Title : Toll-like receptor 3 L 412 F polymorphism promotes a persistent clinical phenotype in pulmonary sarcoidosis

Background: Sarcoidosis is a multisystemic disorder of unknown etiology, characterised by the presence of non-caseating granulomas in target organs. In ninety percent of cases, there is thoracic involvement. Fifty to seventy percent of pulmonary sarcoidosis patients will experience acute, self-limiting disease. For the subgroup of patients who develop persistent disease, no targeted therapy is currently available. Aim: To investigate the potential of the single nucleotide polymorphism (SNP), Toll-like receptor 3 Leu412Phe (TLR3 L412F; rs3775291), as a causative factor in the development of, and in disease persistence in pulmonary sarcoidosis. To investigate the functionality of TLR3 L412F in vitro in primary human lung fibroblasts from pulmonary sarcoidosis patients. Methods: Cohorts of Irish sarcoidosis patients (n=228), healthy Irish controls (n = 263) and a secondary cohort of American sarcoidosis patients (n=123) were genotyped for TLR3 L412F. Additionally, the effect of TLR3 L412F in primary lung fibroblasts from pulmonary sarcoidosis patients was quantitated following TLR3 activation in the context of cytokine and type I interferon production, TLR3 expression, and apoptoticand fibroproliferative-responses. Results: We report a significant association between TLR3 L412F and persistent clinical disease in two cohorts of Irish and American Caucasians with pulmonary sarcoidosis. Furthermore, activation of TLR3 in primary lung fibroblasts from 412F-homozygous pulmonary sarcoidosis patients resulted in reduced IFN-β and TLR3 expression, reduced apoptosisand dysregulated fibroproliferative-responses compared with TLR3 wild-type patients. Conclusions: This study identifies defective TLR3 function as a previously unidentified factor in persistent clinical disease in pulmonary sarcoidosis and reveals TLR3 L412F as a candidate biomarker. Downloaded from https://academic.oup.com/qjmed/advance-article-abstract/doi/10.1093/qjmed/hcx243/4725112 by University of Tasmania Library user on 04 January 2018 INTRODUCTION Sarcoidosis is a multi-systemic disorder of unknown cause which is characterised by the presence of non-caseating granulomas in target organs. Ninety percent of sarcoidosis cases have thoracic involvement (1, 2). The highest annual incidence of sarcoidosis has been observed in northern European countries (5 to 40 cases per 100,000 people) (3). Phenotypically, sarcoidosis follows either an acute or chronic course. Up to seventy percent of patients present with acute sarcoidosis and experience self-limiting disease which will resolve within 1-2 years (4). In contrast, thirty to fifty percent of patients will develop persistent pulmonary sarcoidosis for which no approved treatments are currently available and corticosteroid use is the standard, nonspecific treatment method (4). Although mechanisms underlying the development of sarcoidosis are currently unknown, it hypothesized to be caused by an aberrant host immune response to unknown environmental antigens in genetically predisposed individuals. A number of alterations in sarcoidosis patients’ immune responses has been reported including, an exaggerated Th1 response, increased Th17 activity, attenuated regulatory T cell responses and oligoclonal expansion of CD4+ T cell responses, which is consistent with chronic antigenic stimulation (5-9). To date, no specific pathogen has been identified as a causative factor in sarcoidosis. Several studies have a role for mycobacterial or propionibacterial organisms in the pathogenesis of sarcoidosis. Specifically, a meta-analysis of studies carried out between 1980 and 2006 demonstrated that 26% of all sarcoidosis tissues had evidence of mycobacterial nucleic acids (10). In the context of viral infection, seroepidemiological studies have demonstrated significant levels of antibodies to Epstein-Barr virus (EBV), rubella and parainfluenza 3 in sarcoidosis (11). However, no correlation could be made between viral antibody titre and stage of disease or activity (11). In this study, we investigated the role of defective TLR3 in the development of persistent clinical disease in pulmonary sarcoidosis. TLR3 has previously been shown to bind dsRNA from viruses, bacteria and helminths, respectively, in addition to mRNA released from necrotic cells (2, 12-15). Specifically, here we investigated the role of the TLR3 polymorphism, Leu412Phe (TLR3 