J. Distler

TNF-induced structural joint damage is mediated by IL-1

Blocking TNF effectively inhibits inflammation and structural damage in human rheumatoid arthritis (RA). However, so far it is unclear whether the effect of TNF is a direct one or indirect on up-regulation of other mediators. IL-1 may be one of these candidates because it has a central role in animal models of arthritis, and inhibition of IL-1 is used as a therapy of human RA. We removed the effects of IL-1 from a TNF-mediated inflammatory joint disease by crossing IL-1α and β-deficient mice (IL-1−/−) with arthritic human TNF-transgenic (hTNFtg) mice. Development of synovial inflammation was almost unaffected on IL-1 deficiency, but bone erosion and osteoclast formation were significantly reduced in IL-1−/−hTNFtg mice, compared with hTNFtg mice based on an intrinsic differentiation defect of IL-1-deficient monocytes. Most dramatically, however, cartilage damage was absent in IL-1−/−hTNFtg mice. Chimera studies revealed that protection of cartilage is based on the loss of IL-1 on hematopoietic, but not mesenchymal, cells, leading to decreased expression of ADAMTS-5 and MMP-3. These data show that TNF-mediated cartilage damage is completely and TNF-mediated bone damage is partially dependent on IL-1, suggesting that IL-1 is a crucial mediator for inflammatory cartilage and bone degradation.

Association of the TNFAIP3 rs5029939 variant with systemic sclerosis in the European Caucasian population

Background TNFAIP3 encodes the ubiquitin-modifying enzyme, a key regulator of inflammatory signalling pathways. Convincing associations between TNFAIP3 variants and autoimmune diseases have been reported. Objective To investigate the association of TNFAIP3 polymorphisms with systemic sclerosis (SSc). Methods Three single nucleotide polymorphisms (SNPs) in a set of 1018 patients with SSc and 1012 controls of French Caucasian origin were genotyped. Two intergenic SNPs, rs10499194 and rs6920220, and one located in TNFAIP3 intron 2, rs5029939, were selected. The TNFAIP3 rs5029939 found to be associated with SSc in this first set was then genotyped in a second set of 465 patients with SSc and 182 controls from Germany and 184 patients with SSc and 124 controls from Italy. Pooled odd ratios were calculated by Mantel–Haenszel meta-analysis. Results The rs5029939 G allele was found to be significantly associated with SSc susceptibility (pooled OR=2.08 (95% CI 1.59 to 2.72); p=1.16×10−7), whereas the rs10499194 and rs6920220 variants displayed no association. Only one of the predicted haplotypes investigated in the French sample was significantly associated with SSc (p=8.91×10−8), and this haplotype was discriminating only in the presence of the rs5029939 risk allele, suggesting that this SNP tags the association signal. The strongest associations of rs5029939 with subphenotypes, having large magnitudes for complex genetic disorders, were observed for diffuse cutaneous SSc (pooled OR=2.71 (1.94 to 3.79), p=5.2×10−9), fibrosing alveolitis (pooled OR=2.26 (1.61 to 3.17), p=2.5×10−6) and pulmonary arterial hypertension (pooled OR=3.11 (1.86 to 5.17), p=1.3×10−5). Conclusion These results suggest that TNFAIP3 is a genetic susceptibility factor for SSc.

BANK1 is a genetic risk factor for diffuse cutaneous systemic sclerosis and has additive effects with IRF5 and STAT4

