Tacrolimus could induce dermatitis mediated mainly by lgE-independent MCs degranulation in vivo
WT mice were applied by different concentrations of tacrolimus and vehicle for 1 day on dorsal skin to mimic tacrolimus clinical application. Although no visible erythema and scaling difference were observed between control group and tacrolimus treated groups (Figure 1A), skin inflammatory cells infiltration (Figure 1B) and degranulation of MCs were more significant in treated groups (Figure 1C). Moreover, scratching bouts increased dose-dependently in tacrolimus group (Figure 1D). Nevertheless, this rapid MCs activation wasn’t accompanied by remarkable elevated IgE levels between control group and treated groups (Figure 1D). However, the levels of tryptase, MCP-1 and TNF-α were upregulated dose-dependently (Figure 1E). Hence, initial topical tacrolimus application induced itching and inflammatory reaction without IgE change.
MCs play an important role in inflammation and pruritus. In order to further verify effect of tacrolimus on MCs, hind paw swelling and extravasation experiment was used. Some researchers have found even 0.01 % (125 μM) tacrolimus could enhance neuropeptide (SP) homogenate concentrations in healthy tissue21. Hence, descending concentrations of tacrolimus solution were used in hind paw swelling and extravasation experiment. We found an obvious swelling in left hind paw injected with tacrolimus or C48/80 in WT mice, compared with right hind paw treated with saline solution (Figure 1F). Besides, paw thickness increase rate for tacrolimus injection was dose-dependent, and tacrolimus increased vascular permeability in vivo , as assayed by Evans Blue dye (EBD) extravasation, which was also dose-dependent (Figure 1G). Here, we demonstrated that tacrolimus could activate MCsin vivo .
KitW-sh/W-sh mice, MCs deficient mice, were used to explore the role of MCs in tacrolimus mediated dermatitis, which were applied with tacrolimus on dorsal skin for one day. There were no obvious erythema and scaling in KitW-sh/W-sh mice group (Figure 2A). Also, skin inflammatory cells infiltration (Figure 2B), scratching behaviors, and the levels of tryptase, MCP-1 and TNF-α in serum didn’t increase after one-day tacrolimus application in KitW-sh/W-sh mice group compared with WT mice (Figure 2C). There was no significant different IgE level in serum between any two groups (Figure 2C). Moreover, in hind paw swelling and extravasation experiment, KitW-sh/W-sh mice showed no prominent difference in paw swelling and EBD exudation between left (injected tacrolimus) and right (injected saline solution) hind paws of themselves, whereas they both showed decreased paw swelling and extravasation when compared to WT mice (injected tacrolimus) (Figure 2D).
Collectively, topical tacrolimus application triggered dermatitis and transient itching via non-IgE dependent MCs activation.
MRGPRB2/X2 participates in tacrolimus induced MCs activation
Further, we set out to detect whether MrgprB2 participates in MCs activation induced by tacrolimus as mentioned above. MrgprB2 deficient mice (MUT mice) were employed here, in hind paw swelling and extravasation experiment, MUT mice showed no significant difference in paw swelling and EBD exudation between left (injected tacrolimus) and right (injected saline solution) hind paws of themselves in hind paw swelling and extravasation experiment, whereas they both exhibited decreased paw swelling and extravasation when compared to WT mice (injected tacrolimus) (Figure 3A). Therefore, we concluded that tacrolimus could activate MrgprB2 leading to local inflammatory reaction induced by MCs.
Besides, MUT mice were also applied with tacrolimus on dorsal skin to mimic tacrolimus clinical use. After one day application of tacrolimus on dorsal skin of WT mice and MUT mice, no visible erythema and scaling were observed in all groups (Figure 3B). However, inflammatory cells infiltration was more obvious in WT group compared with MUT mice (Figure 3C). With avidin staining, no obvious MCs degranulation could be observed in MUT mice (Figure 3D). Significantly, MUT mice didn’t show increased scratching behaviors, as well as serum tryptase, MCP-1 and TNF-α after one-day tacrolimus application (Figure 3E). IgE level in serum didn’t differ significantly between any two groups (Figure 3E). Hence, MrgprB2 plays a vital role in initial dermatitis and MCs stimulation triggered by topical tacrolimus.
Considering the rapid response of MrgprB2 to tacrolimus leading to MCs degranulation in vivo , we set out to further explore whether short-term tacrolimus exposure could activate MCs via MRGPRX2 in vitro . We found calcium influx of LAD2 cells, the early phase of MCs activation, could be altered by short-term tacrolimus priming (Figure 4A), C48/80 (a MRGPRX2 agonist) as a positive group (Figure 4B). Further studies revealed that β hexosaminidase release increased (Figure 4C), as well as tryptase, MCP-1 and IL-8 increased dose-dependently after treated with tacrolimus in LAD2 cells for 30 minutes (Figure 4D). Furthermore, we treated MrgprB2-HEK293 cells with tacrolimus, which resulted in increased intracellular Ca2+ concentration (Figure 4E), but tacrolimus did not alter the intracellular Ca2+ concentration in normal control (NC)-HEK293 cells (Figure 4F). Intriguingly, lower release of inflammatory mediators (β hexosaminidase, tryptase, MCP-1 and IL-8) was observed obviously in MRGPRX2-knockdown group compared to NC-LAD2 cells (Figure 4G, H). Thus, tacrolimus mimics the behavior of C48/80 in LAD2 cells, supporting action at the same receptor MRGPRX2.