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 .