Involvement of X-box binding protein 1 and reactive oxygen species pathways in the pathogenesis of tumour necrosis factor receptor-associated periodic syndrome.
OBJECTIVE
To investigate convergence of endoplasmic reticulum stress pathways and enhanced reactive oxygen species (ROS) production, due to intracellular retention of mutant tumour necrosis factor receptor 1 (TNFR1), as a disease mechanism in TNFR-associated periodic syndrome (TRAPS).


METHODS
Peripheral blood mononuclear cells from patients with TRAPS (n=16) and healthy controls (HC) (n=22) were studied alongside HEK293T cells expressing wild type-TNFR1 or TRAPS-associated mutations. Unfolded protein response (UPR)-associated proteins (protein kinase-like endoplasmic reticulum kinase, PERK), phosphorylated-PERK (p-PERK), phosphorylated inositol-requiring enzyme 1α (p-IRE1α) and spliced X-box binding protein 1 (sXBP1)) were measured by flow cytometry. XBP1 splicing and UPR-associated transcript expression were assessed by reverse transcription PCR/quantitative real-time PCR. ROS levels were measured using CM-H(2)DCFDA and MitoSOX Red 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 transcripts (p<0.01) compared with HC. Raised p-PERK protein was seen, indicative of an UPR, but other UPR-associated transcripts were normal. Increased ROS levels were observed in TRAPS monocytes compared with HCs (p<0.02); these increased further upon IL-6 stimulation (p<0.01). Lipopolysaccharide-stimulated peripheral blood mononuclear cells of patients with TRAPS, but not HCs, demonstrated increased sXBP1 levels (p<0.01), which were reduced by antioxidant treatment (p<0.05).


CONCLUSIONS
Patients with TRAPS have evidence of increased sXBP1 and PERK expression but without other signs of classical UPR, and also with high ROS generation that may contribute to the pro-inflammatory state associated with TRAPS. The authors propose a non-traditional XBP1 pathway with enhanced sXBP1 as a novel disease-contributing mechanism in TRAPS.
Experimental Rheumatology, National Institute for Health Research–Leeds Musculoskeletal Biomedical Research Unit, Leeds Institute of Molecular Medicine, Leeds, West Yorkshire, UK.
Laura J
LJ
Dickie
Azad M
AM
Aziz
Sinisa
S
Savic
Orso M
OM
Lucherini
Luca
L
Cantarini
Janina
J
Geiler
Chi H
CH
Wong
Robert
R
Coughlan
Thirusha
T
Lane
Helen J
HJ
Lachmann
Philip N
PN
Hawkins
Philip A
PA
Robinson
Paul
P
Emery
Dennis
D
McGonagle
Michael F
MF
McDermott
D000293:Adolescent; D000328:Adult; D000368:Aged; D017398:Alternative Splicing / Q000502:physiology; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D059865:Endoplasmic Reticulum Stress / Q000502:physiology*; D005260:Female; D057809:HEK293 Cells; D056660:Hereditary Autoinflammatory Diseases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D017382:Reactive Oxygen Species / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D056811:Unfolded Protein Response / Q000502:physiology; D000071717:X-Box Binding Protein 1; D055815:Young Adult; D019892:eIF-2 Kinase / Q000378:metabolism
D000975:Antioxidants; D004268:DNA-Binding Proteins; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D047888:Receptors, Tumor Necrosis Factor, Type I; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D000071717:X-Box Binding Protein 1; D019892:eIF-2 Kinase; C506235:TNFRSF1A protein, human; C000605543:XBP1 protein, human