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

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

MZ B cells show regulatory features.

<p>(A) MZ and FO B cells from PBS-uninfected and PBS-infected were sorted using flow cytometry and cultured for 5 days in the presence of SEA for IL-10 production as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030883#pone-0030883-g002" target="_blank">Fig. 2A</a>. (B) Irradiated MZ B cells or FO B cells (1×10⁵) were co-cultured with CD4⁺CD25⁻ T cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030883#pone-0030883-g003" target="_blank">Fig. 3A</a>. Treg cell induction by MZ or FO B cells from uninfected mice was set at one. Subsequently, fold change in Treg cell induction by MZ or FO B cells from OVA-uninfected and OVA-infected mice was calculated. Each graph contains three independent experiments with five mice per group.</p

IL-10 production by B cells from different organs during chronic schistosomiasis and their role in protection against AAI.

<p>(A) WT mice 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>. Splenic, pulmonary or mesenteric B cells (1×10⁵) were isolated and cultured in the presence of SEA (20 µg/ml) for five days. IL-10 production was measured using ELISA and medium value was subtracted. (B) OVA-sensitized recipient mice received 5×10⁶ B cells from different organs. After challenge, BAL cell numbers and eosinophils were determined (C) BAL numbers and eosinophilia of mice that received 250 µg isotype control or anti-IL-10R abs per mouse one day before the adoptive transfer. (D) The percentage of CD4⁺CD25⁺FoxP3⁺ Treg cells was determined in the lungs of recipient mice. Each graph represents three independent experiments, consisting of five mice per group.</p

Role of IL-10-producing B cells on OVA-specific AAI during chronic <i>S. mansoni</i> infection.

<p>Chimeric WT and IL-10^−/− B cell mice were infected with <i>S. mansoni</i> and sensitized and challenged for OVA at week 11 and 12 after the start of infection. BAL fluid was collected and total BAL cells (A) and eosinophils were determined. (B) In the BAL fluid, IL-5 and IL-13 were measured by ELISA. (C) MedLN cells were collected and restimulated by OVA (10 µg/mL) for four days and IL-4, IL-5, IL-10, and, IL-13 production was determined using ELISA. Results are from two independent experiments and each group consists of 6 PBS- and 6 OVA-uninfected, 8 PBS- and OVA-infected mice.</p

Demographic characteristics and infection status of Gabonese children.

<p>Co-infections are depicted as number of participants infected out of total number of participants tested.</p

MZ B cells are important in the protection against AAI.

<p>(A) Mock-depleted splenic and the MZ-depleted B cells were injected in OVA-sensitized recipient mice. After challenge, total BAL cell count and the number of BAL eosinophils (B) and the percentage of CD4⁺CD25⁺FoxP3⁺ T cells in the lungs (C) was measured. Figure is representative of two independent experiments, consisting of five mice per group.</p