Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with an unmet need of biomarkers that can aid in the diagnostic and prognostic assessment of the disease and response to treatment. In this two-part explorative proteomic study, we demonstrate how proteins associated with tissue remodeling, inflammation and chemotaxis such as MMP7, CXCL13 and CCL19 are released in response to aberrant extracellular matrix (ECM) in IPF lung. We used a novel ex vivo model where decellularized lung tissue from IPF patients and healthy donors were repopulated with healthy fibroblasts to monitor locally released mediators. Results were validated in longitudinally collected serum samples from 38 IPF patients and from 77 healthy controls. We demonstrate how proteins elevated in the ex vivo model (e.g., MMP7), and other serum proteins found elevated in IPF patients such as HGF, VEGFA, MCP-3, IL-6 and TNFRSF12A, are associated with disease severity and progression and their response to antifibrotic treatment. Our study supports the model’s applicability in studying mechanisms involved in IPF and provides additional evidence for both established and potentially new biomarkers in IPF.

Background and Objective: Studies of autoimmunity and anti‐citrullinated protein antibodies (ACPA) in idiopathic pulmonary fibrosis (IPF) have been confined to investigations of anti‐cyclic citrullinated peptide (anti‐CCP) antibodies which utilize synthetic peptides as surrogate markers for in vivo citrullinated antigens. We studied immune activation by analysing the prevalence of in vivo anti‐modified protein antibodies (AMPA) in IPF.Methods: We included patients with incident and prevalent IPF (N = 120), sex and smoking‐matched healthy controls (HC) (N = 120) and patients with RA (N = 104). Serum (median time: 11 months [Q1–Q3: 1–28 months] from diagnosis) was analysed for presence of antibodies towards native and posttranslational modified (citrullinated [Cit, N = 25]; acetylated [Acet, N = 4] and homocitrullinated [Carb, N = 1]) peptides derived from tenascin (TNC, N = 9), fibrinogen (Fib, N = 11), filaggrin (Fil, N = 5), histone (N = 8), cathelicidin (LL37, N = 4) and vimentin (N = 5) using a custom‐made peptide microarray.Results: AMPA were more frequent and in increased levels in IPF than in HC (44% vs. 27%, p < 0.01), but less than in RA (44% vs. 79%, p < 0.01). We specifically observed AMPA in IPF towards certain citrullinated, acetylated and carbamylated peptides versus HC: tenascin (Cit₍₂₀₃₃₎‐TNC_2025–2040; Cit₍₂₁₉₇₎‐TNC_2177–2200; Cit₍₂₁₉₈₎‐TNC_2177–2200)_, fibrinogen (Cit_(38,42)‐Fibα_36–50; Cit₍₇₂₎‐Fibβ_60–74) and filaggrin (Acet‐Fil_307–324, Carb‐Fil_307–324). No differences in survival (p = 0.13) or disease progression (p = 0.19) between individuals with or without AMPA was observed in IPF. However, patients with incident IPF had better survival if AMPA were present (p = 0.009).Conclusion: A significant proportion of IPF patients present with specific AMPA in serum. Our results suggest autoimmunity as a possible characteristic for a subgroup of IPF that may affect disease outcome.

Background: Presence of autoantibodies is a hallmark of connective tissue diseases (CTD). Exclusion of CTD is required for diagnosis of idiopathic pulmonary fibrosis (IPF). We studied the prevalence of autoantibodies in IPF-patients in Sweden. Methods: Patients with positive autoantibody tests (anti-nuclear antibodies, ANA; rheumatoid factor (RF); anti-citrullinated protein antibodies (ACPA) or anti-neutrophil cytoplasmic antibodies (ANCA)) were compared with patients without in the Swedish IPF-registry. Information on demographics, comorbidities and lung function at inclusion were analyzed. Results: Autoantibodies were analyzed in 348 patients. One or more positive tests were detected in 115 patients (33%). No difference in age (mean 72 years) was observed. More women than men were positive (40% vs. 30%, p=0.037). The most frequent autoantibody was ANA (25% of ANA-tests) followed by RF (9%), ACPA (4%) and ANCA (3%). Sixty-three percent of patients with autoantibodies were ex-smokers and 30% never-smokers. Lung function did not differ in patients with and without autoantibodies. Forced vital capacity (FVC% predicted) was 74.1±18.0 and 71.8±16.6 (mean±SD) respectively, total lung capacity (TLC%) 65.2±12.3 vs. 65.0±13.4 and diffusing capacity for carbon monoxide (DLCO%) 46.5±13.8 vs. 49.7±14.6. No difference in occurrence of comorbidities between the groups was observed. Conclusion: One third of IPF-patients had positive autoantibodies. A majority had a smoking history. No difference in lung function or occurrence of comorbidities was observed. The prognostic importance of positive autoantibodies will be evaluated in longitudinal studies.

