Background: Serum fibrosis markers for systemic sclerosis (SSc) remain limited. The Enhanced Liver Fibrosis (ELF) score is a collagen marker set consisting of procollagen type III amino terminal propeptide (PIIINP), tissue inhibitor of metalloproteinases 1 (TIMP-1), and hyaluronic acid (HA). This longitudinal study aimed to examine the performance of the ELF score and its single analytes as surrogate outcome measures of fibrosis in SSc.Methods: Eighty-five SSc patients fulfilling the 2013 ACR/EULAR criteria with the absence of chronic liver diseases were enrolled. Serum PIIINP, TIMP-1, HA, and the ELF score were measured and correlated with clinical variables including the modified Rodnan skin score (mRSS) and interstitial lung disease (ILD). Twenty SSc patients underwent a follow-up serological testing and mRSS evaluation during treatment with immunosuppressants and/or anti-fibrotic drugs.Results: Serum PIIINP, TIMP-1, and ELF score were significantly higher in patients with SSc than in healthy controls [PIIINP: 10.31 (7.83-14.10) vs. 5.61 (4.69-6.30), p < .001; TIMP-1: 110.73 (66.21-192.45) vs. 61.81 (48.86-85.24), p < .001; ELF: 10.34 (9.91-10.86) vs. 9.68 (9.38-9.99), p < .001]. Even higher levels of PIIINP, TIMP-1, and ELF score were found in patients with diffuse cutaneous SSc than those with limited cutaneous SSc. At baseline, both PIIINP and ELF score showed good correlation with mRSS (PIIINP: r = .586, p < .001; ELF: r = .482, p < .001). Longitudinal analysis showed that change in PIIINP positively correlated with change in mRSS (r = 0.701, p = .001), while change in ELF score were not related, in a statistical context, to the change in mRSS (ELF: r = .140, p = .555). Serum TIMP-1 was significantly higher in SSc patients with ILD, compared to the matched group of patients without ILD [109.45 (93.05-200.09) vs. 65.50 (40.57-110.73), p = 0.007].Conclusion: In patients with SSc, the ELF score well correlates with the extent of skin fibrosis, while serum PIIINP is a sensitive marker for longitudinal changes of skin fibrosis. In the future, circulating collagen metabolites may potentially be used to evaluate therapeutic effects of anti-fibrotic treatments in the disease.

To the Editor: Systemic sclerosis (SSc) is an autoimmune disease characterized by progressive skin and visceral fibrosis, microvasculopathy, and autoimmunity. Circulating auto-antibodies (AAbs) are detectable in 90% to 95% of patients with SSc.[1] It is reported that 60% to 80% of SSc patients are positive for anti-topoisomerase I antibody (ATA), anti-centromere antibody (ACA), and anti-RNA polymerase III antibody (ARA).[2] These three AAbs are the most prevalent SSc-associated AAbs, with high specificity for the diagnosis of SSc; so they have been included in the classification criteria for SSc defined by the American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) in 2013. Currently, SSc-associated AAbs have been widely used in clinical practice, and the research on the clinical significance of AAbs is still in progress. In our present study, we analyzed the correlations between the SSc-associated auto-antibody profile and clinical manifestations in a well-characterized Chinese SSc cohort. One hundred and forty-four patients enrolled between June 2018 and August 2020 in our center were included in this cross-sectional study. The inclusion criteria were as follows: (1) patients diagnosed with SSc according to 2013 ACR/EULAR classification criteria; (2) patients who had undergone chest high-resolution computed tomography (HRCT) scan; and (3) patients tested for ATA, ACA, and AAbs to nuclear-ribonuclear-protein (nRNP). This research was conducted according to the Declaration of Helsinki, and all procedures involving study participants were approved by the ethics committee of Huashan Hospital, Fudan University (No. 2019-191). Each participant signed an informed consent form before the research. Through careful medical history inquiry and physical examination, we collected patient demography (age at onset, sex, smoking history, and disease duration) and clinical characteristics (disease subset, Raynaud's phenomenon [RP], digital ulcer, telangiectasia, puffy finger, arthralgia, and myalgia). The onset of disease was defined as the time when the first non-RP symptom of SSc (skin thickening, sclerodactyly, puffy finger, digital ulcer, or organ involvement) appeared. The skin fibrosis was scored according to the modified Rodnan skin thickness score (mRSS), a widely used clinical assessment of skin thickness where the examining rheumatologist records the degree of skin thickening on a scale of 0 (no involvement) to 3 (severe thickening) in 17 body areas (total score