Proceedings of the 24th Paediatric Rheumatology European Society Congress: Part three

From Springer Nature via Jisc Publications Router.Publication status: PublishedHistory: collection 2017-09, epub 2017-09-0

Absence of fibromodulin affects matrix composition, collagen deposition and cell turnover in healthy and fibrotic lung parenchyma.

The ECM exerts great effects on cells, and changed composition may therefore have profound impact. Small leucine-rich proteoglycans, e.g. fibromodulin, are essential in collagen assembly. Our aim was to investigate the role of fibromodulin in healthy and fibrotic lung parenchyma, theorizing that fibromodulin-deficient animals would be protected against fibrosis. Repeated subcutaneous bleomycin-injections were given to wild type and fibromodulin-deficient mice, inducing pulmonary fibrosis. Development of fibrosis, ECM composition, cell turnover and inflammatory responses were investigated. Fibromodulin-deficient animals were not protected from fibrosis, but the composition of the matrix was affected, with decreased Collagen I in fibromodulin-deficient animals, both in controls (0.07 ± 0.04% vs. 0.18 ± 0.07% tissue area) and after bleomycin (0.37 ± 0.16% vs. 0.61 ± 0.21% tissue area). Biglycan was increased in fibromodulin-deficient animals, whereas decorin was decreased. Furthermore, bleomycin increased cell turnover in wild type, but only proliferation in fibromodulin-deficient animals, resulting in hyperplasia. In addition, the bleomycin-induced immune response was affected in fibromodulin-deficient animals. We thus conclude that fibromodulin has a profound effect on ECM, both in healthy and fibrotic lung parenchyma, and may be providing a permissive microenvironment affecting cell turnover. Furthermore, this study highlights the need to acknowledge specific ECM components, when assessing tissue properties and ultimately cell behaviour

The association between changes in skin echogenicity and the fibroblast production of biglycan and versican in systemic sclerosis

Objective To investigate a possible association between the longitudinal changes in skin involvement and the fibroblast production of proteoglycans in vitro, among patients with early and untreated systemic sclerosis (SSc). Methods In 11 patients, 6 with diffuse cutaneous systemic sclerosis (dSSc) and 5 with limited cutaneous systemic sclerosis (lSSc), and in 6 controls skin thickness and skin echogenicity, of the forearm was measured by high frequency (20 MHz) ultrasound, A skin biopsy was taken from the area of the ultrasound measurements, and from cultivated fibroblasts the production of the proteoglycans versican, perlecan, biglycan and decorin were measured. To investigate longitudinal changes in skin involvement, the ultrasound examination was repeated after 1-3 years. Results Compared to controls, SSc Patients had increased skin thickness at the first evaluation. Patients with dSSc had lower skin echogenicity than both patients with ISSc and the controls. Patients with greater changes in skin thickness and skin echogenicity produced more versican, whereas the production of biglycan and decorin was higher only, in patients with greater changes in skin echogenicity. There was a negative correlation between fibroblast production of biglycan and disease duration. Conclusion High fibroblast synthesis of the proteoglycans versican and biglycan is associated with changes in skin echogenicity and may predict more progressive skin sclerosis in SSc

Limited impact of fibromodulin deficiency on the development of experimental skin fibrosis

Excessive production of collagen is the hallmark of fatal diseases of fibrosis such as systemic sclerosis. Overexpression of the proteoglycan fibromodulin (FMOD) has been associated with improved wound healing and scarless repair. In this study we have investigated the consequences of FMOD deficiency on the development of experimental skin fibrosis. Using immunohistochemistry, we identified FMOD in both human and murine fibrotic skin. In the bleomycin model of skin fibrosis, FMOD(-/-) mice developed skin fibrosis to a similar degree compared to FMOD(+/+) mice. Analysis of skin ultrastructure using transmission electron microscopy revealed a significant reduction in collagen fibril diameter in FMOD(-/-) but not FMOD(+/+) mice following fibrosis. We conclude that impact of FMOD deficiency on the development of experimental skin fibrosis is limited. This article is protected by copyright. All rights reserved

Extracellular matrix alterations and acute inflammation; developing in parallel during early induction of pulmonary fibrosis.

