Review: Serum Biomarkers of Lung Fibrosis in Interstitial Pneumonia with Autoimmune Features—What Do We Already Know?

Interstitial pneumonia with autoimmune features (IPAF) belongs to a group of diseases called interstitial lung diseases (ILDs), which are disorders of a varied prognosis and course. Finding sufficiently specific and sensitive biomarkers would enable the progression to be predicted, the natural history to be monitored and patients to be stratified according to their treatment. To assess the significance of pulmonary fibrosis biomarkers studied thus far, we searched the PubMed, Medline and Cochrane Library databases for papers published between January 2015 and June 2021. We focused on circulating biomarkers. A primary review of the databases identified 38 articles of potential interest. Overall, seven articles fulfilled the inclusion criteria. This review aims to assess the diagnostic and prognostic value of molecules such as KL-6, SP-A, SP-D, circulating fibrocytes, CCL2, CXCL13, CXCL9, CXCL10 and CXCL11. All of these biomarkers have previously been studied in idiopathic pulmonary fibrosis (IPF) and connective tissue disease-associated interstitial lung disease (CTD-ILD). IPAF is a disorder of a heterogeneous nature. It explains the lack of coherent observations in terms of correlations with functional parameters. There is still no meta-analysis of pulmonary fibrosis biomarkers in IPAF. This is mainly due to the heterogeneity of the methodology and groups analysed in the research. More research in this area is needed.

Blood fibrocytes are associated with severity and prognosis in COVID-19 pneumonia

Increased blood fibrocytes are associated with a poor prognosis in fibrotic lung diseases. We aimed to determine whether the percentage of circulating fibrocytes could be predictive of severity and prognosis during Coronavirus disease 2019 (COVID-19) pneumonia. Blood fibrocytes were quantified by flow cytometry as CD45+/CD15-/CD34+/Collagen-1+cells in patients hospitalized for COVID-19 pneumonia. In a subgroup of patients admitted in ICU, fibrocytes were quantified in blood and broncho-alveolar lavage (BAL). Serum amyloid P (SAP), TGF-b1,CXCL12, CCL2, and FGF2 serum concentration were measured in serum. We included 57 patients in the Hospitalized group (median age 59 years [23-87]) and 16 Healthy controls. The median percentage of circulating fibrocytes was higher in patients compared to controls (3.6% [0.2-9.2] vs. 2.1% [0.9-5.1], p=0.04). Blood fibrocyte count was lower in the 6 patients who died compared to survivors (1.6% [0.2-4.4] vs. 3.7% [0.6-9.2], p=0.02). Initial fibrocyte count was higher in patients showing a complete lung CT resolution at 3 months. Circulating fibrocyte count was decreased in the ICU group (0.8% [0.1-2.0]) whereas BAL fibrocyte count was 6.7% [2.2-15.4]. Serum SAP and TGF-b1 concentrations were increased in Hospitalized patients. SAP was also increased in ICU patients. CXCL12 and CCL2 were increased in ICU patients, and negatively correlated with circulating fibrocyte count. We conclude that circulating fibrocytes were increased in patients hospitalized for COVID-19 pneumonia and a lower fibrocyte count was associated with an increased risk of death and a slower resolution of lung CT opacities.

Association of Circulating Fibrocytes With Fibrostenotic Small Bowel Crohn’s Disease