range: 0–51). Interstitial lung disease (ILD) was defined by HRCT. Pulmonary arterial hypertension (PAH) was defined as mean systolic pulmonary arterial pressure ≥40 mmHg detected by echocardiography. Scleroderma renal crisis (SRC) was defined as the acute deterioration of renal function accompanied by hypertension or corresponding renal biopsy results. All patients were classified into four disease subsets, including diffuse cutaneous SSc (dcSSc), limited cutaneous SSc (lcSSc), overlap syndrome (overlap) subsets based on the classification of LeRoy et al, and SSc sine scleroderma subset characterized by typical visceral involvement, vasculopathy, and serologic abnormalities without skin alterations. AAbs were tested by immunoblotting. All the patients were tested for ATA, ACA, and AAbs to Ku, nRNP, and polymyositis (PM)-Scl. Sixty-four patients were also tested for ARA and AAbs to fibrillarin, Th/To, NOR90, and platelet-derived growth factor receptor (PDGFR). Of the 64 patients, those positive for any of the above-mentioned ten AAbs were defined to be AAb-positive SSc (SSc-AAbs [+]) patients, whereas the patients who were negative for the above-mentioned ten AAbs were defined to be AAb-negative SSc (SSc-AAbs [−]) patients. Data were analyzed by GraphPad Prism (version 8.0.2 for Windows, GraphPad Software, San Diego, California, USA). Quantitative data were analyzed using t test, Welchtest, and Mann–Whitney test where appropriate. The Chi-squared test or Fisher's exact test was used to evaluate categorical data. P value < 0.05 was considered statistically significant. A total of 144 patients were included in our SSc cohort with a mean age at onset of 47.8 ± 13.9 years. Of all, 117 (93.5%) were female and 27 (6.5%) were male. SSc disease duration from RP onset and non-RP onset was 4.0 (1.5– 9.6) and 2.0 (1.0–5.0) years, respectively. The mean mRSS was 6.0 (2.0–12.0). There were 58 (41.1%), 61 (42.4%), 8 (5.6%), and 17 (11.8%) patients diagnosed as dcSSc, lcSSc, SSc sine scleroderma, and overlap, respectively. Ninety-three patients (64.6%) were determined with ILD. The patients with SSc-associated AAbs in our cohort is as follows in the order: ATA 61 (42.4%), ACA 34 (23.6%), ARA 11/64 (17.2%), anti-nRNP 15 (10.6%), anti-NOR90 6/64 (9.4%), anti-fibrillarin 4/64 (6.3%), anti-Ku 5 (3.5%), anti-Th/To 2/64 (3.1%), anti-PM-Scl 3 (2.8%), and anti-PDGFR 0/64 (0%). In addition, 11/64 (17.2%) of the patients were SSc-AAbs (–) [Supplementary Table 1, https://links.lww.com/CM9/A875 and Supplementary Figure 1, https://links.lww.com/CM9/A875]. Due to the limited number of anti-fibrillarin, anti-NOR90, anti-Th/To, anti-PDGFR, and anti-PM-Scl subgroups (n< 10), the correlation analysis may not be convincing. Therefore, clinical associations were analyzed only among the SSc patients positive for ATA, ACA, ARA, anti-nRNP and SSc-AAbs (–) subgroups. In our cohort, compared with the patients negative for the corresponding antibodies, patients with ATA had earlier disease onset (P = 0.02), higher mRSS level (P = 0.003), and higher proportions of ILD (P < 0.0001); patients with ACA had lower mRSS level (P = 0.005), lower proportions of male (P = 0.04), myalgia (P = 0.02), and ILD (P < 0.0001) or overlap (P < 0.0001); patients with ARA had later disease onset (P = 0.04); and patients with anti-nRNP had lower mRSS level (P = 0.009). Patients who are negative for SSc-AAbs manifested as more dcSSc than lcSSc (P = 0.009); patients who are positive for ACA manifested as more lcSSc than dcSSc (P < 0.0001) or overlap (P < 0.0001); and patients who are positive for anti-nRNP manifested as more overlap than dcSSc (P = 0.0003) or lcSSc (P = 0.001). No significant correlations were found between SSc-associated AAbs and disease duration, RP, digital ulcer, arthralgia, puffy finger, telangiectasia, PAH, or SRC [Table 1, Supplementary Figure 2, https://links.lww.com/CM9/A875 and Supplementary Tables 1–3, https://links.lww.com/CM9/A875]. Table 1 - Correlations of quantitative clinical variables with SSc-associated AAbs. Quantitative data Positive Negative N+ N– P values Age at onset (years), mean ± SD ATA 44.8 ± 13.3 50.0 ± 14.0 61 83 0.020∗ ACA 50.9 ± 10.3 46.8 ± 14.8 34 110 ns∗ ARA 56.0 ± 13.4 45.9 ± 15.0 11 53 0.040† Anti-nRNP 45.6 ± 15.5 48.0 ± 13.8 15 129 ns SSc-AAbs (+) 47.4 ± 15.8 48.8 ± 11.8 53 11 ns† mRSS, median (IQR) ATA 8.0 (4.0–18.0) 4.0 (2.0–10.3) 57 64 0.003∗ ACA 3.0 (2.0–8.0) 7.0 (3.0–14.5) 29 92 0.005∗ ARA 9.0 (4.0–16.0) 6.0 (2.0–10.3) 11 48 ns∗ Anti-nRNP 2.0 (0.5–4.0) 7.0 (2.0–12.8) 11 110 0.009∗ SSc-AAbs (+) 6.0 (2.0–11.0) 8.5 (5.0–11.5) 51 8 ns∗ Disease duration (years), median (IQR) RP disease duration ATA 4.0 (1.1–7.9) 3.5 (1.5–9.9) 59 82 ns∗ ACA 4.0 (1.5–10.0) 3.0 (1.4–8.0) 33 108 ns∗ ARA 8.0 (0.7–12.0) 2.5 (1.0–7.0) 11 53 ns∗ Anti-nRNP 4.0 (2.0–9.2) 3.0 (1.0–9.5) 14 127 ns∗ SSc-AAbs (+) 3.0 (1.0–9.0) 2.0 (0.6–4.5) 53 11 ns∗ Non-RP disease duration ATA 2.0 (0.8–5.0) 2.0 (1.0–5.0) 61 83 ns∗ ACA 2.9 (1.0–5.0) 2.0 (0.9–5.0) 34 110 ns∗ ARA 0.8 (0.7–2.8) 2.0 (1.0–5.0) 11 53 ns∗ Anti-nRNP 2.0 (0.6–3.5) 2.0 (1.0–5.0) 15 129 ns∗ SSc-AAbs (+) 1.5 (1.0–5.0) 1.0 (0.8–2.0) 53 11 ns∗ ∗t test.