Pulmonary fibrosis is a hallmark of several systemic diseases such as systemic sclerosis. Initiation and early development is not well characterized, as initiation usually is unnoticed in patients, yet fibrosis has been considered a late event, occurring after an inflammatory phase. By utilizing an animal model, the starting point can be defined and the initiation process and early development thoroughly investigated. To investigate these processes from a systemic perspective, we choose a systemic administration route, instead of the more commonly used local administration. The aim of this work was to study the initiation of pulmonary fibrosis in an animal model and to investigate early alterations in connective tissue, cell turnover and acute immune response in lung parenchyma. Animals were injected subcutaneously with bleomycin, three times a week (w) for 1-4w (controls received saline). Total collagen was histologically assessed by Picro Sirius Red and Masson's Trichrome, collagen production by antibodies directed against N-terminal of procollagens I and III, proliferation by labeling with proliferating cell nuclear antigen, apoptosis by TUNEL and innate immunity by detecting neutrophils and macrophages. Total collagen was significantly increased at 1, 2 and 4w compared with controls. Procollagen I, was increased at 1w and remained increased, whereas procollagen III-staining was increased at 2w, compared with controls. Myofibroblasts were increased at all times as were proliferation, whereas apoptosis was increased from 2w. Neutrophils peaked at 1w (2779±820 cells/mm(2)) and gradually decreased, whereas macrophages peaked at 2w (135±29 cells/mm(2)). Subcutaneously administered bleomycin induces rapid alterations in connective tissue and cell turnover, suggesting a plasticity of the connective tissue. A transient neutrophilia is detected and increased number of macrophages likely represents a clearance process of said neutrophils. The study suggests fibrosis initiation and acute inflammation to occur in parallel in this model.Laboratory Investigation advance online publication, 2 April 2012; doi:10.1038/labinvest.2012.57

Biomarkers from bronchoalveolar lavage fluid in systemic sclerosis patients with interstitial lung disease relate to severity of lung fibrosis.

OBJECTIVES: Decision on treatment of systemic sclerosis (SSc) related interstitial lung disease (ILD) largely relies on the findings on high resolution computed tomography (HRCT) and there is a need for improvement in assessment of the fibrotic activity. The objectives of this study were to study biomarkers in bronchoalveolar lavage fluid (BALF) from SSc patients with ILD and to relate the findings to the severity and activity of lung fibrosis. METHODS: Fifteen patients with early SSc and 12 healthy controls were subjected to BAL. Cell counts and analyses of CXCL5, CXCL8 and S100A8/A9 were performed in BALF and serum. COMP and KL-6 were measured in serum. HRCT of lungs was quantified for ground glass opacities (GGO), reticulation and traction bronchiectases. RESULTS: BALF concentrations of CXCL8 (p < 0.001), CXCL5 (p = 0.002) and S100A8/A9 (p = 0.016) were higher in patients than controls. Serum KL-6 (p < 0.001) was increased in SSc patients and correlated with BALF concentration of eosinophils (rS = 0.57, p = 0.027). Patients with more widespread GGO on HRCT were characterised in BALF by a higher eosinophil count (p = 0.002) and in serum by higher KL-6 (p = 0.008). Patients with more fibrosis were characterised in BALF by higher eosinophil count (p = 0.014), higher CXCL8 (p = 0.005) and S100A8A/A9 (p = 0.014) concentration and in serum by a higher serum COMP (p = 0.023). CONCLUSIONS: In SSc related ILD, biomarkers from BALF and serum correlate to findings on HRCT suggesting usefulness as markers of presence and extent of lung fibrosis

Anti-Ro52 positivity is associated with progressive interstitial lung disease in systemic sclerosis-an exploratory study.

