Abstract

Introduction

Fibrosis synchronously affecting skin and internal organs is the defining hallmark of systemic sclerosis (SSc).^1,2 Multiple organ fibrosis in SSc has unknown pathogenesis and no effective treatments.^3,4 Myofibroblasts, the key effector cells of organ fibrosis, secrete collagens and profibrotic mediators including transforming growth factor-β (TGF-β) and interleukin 6 (IL6). ³ Recent findings demonstrate prominent JAK/STAT activation in SSc fibroblasts, SSc biopsies, and in models of disease.^5–7 Furthermore, genetic studies revealed strong association of STAT locus variants with SSc. ⁸ These observations suggest a critical role for pathogenic JAK/STAT signaling in SSc.

JAKs (Janus kinases) are non-receptor tyrosine kinases with central roles in cytokine and growth factor signaling.^9,10 Upon binding of IL6 and related cytokines to their surface receptors, JAK kinases become activated and phosphorylate tyrosine residues in the cytoplasmic region of the receptor.^9,10 STAT proteins that are also activated by JAKs dimerize and translocate into the nucleus, where they induce transcription of several target genes. The IL6/JAK/STAT signaling pathway is implicated in the pathogenesis of numerous inflammatory and autoimmune diseases, including rheumatoid arthritis, psoriasis, and inflammatory bowel disease. Furthermore, mutations in JAK and STAT genes cause a number of immunodeficiency syndromes, and polymorphisms in these genes are associated with autoimmune diseases. ⁹ IL6-targeted therapy in SSc appears to show modest and variable clinical benefit.^11,12 Selective inhibition of intracellular receptor–associated kinases using orally available small molecules is a highly promising novel therapeutic approach for SSc and fibrosis. Tofacitinib is a synthetic kinase inhibitor that primarily targets the activity of JAK1 and JAK3 and, to a lesser extent, JAK2.^13,14 Most importantly, tofacitinib is the first Jakinib approved for the treatment of autoimmune conditions, including rheumatoid arthritis and psoriasis. ¹⁵

The aim of this study was to explore the potential pathogenic role of JAK and STAT signaling in the context SSc pathology and the effect of the selective JAK inhibitor tofacitinib on experimental models of dermal and pulmonary fibrosis. Our results demonstrate evidence of increased IL6-JAK/STAT pathway activity in subsets of SSc skin and lung biopsies. Targeted pharmacological blockade of JAK/STAT signaling using tofacitinib prevented progression of experimental organ fibrosis in multiple distinct disease models. Taken together, our findings provide strong evidence supporting a pathogenic role of JAK/STAT signaling in SSc and suggest that drug repurposing using tofacitinib might be an attractive antifibrotic strategy for treating SSc.

Results

Initial studies sought to evaluate the IL6/JAK/STAT3 signaling axis in SSc skin biopsies. The Gene Set Enrichment Analysis (GSEA) method was used using software from Broad Institute (http://software.broadinstitute.org/gsea/index.jsp). The IL6/JAK/STAT3 signature consisting of 87 prior defined set of genes (http://software.broadinstitute.org/gsea/msigdb/cards/HALLMARK_IL6_JAK_STAT3_SIGNALING.html) was evaluated in a publicly available SSc transcriptome data set (GSE9285, GSE32413, GSE45485, and GSE59785). ¹⁶ Gene expression profiling showed elevated IL6/JAK/STAT3 signaling in skin biopsies from the previously defined inflammatory subset of diffuse cutaneous systemic sclerosis (dcSSc) compared to healthy controls (Figure 1(a)). The GSEA method further confirmed enrichment of the IL6/JAK/STAT3 signature in the inflammatory intrinsic subset (nominal p<0.005, false discovery rate (FDR) q<0.01, family-wise error rate (FWER) p<0.05). We next examined SSc skin biopsies for the tofacitinib gene signature, generated as described in the “Materials and methods” section. Unbiased gene expression profiling showed elevated tofacitinib gene signature in skin biopsies from patients mapping to the inflammatory intrinsic dcSSc subset compared to healthy controls (Figure 1(b)). In addition, the GSEA method showed enrichment of tofacitinib signature in the inflammatory subset of dcSSc (nominal p<0.01, FDR q<0.01, FWER p<0.005). Next, to examine the activation of JAK/STAT pathway in SSc, we measured levels of phosphor-JAK1, JAK2, JAK3, and STAT3 in skin biopsies from SSc patients and healthy controls. In the epidermis, we found comparable expression levels between control subjects and patients with SSc. In contrast, in the dermis, expression of pY-JAK1, pY-JAK2, pY-JAK3, and STAT3 was significantly elevated in SSc biopsies of interstitial cells compared with control biopsies (Figure 2). Patients with late-stage disease showed increased expression of p-JAK2 and p-STAT3 compared with those with early-stage disease (p-JAK2, p=0.022; p-STAT3, p=0.05). There was no association of JAK/STAT pathway activation with the skin score (p>0.05).

