Tacrolimus inhibited MRGPRX2/B2-elicited MCs activation and inflammatory reaction
In order to further assess MrgprB2-elicited degranulation after tacrolimus long-term treatment, C48/80 was used for subcutaneous injection. Surprisingly, scratching bouts has been largely reduced after 7- and 11-day exposure to tacrolimus when compared to vehicle group (Figure 7A) with no significant difference on IgE levels (Figure 7C). In order to investigate the effect of chronic tacrolimus application on the inflammatory reaction by MrgprB2 activation, we detected the skin inflammatory cell infiltration by H&E staining of dorsal skin, which showed reduced inflammatory cells treated by C48/80 in tacrolimus group, and the number of aggregated inflammatory cells both decreased on day 7 and day 11 (Figure 7B). Moreover, MrgprB2-elicited MCs degranulation was exhibited by avidin immunofluorescence staining (Figure 7D). We found that the number of local MCs has not changed after tacrolimus long-term application. Conversely, MCs degranulation and the rate of MCs degranulation treated by C48/80 has been significantly suppressed by tacrolimus (Figure 7D). Further, we detected the skin inflammatory cytokines released by MCs. We discovered that the MrgprB2-triggered MCs degranulation was severely hampered by long-term tacrolimus use, as exhibited by the decreased levels of skin tryptase, IL-8, TNF-α,and MCP-1 (Figure 7E), and decreased mRNA expression similarly (Figure 7F). Consistent with previous results, long-term tacrolimus application eliminates MrgprB2 in vivo , and thereby restrains MrgprB2-triggered MCs degranulation.
What’s more, our study measured long-term pretreatment of tacrolimus to modify the capacity of LAD2 cells degranulation induced by C48/80in vitro . When LAD2 cells were incubated with tacrolimus for 3 d, MRGPRX2 mRNA (Figure 8A) and C48/80 stimulated release of β-hexosaminidase (Figure 8B) from LAD2 (10 μg/L) decreased significantly. On day 3, MRGPRX2 protein expression diminished dramatically (Figure 8C). Similarly, release of tryptase, TNF-α, MCP-1 and IL-8 from LAD2 cells by different concentrations of C48/80 reduced dramatically (Figure 8D).
Collectively, chronic tacrolimus treatment could inhibit MCs induced inflammatory reaction by dampening MGRPRX2/B2 in vitro andin vivo .