TAK-242 ameliorates contact dermatitis exacerbated by IL-36 receptor antagonist deficiency

Loss-of-function mutations in IL36RN cause generalized pustular psoriasis (GPP), which is characterized by neutrophil-infiltrated lesions. Neutrophils are important during contact hypersensitivity in mice. However, it has never been determined whether interleukin-36 receptor antagonist (IL-36Ra) deficiency is an exacerbating factor in contact dermatitis. We examined whether a loss-of-function IL36RN mutation exacerbates contact dermatitis and evaluated the changes in contact dermatitis-related cytokines. Wild-type and Il36rn−/− mice were treated with 1-fluoro-2,4-dinitorobenzene (DNFB) and evaluated for ear thickness, histopathological features, numbers of infiltrated neutrophils, and numbers of CD4 + and CD8 + T cells. Furthermore, mRNA levels of contact dermatitis-related cytokines were measured by real-time polymerase chain reaction, and effects of TAK-242, a toll-like receptor 4 (TLR4) inhibitor, on the contact hypersensitivity (CHS) response were evaluated. We found that the ear thickness, cytokine expression, and neutrophil infiltration significantly increased in Il36rn−/− mice compared with that in wild-type mice. TAK-242 alleviated CHS and prevented neutrophil infiltration, cytokine expression, and ear thickening in Il36rn−/− mice. These data indicate that Il36rn−/− mutations are an exacerbating factor for CHS and that TAK-242 can reduce the inflammatory responses that are associated with the CHS response.

Effect of TAK-242 on CHS response. The wild-type mice showed an increase in neutrophil infiltration more than the Il36rn −/− mice, indicating that, acquired immunity plays a central role in the CHS response, but we speculated that the innate immune system plays a key role in enhancing the contact dermatitis response in Il36rn −/− mice. A previous study reported that TAK-242 administration suppressed neutrophil infiltration in an imiquimod-induced psoriasis model 8 , so we considered that suppressing the innate immune system and infiltration of neutrophils by suppressing downstream of TLR4 could suppress this CHS response even in the CHS model. Thus, we performed a treatment experiment using TAK-242, which, selectively inhibits TLR4.
We examined the effect of the TLR4 inhibitor, TAK-242, on the elevated CHS response observed in Il36rn −/− mice. TAK-242 (0.5, 5.0, or 10 mg/kg/day) or the same amount of vehicle was intraperitoneally administered for 6 days (day 0-5) before DNFB challenge and CHS response was assessed in the different mice lines (Fig. 3A).
Thus, administration of TAK-242 before DNFB challenge diminishes CHS responses in mice with or without IL-36Ra deficiency. Furthermore, TAK-242 showed a dose-dependent effect up to 5.0 mg/kg/day. However, there was no significant difference in the effect between TAK-242 doses of 5.0 mg/kg/day and 10 mg/kg/day.
CHS response and the number of inflammatory cells were similar in TAK-242-treated wild-type and Il36rn −/− mice. Thus, increased CHS response and inflammatory cell recruitment was completely inhibited by TAK-242 treatment, regardless of the genetic background.
TAK-242 treatment changes cytokine and chemokine mRNA expression in mouse ear tissue.