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Figure 1.

JAK/STAT signature is elevated in SSc skin biopsies. Expression microarray data sets (GSE9285, GSE32413, GSE45485, and GSE59785) were queried for IL6/JAK/STAT3 and tofacitinib signature as described in the “Materials and methods” section. (a) Left panel, IL6/JAK/STAT3 signature; right panel, Gene Set Enrichment Analysis (GSEA). (b) Left panel, tofacitinib signature; right panel, Gene Set Enrichment Analysis (GSEA).

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Figure 2.

Phospho-JAK and phosphor-STAT levels elevated in SSc skin. Skin and lung tissues from patients with SSc were analyzed by immunohistochemistry (IHC) microscopy using antibodies to pY-JAK1, pY-JAK2, pY-JAK3, and pY-STAT3. Skin biopsies from patients with SSc and healthy controls were immunostained with indicated antibodies. Left panels, Representative images; dotted lines indicate the dermal–epidermal junction. Scale bar, 10μm. Right panels, Dot plots of positive cell population. The proportion of immunopositive cells was determined from 5hpf/section in each biopsy. The p values shown are results of the Mann–Whitney U test.

Pulmonary fibrosis is a leading complication of SSc. ¹⁷ To explore JAK/STAT activation in SSc-ILD, we measured pY-JAK1, pY-JAK2, pY-JAK3 and STAT3 in lung biopsies from SSc-ILD (n=7) and control lung biopsies (n=4). Non-SSc donor lungs showed only a low level of pY-JAK1, pY-JAK2, pY-JAK3, and STAT3 expressions by immunofluorescence (Figure 3). In these biopsies, pY-JAK1, pY-JAK2, pY-JAK3, and pY-STAT3 expressions were largely restricted to alveolar epithelial lining cells and scattered intra-alveolar macrophages. In marked contrast, strong intracellular pY-JAK1, pY-JAK2, pY-JAK3, and pY-STAT3 expression was noted in each SSc-ILD biopsy. JAK/STAT activation was most prominent at fibrotic loci (Figure 3).

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Figure 3.

Elevated phospho-JAK/STAT3 signaling in SSc lungs. Lung biopsies from patients with SSc-ILD (n=7) and controls (n=4) were immunostained using antibodies to pY-JAK1, pY-JAK2, pY-JAK3, and pY-STAT3. Nuclei were identified by DAPI (blue color). Left panels, Representative confocal microscopic images; scale bar, 25μm. Right panels, Dot plots of positive cell population. The proportion of immunopositive cells was determined from 5 hpf/section in each biopsy. The p values shown are results of the Mann–Whitney U test.

We next sought to investigate whether activated JAK/STAT signaling contributes to pathology in SSc. For this purpose, we evaluated the antifibrotic effects of tofacitinib on preclinical disease models. Eight-week-old C57BL/6J mice were given daily SC (subcutaneous) injections of bleomycin for 2weeks (5days/week), along with concurrent tofacitinib (30mg/kg, bid), a dose selected based on our preliminary experiment or vehicle by daily IP (intraperitoneal) injections (5days/week). Mice were sacrificed at day 22, and lung and lesional skin was harvested. Tofacitinib was well tolerated with no significant weight loss or other signs of toxicity noted. The thickness of the dermis and collagen deposition was markedly increased in bleomycin-treated mice (Figure 4). Both collagen and dermal thickness were markedly reduced when tofacitinib was administered concomitantly with bleomycin. Loss of the SC adipose layer accompanying dermal fibrosis was substantially attenuated in mice treated with tofacitinib (Figure 4(a)). Furthermore, increased expression of the fibrotic markers Col1a1 and Col1a2 and the number of alpha-smooth muscle actin (ASMA)-positive interstitial myofibroblasts, as well as infiltration with macrophages, were all significantly reduced in the skin in tofacitinib-treated mice (Figure 4(b) and (​(c)c) and Supplemental Figure 2A). The percent of bleomycin-induced increase in F4/80-positive macrophages was significantly reduced in tofacitinib-treated mice (Supplemental Figure 2). Dermal fibrosis was associated with a sharp increase in STAT3 phosphorylation in interstitial cells, which was markedly attenuated when mice were treated with tofacitinib (Figure 4(d)). Next, to determine the effect of JAK/STAT inhibition on fibrosis regression, tofacitinib treatment was started at day 8 when dermal fibrosis is already initiated. The results indicated no significant reduction in skin fibrosis under this condition (Supplemental Figure 3A).

