anti-IL-10R or isotype control antibodies. Percentage of Treg cells induced in the presence of isotype control was arbitrarily set at 1. Fold change in Treg cell induction in the presence of anti-IL-10R was calculated. Graph represents three independent experiments.</p