Background Fibrocytes are hematopoietic cells with features of mesenchymal cells found in the circulation and inflammatory sites implicated in promoting fibrosis in many fibroinflammatory diseases. However, their role(s) in the development of intestinal fibrosis is poorly understood. Here, we investigated a potential role of fibrocytes in the development of fibrosis in Crohn’s disease (CD) and sought factors that may impact their development and function. Methods Plasma and mononuclear cells were collected from patients with and without fibrostenotic CD. Fibrocytes defined as CD11b+, CD34+, and Collagen 1+ were correlated with clinical assessments of fibrosis, including evaluation using intestinal ultrasound. We measured the levels of relevant circulating molecules via Luminex and studied the effect of patient plasma proteins on fibrocyte differentiation. Results Fibrocyte numbers were increased in CD patients with stricturing Crohn’s disease compared with patients with an inflammatory phenotype (P = .0013), with strong correlation between fibrocyte numbers and acoustic radiation force impulse (ARFI), a measure of bowel elasticity on intestinal ultrasound (R = .8383, P = .0127). Fibrostenotic plasma was a more potent inducer of fibrocyte differentiation in both primary human monocytes and cell line and contained increased levels of cytokines implicated in fibrocyte differentiation compared with plasma from inflammatory patients. Interestingly, increased fibrocyte numbers at time of ultrasound were associated with escalation of medical therapy and endoscopic/surgical management of small bowel strictures at 30 months follow-up. Conclusions Circulating fibrocytes strongly correlate with fibrostenotic disease in CD, and they may serve as predictors for escalation of medical +/- surgical therapy.

Nintedanib downregulates the transition of cultured systemic sclerosis fibrocytes into myofibroblasts and their pro-fibrotic activity

Background Circulating fibrocytes are an important source of fibroblasts and myofibroblasts, which are involved in fibrotic processes, including systemic sclerosis (SSc). The study aimed to investigate the effect of nintedanib (a tyrosine kinase inhibitor) in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity. Methods Circulating fibrocytes were obtained from 18 SSc patients and 5 healthy subjects (HSs). Cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1 and 1 μM for 3 and 24 h. Fibroblast-specific protein-1 (S100A4) and α-smooth muscle actin (αSMA), as markers of fibroblast/myofibroblast phenotype, together with type I collagen (COL1) and fibronectin (FN), were investigated by qRT-PCR and Western blotting. Non-parametric tests were used for statistical analysis. Results Significantly elevated gene and protein expressions of αSMA, S100A4, COL1, and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: αSMA p < 0.001; others p < 0.0001; protein: all p < 0.05). Interestingly, an increased gene and protein expression of αSMA and S100A4 was found in fibrocytes from SSc patients positive for anti-Scl70 and with interstitial lung disease (ILD) (Scl70+ILD+) compared to Scl70−ILD− patients (S100A4: gene: p < 0.01; protein: p < 0.05), whereas no differences were observed for COL1 and FN. Nintedanib reduced gene and protein expression of αSMA, S100A4, COL1, and FN in SSc fibrocytes compared to untreated ones with different statistical significance. Noteworthy, nintedanib significantly downregulated gene and protein expression of αSMA, S100A4, COL1, and FN in Scl70+ILD+ fibrocytes (all p < 0.05), whereas only that of S100A4 and FN was significantly downregulated (p < 0.05) in Scl70−ILD− fibrocytes compared to the related untreated cells. Conclusions Nintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70+ILD+ SSc patients.

POS0330 NINTEDANIB (TYROSINE KINASE INHIBITOR) DOWNREGULATES THE TRANSITION OF CULTURED SYSTEMIC SCLEROSIS FIBROCYTES INTO MYOFIBROBLASTS AND THEIR PRO-FIBROTIC ACTIVITY

