Background TRAPS is caused by autosomal dominant mutations in the TNFR1 gene . Many TRAPS mutations are associated with reduced cell surface TNFR1 expression leading to the proposed hypothesis of impaired receptor trafficking , 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 , which is of relevance to understanding TRAPS pathogenesis as patients’ cells are hyper-responsive to LPS, a TLR4 agonist . Excessive reactive oxygen species (ROS) has also been linked to elevated proinflammatory cytokines in TRAPS .
Objectives To investigate convergence of ER stress pathways and enhanced ROS production as a disease mechanism in TRAPS.
Methods 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.
Results Mutant TNFR1-expressing HEK293T cells had increased TNFR1 expression associated with intracellular aggregation. TRAPS patients had increased sXBP1 transcript (p<0.01) compared to HC. Significantly increased PERK (p<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<0.02); these increased upon IL-6 stimulation (p<0.05) whereas HC monocytes only increased ROS in response to TNF (p<0.02). Only LPS-stimulated TRAPS PBMC demonstrated increased sXBP1 levels (p<0.01) which could be reduced with DPI antioxidant co-treatment (p<0.05).
Conclusions 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.
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Disclosure of Interest None Declared
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