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 <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >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-1ß 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.

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

Background: Psoriasis 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. Objectives: The 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. Methods: PBMCs 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. Results: Numbers 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). Conclusion: DNA 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.

RIP2-deficiency induces inflammation in response to SV40 Large T induced genotoxic stress through altered ROS homeostasis.

Deciphering signaling pathways that regulate the complex interplay between inflammation and cell death is a key challenge in understanding innate immune responses. Over recent years, receptor interacting protein (RIP) kinases have been described to regulate the interplay between inflammation and cell death. Whereas RIP1 and 3, the most well described members of the RIP kinase family, play important roles in necroptosis, RIP2's involvement in regulating inflammation, cell death processes and cancer is less well described and controversially discussed. Here, we demonstrate that RIP2 exerts immune regulatory functions by regulating mitochondrial damage and mitochondrial superoxide production in response to SV40 LT-induced genotoxic stress by the induction of ULK1-phosphorylation, therefore regulating the expression of interferon stimulated genes (ISGs) and NLRP3-inflammasome dependent IL-1ß release. Because RIP2 is upregulated and/or activated in autoimmune/inflammatory disease and cancer, observations from this study promise implications of RIP kinases in human disease.

DNA Methylation Patterns in CD8+ T Cells Discern Psoriasis From Psoriatic Arthritis and Correlate With Cutaneous Disease Activity.

Background: Psoriasis is a T cell-mediated chronic autoimmune/inflammatory disease. While some patients experience disease limited to the skin (skin psoriasis), others develop joint involvement (psoriatic arthritis; PsA). In the absence of disease- and/or outcome-specific biomarkers, and as arthritis can precede skin manifestations, diagnostic and therapeutic delays are common and contribute to disease burden and damage accrual. Objective: Altered epigenetic marks, including DNA methylation, contribute to effector T cell phenotypes and altered cytokine expression in autoimmune/inflammatory diseases. This project aimed at the identification of disease-/outcome-specific DNA methylation signatures in CD8+ T cells from patients with psoriasis and PsA as compared to healthy controls. Method: Peripheral blood CD8+ T cells from nine healthy controls, 10 psoriasis, and seven PsA patients were collected to analyze DNA methylation marks using Illumina Human Methylation EPIC BeadChips (>850,000 CpGs per sample). Bioinformatic analysis was performed using R (minfi, limma, ChAMP, and DMRcate packages). Results: DNA methylation profiles in CD8+ T cells differentiate healthy controls from psoriasis patients [397 Differentially Methylated Positions (DMPs); 9 Differentially Methylated Regions (DMRs) when ≥CpGs per DMR were considered; 2 DMRs for ≥10 CpGs]. Furthermore, patients with skin psoriasis can be discriminated from PsA patients [1,861 DMPs, 20 DMRs (≥5 CpGs per region), 4 DMRs (≥10 CpGs per region)]. Gene ontology (GO) analyses considering genes with ≥1 DMP in their promoter delivered methylation defects in skin psoriasis and PsA primarily affecting the BMP signaling pathway and endopeptidase regulator activity, respectively. GO analysis of genes associated with DMRs between skin psoriasis and PsA demonstrated an enrichment of GABAergic neuron and cortex neuron development pathways. Treatment with cytokine blockers associated with DNA methylation changes [2,372 DMPs; 1,907 DMPs within promoters, 7 DMRs (≥5 CpG per regions)] affecting transforming growth factor beta receptor and transmembrane receptor protein serine/threonine kinase signaling pathways. Lastly, a methylation score including TNF and IL-17 pathway associated DMPs inverse correlates with skin disease activity scores (PASI). Conclusion: Patients with skin psoriasis exhibit DNA methylation patterns in CD8+ T cells that allow differentiation from PsA patients and healthy individuals, and reflect clinical activity of skin disease. Thus, DNA methylation profiling promises potential as diagnostic and prognostic tool to be used for molecular patient stratification toward individualized treatment.

Cyclic AMP Response Element Modulator-α Suppresses PD-1 Expression and Promotes Effector CD4+ T Cells in Psoriasis.

