Combination of specific allergen and probiotics induces specific regulatory B cells and enhances specific immunotherapy effect on allergic rhinitis

Abstract

The therapeutic efficacy of allergen specific immunotherapy (SIT) on allergic diseases is to be improved. Probiotics can regulate immune response. This study aims to promote the effect of SIT on allergic rhinitis (AR) by co-administration with Clostridium butyricum (Cb). In this study, patients with AR sensitized to mite allergens were enrolled to this study, and treated with SIT or/and Cb. The therapeutic efficacy was evaluated by the total nasal symptom scores (NSS), medication scores, serum specific IgE levels and T helper (Th)2 cytokine levels. The improvement of immune regulation in the AR patients was assessed by immunologic approaches. The results showed that treating AR patients with SIT alone markedly reduced NSS and medication scores; but did not alter the serum specific IgE, Th2 cytokines and skin prick test (SPT) index. The clinical symptoms on AR in SIT group relapsed one month after stopping SIT. Co-administration of Cb significantly enhanced the efficacy of SIT on AR as shown by suppression of NSS, medication scores, serum specific IgE, Th2 cytokines and SPT index; the regulatory B cell frequency was also markedly increased. Such an effect on AR was maintained throughout the observation period even after stopping the treatment. Butyrate blocked the activation of histone deacetylase-1, the downstream activities of epsilon chain promoter activation, and the IgE production in the antigen specific B cells. On the other hand, butyrate induced the IL-10 expression in B cells with a premise of the B cell receptor activation by specific antigens. In conclusion, administration with Cb can markedly enhance the efficacy of SIT on AR.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; allergic rhinitis; immunoglobulin E; probiotics; regulatory B cells; specific immunotherapy.

Figure 1. Assessment of AR status

AR patients were treated as denoted on the X axis of the bar graphs. Placebo: Patients were treated with placebo (n = 20), or SIT (n = 46; group1), or SIT/Cb (Cb = C. butyric; n = 44; group2), or Cb (n = 48) respectively. A. The NSS were recorded once a week. The bars indicate the averaged NSS of 2 weeks prior to the indicated time-points. B. The bars indicate the medical scores of 2 weeks prior to the indicated time-points. C.-H. The sera were collected from each patient at month 0, 6, 7 and 12 respectively and analyzed by ELISA. I. The bars indicate the SPT results at the indicated time-points. Data of bars are presented as mean ± SD. *, p < 0.01, compared to the placebo group. #, p < 0.01, compared with the SIT group.

Figure 2. SIT/Cb promotes generation of Bregs in AR patients

The number of patient and the patient information is the same as Figure 1. The peripheral mononuclear cells (PBMC) were collected from each patient and analyzed by flow cytometry. A. The gated cells show the CD4⁺ T cells in LPMCs. B.-F. The gated cells show Tregs in the CD4⁺ T cells of panel A. G. The bars indicate the summarized data of B-F. H. The bars indicate the frequency of IL-10⁺ B cells in PBMCs. The data of bars are presented as mean ± SD. *, p < 0.01, compared with group B (G), or the healthy group (H). Samples from individual patients were processed separately. Cb: C. butyricum.

Figure 3. Specific antigens and butyrate differentially modulate antigen specific B cell properties

DerpsBCs were isolated from 12 AR patients (samples from each patient were used for one test). A.-D. The dot plots show the frequencies of IgE or/and IL-10 positive B cells in response to the treatment (denoted above each panel; anti-CD40 was added to the culture at 20 ng/ml). BS: Butyrate sodium (1 mg/ml). The bars indicate the summarized data of A-D. E. The histograms indicate Teff cell proliferation, F. The gated dot plots indicate the frequency of apoptotic Teff cells, in response to the treatment (denoted above each panel). Teff: Breg (or naive B cell; nBC) = 10⁴:10⁴/ml. Teff cells were activated by the presence of anti-CD3 (2 μg/ml)/CD28 (5 μg/ml). Bregs were activated by the specific antigen, Der p 1 (1 μg/ml). Ab: Anti-IL-10 (1 μg/ml). IgG: Isotype IgG (1 μg/ml; control Ab).

Figure 4. Exposure to Der p 1 and butyrate modulates IgE expression in DerpsBCs

DerpsBCs were isolated from 30 AR patients. The cells were stimulated with Der p 1 or/and BS in the culture for 48 h. The cells were collected and analyzed by ChIP, RT-qPCR and Western blotting. A.-C. The bars indicate the pHDAC1 (A), pp300 (B) and pSTAT3 (C) at the IgE promoter locus. D.-E. The bars indicate the mRNA levels and the immune blots show the protein levels of IgE in the DerpsBCs. F.-G. the bars indicate the mRNA levels and the immune blots show the protein levels of IL-10 in the DerpsBCs. a, b, c: Btk (a; PCI-32765 = 1 mM), p300 inhibitor (b; C646; 1 mM) and STAT3 inhibitor (c; SC-1; 15 mM) were added to the culture. Der p 1: 1 μg/ml. BS: 1 mg/ml. *, p < 0.01, compared with the medium group.