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Figure 4.

Tofacitinib treatment prevents skin fibrosis. C57/BL6 mice received daily SC injections of PBS or bleomycin alone, or together with tofacitinib (30mg/kg, IP) or vehicle. Mice were sacrificed at day 22, and skin was harvested for analysis. (a) Left panel, H&E stain. Representative images. Bar=50μm. Right panel, Dermal thickness (means±SD of eight determinations/hpf from five mice/group). One-way analysis of variance followed by Sidak’s multiple comparison test. (b) Real-time quantitative PCR. Results, normalized with GAPDH, are means±SD of triplicate determinations from three or four mice/group; one-way analysis of variance followed by Sidak’s multiple comparison test. Immunohistochemistry using antibodies to ASMA, F4/80, and p-STAT3. (c) Immunohistochemistry using antibodies to ASMA. Representative images. Bar=25μm. (d) pY-STAT3 IHC. Representative images. Bar=50μm.

In order to evaluate the effect of selective JAK inhibition on an inflammation-independent skin fibrosis model, we treated TSK1/+ mice with tofacitinib or vehicle in parallel starting at 5weeks of age, when skin fibrosis is already apparent. ¹⁸ At 10weeks, vehicle-treated TSK1/+ mice displayed a substantial increase in hypodermal thickness (Figure 5(a)). In contrast, TSK1/+ mice that received tofacitinib showed significantly attenuated hypodermal thickening and STAT3 phosphorylation (Figure 5(b)).

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Figure 5.

Tofacitinib treatment prevents fibrosis in Tsk1/+ mice. Five-week-old Tsk1/+ mice received tofacitinib (IP; 30mg/kg) daily for 5weeks. Mice were sacrificed at 10weeks of age, and dorsal skin was harvested for analysis. (a) Left panel, H&E stain (dotted line indicates hypodermis). Right panel, Hypodermal thickness (means±SD of eight determinations/hpf from eight or nine mice/group). Representative images. Bar=200μm. Student’s t test. (b) Immunohistochemistry using antibodies to pY-STAT3. Representative images. Bar=50μm.

In view of the pronounced activation of the JAK/STAT pathway seen in SSc-ILD (Figure 3), subsequent experiments sought to explore the effect of tofacitinib on lung fibrosis. Chronic SC bleomycin elicited prominent architectural changes in the lungs, with an influx of inflammatory cells accompanied by appearance of fibrotic foci primarily in the subpleural area, along with sparse perivascular and interstitial fibrosis (Figure 6). These changes were associated with substantial collagen accumulation, increase in the fibrosis score, and elevated ASMA, F4/80, and CD3 expressions (Figure 6). Each of these parameters showed substantial attenuation in mice with concurrent tofacitinib treatment (Figure 6). Moreover, tofacitinib effectively reduced the sharp increase in pulmonary STAT3 phosphorylation (Figure 6(c)). We confirmed the antifibrotic effect of tofacitinib on IL6-treated explanted skin fibroblasts. Incubation with IL6 induced stimulation of type I collagen and ASMA levels, and STAT3 phosphorylation (Supplemental Figure 1). Pretreatment of the cultures with tofacitinib completely abrogated STAT3 phosphorylation at both early and late time-points and reduced the levels of type I collagen and ASMA at late time-point. However, no significant effect on regression of lung fibrosis was observed when tofacitinib treatment started at day 8 and continued until harvest at day 29 (Supplemental Figure 3B).

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Figure 6.

