The recombinant fusion protein of cholera toxin B and neutrophil-activating protein expressed on Bacillus subtilis spore surface suppresses allergic inflammation in mice

Abstract

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) has been identified as a modulator with anti-Th2 inflammation activity, and cholera toxin B (CTB) is a mucosal adjuvant that can also induce antigen tolerance. In this study, we constructed a CTB-NAP fusion protein on the surface of Bacillus subtilis spore and evaluate the efficiency of oral administration of the recombinant CTB-NAP spores in preventing asthma in mice. Oral administration of recombinant CTB or CTB-NAP spores significantly decreased serum ovalbumin (OVA)-specific IgE (p < 0.001) and increased fecal IgA (p < 0.01) compared to the treatment with non-recombinant spores. Oral administration of recombinant CTB or CTB-NAP spores induced IL-10 and IFN-γ expression and reduced IL-4 levels in bronchoalveolar lavage fluid (BALF). Moreover, CTB and CTB-NAP spores reduced the eosinophils in BALF and inflammatory cell infiltration in the lungs. Furthermore, CD4CD25Foxp3 Tregs in splenocytes were significantly increased in mice treated with recombinant CTB or CTB-NAP spores. The number of CD4CD25Foxp3 Tregs caused by CTB-NAP was higher than that by CTB alone. Our study indicated that B. subtilis spores with surface expression of subunit CTB or CTB-NAP could inhibit OVA-induced allergic inflammation in mice. The attenuated inflammation was attributed to the induction of CD4CD25Foxp3 Tregs and IgA. Moreover, the fusion protein CTB-NAP demonstrated a better efficiency than CTB alone in inhibiting the inflammation.

Allergenicity reduction and rheology property of Lactobacillus‐fermented soymilk

“…As FSM can reduce IgE synthesis and increase IgG synthesis, it can shift the Th1/Th2 balance toward Th1 by regulating B cells to produce IgG instead of IgE. Dong et al . also found that the allergen‐specific IgG2a and IFN‐ γ increased and IgE levels decreased after the immunomodulating properties of the antigen were modified.…”

Oral administration of recombinant Bacillus subtilis spores expressing Helicobacter pylori neutrophil‐activating protein suppresses peanut allergy via up‐regulation of Tregs

“…Peanut allergy is an IgE‐mediated food allergy that can cause an increase in IL‐4, IL‐5 and IL‐13 expression . Our previous study indicated that CNAP spores could inhibit OVA‐induced asthma and reduce IL‐4 and IL‐5 expressions in a murine model . In the present study, recombinant NAP or CNAP spore treatment reduced IL‐4 and IL‐5 expression and increased IFN‐γ expression in the spleen cells of peanut‐sensitized mice.…”

“…Bacillus subtilis spore coat protein C (B.s-CotC), B.s-CotC-CTB, B.s-CotC-NAP and B.s-CotC-CNAP were constructed according the protocol mentioned in our previous study. 11,14 Recombinant spores were cultured in Difco sporulation medium (DSM; BD Biosciences). After incubation with shaking at 250 rpm for 24 hours, spores were harvested by centrifugation at 8000 g for 10 minutes and then treated with 4 mg/ mL lysozyme at 35°C for 30 minutes to disrupt residual sporangial cells.…”

“…10 We previously successfully constructed recombinant B subtilis spores expressing NAP or cholera toxin B (CTB)-conjugated NAP (CNAP) in a previous study. 11 These recombinant spores showed good immunogenicity and could increase faecal IgA levels. In addition, the co-administration of CTB and the antigen can promote mucosal tolerance in allergic diseases.…”

“…13 Our previous study showed that administration with B subtilis spores expressing NAP can induce Treg activation and suppress OVA-induced anaphylactic responses in mice. 11 This study is the first to utilize recombinant NAP B subtilis spores for the treatment of a food allergy. We sought to determine whether oral administration of recombinant NAP spores could inhibit allergic inflammation in peanut allergy-induced mice via the up-regulation of CD4 + CD25 + Foxp3 + Tregs.…”