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.