OBJECTIVE To determine whether the functional BANK1 variants rs3733197 and rs10516487 are associated with systemic sclerosis (SSc) in 2 European Caucasian populations and to investigate the putative gene-gene interactions between BANK1 and IRF5 as well as STAT4. METHODS BANK1 single-nucleotide polymorphisms were genotyped in a total population of 2,432 individuals. The French cohort consisted of 874 SSc patients and 955 controls (previously genotyped for both IRF5 rs2004640 and STAT4 rs7574865). The German cohort consisted of 421 SSc patients and 182 controls. RESULTS The BANK1 variants were found to be associated with diffuse cutaneous SSc (dcSSc) in both cohorts, providing an odds ratio (OR) of 0.77 for the rs10516487 T rare allele in the combined populations of dcSSc patients as compared with the combined populations of controls (95% confidence interval [95% CI] 0.64-0.93) and an OR of 0.73 (95% CI 0.61-0.87) for the rs3733197 A rare allele. BANK1 haplotype analysis found the A-T haplotype to be protective in dcSSc patients (OR 0.70 [95% CI 0.57-0.86], P = 3.39 x 10(-4)) and the G-C haplotype to be a risk factor (OR 1.25 [95% CI 1.06-1.47], P = 0.008). Significant differences were also observed when the limited cutaneous subset of SSc was compared with the dcSSc subset, both for the rare alleles and for the haplotypes. The BANK1, IRF5, and STAT4 risk alleles displayed a multiplicatively increased risk of dcSSc of 1.43-fold. CONCLUSION Our results establish BANK1 as a new SSc genetic susceptibility factor and show that BANK1, IRF5, and STAT4 act with additive effects.

Interleukin-1 is essential for systemic inflammatory bone loss

Objectives: Chronic inflammation is a major risk factor for systemic bone loss leading to osteoporotic fracture and substantial morbidity and mortality. Inflammatory cytokines, particularly tumour necrosis factor (TNF) and interleukin-1 (IL1), are thought to play a key role in the pathogenesis of inflammation-induced bone loss, but their exact roles are yet to be determined. Methods: To determine whether TNF directly triggers bone loss or requires IL1, human TNFα mice (hTNFtg) were crossed with mice lacking IL1α and IL1β (IL1−/−hTNFtg). Systemic bone architecture was evaluated using CT scanning, static and dynamic bone histomorphometry and serum markers of bone metabolism. Results: hTNFtg mice developed severe bone loss accompanied by a severe distortion of bone microarchitecture. Bone trabeculae were thinner and decreased in numbers, resulting in increased trabecular separation. Histomorphometric analyses revealed strongly increased bone resorption in hTNFtg mice compared with wild-type mice. In contrast, IL1−/−hTNFtg mice were fully protected from systemic bone loss despite still developing inflammation in their joints. Lack of IL1 completely reversed increased osteoclast formation and bone resorption in hTNFtg mice and the increased levels of RANKL in these mice. Structural parameters and osteoclast and osteoblast numbers were indistinguishable from wild-type mice. Conclusions: These data indicate that IL1 is essential for TNF-mediated bone loss. Despite TNF-mediated inflammatory arthritis, systemic bone is fully protected by the absence of IL1, which suggests that IL1 is an essential mediator of inflammatory osteopenia.

Animal models for arthritis: innovative tools for prevention and treatment

The development of novel treatments for rheumatoid arthritis (RA) requires the interplay between clinical observations and studies in animal models. Given the complex molecular pathogenesis and highly heterogeneous clinical picture of RA, there is an urgent need to dissect its multifactorial nature and to propose new strategies for preventive, early and curative treatments. Research on animal models has generated new knowledge on RA pathophysiology and aetiology and has provided highly successful paradigms for innovative drug development. Recent focus has shifted towards the discovery of novel biomarkers, with emphasis on presymptomatic and emerging stages of human RA, and towards addressing the pathophysiological mechanisms and subsequent efficacy of interventions that underlie different disease variants. Shifts in the current paradigms underlying RA pathogenesis have also led to increased demand for new (including humanised) animal models. There is therefore an urgent need to integrate the knowledge on human and animal models with the ultimate goal of creating a comprehensive ‘pathogenesis map’ that will guide alignment of existing and new animal models to the subset of disease they mimic. This requires full and standardised characterisation of all models at the genotypic, phenotypic and biomarker level, exploiting recent technological developments in ‘omics’ profiling and computational biology as well as state of the art bioimaging. Efficient integration and dissemination of information and resources as well as outreach to the public will be necessary to manage the plethora of data accumulated and to increase community awareness and support for innovative animal model research in rheumatology.