Background:Fibroblast-to-myofibroblast transition is one of the fundamental steps involved in the fibrotic process that characterise systemic sclerosis (SSc) [1]. Myofibroblasts are α-smooth muscle actin (αSMA) positive cells that contribute to fibrosis through the excessive synthesis and deposition of extracellular matrix (ECM) proteins, primarily fibronectin (FN) and type I collagen (COL1) [2].Among the cells involved in the fibrotic process of SSc, circulating fibrocytes seem to have an emerging role as an important source of fibroblasts and myofibroblasts [3].Nintedanib is a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis that interferes with the signalling pathways involved in the pathogenesis of fibrosis (4). Nintedanib was recently demonstrated to have a beneficial effect in patients with interstitial lung disease (ILD) associated with SSc (5).Objectives:To investigate nintedanib effect in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity.Methods:Circulating fibrocytes were obtained from 14 SSc patients (mean age 64±14 years), who fulfilled the 2013 ACR/EULAR criteria for SSc and that underwent complete disease staging in a day-hospital setting at the Rheumatology Division of Genoa University. Five age-matched healthy subjects (HSs) were also analysed. All SSc patients and HSs signed the informed consent and the local EC approved the study. Peripheral blood mononuclear cells were isolated by density gradient centrifugation and plated on FN-coated dishes. After overnight culture, non-adherent cells were removed, and adherent cells were maintained in growth medium for 8 days (T8) to obtain fibrocytes [6]. T8-cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1μM and 1μM for 3 and 24 hours. Fibroblast specific protein-1 (S100A4) and αSMA, as markers of fibroblast/myofibroblast phenotype, together with COL1 and FN, were investigated by qRT-PCR and Western blotting. Non-parametric Mann-Whitney and Wilcoxon tests were used for the statistical analysis.Results:Significantly elevated gene and protein expressions of αSMA, S100A4, COL1 and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: αSMA p<0.001; others p<0.0001; protein: all p<0.05). In accordance with the antibody positivity for Scl70 and the presence or absence of ILD at CT scan, SSc patients were grouped as either Scl70 positive patients with ILD (Scl70+ILD+) or Scl70 negative patients without ILD (Scl70-ILD-). Significant αSMA, S100A4, COL1 and FN gene expressions were found in fibrocytes from Scl70+ILD+ compared to HS fibrocytes (αSMA p<0.001; others p<0.0001). Moreover, fibrocytes from Scl70+ILD+patients showed a more significant gene expression of fibroblasts/myofibroblasts markers compared to Scl70-ILD-patients (p<0.01 for S100A4), whereas no differences were observed for ECM gene expression.Nintedanib reduced the gene and protein expression of αSMA, COL1 and FN in SSc fibrocytes compared to untreated ones with different statistical significance.Noteworthy, nintedanib significantly downregulated αSMA, S100A4, COL1 and FN gene expression (all p<0.05) in Scl70+ILD+fibrocytes, whereas only that of S100A4 and FN was significantly downregulated (p<0.05) in Scl70-ILD- fibrocytes compared to untreated cells.Conclusion:Nintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70+ILD+SSc patients.References:[1]Cutolo M et al. Exp Rev Clin Immunol. 2019;15:753-64.[2]Van Caam A et al. Front. Immunol. 2018;9:2452.doi:10.3389/fimmu.2018.02452.[3]Distler JH et al. Arthritis Rheumatol. 2017;69:257-67.[4]Distler O et al. New Eng J Med. 2019; 380:2518-28.[5]Maher TB et al. Arthritis Rheumatol.2020.doi:10.1002/art.41576.[6]Cutolo M et al. Arthritis Res Ther. 2018;20:157.doi:10.1186/s13075-018-1652-6.Acknowledgements:We thank Stefano-Lutz Willing for the scientific support through the study.Disclosure of Interests:Stefano Soldano: None declared, Paola Montagna: None declared, Emanuele Gotelli: None declared, Samuele Tardito: None declared, Sabrina Paolino: None declared, Claudio Corallo: None declared, Carmen Pizzorni: None declared, Alberto Sulli: None declared, Carlotta Schenone: None declared, Greta Pacini: None declared, Vanessa Smith: None declared, Maurizio Cutolo Grant/research support from: I received grant/research support from Bristol-Myers Squibb, Boehringer, Celgene

POS0377 FIBROCYTES IN EARLY AND LONGSTANDING RHEUMATOID ARTHRITIS: A 6-MONTH TRIAL WITH REPEATED SYNOVIAL BIOPSY, IMAGING, AND LUNG FUNCTION TEST

