Topical application of toll-like receptor 3 inhibitors ameliorates chronic allergic skin inflammation in mice

Atopic dermatitis (AD) is a chronic skin disorder, which is characterized by repeated cycles of exacerbation and remission of eczematous lesions and pruritus. Evidence suggests that skin barrier dysfunction and increased innate and adaptive immune responses cause AD pathology [ [1] Leung D.Y.M. Berdyshev E. Goleva E. Cutaneous barrier dysfunction in allergic diseases. J. Allergy Clin. Immunol. 2020; 145: 1485-1497 Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar ]. Toll-like receptors (TLRs) evoke and enhance immune responses by recognizing pathogen-specific molecular structures. TLR3 recognizes viral double-stranded RNA and polyinosinicpolycytidylic acid (poly(I:C)) and induces the production of type I interferons and the expression of proinflammatory cytokines [ [2] Tamagawa-Mineoka R. Ueta M. Katoh N. TLR3 and inflammatory skin disease: from environmental factors to molecular opportunities. in: Wondrak G.T. Skin Stress Response Pathways. Springer, Switzerland, Gewerbestrasse2016: 235-249 Crossref Scopus (5) Google Scholar ]. We previously found that TLR3 was strongly expressed in the epidermal cells of both healthy controls and AD patients [ [3] Nakamura N. Tamagawa-Mineoka R. Ueta M. Konishi E. Yasuike R. Masuda K. Matsunaka H. Murakami Y. Yokosawa E. Katoh N. Stratum corneum toll-like receptor 3 expressions correlate with the severity of atopic dermatitis lesions. J. Dermatol. Sci. 2019; 94: 354-357 Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar ]. In addition, higher stratum corneum TLR3 expression levels were detected in the affected skin of AD patients than in the unaffected skin of AD patients or healthy individuals [ [3] Nakamura N. Tamagawa-Mineoka R. Ueta M. Konishi E. Yasuike R. Masuda K. Matsunaka H. Murakami Y. Yokosawa E. Katoh N. Stratum corneum toll-like receptor 3 expressions correlate with the severity of atopic dermatitis lesions. J. Dermatol. Sci. 2019; 94: 354-357 Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar ]. Furthermore, using murine models we demonstrated that TLR3 signaling, especially that of epithelial cells, is closely associated with the pathomechanisms of allergic inflammation [ 4 Yasuike R. Tamagawa-Mineoka R. Ueta M. Nakamura N. Kinoshita S. Katoh N. The role of toll-like receptor 3 in chronic contact hypersensitivity induced by repeated, elicitation. J. Dermatol. Sci. 2017; 88: 184-191 Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar , 5 Nakamura N. Tamagawa-Mineoka R. Ueta M. Kinoshita S. Katoh N. Toll-like receptor 3 increase allergic and irritant contact dermatitis. J. Invest. Dermatol. 2015; 135: 411-417 Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar , 6 Ueta M. Uematsu S. Akisa S. Kinoshita S. Toll-like receptor 3 enhances late-phase reaction of experimental allergic conjunctivitis. J. Allergy Clin. Immunol. 2009; 123: 1187-1189 Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar ]. These findings imply that blocking TLR3 signaling might suppress allergic skin inflammation. Here, we examined the effects of the topical application of TLR3 inhibitors in a murine model of chronic allergic dermatitis, which exhibited similar features to those seen in AD patients.; We repeatedly applied a hapten, 2,4,6-trinitro-1-chlorobenzene (TNCB), to the skin of sensitized mice followed by one of two TLR3 inhibitors (50 μM), a commercially available TLR3 inhibitor (TLR3/dsRNA complex inhibitor, Calbiochem, Darmstadt, Germany) and a novel TLR3 inhibitor that was identified by screening a compound library, to determine the therapeutic effects of the TLR3 inhibitors (Fig. 1a). Addition of the latter TLR3 inhibitor reduces the levels of several inflammatory mediators, which were released from keratinocytes by stimulation with poly(IC), a TLR3 ligand (Supplementary Fig. 1). The preliminary experiments showed that topical application of TLR3 inhibitors (5 μM) every day or topical application of TLR3 inhibitors (50 μM) every other day did not significantly reduce skin inflammation by repeated application of TNCB (data not shown).; A histological examination of the ear skin of the mice was performed at 6 h after the challenge on day 8, the point when cutaneous responses peaked, as reported previously [ [4] Yasuike R. Tamagawa-Mineoka R. Ueta M. Nakamura N. Kinoshita S. Katoh N. The role of toll-like receptor 3 in chronic contact hypersensitivity induced by repeated, elicitation. J. Dermatol. Sci. 2017; 88: 184-191 Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar ]. TNCB-mediated ear swelling was attenuated by the topical application of either TLR3 inhibitor (Fig. 1b). A hyperplastic epidermis and marked leukocyte infiltration into the dermis were seen in the mice applied repeatedly with TNCB (Fig. 1c). Interestingly, in the mice treated with TLR3 inhibitors, both TLR3 inhibitors inhibited such skin inflammation (Fig. 1c). In addition, the topical TLR3 inhibitors significantly reduced the number of leukocytes that infiltrated into the inflamed skin (Fig. 1d). These findings suggest that the inhibition of TLR3 signaling effectively suppresses the development of cutaneous chronic inflammation.; Quantitative real-time PCR was performed to investigate the expression of cytokines associated with allergic skin inflammation. We examined the release of type-1 (interferon [IFN]-ɤ and (interleukin [IL]-2) and type-2 cytokines (IL-4, IL-10, and IL-33) in the whole ears of the mice at 6 h after