†Mann–Whitney test. N+ = The number of positive group patients; N– = The number of negative group patients. AAbs: Auto-antibodies; ACA: Anti-centromere antibody; ARA: Anti-RNA polymerase III antibody; ATA: Anti-topoisomerase I antibody; IQR: Interquartile range; mRSS: Modified Rodnan skin score; ns: Not significant; RP: Raynaud's phenomenon; SD: Standard deviation; SSc: Systemic sclerosis; SSc-AAbs (+): AAb-positive-SSc. The present study compared the prevalence of SSc-associated AAbs in a well-described Chinese cohort and analyzed the associations between SSc-associated AAbs and clinical features, such as disease subsets and critical organ involvement. Considering that fibrosis is one of the most important characteristics of SSc, we applied various ways to evaluate the degree of fibrosis in patients, including mRSS, disease subsets according to the extent of skin fibrosis and ILD. In our study, the proportion of lcSSc in ACA-positive patients was much higher than that of dcSSc and overlap; also, the mRSS was significantly lower in the patients who are positive for ACA than in those who are negative for ACA. We also found that mRSS was higher in ATA-positive patients and that the subset of dcSSc was more frequently seen in SSc patients who are positive for ATA. The proportion of ILD was higher in ATA-positive patients and lower in ACA-positive patients. Since ILD is one of the major causes of death in SSc at present, and normally indicative of severer disease and worse prognosis, our findings confirmed the view that early screening for ILD is highly recommended in ATA-positive patients.[3] Importantly, it was found in our cohort that among SSc-AAbs (-) patients, the proportion of dcSSc was higher and that of lcSSc was lower, which appeared even more remarkable than in ATA-positive patients. We speculated that there could be some existing but still unknown AAbs underlying, which could be the direction of our further research. In summary, we have applied different methods to analyze the correlation between SSc-associated auto-antibody profile and fibrosis, and gotten consistent results, which may provide supportive and supplementary data for the previous similar study in a Chinese SSc cohort.[4] Besides the correlation with fibrosis, we also found other clinical correlations of SSc-associated AAbs in our cohort. We found that the onset age of ATA-positive patients was relatively earlier in our cohort and that ACA was more commonly seen in females, consistent with the findings of Mierau et al.[5] In terms of musculoskeletal involvement, we found that myalgia is less common in ACA-positive patients, and another study[5] also reported that ACA-positive patients had less musculoskeletal involvement. In the future, we need to add more detailed musculoskeletal parameters (such as myodynamia, electromyography, muscle biopsy, and creatine kinase level) to obtain more thorough observation on the clinical relationship between AAbs and muscle involvement. ARA-positive patients had later disease onset. None of the ARA-positive patients in our cohort developed renal crisis so far, although ARA was reported to be highly correlated with SRC.[1] Since the number of ARA-positive patients was relatively small (n = 15), these findings may need further observation in a larger cohort. Anti-nRNP is usually associated with SSc overlap,[1] which was also confirmed in our cohort. The limitation of our current study mainly lies in the limited sample size in this cohort. A well-described cohort with larger sample size from multiple centers may help us to study the clinical associations with AAbs more comprehensively and thoroughly. Acknowledgments The authors appreciate and acknowledge all members of the Division of Rheumatology of Huashan Hospital; they made considerable effort in the evaluation of the patient's condition, data collection, statistical analysis, etc. The authors thank the patients for their cooperation in providing descriptions of their conditions and blood samples. Funding This work was supported by grants from the Youth Program of National Natural Science Foundation of China (No. 81501391), and medical and health research projects from Shanghai Baoshan Science and Technology Commission (No. 20-E-3). Conflicts of interest None.