Discussion
The results of our study demonstrate that the CHS response is enhanced in Il36rn −/− mice.
The results of histopathological examination showed that the number of neutrophils and lymphocytes in the CHS lesion sites significantly increased in Il36rn −/− mice compared to that in wild-type mice. Moreover, expression of cytokines and chemokines in the CHS lesions revealed that IL-1β, IL-17A, TNF-α, CXCL1, CCL4, IL-36γ, IL-23p19, and EBI3 significantly increased in Il36rn −/− mice compared with that in wild-type mice. The treatment experiments with TAK-242, which has proven safe for human use 8,24 , showed a decrease in the CHS response in both wild-type and Il36rn −/− mice. Furthermore, it was revealed that in treated CHS lesion, the expression of cytokines and chemokines such as IL-1β, IL-4, IL-6, IL-10, IFN-α IL-17A, IL-12p40, CXCL1, and CXCL2 decreased in both wild-type and Il36rn −/− mice compared with levels in the vehicle control. However, there were no significant differences in the expression levels of IL-36α between vehicle control and treated group in both wild-type and Il36rn −/− mice, consistent with a previous report that IL-36α is not essential for induction of local inflammation during DNFB-induced CHS 25 . Collectively, the results of this study suggest that mutations in Il36rn enhance the CHS response by acting on various cytokines and chemokines involved in neutrophil migration, and that inhibiting TLR4 is likely to affect the production of these cytokines and chemokines.
The CHS response was enhanced in Il36rn −/− mice, and we considered that this is attributable to the increase in neutrophils. Actually, several recent studies have reported that neutrophils play an important role in both the sensitization and elicitation phases of contact dermatitis [21][22][23] . When there is a deficiency in IL-36Ra function, the signal transduction continues via IL-36R. Because IL-36R induces the production of inflammatory cytokines by activating NF-κB and MAPK 9,10 , IL-1β, TNF-α, and IL-36γ are increased in the CHS lesion and result in the sustained activation of the receptor. The increase in TNF-α upregulates the production of IL-17A from Th17 cells, which subsequently increases chemokines such as CXCL1 and CXCL2 [26][27][28][29] . In fact, it has been reported that IL-17A regulates epidermal keratinocytes to produce chemokines such as CXCL1 and CXCL2 to induce neutrophils. In addition, it has also been reported that IL-39, which is a hetero dimer of IL-23p19 and EBI3, is downstream of IL-36γ and involved in the induction of neutrophils 30 . When IL-36Ra is lost, neutrophil-induced pathways, triggered by increased chemokines and IL-39, are further amplified through a positive feedback loop, which is consistent with the increase in neutrophils which we observed in our histopathological investigations.
We considered that TAK-242 regulated both the sensitisation and elicitation phases of contact dermatitis by inhibiting the CHS response. In the sensitisation phase, the CHS response can be suppressed by inhibiting the activation of the innate immune system 31 . In this study, the gene expression of cytokines such as IL-6, IFN-γ, www.nature.com/scientificreports www.nature.com/scientificreports/ IL-4, IL-17, and IL-10 was reduced in the CHS lesions of both wild-type and Il36rn −/− mice after TAK-242 treatment. Other studies have shown that these cytokines are produced by macrophages and effector T cells 32,33 , and that TLR4 increases the sensitivity of contact dermatitis. TLR4 is expressed in several non-immune cells such as keratinocytes and sebocytes, and antigen-presenting cells such as macrophages and dendritic cells (DCs) [33][34][35] , and is required for the activation of the innate immune system, which is necessary for the sensitization of allergens 34,[36][37][38] . When the innate immune system is activated, the migration of dermal DCs to regional lymph nodes, which is the sensitisation phase of contact dermatitis, is initiated. Thus, if the sensitisation phase is inhibited by TAK-242, CHS is reduced in both wild-type and Il36rn −/− mice. www.nature.com/scientificreports www.nature.com/scientificreports/ In the induction phase of contact dermatitis, the CHS response can be inhibited primarily by suppressing the activation of both effector T cells and TNF and iNOS-producing DCs (Tip-DC) 31 . By suppressing the activation of effector T cells, the production of IL-17A in Th17 cells decreases, followed by a decrease in the production of www.nature.com/scientificreports www.nature.com/scientificreports/ CXCL1 and CXCL2 chemokines. In fact, the mRNA expression of IL-17A, CXCL1, and CXCL2 decreased in CHS lesions of both wild-type and Il36rn −/− mice in this experiment. Histopathologically, the number of neutrophils in tissues reduced, reflecting a decrease in the expression of CXCL1 and CXCL2.
Dysfunction of IL-36Ra upregulates inflammatory cytokines such as TNF-α. Subsequently, these cytokines regulate Tip-DCs and promote IL-17A infiltration. The decrease in IL-36R signalling can be explained by the decrease in the expression of IL-39 downstream of IL-36γ. We found that the expression of TNF-α in the CHS lesions of Il36rn −/− mice increased. Furthermore, the present study showed that a decrease, in TNF-α expression levels was observed only in Il36rn −/− mice treated with TAK-242. Moreover, it has been reported that overexpression of IL-36 cytokines in allergic contact dermatitis patients can induce Th17 cytokines 39 . From these reasons, we considered this to be one of the characteristics of Il36rn deficiency. Thus, we concluded that TAK-242 blocks TNF-α induction by inhibiting TLR4 expression on the cell surface of Tip-DCs and it suppresses effector T cell activation.
In summary, this study demonstrates that the activation and intensity of CHS response depend on the deficiency of IL-36Ra. Furthermore, we demonstrated that blocking TLR4 function with TAK-242 inhibits the CHS response in both Il36rn −/− and wild-type mice. The Il36rn mutation increased the CHS response by eliciting excessive infiltration of neutrophils into the skin, which was due to the activation of IL-36 receptor-mediated sustained inflammatory signalling. These results suggest that a deficiency in IL-36Ra intensifies the CHS response and that blocking TLR4 signals by TAK-242 is a promising therapeutic strategy for treating contact dermatitis. Mice. Gender matched female wild-type (C57BL/6NCr1) and Il36rn −/− mice (Aged 6-12w) were used for all experiments. Il36rn −/− mice were generated as previously reported 8 and genotypically confirmed by allele-specific PCR. Control C57BL/6NCr1 animals were obtained from Charles River Laboratories (Charles River Laboratories, Inc., Wilmington, Massachusetts, USA). All experiments were repeated thrice using healthy and fertile mice that did not display any evidence of infection or disease. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens.

Materials and Methods
Induction of contact hypersensitivity. The CHS mouse model was induced with DNFB (Wako Pure Chemicals, Tokyo, Japan) as previously reported 40 . Briefly, age-matched mice were sensitised with 25 µl 0.5% DNFB in acetone/olive oil (4:1) on a shaved back on day 0. On day 5, sensitised mice were topically challenged with 15 µl 0.2% DNFB in acetone/olive oil (4:1) on each side of both ears. Ear thickness was measured with dial thickness gauges (Peacock, Ozaki MFG. CO., Ltd, Chiba, Japan) before DNFB challenge and 24 h and 48 h after DNFB challenge. Each ear lobe was measured three times and the mean of those values was used for analysis.
Histological analysis of ear sections. Mice ears were harvested 48 h after DNFB challenge; central strips of the ears were fixed in 3.5% paraformaldehyde and embedded in paraffin. From these preparations, 6-µm paraffin sections were stained with haematoxylin and eosin (H&E) for conventional histological evaluation. Dermal neutrophil infiltration was evaluated by counting the number of neutrophils present in 12 high-power fields (0.07 mm 2 ). Each section was examined independently by two investigators in a blind study and the mean of their measurements was used for analysis.