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

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.

positive control and saline was used as a negative control. The skin test index was the ratio between the response to an allergen and the response to histamine.

ELISA (enzyme-linked immunosorbent assay)
The sera were separated from the blood samples collected from each subject. The levels of DEM-specific IgE, DEM-specific IgG4, IL-4, IL-5, IL-13 and IFN-γ in the sera were determined by ELISA with commercial reagent kits following the manufacturers' instructions.

Isolation of immune cells
The peripheral blood was collected from AR patients and healthy volunteer subjects (40 ml/person). The peripheral mononuclear cells (PBMC) were isolated by the gradient density centrifugation. The immune cells were purified from PBMCs by the magnetic cell sorting (MACS) with commercial reagent kits following the manufacturer's instructions. The purity of the isolated cells was assessed by flow cytometry. If the purity did not reach 95%, MACS was performed again with the cells. The viability of the cells was greater than 98% as checked by Trypan blue exclusion assay.

Cell culture
The immune cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine. For B cell culture, anti-CD40 (20 ng/ml) was added to the culture to avoid apoptosis throughout the study. The viability was greater than 98% before using for further experiments as checked by Trypan blue exclusion assay.

Flow cytometry
Cells were stained with fluorochrome-labeled antibodies (or isotype IgG) at 0.5 µg/ml for 1 h at room temperature. The expression of surface markers was assessed with a flow cytometer (FACSCanto I, BD Biosciences). In the case of intracellular staining, the cells (fresh or stained for the surface markers) were fixed with 1% paraformaldehyde mixed with 0.5% saponin for 1 h.
After washing, the cells were stained with fluorochrome-labeled antibodies (or isotype IgG) for 1 h at room temperature and then analyzed with a flow cytometer. The data were analyzed with the software flowjo. The data of isotype IgG staining were used as a reference to set the gates. In the case of cell proliferation assay, the cells of interest were labeled with CFSE (Carboxyfluorescein succinimidyl ester) and cultured with appropriate procedures, and analyzed by the CFSE-dilution assay.

Assessment of apoptosis
After appropriate treatment, the cells were collected from the culture, stained with Annextin V reagent and propidium iodide (PI), and analyzed by flow cytometry. The Annexin V positive cells, or both Annexin V/PI positive cells were regarded as apoptotic cells.

Real time quantitative RT-PCR (RT-qPCR)
The total RNA was extracted from the cells of interest. A cDNA was synthesized with the RNA using a reverse transcription kit. The qPCR was performed on a real time PCR device (MiniOpticon, Bio-Rad) with the SYBR Green Master Mix. The results were calculated with the 2 -∆∆Ct method. The results of mRNA are presented as relevant changes against controls. The primers using in this study include: IgE, tagtgactctgatgccaccc and ccccagaggtccaagtaaca. IL-10, gttctttggggagccaacag and gctccctggtttctcttcct. β-actin, cgcaaagacctgtatgccaa and cacacagagtacttgcgctc.

Preparation of cytosolic and nuclear extracts
Cells were incubated with lysis buffer (10 mM HEPES, pH 7.4, 10 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.2% Nonidet P-40, and 0.2 mM PMSF) at 4C for 15 min, and centrifuged at 500 g for 10 min at 4°C. The supernatant was collected as the cytosolic extract. The pellet was added with nuclear extract buffer (20 mM HEPES-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, and 1 protease inhibitor cocktail) and incubated for 15 min at 4°C, followed by centrifugation at 13,000 g for 10 min at 4°C. The supernatant was collected as the nuclear extract. The protein concentrations were determined by the Bradford method.

Western blotting
Proteins were extracted from the cells. The proteins were fractioned with sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAGE) and transferred onto a PVDF membrane.
After blocking with 5% skim milk for 30 min, the membrane was incubated with the primary antibodies at 300 ng/ml overnight at 4°C and followed by incubating with the second antibodies (labeled with peroxidase) for 1 h at room temperature. Washing with TBST (Tris-buffered saline Tween 20) was performed after incubation. The membrane was developed with enhanced chemiluminescence (ECL). The results were photographed with an imaging device (UVI system; Beijing, China).

Validation of commercial DME (DME#)
To validate the commercial DME, ELISA was performed with DME# or DME (prepared from dust mites). The immune response of DME and DME# is presented below (Fig. S2). Figure S1. Immune response of dust mite extracts (DME). DME was prepared with dust mite by us. DME# is commercial DME, which was used in SIT of the present study. The 96 well plates were coated with 100 µl of DME or DME# at a concentration of 1 µg/mL in carbonate buffer and stored at 4°C overnight with 3% BSA. The plates were incubated with monoclonal antibodies of subtypes of mite allergens that were prepared by us as denoted on the X axis at 37 °C for 2 h. Then the plates were added with 100 µl of HRP labeled anti-mouse antibody (1:2000 v/v) and incubated at 37°C for 2 h. The plates were subjected to color development 6 with TMB at 37°C for 10 minutes, and the reactions were terminated by adding 2 mol/L H 2 SO 4 .
The plates were read with a microplate reader at 450 nm. The bars indicate the immune response of DME and DME#. Data of bars are presented as mean ± SD. There is no significant difference in the immune response between DME and DME#. The data were summarized from 3 independent experiments.