Effector CD4+ T lymphocytes contribute to inflammation and tissue damage in psoriasis, but the underlying molecular mechanisms remain poorly understood. The transcription factor CREMα controls effector T cell function in people with systemic autoimmune diseases. The inhibitory surface coreceptor PD-1 plays a key role in the control of effector T cell function and its therapeutic inhibition in patients with cancer can cause psoriasis. In this study, we show that CD4+ T cells from patients with psoriasis and psoriatic arthritis exhibit increased production of IL-17 but decreased expression of IL-2 and PD-1. In genetically modified mice and Jurkat T cells CREMα expression was linked to low PD-1 levels. We demonstrate that CREMα is recruited to the proximal promoter of PDCD1 in which it trans-represses gene expression and corecruits DNMT3a-mediating DNA methylation. As keratinocytes limit inflammation by PD-1 ligand expression and, in this study, reported reduced expression of PD-1 on CD4+ T cells is linked to low IL-2 and high IL-17A production, our studies reveal a molecular pathway in T cells from people with psoriasis that can deserve clinical exploitation.

CASP1 variants influence subcellular caspase-1 localization, pyroptosome formation, pro-inflammatory cell death and macrophage deformability.

CASP1 variants result in reduced enzymatic activity of procaspase-1 and impaired IL-1ß release. Despite this, affected individuals can develop systemic autoinflammatory disease. These seemingly contradictory observations have only partially been explained by increased NF-κB activation through prolonged interaction of variant procaspase-1 with RIP2. To identify further disease underlying pathomechanisms, we established an in vitro model using shRNA-directed knock-down of procaspase-1 followed by viral transduction of human monocytes (THP-1) with plasmids encoding for wild-type procaspase-1, disease-associated CASP1 variants (p.L265S, p.R240Q) or a missense mutation in the active center of procaspase-1 (p.C285A). THP1-derived macrophages carrying CASP1 variants exhibited mutation-specific molecular alterations. We here provide in vitro evidence for abnormal pyroptosome formation (p.C285A, p.240Q, p.L265S), impaired nuclear (pro)caspase-1 localization (p.L265S), reduced pro-inflammatory cell death (p.C285A) and changes in macrophage deformability that may contribute to disease pathophysiology of patients with CASP1 variants. This offers previously unknown molecular pathomechanisms in patients with systemic autoinflammatory disease.

Enzymatically Inactive Procaspase 1 stabilizes the ASC Pyroptosome and Supports Pyroptosome Spreading during Cell Division.

Caspase-1 is a key player during the initiation of pro-inflammatory innate immune responses, activating pro-IL-1ß in so-called inflammasomes. A subset of patients with recurrent febrile episodes and systemic inflammation of unknown origin harbor mutations in CASP1 encoding caspase-1. CASP1 variants result in reduced enzymatic activity of caspase-1 and impaired IL-1ß secretion. The apparent paradox of reduced IL-1ß secretion but systemic inflammation led to the hypothesis that CASP1 mutations may result in variable protein interaction clusters, thus activating alternative signaling pathways. To test this hypothesis, we established and characterized an in vitro system of transduced immortalized murine macrophages expressing either WT or enzymatically inactive (p.C284A) procaspase-1 fusion reporter proteins. Macrophages with variant p.C284A caspase-1 did not secrete IL-1ß and exhibited reduced inflammatory cell death, referred to as pyroptosis. Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) formed cytosolic macromolecular complexes (so-called pyroptosomes) that were significantly increased in number and size in cells carrying the p.C284A caspase-1 variant compared with WT caspase-1. Furthermore, enzymatically inactive caspase-1 interacted with ASC longer and with increased intensity compared with WT caspase-1. Applying live cell imaging, we documented for the first time that pyroptosomes containing enzymatically inactive variant p.C284A caspase-1 spread during cell division. In conclusion, variant p.C284A caspase-1 stabilizes pyroptosome formation, potentially enhancing inflammation by two IL-1ß-independent mechanisms: pyroptosomes convey an enhanced inflammatory stimulus through the recruitment of additional proteins (such as RIP2, receptor interacting protein kinase 2), which is further amplified through pyroptosome and cell division.