Tofacitinib treatment prevents lung fibrosis. C57/BL6 mice received daily SC injections of PBS or bleomycin alone, or together with tofacitinib (30mg/kg) or vehicle. Mice were sacrificed at day 22, and lungs were harvested. (a) Left panel, Masson’s Trichome stain. Representative images. Bar=25μm. Right panel, Ashcroft score. Results are means±SD from 5hpf per mice from at least three mice per experimental group. One-way analysis of variance followed by Sidak’s multiple comparison test. (b) Real-time quantitative PCR. Results, normalized with GAPDH, are means±SD of triplicate determinations from three or four mice/group; one-way analysis of variance followed by Sidak’s multiple comparison test. (c) Upper panels, immunofluorescence using antibodies to ASMA, ProcolI, CD3, and F4/80 and immunohistochemistry using pY-STAT3. Representative images. Bar=50 or 25μm as indicated. For ASMA, percent of relative fluorescence intensities (means from four randomly selected fields/sample). For CD3, F4/80, and pY-STAT3, the percent of immunopositive cells was determined from 5hpf/section. One-way analysis of variance followed by Sidak’s multiple comparison test.

Discussion

The pathogenesis of SSc remains incompletely understood, and the disease is highly variable in its clinical presentation and course.^1,19 Genome-wide molecular profiling has identified four “intrinsic” molecular SSc subsets called the fibroproliferative, inflammatory, limited, and normal-like subsets. ²⁰ Each of these molecular subsets shows deregulation of distinct signaling pathways; however, the full set of pathways contributing to this differential gene expression has not been fully elucidated. ¹⁶ Recent studies have called attention to the presence of prominent JAK/STAT activation in SSc biopsies and in models of disease.^5–7 Our analysis of SSc transcriptomes showed that skin biopsies mapping to the inflammatory intrinsic gene subset, accounting for approximately 40% of all biopsies, have significantly elevated levels of a consensus IL6/JAK/STAT3 signature which was derived on the basis based on experimentally derived gene expression. Moreover, immunohistochemistry similarly revealed the presence of activated JAKs and of activated STAT3 in SSc skin and lung biopsies. In addition, we found evidence of a repressed tofacitinib signature in these skin biopsies. Based on these combined transcriptome and protein data, we conclude that the disease process in particular subsets of SSc patients is directly driven by activation of the JAK/STAT3 pathway, and that these patients might show a favorable response to therapeutic interventions aimed at inhibiting the pathway, with normalization of aberrant gene signatures and resolution of tissue fibrosis.

A number of other JAK inhibitors are currently under development or in phase II and III clinical trials for the treatment of a variety of autoimmune inflammatory diseases. ²¹ Tofacitinib, which shows some selectivity for JAK1 and JAK3, is the first JAK inhibitor approved by the Food and Drug Administration (FDA) for the treatment of rheumatoid arthritis and psoriasis.^15,22,23 The efficacy of tofacitinib for the treatment of fibrotic processes remains unknown. In contrast, previous studies have provided evidence that inhibiting JAKs and STAT3 might have antifibrotic effect. In particular, previous studies have shown that JAK/STAT inhibitor mitigates fibrosis in experimental models of skin and lung fibrosis.^5–7 Therefore, drug repurposing using FDA-approved tofacitinib to selectively target JAK/STAT signaling is an attractive therapeutic antifibrotic strategy for SSc patients demonstrating evidence of activated JAK/STAT signaling in the skin. In the present studies, treatment with tofacitinib exerted potent antifibrotic effects on both bleomycin-induced skin fibrosis that recapitulate the inflammatory stage of SSc in fibrotic skin ²⁴ and on Tsk1/+ mice that phenocopy non-inflammatory SSc skin fibrosis. ²⁴ The beneficial effects of tofacitinib on bleomycin-induced fibrosis were restricted to preventive, and not therapeutic, application. This suggests that JAK/STAT3 signaling is being consistent with the recent demonstration that in established fibrosis, matrix stiffness itself is sufficient to drive STAT3 nuclear localization and activation independent of exogenous soluble ligands prompted reconsideration of the potential role of STAT3 in persistent myofiboblast mechanoactivation, ²⁵ and STAT3 activity is essential for transcription of TGF-β-induced COL1A2 in fibroblast. ²⁶

Together, our results indicate that tofacitinib, by selectively targeting JAK/STAT3 signaling, prevented organ fibrosis in preclinical disease models and abrogated fibrotic responses in normal fibroblasts in vitro. These studies can facilitate the development of predictive biomarkers for identifying SSc patients likely to show responses to JAK/STAT3 inhibition treatment and as pharmacodynamics markers to evaluate molecular responses in sequential skin biopsies from patients undergoing inhibitor treatment.