Tyrosine kinase inhibitors for the treatment of fibrotic diseases such as systemic sclerosis: towards molecular targeted therapies

Systemic sclerosis (SSc) is a fibrosing connective tissue disease with significantly increased mortality. Therapeutic options to treat fibrosis are limited. The small molecule tyrosine kinase inhibitor imatinib and related drugs such as dasatinib and nilotinib target simultaneously two of the major profibrotic pathways, TGFβ- and PDGF- signaling. Imatinib, dasatinib and nilotinib inhibit collagen synthesis in cultured fibroblasts and have potent anti-fibrotic effects in animal models of different fibrotic diseases. Moreover, several case reports, case series and uncontrolled studies on patients with SSc, mixed connective tissue disease, nephrogenic systemic fibrosis and in particular sclerodermatous graft versus host disease (cGvHD) report regression of fibrosis and good tolerability. However, the results of larger controlled trials, which are currently ongoing, are needed before any conclusions on efficacy and tolerability can be drawn. Until the results of these trials are available, we discourage off-label use of Imatinib in single patients.

Monocyte chemoattractant proteins in the pathogenesis of systemic sclerosis

Activation of the immune system and increased synthesis of extracellular matrix proteins by fibroblasts are hallmarks in the pathogenesis of SSc. The molecular mechanisms underlying the infiltration of inflammatory cells into the skin and the subsequent activation of fibroblasts are still largely unknown. Chemokines are a family of small molecules that are classified according to the position of the NH(2)-terminal cysteine motif. Recent data indicate that chemokines and in particular two members of the subfamily of monocyte chemoattractant proteins, MCP-1 (CCL-2) and MCP-3 (CCL-7), might be involved in the pathogenesis of SSc. MCP-1 and -3 are overexpressed by SSc fibroblasts and in skin lesions from SSc patients compared to healthy controls. MCP-1 and -3 are chemotactic for inflammatory cells and stimulate their migration into the skin. In addition to their pro-inflammatory effects, MCP-1 and -3 contribute to tissue fibrosis by activating the synthesis of extracellular matrix proteins in SSc fibroblasts. Therapeutic strategies targeting MCP-1 have revealed promising results in several animal models of SSc. Antagonists against the receptor CCR2 are currently tested in clinical trials of a variety of diseases and also represent interesting candidates for target-directed therapy in SSc.

Association of the CD226 Ser307 variant with systemic sclerosis: Evidence of a contribution of costimulation pathways in systemic sclerosis pathogenesis

OBJECTIVE The nonsynonymous polymorphism rs763361 of the CD226 gene, which encodes DNAX accessory molecule 1, which is involved in T cell costimulation pathways, has recently been identified as a genetic risk factor for autoimmunity. The purpose of this study was to test for association of the CD226 rs763361 polymorphism with systemic sclerosis (SSc) in European Caucasian populations. METHODS CD226 rs763361 was genotyped in 3,632 individuals, consisting of a discovery sample (991 SSc patients and 1,008 controls) and a replication sample (999 SSc patients and 634 controls). All study subjects were of European Caucasian origin. Expression of CD226 was assessed on peripheral blood mononuclear cells obtained from 21 healthy donors genotyped for CD226 rs763361. RESULTS The CD226 rs763361 T allele was found to be associated with SSc in both the discovery and the replication samples, showing the following results in the combined populations: odds ratio (OR) 1.22 (95% confidence interval [95% CI] 1.10-1.34), P = 5.69 × 10(-5) . The CD226 T allele was also associated with various SSc subsets, highlighting a potential contribution to disease severity. The most remarkable associations of the CD226 TT risk genotype were observed with the diffuse cutaneous SSc subtype, the anti-topoisomerase I antibody-positive, and SSc-related fibrosing alveolitis subsets: OR 1.86 (95% CI 1.42-2.43), P = 5.15 × 10(-6) , OR 1.82 (95% CI 1.38-2.40), P = 2.16 × 10(-5) , and OR 1.61 (95% CI 1.25-2.08), P = 2.73 × 10(-4) , respectively. CD226 expression was not significantly influenced by CD226 rs763361 genotypes whatever the T cell subtype investigated. CONCLUSION Our results establish CD226 as a new SSc genetic susceptibility factor underlying the contribution of costimulation pathways in the pathogenesis of SSc. Further work is nevertheless needed to define the causal variant at the CD226 locus as well as the functional consequences.

