Ruxolitinib Attenuates Cutaneous Lupus Development in a Mouse Lupus Model

discoid lupus erythematosus Food and Drug Administration systemic lupus erythematosus signal transducer and activator of transcription TO THE EDITOR Systemic lupus erythematosus (SLE) is an autoimmune disease that is classically associated with fatigue, fever, joint pains, and skin involvement, although any organ system can be affected. Almost all patients with SLE experience cutaneous manifestations at some point in their disease course, and an additional population of patients exists who experience cutaneous lupus but do not meet the criteria for SLE. Discoid lupus erythematosus (DLE), a common lupus-specific skin manifestation, is often a source of disfigurement and scarring alopecia. Currently, there are no Food and Drug Administration (FDA)–approved therapies for DLE. Treatment for DLE is empiric; DLE is treated with broad-spectrum immunosuppressants that have the potential for deleterious side effects and often require frequent monitoring. There is therefore an unmet need for effective treatments for DLE with favorable side-effect profiles (Jessop et al., 2000Jessop S. Whitelaw D. Jordaan F. Drugs for discoid lupus erythematosus.Cochrane Database Syst Rev. 2000; 4: CD002954Google Scholar). The IFN signature has been described in both SLE and in cutaneous lupus, inviting studies targeting this pathway for therapeutic purposes. Given the significantly increased IFN signature in human DLE lesions (Jabbari et al., 2014Jabbari A. Suárez-Fariñas M. Fuentes-Duculan J. et al.Dominant Th1 and minimal Th17 skewing in discoid lupus revealed by transcriptomic comparison with psoriasis.J Invest Dermatol. 2014; 134: 87-95Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), we hypothesized that blocking proximal signaling molecules downstream of the type I and II IFN receptors may attenuate the disease. Ligation of these receptors results in the activation of JAK1 and TYK2 or the activation of both JAK1 and JAK2 (for the type I IFN receptor or the type II IFN receptor, respectively) (Platanias, 2005Platanias L.C. Mechanisms of type-I- and type-II-interferon-mediated signalling.Nat Rev Immunol. 2005; 5: 375-386Crossref PubMed Scopus (2318) Google Scholar). Ruxolitinib is a small-molecule tyrosine kinase inhibitor with relative specificity for JAK1 and JAK2, approved in 2012 for the treatment of myelofibrosis (Harrison et al., 2012Harrison C. Kiladjian J-J Al-Ali H.K. et al.JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis.N Engl J Med. 2012; 366: 787-798Crossref PubMed Scopus (1336) Google Scholar; Verstovsek et al., 2012Verstovsek S. Mesa R.A. Gotlib J. et al.A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis.N Engl J Med. 2012; 366: 799-807Crossref PubMed Scopus (1475) Google Scholar). Ruxolitinib was administered by oral gavage to female MRL/lpr mice, prone to spontaneously develop cutaneous and systemic lupus, before development of severe skin involvement. This treatment indeed attenuated the development of severe skin lesions by the MRL/lpr mice (Figure 1a). Ruxolitinib-treated mice exhibited significantly reduced lesion severity scores by week 4 of treatment compared with control treatment (Figure 1b). Histopathologic analysis showed a significant reduction in epidermal hyperplasia and inflammatory infiltrate with ruxolitinib treatment (Figure 1c and d). The MRL/lpr mouse model system also exhibits other manifestations of SLE including autoantibody production, renal disease, and immune complex deposition. Assessments of renal endocapillary proliferation, crescent formation, and interstitial inflammation did not identify significant differences between ruxolitinib and vehicle control–treated mice (Figure 1e). Immune complex deposition in kidneys was also unchanged with ruxolitinib treatment as assessed by staining for IgG and C3 (Figure 1e). Examinations of other lupus manifestations did not detect any change with ruxolitinib treatment. Autoantibody levels (Supplementary Figure S1 online), lymphadenopathy, and splenomegaly (Supplementary Table S1 online) showed no consistent differences between groups. In sum, only the skin involvement was significantly alleviated in mice that received ruxolitinib compared with those that received vehicle control. Download .pdf (.08 MB) Help with pdf files Supplementary Material Prevention of the development of cutaneous manifestations of lupus with continued progression of other disease manifestations was surprising. One hypothesis that may account