Objective CXCL4, a chemokine with antiangiogenic property, is reported to be involved in systemic sclerosis (SSc) related pulmonary arterial hypertension (PAH). We investigated the contribution of CXCL4 to SSc development by focusing on the correlation of circulatory CXCL4 levels with their peripheral vasculopathy, as well as the effect of CXCL4 on endothelial cell dysfunction and angiogenesis disturbance in SSc and the potential signaling.MethodsWe measured the serum CXCL4 levels in 58 patients with SSc, 10 patients with the very early diagnosis of SSc (VEDOSS), and 80 healthy controls. Then, CXCL4 levels were correlated with their clinical features, especially the peripheral vasculopathy. These observations were further validated in an additional cohort including 50 SSc patients, 12 VEDOSS patients, and 80 healthy controls. Moreover, we studied the anti-angiogenesis effects and the underlying signaling of CXCL4 in human umbilical vein endothelial cells (HUVECs) in vitro. ResultsCirculating levels of the CXCL4 were 103.62% higher in patients with SSc and 201.51 % higher in patients with VEDOSS than matched HCs, and these observations were confirmed in two independent cohorts. CXCL4 levels were closely associated with digital ulcers (DU) and nailfold video capillaroscopy (NVC) abnormalities in SSc. The proliferation, migration, and tube formation of HUVECs were significantly inhibited by recombinant human CXCL4 or SSc derived serum, which reversed by CXCL4 neutralizing antibody, but not CXCR3 inhibitor. CXCL4 downregulated the transcription factor Friend leukaemia integration factor‐1 (Fli-1) via c-Abl signaling. Furthermore, CXCL4 blocked the transforming growth factor (TGF) -β or platelet-derived growth factor (PDGF) induced cell proliferation of HUVECs. Conclusions CXCL4 may contribute to peripheral vasculopathy in SSc by downregulating Fli-1 via c-Abl signaling in endothelial cells and interfering angiogenesis.

ObjectiveTo investigate the role of the inflammatory lipid mediator leukotriene B4 (LTB4) and its receptor, BLT1, in the development and progression of systemic sclerosis (SSc).MethodsSerum levels of LTB4 were compared in 64 patients with SSc and 80 healthy controls. Skin and lung tissue sections from patients with SSc and healthy donors were immunostained for leukotriene A4 hydrolase (LTA4H), the critical enzyme for LTB4 synthesis, and BLT1, in combination with different cell markers. In mouse models of SSc using bleomycin or angiotensin II challenge or immunization with the DNA topoisomerase I, genetic or pharmacologic interruption of the LTB4–BLT1 axis in mice was carried out to assess its effects on systemic disease features and myofibroblast markers. Immunoblotting was performed to examine the signaling pathway in fibroblasts and endothelial cells following stimulation with LTB4 or with serum from SSc patients.ResultsSerum LTB4 levels were 44.93% higher in patients with SSc than in matched healthy controls (mean ± SD 220.3 ± 74.75 pg/ml versus 152.0 ± 68.05 pg/ml; P < 0.0001), and this was associated with the patient subsets of SSc‐associated interstitial lung disease and diffuse cutaneous SSc. Levels of LTA4H and BLT1 were increased in lesional areas of the skin and lungs of SSc patients, and both were abundant in myofibroblasts and endothelial cells. Interruption of the LTB4–BLT1 axis in mouse models of SSc significantly mitigated dermal and pulmonary fibrosis, with 54.00% and 52.65% fewer α‐smooth muscle actin–positive myofibroblasts accumulating in the skin and lungs of mice, respectively, after bleomycin challenge. Immunoblotting of cultures with recombinant LTB4–stimulated fibroblasts and endothelial cells or with serum from SSc patients showed that fibroblast–myofibroblast and endothelial–mesenchymal transitions were promoted via BLT1, and that this was dependent on activation of the phosphatidylinositol 3‐kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway but independent of the release of transforming growth factor β (TGFβ) by fibroblasts or endothelial cells.ConclusionThe LTB4–BLT1 axis may contribute to fibrosis in SSc by directly promoting myofibroblast differentiation via the PI3K/Akt/mTOR pathway, and this appears to operate independently of autocrine secretion of TGFβ.