Materials and methods

Cell culture and reagent

Skin and lung biopsies were performed after obtaining written informed consent and in accordance with protocols approved by the Institutional Review Board for Human Studies at Northwestern University, University of Pittsburgh School of Medicine. Information of patients is shown in Table 1. Primary cultures of fibroblasts were established by explantation from neonatal foreskin. ¹⁸ Low-passage fibroblasts were grown in monolayers in plastic dishes and studied at early confluence. Cultures were maintained in Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, NY), 1% vitamin solutions, and 2-mM l-glutamine. All other tissue culture reagents were from Lonza (Basel, Switzerland). For experiments, cultures were placed in serum-free media containing 0.1% bovine serum albumin overnight, followed by tofacitinib (Selleck Chemical, 1µM) for indicated periods. Tofacitinib was added 60min prior to IL6 (R&D, 100ng/mL).

Table 1.

Clinical characteristics of subjects.

Study code	Age	Sex	Race	Disease subtype	Early/late
Subject information for p-JAK1 and p-JAK3 IHC
SPARC_SSc_02	31	F	W	dcSSc	Late
SPARC_SSc_06	70	M	W	dcSSc	Early
SPARC_SSc_07	50	F	W	dcSSc	Early
SPARC_SSc_09	30	F	W	lcSSc	Late
SPARC_SSc_10	41	F	W	dcSSc	Late
SPARC_SSc_13	45	F	W	dcSSc	Late
SPARC_SSc_19	36	F	W	dcSSc	Early
SPARC_SSc_20	46	F	W	VEDOSS	Early
SPARC_SSc_21	51	M	W	lcSSc	Late
SPARC_SSc_22	51	F	W	lcSSc	Late
SPARC_SSc_24	69	M	W	dcSSc	Late
SPARC_SSc_29	51	F	W	dcSSc	Late
SPARC_SSc_30	48	F	W	lcSSc	Late
SPARC_SSc_32	42	F	W	lcSSc	Late
SPARC_SSc_33	49	M	W	dcSSc	Early
SPARC_SSc_34	64	M	W	dcSSc	Early
SPARC_SSc_35	62	M	W	dcSSc	Early
SPARC_SSc_36	49	F	W	VEDOSS	Early
SPARC_SSc_38	51	M	W	dcSSc	Late
Subject information for p-JAK2 and p-STAT3 IHC
SPARC_SSc_01	60	F	W	dcSSc	Late
SPARC_SSc_02	31	F	W	dcSSc	Late
SPARC_SSc_03	51	F	W	dcSSc	Late
SPARC_SSc_05	60	M	W	lcSSc	Late
SPARC_SSc_07	50	F	W	dcSSc	Early
SPARC_SSc_08	59	F	B	dcSSc	Late
SPARC_SSc_10	40	F	W	dcSSc	Late
SPARC_SSc_11	64	F	W	dcSSc	Early
SPARC_SSc_12	45	F	W	dcSSc	Late
SPARC_SSc_13	44	F	W	dcSSc	Late
SPARC_SSc_14	39	F	A	dcSSc	Early
SPARC_SSc_15	30	M	W	lcSSc	Early
SPARC_SSc_16	45	F	B	dcSSc	Late
SPARC_SSc_17	63	F	W	dcSSc	Early
SPARC_SSc_19	36	F	W	dcSSc	Early
SPARC_SSc_23	37	M	W	dcSSc	Early
SPARC_SSc_24	69	M	W	dcSSc	Early
SPARC_SSc_26	45	F	W	dcSSc	Late
SPARC_SSc_29	50	F	W	dcSSc	Early
SPARC_SSc_31	60	F	W	dcSSc	Early

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JAK: Janus kinases; IHC: immunohistochemistry; SSc: systemic sclerosis; VEDOSS: very early diagnosis of systemic sclerosis; dcSSc: diffuse cutaneous systemic sclerosis; lcSSc: limited cutaneous systemic sclerosis.

Subjects providing skin biopsy samples for IHC. Early, <3years from the first non-Raynaud disease manifestation; late, >3years from the first non-Raynaud manifestation. Controls were healthy adults (70% female; median age: 32years; range: 26–57years).

Supplemental Material

Acknowledgments

Footnotes

References