Regulation of allergic reactions of mast cells by intestinalbacteria and probiotics
Kyoko TAKAHASHI
College of Bioresource Sciences, Nihon University

Research aims
People suffering from allergic diseases have dramatically increased in recent years.
Mast cells, which are responsible for allergic inflammation, express the high affinity IgE
receptor (FcεRI). Crosslinking of FcεRI with IgE and allergen induces degranulation, whichis the release of inflammatory mediators such as histamine stored in intracellular granules.
Mast cells are differentiated in the bone marrow, released into blood as immature
cells, and finally differentiated into mature cells after reaching peripheral tissues such as themucous membrane and skin. The intestinal tract, which has a vast mucosal area andfunctions as a reservoir of mast cells in vivo, is inhabited by an enormous number ofcommensal bacteria. This study aimed to clarify novel mechanisms of the anti-allergic actionof intestinal bacteria on mast cells for application to preventing or improving allergic
symptoms through diet.
Methods①Regulation of granule formation in mast cells by intestinal bacteria
We have reported that Lactobacillus casei JCM1134 (LC) suppresses granuleformation in mast cells through increased expression of the transcription factor C/EBPα.1 To
clarify the underlying molecular mechanisms, following experiments were performed. Bonemarrow cells from femurs of female C57BL/6 mice at 6 weeks of age were cultured for 4 to
8 weeks in medium supplemented with IL-3 to obtain bone marrow-derived mast cells(BMMCs). BMMCs were stimulated with LC, and the expression of molecules involved ingranule formation was analyzed by qRT-PCR. Alternatively, an expression plasmid forC/EBPα was introduced into BMMCs by electroporation and similar analyses wereperformed. In addition, BMMCs were cultured in the presence of fractionated components ofLC for 24 h, followed by measurement of C/EBPα expression by qRT-PCR. The
fractionation method is shown in Fig. 1②Regulation of IgE receptor expression on mast cells by intestinal bacteriaWe have found that Bacteroides acidifaciens type A43 (BA) suppresses the cellsurface expression of FcεRI on BMMCs among a variety of heat-killed intestinal bacteria.
To clarify the underlying mechanism, the effects of BA on mRNA and total protein
expression of each subunit of FcεRI and the cell surface expression of FcεRI were analyzed
in BMMCs by qRT-PCR, western blotting, and flow cytometry, respectively. The influenceof BA on activation of signal molecules such as MAPK in BMMCs was also analyzed bywestern blotting.
Results①Regulation of granule formation in mast cells by intestinal bacteria
The expression of HDC, serglycin, and Ctr2 was decreased in BMMCs treated withLC. Overexpression of C/EBPα in BMMCs resulted in decreased expression of serglycin,
but not that of HDC and Ctr2. Therefore, serglycin expression was suppressed depending on
C/EBPα, whereas suppression of HDC and Ctr2 expression was independent of C/EBPα.
These results are summarized in Table 1. It was suggested that suppression of granuleformation by bacterial stimulation is achieved by reduction in proteoglycans such as
chondroitin sulfate and heparin that constitute the granules. In addition, the cell wall fractionof LC enhanced the C/EBPα expression, and teichoic acid particularly showed a strongenhancing activity in the cell wall fraction.
②Regulation of IgE receptor expression on mast cells by intestinal bacteria
Lasting stimulation of BMMCs with BA decreased the cell surface expression ofFcεRI on BMMCs. On the other hand, mRNA expression of FcεRI α and β chains decreasedtransiently at the early stage of stimulation, although a statistically significant difference wasnot observed. In addition, during the stimulation period, there was no change in the total
protein expression level including the intracellular content of the β chain. Furthermore, Erkphosphorylation tended to be suppressed at the early stage of stimulation. Addition of an Erk
inhibitor to BMMCs also decreased the expression of FcεRI on the cell surface. Collectively,
it was thought that BA decreases the expression of FcεRI on mast cells mainly by
posttranslational control via inhibition of Erk activation.
Conclusion
The following regulatory mechanisms of mast cells by intestinal bacteria wereshown. (1) Specific bacterial components suppress granule formation in mast cells by
reducing the proteoglycans constituting the granules in a C/EBPα-dependent manner. (2)Specific intestinal bacteria suppress the cell surface expression of FcεRI on mast cells
mainly at the post-translational stage through inhibition of Erk activation. These mechanisms
will provide novel targets for evaluating the anti-allergic activity of probiotics.

References
1. Kasakura, K. et al. (2014) C/EBPα controls mast cell function. FEBS Lett. 588:
4645-4653.