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. 2018 Apr 1;111(4):217-224.
doi: 10.1093/qjmed/hcx243.

Toll-like receptor 3 L412F polymorphism promotes a persistent clinical phenotype in pulmonary sarcoidosis

G Cooke  1 , I Kamal  2   3 , M Strengert  2 , E Hams  4   5 , L Mawhinney  4 , A Tynan  4 , C O'Reilly  4 , D N O'Dwyer  2   3 , S L Kunkel  6 , U G Knaus  2 , D C Shields  7 , D R Moller  8 , A G Bowie  9 , P G Fallon  4   5 , C M Hogaboam  6 , M E Armstrong  4 , S C Donnelly  4   10
Affiliations

Toll-like receptor 3 L412F polymorphism promotes a persistent clinical phenotype in pulmonary sarcoidosis

G Cooke et al. QJM. .

Abstract

Background/introduction: Sarcoidosis is a multi-systemic disorder of unknown etiology, characterized by the presence of non-caseating granulomas in target organs. In 90% 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.

Design: SNP-genotyping and cellular assays, respectively, were used to investigate the role of TLR3 L412F in the development of persistent pulmonary sarcoidosis.

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 apoptotic- and 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 412 F-homozygous pulmonary sarcoidosis patients resulted in reduced IFN-β and TLR3 expression, reduced apoptosis- and dysregulated fibroproliferative-responses compared with TLR3 wild-type patients.

Discussion/conclusion: 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.

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Figures

Figure 1
Figure 1
Effect of TLR3 L412F on TLR3-induced IL-8, IFN-β mRNA and TLR3 mRNA in Leu/Leu and Phe/Phe primary human lung fibroblasts from pulmonary sarcoidosis patients. TLR3 L412F attenuates Poly(I:C)-driven (A) IL-8 production, (B) IFN-β mRNA and (C) TLR3 mRNA expression in primary human lung fibroblasts from Phe/Phe sarcoidosis patients compared with Leu/Leu patients at 24 h post-treatment, as quantitated by ELISA and QPCR analysis, respectively. (A–C) *P < 0.05, **P < 0.01, ***P < 0.001: Poly(I:C) 100 μg/ml compared with Medium-only at 24 h post-treatment.  ++P < 0.01: Poly(I:C) 100 μg/ml in fibroblasts from Phe/Phe (n = 3) compared with Leu/Leu patients (n = 3). Results shown are the mean ± SEM of (B, C) three or (A) six replicates from a representative of (B) two or (A, C) three separate experiments.
Figure 2
Figure 2
Effect of TLR3 L412F on TLR3-induced apoptotic responses in Leu/Leu and Phe/Phe primary human lung fibroblasts from pulmonary sarcoidosis patients. A significant increase in Poly(I:C)-induced (A, B) late-phase apoptosis in Leu/Leu, but not Phe/Phe, fibroblasts following 24 h treatment, as quantitated by Annexin V/Propidium Iodide staining using FACS analysis. (B) *P < 0.05, Poly(I:C) 100 μg/ml compared with Medium-only.  ++P < 0.01: Treatment with 100 μg/ml Poly(I:C) in fibroblasts from Phe/Phe (n = 3) compared with Leu/Leu (n = 3). Results shown are the mean ± SEM of (B) six or (A) five replicates from a representative of (A, B) three separate experiments.
Figure 3
Figure 3
Effect of TLR3 L412F on TLR3-induced proliferative responses in Leu/Leu and Phe/Phe primary human lung fibroblasts from pulmonary sarcoidosis patients. (A) Poly(I:C) treatment significantly reduces proliferation of Leu/Leu fibroblasts following 24 h treatment with 1-100 μg/ml Poly(I:C), as quantitated using 3 H-thymidine incorporation. The fold-decrease in Poly(I:C)-induced proliferation at 100 μg/ml at 24 h is significantly more in Leu/Leu compared with Phe/Phe fibroblasts, as quantitated using 3 H-thymidine incorporation. (B) Reconstitution of Phe/Phe cells with recombinant human IFN-β (1000 IU/ml) leads to an equivalent reduction in cell proliferation in Leu/Leu and Phe/Phe cells. (A, B) *P < 0.05, **P < 0.01, ***P < 0.001: Poly(I:C) 100 μg/ml compared with Medium-only.  ++P < 0.01: Treatment with 100 μg/ml Poly(I:C) in fibroblasts from Phe/Phe (n = 3) compared with Leu/Leu patients (n = 3). Results shown are the mean ± SEM of (B) six or (A) five replicates from a representative of (A, B) three separate experiments.
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