BACKGROUND: Interstitial lung disease (ILD) is the most common cause of death in patients with systemic sclerosis (SSc). Prognostic biomarkers are needed to identify SSc-ILD patients at risk for progressive pulmonary fibrosis. This study investigates autoantibodies measured in bronchoalveolar lavage (BAL) fluid and in serum in reference to the clinical disease course of SSc-ILD. METHODS: Fifteen patients with new onset SSc-ILD underwent bronchoscopy. Autoantibody levels were analyzed using addressable laser bead immunoassay from BAL fluid and the serum. In a separate longitudinal cohort of 43 patients with early SSc-ILD, autoantibodies in serum were measured at baseline and pulmonary function tests were performed at least 2 times over the course of at least 2 or more years. Linear mixed effect models were created to investigate the relationship between specific autoantibodies and progression of SSc-ILD. Finally, lung tissue from healthy controls and from subjects with SSc was analyzed for the presence of the Ro52 antigen using immunohistochemistry. RESULTS: Among SSc-ILD patients who were positive for anti-Ro52 (N = 5), 3 (60%) had enrichment of anti-Ro52 in BAL fluid at a ratio exceeding 50x. In the longitudinal cohort, 10/43 patients (23%) were anti-Ro52 positive and 16/43 (37%) were anti-scl-70 positive. Presence of anti-Scl-70 was associated with a lower vital capacity (VC) at baseline (-12.6% predicted VC [%pVC]; 95%CI: -25.0,&nbsp;-0.29; p = 0.045), but was not significantly associated with loss of lung function over time (-1.07%pVC/year; 95%CI: -2.86, 0.71; p = 0.230). The presence of anti-Ro52 was significantly associated with the loss of lung function over time (-2.41%pVC/year; 95% CI: -4.28,&nbsp;-0.54; p = 0.013). Rate of loss of lung function increased linearly with increasing anti-Ro52 antibody levels (-0.03%pVC per arbitrary units/mL and year; 95%CI: -0.05,&nbsp;-0.02; p &lt; 0.001). Immunohistochemical staining localized the Ro52 antigen to alveolar M2 macrophages in peripheral lung tissue both in subjects with and without SSc. CONCLUSIONS: This study suggests that antibodies targeting Ro52 are enriched in the lungs of patients with new-onset SSc-ILD, linking Ro52 autoimmunity to the pulmonary pathology of SSc. Clinical and immunohistochemical data corroborates these findings and suggest that anti-Ro52 may serve as a potential biomarker of progressive SSc-ILD

Anti-Ro52 positivity is associated with progressive interstitial lung disease in systemic sclerosis—an exploratory study

Abstract Background Interstitial lung disease (ILD) is the most common cause of death in patients with systemic sclerosis (SSc). Prognostic biomarkers are needed to identify SSc-ILD patients at risk for progressive pulmonary fibrosis. This study investigates autoantibodies measured in bronchoalveolar lavage (BAL) fluid and in serum in reference to the clinical disease course of SSc-ILD. Methods Fifteen patients with new onset SSc-ILD underwent bronchoscopy. Autoantibody levels were analyzed using addressable laser bead immunoassay from BAL fluid and the serum. In a separate longitudinal cohort of 43 patients with early SSc-ILD, autoantibodies in serum were measured at baseline and pulmonary function tests were performed at least 2 times over the course of at least 2 or more years. Linear mixed effect models were created to investigate the relationship between specific autoantibodies and progression of SSc-ILD. Finally, lung tissue from healthy controls and from subjects with SSc was analyzed for the presence of the Ro52 antigen using immunohistochemistry. Results Among SSc-ILD patients who were positive for anti-Ro52 (N = 5), 3 (60%) had enrichment of anti-Ro52 in BAL fluid at a ratio exceeding 50x. In the longitudinal cohort, 10/43 patients (23%) were anti-Ro52 positive and 16/43 (37%) were anti-scl-70 positive. Presence of anti-Scl-70 was associated with a lower vital capacity (VC) at baseline (-12.6% predicted VC [%pVC]; 95%CI: -25.0, -0.29; p = 0.045), but was not significantly associated with loss of lung function over time (-1.07%pVC/year; 95%CI: -2.86, 0.71; p = 0.230). The presence of anti-Ro52 was significantly associated with the loss of lung function over time (-2.41%pVC/year; 95% CI: -4.28, -0.54; p = 0.013). Rate of loss of lung function increased linearly with increasing anti-Ro52 antibody levels (-0.03%pVC per arbitrary units/mL and year; 95%CI: -0.05, -0.02; p < 0.001). Immunohistochemical staining localized the Ro52 antigen to alveolar M2 macrophages in peripheral lung tissue both in subjects with and without SSc. Conclusions This study suggests that antibodies targeting Ro52 are enriched in the lungs of patients with new-onset SSc-ILD, linking Ro52 autoimmunity to the pulmonary pathology of SSc. Clinical and immunohistochemical data corroborates these findings and suggest that anti-Ro52 may serve as a potential biomarker of progressive SSc-ILD