Update: Cytokine Dysregulation in Chronic Nonbacterial Osteomyelitis (CNO)

Chronic nonbacterial osteomyelitis (CNO) with its most severe form chronic recurrent multifocal osteomyelitis (CRMO) is a non-bacterial osteitis of yet unknown origin. Secondary to the absence of both high-titer autoantibodies and autoreactive T lymphocytes, and the association with other autoimmune diseases, it was recently reclassified as an autoinflammatory disorder of the musculoskeletal system. Since its etiology is largely unknown, the diagnosis is based on clinical criteria, and treatment is empiric and not always successful. In this paper, we summarize recent advances in the understanding of possible etiopathogenetic mechanisms in CNO

Update: cytokine dysregulation in chronic nonbacterial osteomyelitis (CNO

Chronic nonbacterial osteomyelitis (CNO) with its most severe form chronic recurrent multifocal osteomyelitis (CRMO) is a non-bacterial osteitis of yet unknown origin. Secondary to the absence of both high-titer autoantibodies and autoreactive T lymphocytes, and the association with other autoimmune diseases, it was recently reclassified as an autoinflammatory disorder of the musculoskeletal system. Since its etiology is largely unknown, the diagnosis is based on clinical criteria, and treatment is empiric and not always successful. In this paper, we summarize recent advances in the understanding of possible etiopathogenetic mechanisms in CNO

DNA methylation patterns in CD4+ T-cells separate psoriasis patients from healthy controls, and skin psoriasis from psoriatic arthritis

BackgroundPsoriasis is an autoimmune/inflammatory disorder primarily affecting the skin. Chronic joint inflammation triggers the diagnosis of psoriatic arthritis (PsA) in approximately one-third of psoriasis patients. Although joint disease typically follows the onset of skin psoriasis, in around 15% of cases it is the initial presentation, which can result in diagnostic delays. The pathophysiological mechanisms underlying psoriasis and PsA are not yet fully understood, but there is evidence pointing towards epigenetic dysregulation involving CD4+ and CD8+ T-cells.ObjectivesThe aim of this study was to investigate disease-associated DNA methylation patterns in CD4+ T-cells from psoriasis and PsA patients that may represent potential diagnostic and/or prognostic biomarkers.MethodsPBMCs were collected from 12 patients with chronic plaque psoriasis and 8 PsA patients, and 8 healthy controls. CD4+ T-cells were separated through FACS sorting, and DNA methylation profiling was performed (Illumina EPIC850K arrays). Bioinformatic analyses, including gene ontology (GO) and KEGG pathway analysis, were performed using R. To identify genes under the control of interferon (IFN), the Interferome database was consulted, and DNA Methylation Scores were calculated.ResultsNumbers and proportions of CD4+ T-cell subsets (naïve, central memory, effector memory, CD45RA re-expressing effector memory cells) did not vary between controls, skin psoriasis and PsA patients. 883 differentially methylated positions (DMPs) affecting 548 genes were identified between controls and “all” psoriasis patients. Principal component and partial least-squares discriminant analysis separated controls from skin psoriasis and PsA patients. GO analysis considering promoter DMPs delivered hypermethylation of genes involved in “regulation of wound healing, spreading of epidermal cells”, “negative regulation of cell-substrate junction organization” and “negative regulation of focal adhesion assembly”. Comparing controls and “all” psoriasis, a majority of DMPs mapped to IFN-related genes (69.2%). Notably, DNA methylation profiles also distinguished skin psoriasis from PsA patients (2,949 DMPs/1,084 genes) through genes affecting “cAMP-dependent protein kinase inhibitor activity” and “cAMP-dependent protein kinase regulator activity”. Treatment with cytokine inhibitors (IL-17/TNF) corrected DNA methylation patterns of IL-17/TNF-associated genes, and methylation scores correlated with skin disease activity scores (PASI).ConclusionDNA methylation profiles in CD4+ T-cells discriminate between skin psoriasis and PsA. DNA methylation signatures may be applied for quantification of disease activity and patient stratification towards individualized treatment

Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)-LRR oligomerization interface

Background Monogenic autoinflammatory disorders are characterized by dysregulation of the innate immune system, for example by gain-of-function mutations in inflammasome-forming proteins, such as NOD-like receptor family CARD-containing 4 protein (NLRC4). Objective Here we investigate the mechanism by which a novel mutation in the leucine-rich repeat (LRR) domain of NLRC4 (c.G1965C, p.W655C) contributes to autoinflammatory disease. Methods: We studied 2 unrelated patients with early-onset macrophage activation syndrome harboring the same de novo mutation in NLRC4. In vitro inflammasome complex formation was quantified by using flow cytometric analysis of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 techniques and lentiviral transduction were used to generate THP-1 cells with either wild-type or mutant NLRC4 cDNA. Cell death and release of IL-1β/IL-18 were quantified by using flow cytometry and ELISA, respectively. Results The p.W655C NLRC4 mutation caused increased ASC speck formation, caspase-1–dependent cell death, and IL-1β/IL-18 production. ASC contributed to p.W655C NLRC4–mediated cytokine release but not cell death. Mutation of p.W655 activated the NLRC4 inflammasome complex by engaging with 2 interfaces on the opposing LRR domain of the oligomer. One key set of residues (p.D1010, p.D1011, p.L1012, and p.I1015) participated in LRR-LRR oligomerization when triggered by mutant NLRC4 or type 3 secretion system effector (PrgI) stimulation of the NLRC4 inflammasome complex. Conclusion This is the first report of a mutation in the LRR domain of NLRC4 causing autoinflammatory disease. c.G1965C/p.W655C NLRC4 increased inflammasome activation in vitro. Data generated from various NLRC4 mutations provides evidence that the LRR-LRR interface has an important and previously unrecognized role in oligomerization of the NLRC4 inflammasome complex

P2RX7 gene variants associate with altered inflammasome assembly and reduced pyroptosis in chronic nonbacterial osteomyelitis (CNO).

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF 20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1β and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care

De novoCIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (NOMID): A new member of the expanding family of pyrin-associated autoinflammatory diseases

Neonatal-onset multisystem inflammatory disease (NOMID; also known as chronic infantile neurologic, cutaneous, articular [CINCA] syndrome) is characterized by fever, chronic meningitis, uveitis, sensorineural hearing loss, urticarial skin rash, and a characteristic deforming arthropathy. We investigated whether patients with this disorder have mutations in CIAS1, the gene which causes Muckle-Wells syndrome and familial cold autoinflammatory syndrome, two dominantly inherited disorders with some similarities to NOMID/CINCA syndrome