Background: In this study, we combine proteomics with functional parameters and imaging to examine potential biomarkers that may identify patients at risk of developing persistent lung sequelae following coronavirus disease 2019 (COVID-19).Methods: We performed multiplex profiling of serum and collected clinical data from intensive care unit (ICU)-treated patients with COVID-19 (n=43) at 4 and 10 months post hospitalisation.Results: Four months post discharge, patients with COVID-19 demonstrated lower % predicted forced vital capacity (72.2%versus113% (p<0.0001)) and % predicted forced expiratory volume in 1 s (74.5%versus103% (p5% of lung tissue) on high-resolution computed tomography (HRCT) had higher levels of the proteins lysosomal associated membrane protein-3 (LAMP3) and MMP7 compared with patients with minor (<5%) or no findings on HRCT. Both proteins demonstrated consecutive associations to lung function and parenchymal involvement. Histological evaluation of LAMP3 in lung tissue confirmed LAMP3 localisation to alveolar type 2 cells in more preserved areas of the parenchyma. However, areas of remodelling were devoid of LAMP3 concurrent with the appearance of KRT5+ and KRT17+ basal cells.Conclusion: Despite functional and radiological improvements following COVID-19, persistent upregulation of inflammation and remodelling factors were observed. Similarities in the expression of LAMP3 in COVID-19 and idiopathic pulmonary fibrosis may suggest it as a potential biomarker for chronic lung damage.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with limited treatment options and a considerable diagnostic delay, warranting studies for diagnostic and prognostic biomarkers. In an unpublished study, we discovered elevated levels of inflammatory and oncology-related proteins in a novel ex-vivo model where human healthy lung fibroblasts were cultured on decellularized lung tissue derived from IPF patients or healthy individuals. Aim: Evaluate potential biomarkers found elevated in ex-vivo model in serum from IPF patients. Methods: A panel of 92 proteins were examined with proximity extension assay in serum from 38 IPF patients and in 77 healthy donors. Protein data were correlated to clinical measures of disease severity. Results: We demonstrated that proteins elevated in the ex-vivo model, were also significantly elevated in serum from IPF patients, showing higher levels of CCL19, CXCL13 and MMP7 in comparison to healthy controls. In addition, MMP7 showed a negative correlation to percent predicted forced vital capacity (FVC%, r= -0.48, p=0.0020) and total lung capacity (TLC%, r= -0.49, p=0.0017). Conclusion: We conclude that the ex-vivo model may mimic fibrotic mechanisms in IPF as the fibroblast-released mediators correspond to the elevated protein levels seen in IPF patients. An association with measures of disease severity strengthens our findings. Our results highlight the identified proteins in their roles as important drivers of fibrotic processes and the model’s applicability as an innovative way of studying pathological responses in IPF.

Background: Idiopathic pulmonary fibrosis (IPF) is a debilitating disease with a heterogeneous disease course that is difficult to predict. There is therefore a need to find variables or patterns of variables that can predict survival. Objective: To identify clusters of IPF-patients by using accrued data from the Swedish IPF registry in order to identify groups of patients with shared clinical measures and to explore their cluster related outcome. Methods: Two-step cluster analysis was performed by using 14 variables available from baseline from 175 patients. Kaplan-Meier estimates were used for survival analysis. Results: Three clusters were identified. Cluster 1 (n= 51) consisted exclusively of females with a low-moderate Gender-Age-Physiology (GAP)-stage and a mild composite physiological index (CPI). Cluster 2 (n=90) was exclusively male with a moderate GAP-stage and moderate CPI. Cluster 3 (n=34) consisted of 80% males with low GAP-stage and very mild CPI. There was a significant difference in survival between the clusters (log-rank:p=0.004), with patients in cluster 2 having the highest mortality. Interestingly, no difference in survival was observed between cluster 1 (only females) and cluster 3 (mainly males), despite significant differences in GAP and CPI. Conclusions: Our data indicate that male IPF-patients may have worse survival than females in Sweden. The identified phenotypes by cluster may be used to predict outcome and aid clinicians in a more personalized management approach.