Inositol-Requiring Enzyme 1-Mediated Downregulation of MicroRNA (miR)-146a and miR-155 in Primary Dermal Fibroblasts across Three TNFRSF1A Mutations Results in Hyperresponsiveness to Lipopolysaccharide.

Stephanie R Harrison,Miriam Wittmann,Chi Wong,Thomas Scambler,Lylia Oubussad,Sinisa Savic,Michael F Mcdermott

doi:10.3389/fimmu.2018.00173

Abstract

Tumor necrosis factor (TNF)-receptor-associated periodic fever syndrome (TRAPS) is a rare monogenic autoinflammatory disorder characterized by mutations in the TNFRSF1A gene, causing TNF-receptor 1 (TNFR1) misfolding, increased cellular stress, activation of the unfolded protein response (UPR), and hyperresponsiveness to lipopolysaccharide (LPS). Both microRNA (miR)-146a and miR-155 provide negative feedback for LPS-toll-like receptor 2/4 signaling and cytokine production, through regulation of nuclear factor kappa B (NF-κB). In this study, we hypothesized that proinflammatory cytokine signaling in TRAPS downregulates these two miRs, resulting in LPS-induced hyperresponsiveness in TRAPS dermal fibroblasts (DFs), irrespective of the underlying genetic mutation. Primary DF were isolated from skin biopsies of TRAPS patients and healthy controls (HC). TNFR1 cell surface expression was measured using immunofluorescence. DF were stimulated with LPS, interleukin (IL)-1β, thapsigargin, or TNF, with and without inositol-requiring enzyme 1 (IRE1) inhibitor (4u8C), following which miR-146a and miR-155 expression was measured by RT-qPCR. IL-1β, IL-6, and TNF secretion was measured by enzyme-linked immunosorbent assays, and baseline expression of 384 different miRs was assessed using microfluidics assays. TNFR1 was found to be expressed on the surface of HC DF but expression was deficient in all samples with TRAPS-associated mutations. HC DF showed significant dose-dependent increases in both miR-146a and miR-155 expression levels in response to LPS; however, TRAPS DF failed to upregulate either miR-146a or miR-155 under the same conditions. This lack of miR-146a and miR-155 upregulation was associated with increased proinflammatory cytokine production in TRAPS DF in response to LPS challenge, which was abrogated by 4u8C. Incubation of HC DF with IL-1β led to downregulation of miR-146a and miR-155 expression, which was dependent on IRE1 enzyme. We observed global dysregulation of hundreds of other miRs at baseline in the TRAPS DF. In summary, these data suggest a mechanism whereby IL-1β, produced in response to activation of the UPR in TRAPS DF, downregulates miR-146a and miR-155, by inducing IRE1-dependent cleavage of both these miRs, thereby impairing negative regulation of NF-κB and increasing proinflammatory cytokine production.

Highlights

Tumor necrosis factor (TNF)-receptor-associated periodic fever syndrome (TRAPS) is a rare monogenic autoinflammatory disorder characterized by recurrent attacks of fever and inflammation, predominantly affecting fibroblast-rich tissues such as serosal surfaces, synovial joints, and the deep dermis of the skin [1]
Healthy human dermal fibroblast (DF) regulate inflammatory responses by expressing various cell surface receptors, including toll-like receptor 4 (TLR4) [25]; to determine if TRAPS DF continue to express TLR4 in vitro, and successfully respond to LPS stimulation, we studied the expression of TLR4 on the cell surface in primary DF, from HC (n = 3) and from TRAPS patients, respectively, bearing T50M, C88R, and c.472 + 1 slice site TNFRSF1A mutation
Only one study has reported on TNF-receptor 1 (TNFR1) expression in primary DF, from TRAPS patients bearing the C33Y mutation, which found reduced levels of sTNFR1 and reduced receptor shedding when compared with HC [27, 28]

Summary

Introduction

Tumor necrosis factor (TNF)-receptor-associated periodic fever syndrome (TRAPS) is a rare monogenic autoinflammatory disorder characterized by recurrent attacks of fever and inflammation, predominantly affecting fibroblast-rich tissues such as serosal surfaces, synovial joints, and the deep dermis of the skin [1]. A number of molecular mechanisms have been implicated in the pathogenesis of TRAPS, including abnormal TNF-receptor 1 (TNFR1) receptor cleavage [3], ligand-independent activation of mutant TNFR1 [4,5,6], activation of nuclear factor kappa B (NF-κB)/mitogenactivated protein kinase [6,7,8,9,10], generation of mitochondrial reactive oxygen species [7, 11], and TNFR1 misfolding and retention within the endoplasmic reticulum (ER), leading to activation of the unfolded protein response (UPR) [7] This latter cellular stress response appears to be limited to selective activation of the ER-associated endonuclease, inositol-requiring enzyme 1 (IRE1) [8, 12]; this activation can have significant proinflammatory effects as shown by Martinon et al They demonstrated that lipopolysaccharide (LPS) can activate IRE1, via the TLR4 signaling pathway. These findings corroborate the observations that TRAPS cells are hyperresponsiveness to low-dose LPS, with increased production of proinflammatory cytokines, the release of which leads to clinical manifestations persisting for periods of weeks to months [1, 15, 16]

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Results

Conclusion