the challenge on day 8. Repeated elicitation tended to increase the mRNA expression of IL-4 and IL-10 in the mice (Fig. 2). Lesional IL-4 expression was significantly lower in the mice that had been treated with a commercially available TLR3 inhibitor than in the vehicle-treated mice, while the IL-4 mRNA expression levels of the mice that had been treated with the TLR3 inhibitor we found were lower, but not significantly, than those of the vehicle-treated mice (Fig. 2). We examined the expressions of several chemokines (CCL2, CCL5, CCL20, and CXCL10) in the skin of the mice that had been treated with the TLR3 inhibitors and vehicle, but there was no significant difference of the mRNA expression levels (data not shown).; In this study, we demonstrated for the first time that TLR3 inhibitors markedly suppressed the development of the chronic allergic inflammation induced by repeated elicitation in mice. These findings suggest that inhibiting TLR3 signaling might contribute to the exacerbation of eczema, such that the associated dermatitis becomes chronic and refractory, as is seen in AD patients. Our previous study showed that TLR3 was expressed at higher levels in the epidermal cells of AD lesions than in non-lesion skin [ [3] Nakamura N. Tamagawa-Mineoka R. Ueta M. Konishi E. Yasuike R. Masuda K. Matsunaka H. Murakami Y. Yokosawa E. Katoh N. Stratum corneum toll-like receptor 3 expressions correlate with the severity of atopic dermatitis lesions. J. Dermatol. Sci. 2019; 94: 354-357 Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar ]. We also previously reported that TLR3-deficient mice exhibited lower levels of poly(IC) stimulation-induced chemokine production by keratinocytes and fibroblasts than wild-type mice [ [5] Nakamura N. Tamagawa-Mineoka R. Ueta M. Kinoshita S. Katoh N. Toll-like receptor 3 increase allergic and irritant contact dermatitis. J. Invest. Dermatol. 2015; 135: 411-417 Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar ]. On the other hand, there were no differences in the functions of dendritic cells, lymphocytes, macrophages, or mast cells between TLR3-deficient mice and wild-type mice [ [5] Nakamura N. Tamagawa-Mineoka R. Ueta M. Kinoshita S. Katoh N. Toll-like receptor 3 increase allergic and irritant contact dermatitis. J. Invest. Dermatol. 2015; 135: 411-417 Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar ]. Taken together, these findings suggest that TLR3 signaling by keratinocytes might participate in the development of chronic allergic inflammation. Thus, the topical application of TLR inhibitors to the epidermis could be effective against such symptoms.; TLRs recognize pathogen-associated molecular patterns and can also sense endogenous molecules that are released after cellular or tissue damage, which are known as danger-associated molecular patterns [ [7] Tsan M.F. Gao B. Endogenous ligands of toll-like receptors. J. Leukoc. Biol. 2004; 76: 514-519 Crossref PubMed Scopus (585) Google Scholar ]. Quantitative real-time PCR analyses showed that TLR3 mRNA expression in the epidermis of ear skin after the repeated application with TNCB was significantly elevated compared with that with vehicle (Supplementary Fig. 2). In our previous study, TLR3 expression in keratinocytes was upregulated after stimulation with poly(IC), but not after stimulation with Th2, Th17, or proinflammatory cytokines [ [3] Nakamura N. Tamagawa-Mineoka R. Ueta M. Konishi E. Yasuike R. Masuda K. Matsunaka H. Murakami Y. Yokosawa E. Katoh N. Stratum corneum toll-like receptor 3 expressions correlate with the severity of atopic dermatitis lesions. J. Dermatol. Sci. 2019; 94: 354-357 Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar ]. In addition, it was reported that TLR3 recognized RNA that was released from keratinocytes after UVB exposure and stimulated the production of inflammatory cytokines from keratinocytes [ [8] Bernard J.J. Cowing-Zitron C. Nakatsuji T. Muehleisen B. Muto J. Borkowski A.W. Martinez L. Greidinger E.L. Yu B.D. Gallom R.L. Ultraviolet radiation damages self noncoding RNA and is detected by TLR3. Nat. Med. 2012; 18: 1286-1290 Crossref PubMed Scopus (272) Google Scholar ]. These findings suggest that endogenous TLR3 ligands, such as the self-RNA released from cells that have been damaged by AD inflammation, might stimulate TLR3 in keratinocytes, leading to the exacerbation of AD.; Topical corticosteroids are one of the major drugs used to treat AD patients, but they are known to have adverse effects, e.g., they can cause skin atrophy and telangiectasia. Novel drugs that specifically act on particular molecular targets are expected to have less adverse effects than corticosteroids. It has been shown that TLRs are associated with the pathogeneses of several inflammatory diseases, including AD [ [9] Sun L. Liu W. Zhang L.J. The role of toll-like receptors in skin host defense, psoriasis, and atopic dermatitis. J. Immunol. Res. 2019; Crossref Scopus (25) Google Scholar ]. Our results could lead to the establishment of novel TLR suppression-based treatments for these inflammatory disorders based on the concept of innate immunity. Fig. 2The effects of topical TLR3 inhibitors on cytokine production in TNCB-induced chronic dermatitis. Quantitative real-time PCR analyses of IFN-ɤ, IL-2, IL-4, IL-10, and IL-33 mRNA expression in the whole ears of mice were performed at 6 h after the TNCB challenge on day 8 (n = 3 per group). Data are expressed as the mean ± SD. *P < 0.05. View Large Image Figure Viewer Download Hi-res image