for this difference pertains to the timing of drug administration. Ruxolitinib treatment was started when the animals started to develop the first signs of skin disease and, at this point, had already exhibited appreciable gross lymphadenopathy, renal disease, and autoantibody formation. Ruxolitinib administration may have been able to curtail these extracutaneous markers of end-organ damage if it had been initiated at an earlier age. Alternatively, a distinct pathogenic mechanism may be responsible for the cutaneous lupus as opposed to lupus nephritis. Indeed, it seems likely that IL-17 is a contributor to the pathogenesis of lupus nephritis (Crispín et al., 2008Crispín J.C. Oukka M. Bayliss G. et al.Expanded double negative T cells in patients with systemic lupus erythematosus produce IL-17 and infiltrate the kidneys.J Immunol. 2008; 181: 8761-8766Crossref PubMed Scopus (580) Google Scholar; Zhang et al., 2009Zhang Z. Kyttaris V.C. Tsokos G.C. The role of IL-23/IL-17 axis in lupus nephritis.J Immunol. 2009; 183: 3160-3169Crossref PubMed Scopus (246) Google Scholar), whereas it is likely playing a very limited role in discoid lupus (Jabbari et al., 2014Jabbari A. Suárez-Fariñas M. Fuentes-Duculan J. et al.Dominant Th1 and minimal Th17 skewing in discoid lupus revealed by transcriptomic comparison with psoriasis.J Invest Dermatol. 2014; 134: 87-95Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Further studies will therefore be required to distinguish these possibilities. In order to examine the effects of ruxolitinib on the IFN response, MRL/lpr splenocytes were cultured with IFN-α and -γ along with graded concentrations of ruxolitinib. After 6 hours of co-culture, expression of IFN signature genes was assessed (Figure 2 and Supplementary Figure S2 online). Ruxolitinib diminished the expression of IFN response genes (Figure 2a). In particular, the T-cell chemokines Cxcl9 and Cxcl10, both of which exhibited relatively high upregulation in response to IFNs, were abrogated in expression in the presence of ruxolitinib. Ruxolitinib therefore decreases the production of chemotactic signals for T cells. An assessment of the T-cell infiltrate was performed by counting immunofluorescently stained CD3+, CD4+, and CD8+ cells (Figure 2b and c). The skin from ruxolitinib- versus vehicle-treated mice had significantly fewer CD3+ cells and CD4+ cells. CD8 T cells were a minor part of the immune infiltrate, but the cumulative data did show differences between these groups. These results indicate that ruxolitinib was diminishing the immune infiltrate in cutaneous lupus lesions, likely in part by diminishing the expression of T-cell chemotactic genes. In sum, the use of a small-molecule inhibitor of JAK1 and JAK2, the proximal signaling molecules that transduce the type I and II IFN signal, attenuated the development of cutaneous manifestations in a mouse model of lupus. Interestingly, other manifestations of SLE were not significantly affected, and a striking specificity for alleviating the development of skin lesions was observed. Previously published data examining the effects of ablating type I and II IFN signaling are mixed in their effects on SLE manifestations (Crow, 2014Crow M.K. Type I interferon in the pathogenesis of lupus.J Immunol. 2014; 192: 5459-5468Crossref PubMed Scopus (336) Google Scholar), although skin manifestations have not always been well described in prior studies. Depending on the mouse model used, genetic deficiency of the type I IFN receptor either alleviated (Braun and Demengeot, 2003Braun D. Demengeot J. Type I Interferon controls the onset and severity of autoimmune manifestations in lpr mice.J Autoimmun. 2003; 20: 15-25Crossref PubMed Scopus (157) Google Scholar; Santiago-Raber et al., 2003Santiago-Raber M.L. Baccala R. Haraldsson K.M. et al.Type-I interferon receptor deficiency reduces lupus-like disease in NZB mice.J Exp Med. 2003; 197: 777-788Crossref PubMed Scopus (454) Google Scholar) or exacerbated (Hron and Peng, 2004Hron J.D. Peng S.L. Type I IFN protects against murine lupus.J Immunol. 2004; 173: 2134-2142Crossref PubMed Scopus (174) Google Scholar) SLE manifestations. Type II IFN signaling attenuation, either by genetic ablation (Peng et al., 1997Peng S.L. Mosiehi J. Craft J. Roles of interferon-gamma and interleukin-4 in murine lupus.J Clin Invest. 1997; 99: 1936-1946Crossref PubMed Scopus (277) Google Scholar; Hron and Peng, 2004Hron J.D. Peng S.L. Type I IFN protects against murine lupus.J Immunol. 