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

Cigarette Smoke Modulates Repair and Innate Immunity following Injury to Airway Epithelial Cells

<div><p>Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding. Upon mechanical injury CS caused a delayed recovery in the epithelial barrier integrity and wound closure. Furthermore CS enhanced innate immune responses, as demonstrated by increased expression of the antimicrobial protein RNase 7. These differential effects on epithelial repair and innate immunity were both mediated by CS-induced oxidative stress. Overall, our findings demonstrate modulation of wound repair and innate immune responses of injured airway epithelial cells that may contribute to COPD development and progression.</p></div

EGFR and ERK1/2 signaling in wounded ALI-PBEC.

<p>(A) Intrinsic wound healing of ALI-PBEC was determined in the presence of the EGFR tyrosine kinase inhibitor AG1478 (1 μM) or the MEK1/2 inhibitor U0126 (25 μM) at 6 h after exposure with either air or CS. Data are shown as percentage wound closure compared to t = 0 h. (B) mRNA expression of <i>RNASE7</i> was determined by qPCR. Data are shown as normalized mRNA expression compared to <i>RPL13A</i> and <i>ATP5B</i>. (C) Western blot analysis of EGFR and ERK1/2 phosphorylation of wounded ALI-PBEC exposed to air or CS in the presence of AG1478, U0126, and NAC, at 6 h after exposure. (D) Bands were quantified by densitometry for analysis of EGFR and (E) ERK1/2 phosphorylation and corrected for total-EGFR and total-ERK1/2, respectively. For all graphs data are shown as mean; error bars represent SEM; experiments were conducted in duplicate; n = 3 independent donors; * p < 0.05.</p

p63⁺ cells at the wound edge of ALI-PBEC.

<p>(A) Immunofluorescence staining for p63 (green) and nuclei (blue (DAPI)) of mechanically injured ALI-PBEC. (B) Percentage of p63⁺ cells at the first line of cells directly at the wound edge or in intact areas, in air- versus CS-exposed cells. (C) Number of p63⁺ cells and p63^- cells at the wound edge per 400 μm length of wound edge, in air- versus CS-exposed cells. (D) Internuclear distance in μm between p63⁺ cells located directly at the leading wound edge and the first adjacent p63⁺ cell. All graphs: data are shown as mean; error bars represent SEM, experiments were conducted in duplicate, n = 3 independent donors, * p < 0.05.</p

Proposed model.

<p>EGFR signaling is activated by CS and injury, and this leads to MEK1/2-mediated phosphorylation of downstream ERK1/2. CS furthermore directly causes phosphorylation and activation of ERK1/2 via oxidative stress, which is independent of EGFR signaling. EGFR/ERK1/2-mediated wound repair is suppressed by CS via oxidative stress. In contrast, activation of ERK1/2 due to a combined effect of CS-induced oxidative stress and injury, results in an enhanced innate immune response. Solid lines represent the effect of CS, dashed lines the effect of injury. NAC, AG1478 and U0126 were used to inhibit oxidative stress, EGFR phosphorylation, and ERK/12 phosphorylation respectively.</p

Effect of CS on airway epithelial wound healing and innate immunity.

<p>ALI-PBEC were mechanically injured and subsequently exposed to air (control) or whole cigarette smoke (CS). (A) Phase-contrast light microscopy images were made of air- and CS-exposed ALI-PBEC at 0, 6, 24 and 48 h after exposure. (B) Wound closure is shown in percentage in air- versus CS-exposed cells and (C) wound closure rate in percentage per hour at different time intervals was calculated. n = 8 independent donors. (D) Wound closure rates per hour in air- and CS-exposed ALI-PBEC up to 12 h after exposure were determined using live imaging. n = 7 independent donors. (E) <i>RNASE7</i> mRNA expression was determined in intact or wounded ALI-PBEC exposed to air or CS, at 6 h after exposure. Values shown represent normalized mRNA expression compared to <i>RPL13A</i> and <i>ATP5B</i>. n = 7 independent donors. (F) IL-8 secretion was determined in the basal culture medium. n = 9 independent donors. Data are shown as mean; error bars represent SEM; experiments were conducted in duplicate i, * p < 0.05.</p

Effects of CS on airway epithelial barrier recovery and innate immunity.

<p>Barrier integrity in ALI-PBEC was disrupted using calcium depletion, and cells were subsequently exposed to air or CS. (A) The trans-epithelial electrical resistance (TEER) was subsequently measured at 0, 3, 6, and 24 h after exposure to assess loss and recovery of barrier integrity in air- and CS-exposed cultures. TEER values in ohm (Ω). n = 7 independent donors. (B) At 24 h, mRNA expression of <i>RNASE7</i> was assessed in ALI-PBEC that were incubated with calcium-depleted medium (w/o Ca²⁺) versus control medium (ctrl), and subsequently exposed to either air or CS and further incubated in calcium containing medium. Normalized mRNA expression compared to <i>RPL13A</i> and <i>ATP5B</i> is depicted in the graph. n = 4 independent donors. (C) Secretion of IL-8 in the basal culture medium was assessed by ELISA. n = 5 independent donors. Data are shown as mean; error bars represent SEM; experiments were conducted using duplicate exposures in all donors, * p < 0.05.</p

CD4⁺CD25^hiFOXP3⁺ Cells in Cord Blood of Neonates Born from Filaria Infected Mother Are Negatively Associated with CD4⁺Tbet⁺ and CD4⁺RORγt⁺ T Cells

<div><p>Background</p><p>Children who have been exposed <i>in utero</i> to maternal filarial infection are immunologically less responsive to filarial antigens, have less pathology, and are more susceptible to acquire infection than offspring of uninfected mothers. Moreover children from filaria infected mothers have been shown to be less responsive to vaccination as a consequence of an impairment of their immune response. However, it is not well known how <i>in utero</i> exposure to parasite antigens affects cellular immune responses.</p><p>Methodology</p><p>Here, 30 pregnant women were examined for the presence of microfilaria of <i>Loa loa</i> and <i>Mansonella perstans</i> in peripheral blood. At delivery, cord blood mononuclear cells (CBMC) were obtained and the CD4⁺T cells were phenotyped by expression of the transcription factors Tbet, RORγt, and FOXP3.</p><p>Results</p><p>No significant difference was observed between newborns from infected versus uninfected mothers in the frequencies of total CD4⁺T cells and CD4⁺T cells subsets including CD4⁺Tbet⁺, CD4⁺RORγt⁺ T and CD4⁺CD25^hiFOXP3⁺ T cells. However, there was a negative association between CD4⁺CD25^hiFOXP3⁺T cells and CD4⁺Tbet⁺ as well as CD4⁺RORγt⁺ T cells in the infected group only (B = −0.242, P = 0.002; B = −0.178, P = 0.013 respectively).</p><p>Conclusion</p><p>Our results suggest that filarial infection during pregnancy leads to an expansion of functionally active regulatory T cells that keep TH1 and TH17 in check.</p></div

The relation between CD4⁺CD25^hiFOXP3⁺ T cells and CD4⁺Tbet⁺ (upper panel) as well as CD4⁺CD25^hiFOXP3⁺ T cells and CD4⁺RORγT⁺ T cells (lower panel) of CBMC of neonates from filaria negative (in grey) and filaria positive (in black) mothers assessed by a linear regression analysis.

<p>Each dot shows value from a single subject while the solid lines represent the regression lines of the model. The strength of the association between two variables is given by the value of the regression coefficient beta (β) value in each graph. A positive β value indicates a positive association between the variables in the model while a negative β value indicates a negative association. P values are given to indicate the statistical significance of the associations.</p