Discussion
MRGPRX2, recently discovered receptor, appears to be observed
predominantly in skin MCs, while other members of MRGPR family are found
in peripheral neurons mostly22. Unusually, MRGPRX2 can
be activated broadly by several ligands which include both peptides and
small molecules such as antibiotics, neuropeptides and anesthetics.
Possibly, C48/80 is the most potent of the small molecules and
non-peptidergic MRGPRX2 agonists23. These agonists of
MRGPRX2 are able to stimulate tissue-resident MCs which are found
in close proximity to nerve endings
and blood vessels. Interestingly, MCs activation induced by MRGPRX2 is
non-IgE dependent. And then MCs activation leads to enhanced
inflammatory reaction, as well as engagement in a bidirectional loop
with sensory nerve by releasing MCs-derived
cytokines24. Currently, MRGPRX2 has been identified as
a pruritogenic receptor18. As a result,
MRGPRX2-stimulation will result in inflammatory reaction and pruritus
such as pseudo-allergic reaction (transient side effects by first-time
drug therapy), as well as many chronic, inflammatory and pruritus
dermatoses with over-expressed MRGPRX214,25.
Tacrolimus is an immunosuppressant, and topical tacrolimus has been
widely used in the treatment of chronic and inflammatory skin diseases
such as AD, rosacea, contact dermatitis, and psoriasis for its potent
anti-inflammation effect26. Besides, refractory
itching of these diseases, reported to be IgE-independent, can be
alleviated by topical tacrolimus therapy27. However,
initial skin irritation induced by first-time tacrolimus use is common
and can’t be ignored. We therefore investigated the underlying mechanism
of side effects induced by short-term tacrolimus application and
suppressed inflammatory reactions after chronic tacrolimus exposure,
placing particular emphasis on MRGPRX2.
Intriguingly, we discovered that dose-dependent inflammatory reaction
and scratching behavior could be observed in WT mice instead of
KitW-sh/W-sh mice via non-IgE pathway after tacrolimus
short-term application. This transient skin irritation in vivo is
similar to initial side effect of tacrolimus clinical topical
application, which may be speculated as a non-IgE dependent
pseudo-allergic reaction. Actually, IgE-independent MCs activation plays
a vital role in the underlying mechanism of initial side effect of
topical tacrolimus application. And we confirmed that tacrolimus could
act on MCs in a IgE-independent pathway.
We set out to further explore this non-IgE dependent MCs activation by
tacrolimus, and we found short-term tacrolimus exposure causes MCs
activation via MRGPRX2/B2 in vivo and in intro . Therefore,
we speculated that MRGPRX2/B2 could be a novel target for tacrolimus. We
discovered tacrolimus could bind to MRGPRX2 directly by molecular
docking. To further clarify how tacrolimus regulates MRGPRX2, FK-DNS, a
fluorescently labeled tacrolimus, was constructed. We found FK-DNS,
having similar function to tacrolimus on MRGPRX2, can bind to MRGPRX2
directly exhibiting green fluorescence, and later move into cell to
modulate MRGPRX2. Hence, tacrolimus was acknowledged to regulate MCs
activation via MRGPRX2/B2 directly.
Addressing the MRGPRX2 intracellular signaling events elicited by
short-term tacrolimus in MCs, upregulated phosphorylated PLCγ1, PKC,
ERK1/2 and P38 expression were verified. ERK and p38, part of MAPK
family, mediate intracellular signaling and play important roles in
producing proinflammatory cytokines, which demonstrates the modulatory
role of tacrolimus on MRGPRX2 in inflammation
pathology28,29 . Here, inflammatory cytokines,
tryptase, IL-8, TNF-α and MCP-1, released from MCs upregulated by
tacrolimus short-term treatment in our study, will result in
inflammatory cells recruitment, inflammatory reaction, and pruritus,
even burning16,30,31. Collectively, tacrolimus could
regulate MRGPRX2-mediated inflammatory respond.
Initial inflammatory reaction faded away and MCs activation induced by
long-term tacrolimus decreased when compare to short-term tacrolimus
treatment. Strikingly, we found that chronic tacrolimus treatment can
suppress MRGPRX2/B2-elicited activation by dampening expression of
MRGPRX2/B2. Consequently, we discovered MRGPRX2/B2-elicited MCs
activation and inflammatory mediators, IL-8, TNF-α, MCP-1 and tryptase,
released decreased in the presence of long-term tacrolimus. Besides, the
infiltration of inflammatory cells also decreases. Similarly, the
inhibitive effect of MRGPRX2/B2 expression was increased by tacrolimus
with respect to time. Possibly, these data suggests that tacrolimus may
also inhibit inflammatory response of other immune cells involved in
MRGPRX2/B2-elicited pathology by suppressing inflammatory cytokines
release. What’s more, we speculated dampened MRGPRX2 expression plays an
important role in the disappearance of side effects of tacrolimus
application.
Moreover, tryptase, main mediators of non-IgE dependent pruritis,
drastically reduces from MRGPRX2/B2-stimulated degranulation after
long-term tacrolimus treatment in our study. The declined tryptases
release potentially contributes to itching suppression, for the
receptors of which (Protease activated receptor 2, PAR2) present on
itch-sensory neurons32. PAR2 activated by tryptase may
be a mechanism by which tacrolimus inhibits MRGPRX2-stimulated
neurogenic itching and inflammation32.
In addition to its function on MRGPRX2 which has not been reported
before, topical tacrolimus has additional mechanisms to alleviate
chronic inflammation and pruritus33. Some studies
raised the possibility that tacrolimus can act on the sensory nerves and
cause desensitization33. Moreover, neuroimmune has
been given more attention in chronic inflammation and pruritus, and
MRGPRX2 has its potential role in neurogenic inflammation and
itch14. For example, SP, a vital factor in neurogenic
inflammation and pruritis, has been recently implicated in correlating
with MRGPRX2/B2 activation in many chronic skin
diseases34,35. Interestingly, skin SP immunoreactive
cell counts decreased significantly after tacrolimus treatment in AD
patients36. Hence, tacrolimus may modulate neuroimmune
clusters by MRGPRX2/B2 signaling in skin physiological and itching
processes37. In the future, in pathology of neurogenic
inflammation and itching, inhibition MRGPRX2 and its signaling may be a
potential therapeutic strategy for tacrolimus.
In summary, tacrolimus can regulate IgE-independent MCs activation and
MRGPRX2 is a novel target of tacrolimus. Inhibition of MRGPRX2
activation by short-term tacrolimus may play an important role in
initial side effects induced. Chronic tacrolimus treatment may have
potential to be utilized in the resolution of chronic inflammatory
reaction related to MRGPRX2 particularly.