2004; 173: 2134-2142Crossref PubMed Scopus (174) Google Scholar) or by blocking antibodies (Jacob et al., 1987Jacob C.O. van der Meide P.H. McDevitt H.O. In vivo treatment of (NZB X NZW)F1 lupus-like nephritis with monoclonal antibody to gamma interferon.J Exp Med. 1987; 166: 798-803Crossref PubMed Scopus (361) Google Scholar), delayed or prevented SLE manifestations and, in one study, seemed to mitigate the exacerbated phenotype seen due to type I IFN receptor ablation (Hron and Peng, 2004Hron J.D. Peng S.L. Type I IFN protects against murine lupus.J Immunol. 2004; 173: 2134-2142Crossref PubMed Scopus (174) Google Scholar). Much like this last study, we show that the net effect of inhibiting both pathways with ruxolitinib resulted in a favorable response in a lupus end organ, in this case the skin. Furthermore, the use of a small-molecule inhibitor offers advantages not currently possible for antibody treatments including adaptation into a topical form (Fridman et al., 2011Fridman J.S. Scherle P.A. Collins R. et al.Preclinical evaluation of local JAK1 and JAK2 inhibition in cutaneous inflammation.J Invest Dermatol. 2011; 131: 1838-1844Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). A double-blind clinical trial testing the efficacy of a topical form of ruxolitinib has been shown to have some efficacy in treating lesions of psoriasis (Punwani et al., 2012Punwani N. Scherle P. Flores R. et al.Preliminary clinical activity of a topical JAK1/2 inhibitor in the treatment of psoriasis.J Am Acad Dermatol. 2012; 67: 658-664Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), an autoimmune skin disease in which IFNs contribute to its pathogenesis (Lowes et al., 2014Lowes M.A. Suárez-Fariñas M. Krueger J.G. Immunology of psoriasis.Annu Rev Immunol. 2014; 32: 227-255Crossref PubMed Scopus (973) Google Scholar). The MRL/lpr mouse model is notable for its spontaneous development of cutaneous manifestations of disease (Ghoreishi and Dutz, 2010Ghoreishi M. Dutz J.P. Cutaneous lupus erythematosus: recent lessons from animal models.Lupus. 2010; 19: 1029-1035Crossref PubMed Scopus (14) Google Scholar). Although the MRL/lpr model exhibits many of the features of human cutaneous lupus (Furukawa et al., 1984Furukawa F. Tanaka H. Sekita K. et al.Dermatopathological studies on skin lesions of MRL mice.Arch Dermatol Res. 1984; 276: 186-194Crossref PubMed Scopus (105) Google Scholar; Kanauchi et al., 1991Kanauchi H. Furukawa F. Imamura S. Characterization of cutaneous infiltrates in MRL/1pr mice monitored from onset to the full development of lupus erythematosus-like skin lesions.J Invest Dermatol. 1991; 96: 478-483Abstract Full Text PDF PubMed Google Scholar; Furukawa, 1997Furukawa F. Animal models of cutaneous lupus erythematosus and lupus erythematosus photosensitivity.Lupus. 1997; 6: 193-202Crossref PubMed Scopus (26) Google Scholar), not all aspects of the human condition are replicated, most notable of which may be the lack of an interface dermatitis as a predominant histological feature in the MRL/lpr model. Further investigations will therefore be required before large clinical trials addressing the efficacy of ruxolitinib in human cutaneous lupus. The findings here show that JAK inhibition prevents the development of cutaneous lupus, supporting an important role of JAK signaling in cutaneous lupus pathogenesis with a seemingly diminished role in the pathogenesis of dysfunction in other lupus end organs. Greater elucidation of the role of IFN in cutaneous lupus and DLE development, as well as the role of JAK/signal transducer and activator of transcription (STAT) inhibition, has the potential to identify new treatments for human DLE, and our findings identify JAK1/2 inhibition as a therapeutic strategy worthy of further studies. The Materials and Methods are documented in the Supplementary Data online. The institutional animal care and use committee at the Columbia University Medical Center approved all described studies. We thank Emily Chang and Ming Zhang for excellent technical support and Ryan Langlois for helpful discussions. AJ was supported by a Physician-Scientist Career Development Award from the Dermatology Foundation and also by the Louis V. Gerstner, Jr Scholars Program. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid Correction to: “Journal of Investigative Dermatology” advance online publication, 9 April 2015; doi:10.1038/jid.2015.107Journal of Investigative DermatologyVol. 135Issue 9PreviewRuxolitinib Attenuates Cutaneous Lupus Development in a Mouse Lupus Model Full-Text PDF Open Archive