A synthetic PPAR-γ agonist triterpenoid ameliorates experimental fibrosis: PPAR-γ-independent suppression of fibrotic responses

Background Persistent fibroblast activation initiated by transforming growth factor β (TGF-β) is a fundamental event in the pathogenesis of systemic sclerosis, and its pharmacological inhibition represents a potential therapeutic strategy. The nuclear receptor, peroxisome proliferator-activated receptor γ (PPAR-γ), exerts potent fibrotic activity. The synthetic oleanane triterpenoid, 2-cyano-3,12-dioxo-olean-1,9-dien-28-oic acid (CDDO), is a PPAR-γ agonist with potential effects on TGF-β signalling and dermal fibrosis. Objective To examine the modulation of fibrogenesis by CDDO in explanted fibroblasts, skin organ cultures and murine models of scleroderma. Material and methods The effects of CDDO on experimental fibrosis induced by bleomycin injection or by overexpression of constitutively active type I TGF-β receptor (TgfbR1ca) were evaluated. Modulation of fibrotic gene expression was examined in human skin organ cultures. To delineate the mechanisms underlying the antifibrotic effects of CDDO, explanted skin fibroblasts cultured in two-dimensional monolayers or in three-dimensional full-thickness human skin equivalents were studied. Results CDDO significantly ameliorated dermal fibrosis in two complementary mouse models of scleroderma, as well as in human skin organ cultures and in three-dimensional human skin equivalents. In two-dimensional monolayer cultures of explanted normal fibroblasts, CDDO abrogated fibrogenic responses induced by TGF-β. These CDDO effects occurred via disruption of Smad-dependent transcription and were associated with inhibition of Akt activation. In scleroderma fibroblasts, CDDO attenuated the elevated synthesis of collagen. Remarkably, the in vitro antifibrotic effects of CDDO were independent of PPAR-γ. Conclusions The PPAR-γ agonist triterpenoid CDDO attenuates fibrogenesis by antagonistically targeting canonical TGF-β/Smad and Akt signalling in a PPAR-γ-independent manner. These findings identify this synthetic triterpenoid as a potential new therapy for the control of fibrosis.

Association of a KCNA5 gene polymorphism with systemic sclerosis–associated pulmonary arterial hypertension in the European Caucasian population

OBJECTIVE Pulmonary arterial hypertension (PAH) has emerged as a leading cause of death in systemic sclerosis (SSc). The genetic basis of PAH has been unraveled in recent years, with a major role played by transforming growth factor β receptors; however, some other candidate genes have also been advocated, including potassium voltage-gated channel, shaker-related subfamily, member 5 (KCNA5). We undertook this study to determine whether KCNA5 polymorphisms confer susceptibility to SSc and its vascular phenotype, including PAH. METHODS Four KCNA5 single-nucleotide polymorphisms (SNPs), rs10744676, rs1860420, rs3741930, and rs2284136, were genotyped in a discovery set of 638 SSc patients and 469 controls. In addition, rs10744676 was genotyped in an independent replication sample (938 SSc patients and 564 controls) and in a cohort of 168 patients with different PAH subtypes. RESULTS The KCNA5 rs10744676 variant was found to be associated with SSc in the discovery sample, with an odds ratio (OR) of 0.62 (95% confidence interval [95% CI] 0.48-0.79, adjusted P = 0.0003) in comparison with controls (C allele frequency 11.4% versus 17.2%). When subphenotypes were investigated, an association was found solely for PAH associated with SSc (OR 0.31 [95% CI 0.13-0.71], adjusted P = 0.04). The other KCNA5 SNPs tested were not associated with any SSc subset. The above association with PAH associated with SSc was replicated in the second set. In the combined population, rs10744676 was strongly associated with PAH associated with SSc in comparison with controls (OR 0.36 [95% CI 0.21-0.63], P = 0.0002). In the independent cohort of patients with PAH, after investigating PAH subtypes, only rs10744676 showed an association with PAH associated with SSc. CONCLUSION Our results provide the first evidence for an association between the KCNA5 rs10744676 variant and PAH associated with SSc.