OP0256 Involvement of unfolded protein response and reactive oxygen species pathways in the pathogenesis of tumour necrosis factor receptor- associated periodic syndrome (TRAPS)
Authors: L.J. Dickie, A.M. Aziz, S. Savic, P. Emery, D. McGonagle, M.F. McDermott
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background</h3> TRAPS is caused by autosomal dominant mutations in the TNFR1 gene [1]. Many TRAPS mutations are associated with reduced cell surface TNFR1 expression leading to the proposed hypothesis of impaired receptor trafficking [2], which may cause an unfolded protein response (UPR). There are 3 main signalling cascades activated in a classical UPR through the proteins PERK, IRE1α and ATF6. Spliced XBP1 (sXBP1), downstream of IRE1α has been described to enhance TLR4 responses [3], which is of relevance to understanding TRAPS pathogenesis as patients' cells are hyper-responsive to LPS, a TLR4 agonist [4]. Excessive reactive oxygen species (ROS) has also been linked to elevated proinflammatory cytokines in TRAPS [5]. <h3>Objectives</h3> To investigate convergence of ER stress pathways and enhanced ROS production as a disease mechanism in TRAPS. <h3>Methods</h3> PBMC from TRAPS patients (n=16) and healthy controls (HC) (n=22) were studied alongside HEK293T cells expressing wild type (WT)-TNFR1 or TRAPS-associated mutations. UPR-associated proteins PERK, phosphorylated-PERK (p-PERK), phosphorylated-IRE1α (p-IRE1α) and sXBP1 were measured by flow cytometry. XBP1 splicing and UPR–associated transcript expression was assessed by qRT-PCR. ROS levels were measured using CM-H2DCFDA and MitoSOX Red dyes in patients' monocytes or HEK293T cells by flow cytometry. <h3>Results</h3> Mutant TNFR1-expressing HEK293T cells had increased TNFR1 expression associated with intracellular aggregation. TRAPS patients had increased s<i>XBP1</i> transcript (p&lt;0.01) compared to HC. Significantly increased PERK (p&lt;0.01) and raised p-PERK protein was seen in TRAPS monocytes, yet other UPR-associated transcripts were normal. High ROS levels were observed in TRAPS monocytes compared to HC (p&lt;0.02); these increased upon IL-6 stimulation (p&lt;0.05) whereas HC monocytes only increased ROS in response to TNF (p&lt;0.02). Only LPS-stimulated TRAPS PBMC demonstrated increased <i>sXBP1</i> levels (p&lt;0.01) which could be reduced with DPI antioxidant co-treatment (p&lt;0.05). <h3>Conclusions</h3> The TRAPS UPR was associated with increased sXBP1 and PERK but without other signs of a classical UPR, and also with high ROS generation that may contribute to the pro-inflammatory state associated with TRAPS. We propose that a UPR response, mediated by sXBP1, is a novel disease-contributing mechanism in TRAPS. <h3>References</h3> McDermott MF et al. Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell 1999;97:133-144. Lobito AA et al. Abnormal disulfide-linked oligomerization results in ER retention and altered signaling by TNFR1 mutants in TNFR1-associated periodic fever syndrome (TRAPS). Blood 2006;108:1320-1327. Martinon F et al. TLR activation of the transcription factor XBP1 regulates innate immune responses in macrophages. Nat Immunol 2010;11:411-418. Simon A, Park H, Maddipati R, et al. Concerted action of wild-type and mutant TNF receptors enhances inflammation in TNF receptor 1-associated periodic fever syndrome. Proc Natl Acad Sci U S A 2010;107:9801-9806. Bulua AC et al. Mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in TNFR1-associated periodic syndrome (TRAPS). J Exp Med 2011;208:519-533. <h3>Disclosure of Interest</h3> None Declared