Background:Fibrocytes are bone marrow-derived cells, that express both hematopoietic and stromal markers. In murine collagen-induced arthritis models circulating fibrocytes have been found to home to inflamed joints and enhance arthritis activity. The cell has, therefore, been proposed to be precursor cells for the fibroblast-like synoviocytes (FLS), which are central in the Rheumatoid Arthritis (RA) pathogenesis. Fibrocytes levels are further correlated with disease progression and mortality in interstitial lung disease (ILD) and identified as a new treatment target. ILD is also an extra-articular manifestation of RA (RA-ILD), leading to a reduced lung diffusion capacity and increased mortality.Objectives:To correlate the level of fibrocytes in peripheral blood, synovial tissue, and in vitro culture in RA to changes in disease activity, imaging, and pulmonary function.Methods:Twenty patients with early RA (ERA) and 20 patients with longstanding RA (LRA) were enrolled in a six months prospective study. Sixteen patients undergoing wrist arthroscopy were healthy controls. RA patients underwent pulmonary function tests, ultrasound, and synovial ultrasound-guided needle biopsy of the same wrist, at baseline and six months. Wrist magnetic resonance imaging was performed at baseline (all) and six months (ERA). Circulating fibrocytes were measured by flow cytometry, in vitro by the number of monocytes that were differentiated to fibrocytes, and in synovial biopsies by counting in histological sections.Results:Fibrocyte levels did not decline during the trial despite effective RA treatment. Fibrocytes were primarily located around vessels and in the subintimal area in the synovium (Figure 1A and 1B, fibrocytes marked with arrows). In the ERA group, increased synovitis assessed by ultrasound was moderate and strongly correlated to respectively increase in circulating and synovial fibrocyte levels. Increased synovitis assessed by MRI during the trial in the ERA group, was moderately correlated to both increased numbers of circulating and cultured fibrocytes. Absolute diffusion capacity level was overall weakly negatively correlated to the level of circulating and synovial fibrocytes. The decline in forced ventilatory capacity and diffusion capacity during the trial was moderately correlated to increased levels of synovial fibrocytes (Figure 1C and 1D).Conclusion:Our findings point to fibrocytes as key mediators of RA pathogenesis, and as a possible pathogenic link between the disease process in the synovium and RA lung affection. Studies are needed to investigate if the new therapies targeting fibrocyte differentiation/migration could be a path forward in RA/RA-ILD treatment.Disclosure of Interests:None declared

Circulating fibrocytes are not disease-specific prognosticators in idiopathic pulmonary fibrosis

ObjectiveCirculating fibrocytes are elevated in idiopathic pulmonary fibrosis, but the relationship between fibrocyte level with lung function decline and outcomes is lacking replication in prospective clinical study. We aim to validate the utility of circulating fibrocyte levels as a prognostic biomarker in idiopathic pulmonary fibrosis.MethodsWe tested associations between circulating fibrocyte levels, mortality, disease progression and longitudinal lung function in a well-defined prospective observational study of pulmonary fibrosis (PROFILE; NCT01134822). A subset of recruited participants had blood samples processed for fibrocyte measurement, with flow cytometry based on CD45 and collagen-I gating. Associations were tested using univariable and multivariable generalised linear models. Mortality data were subsequently combined with an independent cohort in a mixed-effect multilevel analysis.ResultsIn 102 participants with idiopathic pulmonary fibrosis, a previously defined mortality risk threshold of 5% circulating fibrocytes was not reproducible. An empirically defined cutpoint of 2.22% was associated with a greater risk of overall mortality in adjusted analysis (Hazard Ratio 2.24 95% CI 1.06-4.72). A 2.5 fold greater risk of mortality was supported in a pooled analysis with a historic cohort for a larger sample of 162 participants with idiopathic pulmonary fibrosis (Hazard Ratio 2.49 95% CI 2.41-2.56). We found no association of fibrocytes with lung function or disease progression.ConclusionsIn a large sample of circulating fibrocytes from people with idiopathic pulmonary fibrosis, levels of 2.22% or above were associated with greater mortality, but not with disease related decline in lung function.