Leu412Phe, L412F; rs3775291) as a causative factor in the development of and in disease persistence in pulmonary sarcoidosis, respectively. Previously, we identified a role for TLR3 L412F in accelerated disease progression and increased risk of mortality in idiopathic pulmonary Downloaded from https://academic.oup.com/qjmed/advance-article-abstract/doi/10.1093/qjmed/hcx243/4725112 by University of Tasmania Library user on 04 January 2018 fibrosis (IPF) (16). TLR3 L412F has also been implicated as a causative factor in a number of autoimmune and inflammatory diseases such as diabetes, systemic lupus erythematosus (SLE) and rheumatoid arthritis (17-19), as well as a variety of cancers (20-24). TLR3 L412F has also been demonstrated to have either a protective or pathogenic effect in viral infection (25, 26). In this study, we report a significant association between development of a persistent clinical phenotype in pulmonary sarcoidosis and the TLR3 L412F variant in cohorts of Irish and American Caucasians, respectively. Furthermore, activation of TLR3 in vitro in primary human lung fibroblasts from 412F-homozygous patients resulted in decreased TLR3 and IFN-β expression, reduced apoptosis and dysregulated proliferation, respectively, compared with fibroblasts from TLR3 wild-type patients. Our findings imply that defective TLR3 promotes a persistent disease phenotype in sarcoidosis and reveals TLR3 L412F as a candidate prognostic biomarker in this interstitial lung disease. Downloaded from https://academic.oup.com/qjmed/advance-article-abstract/doi/10.1093/qjmed/hcx243/4725112 by University of Tasmania Library user on 04 January 2018 MATERIALS AND METHODS Study Subjects A cohort of Irish Caucasian pulmonary sarcoidosis patients (n=228; Table 1) was recruited from St Vincent’s University Hospital, Elm Park, Dublin 4 (SVUH). A cohort of Irish Caucasian healthy volunteers (n = 263) was additionally recruited as a control group. Genomic DNA was obtained from the American cohort of sarcoidosis patients attending the specialized Sarcoidosis Clinic at Johns Hopkins University School, Baltimore, MA, USA (n=123; Table 1). Diagnosis of pulmonary sarcoidosis and classification of persistent disease Irish and American pulmonary sarcoidosis patients were diagnosed at initial hospital presentation by the same physician (S.C.D. and D.R.M), respectively, and followed-up for at least 2 years (see Supplementary Methods). Patients at 2 years follow-up were classified as having either “persistent” disease or “non-persistent” disease based on a modification of a system previously described (2)(see Supplementary Methods). TLR3 L412F genotyping TLR3 L412F genotyping was carried out as described by us previously in a parallel study investigating the role of the TLR3 L412F in IPF (16)(see Supplementary Methods). Analysis of TLR3 L412F functionality in primary human lung fibroblasts from pulmonary sarcoidosis patients Primary fibroblast cell lines were isolated from lung biopsies of sarcoidosis patients (supplied by SVUH) and cultured as described previously (27) (see Supplementary Methods). Methodology pertaining to the analysis of the effects of TLR3 L412F on fibroblast-apoptosis, -proliferation and -cytokine/interferon production, respectively, is detailed in the Supplementary Methods section. Statistical analysis All statistical analyses were carried out using GraphPad Instat Software (GraphPad Software Inc. CA, USA). Statistical analyses of genotype and allele frequencies were performed using twotailed χ2 tests (3 x 2 χ2 tests for independence and trend, respectively, or 2 x 2 χ2 test where Downloaded from https://academic.oup.com/qjmed/advance-article-abstract/doi/10.1093/qjmed/hcx243/4725112 by University of Tasmania Library user on 04 January 2018 appropriate) or 2 x 2 Fisher’s exact tests if the χ2 test was inappropriate. Forward, stepwise logistic regression analysis was carried out to obtain corrected P values for appropriate confounders. One-way analysis of variance (ANOVA) was used to test for statistical significance (two-tailed analysis) between experimental groups of three. Multiple comparisons between groups were then assessed using the Tukey-Kramer post-hoc test (for parametric analysis) or Dunn’s post-hoc test (for non-parametric analysis). Statistical significance was recorded at p<0.05. Downloaded from https://academic.oup.com/qjmed/advance-article-abstract/doi/10.1093/qjmed/hcx243/4725112 by University of Tasmania Library user on 04 January 2018