The proinflammatory enzyme caspase-1 plays an important role in the innate immune system and is involved in a variety of inflammatory conditions. Rare naturally occurring human variants of the caspase-1 gene (CASP1) lead to different protein expression and structure and to decreased or absent enzymatic activity. Paradoxically, a significant number of patients with such variants suffer from febrile episodes despite decreased IL-1β production and secretion. In this study, we investigate how variant (pro)caspase-1 can possibly contribute to inflammation. In a transfection model, such variant procaspase-1 binds receptor interacting protein kinase 2 (RIP2) via Caspase activation and recruitment domain (CARD)/CARD interaction and thereby activates NF-κB, whereas wild-type procaspase-1 reduces intracellular RIP2 levels by enzymatic cleavage and release into the supernatant. We approach the protein interactions by coimmunoprecipitation and confocal microscopy and show that NF-κB activation is inhibited by anti–RIP2-short hairpin RNA and by the expression of a RIP2 CARD-only protein. In conclusion, variant procaspase-1 binds RIP2 and thereby activates NF-κB. This pathway could possibly contribute to proinflammatory signaling.

Effector CD4 + T cells with increased IL-17A and reduced IL-2 production contribute to tissue inflammation and organ damage in systemic lupus erythematosus (SLE). Increased expression of the transcription factor cAMP response element modulator (CREM) a promotes altered cytokine expression in SLE. The aim of this study was to investigate CREMa-mediated events favoring effector CD4 + T cells in health and disease. Using CRISPR/Cas9 genome editing and lentiviral transduction, we generated CREMa-deficient and CREMa-overexpressing Jurkat T cells. Gene expression and regulatory events were assessed using luciferase reporter assays and chromatin immunoprecipitation. Interaction between CREMa and p300 was investigated using proximity ligation assays, coimmunoprecipitation, and knockdown of p300. Gene expression profiles of modified cells were compared with CD4 + T cells from patients with juvenile-onset SLE. We show that CREMa induces dual specificity protein phosphatase (DUSP) 4 in effector CD4 + T cells through corecruitment of p300. The transcriptional coactivator p300 mediates histone acetylation at DUSP4, prompting increased gene expression. Using DUSP4 transfection models and genetically modified CREMdeficient and CREMa-overexpressing T cells, we demonstrate the molecular underpinnings by which DUSP4 induces IL-17A while limiting IL-2 expression. We demonstrate that CD4 + T cells from patients with juvenile-onset SLE share phenotypical features with CREMa-overexpressing CD4 + T cells, including increased DUSP4 expression and imbalanced IL-17A and IL-2 production. Taken together, we describe CREMa-mediated mechanisms that involve the transcriptional upregulation of DUSP4, leading to imbalanced cytokine production by effector T cells. Our findings identify the CREMa/DUSP4 axis as a promising candidate in the search for biomarkers and therapeutic targets in SLE.

Caspase-1 is a key player during the initiation of pro-inflammatory innate immune responses, activating pro-IL-1␤ in socalled 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-1␤ secretion. The apparent paradox of reduced IL-1␤ 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-1␤ 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-1␤-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.Caspase-1 is a key player during innate immune responses. It aids to control pathogens and danger signals by triggering pyroptotic cell death. Together with caspase-11 and -12 in mice and caspase-4, -5, and -12 in humans, caspase-1 belongs to the family of inflammatory caspases, which are produced as inactive forms. After the detection of pathogens or cytoplasmic danger signals, inactive caspase-1 monomers recruit to multiprotein complexes referred to as inflammasomes (1-3). In such inflammasomes, inactive procaspases are autoprocessed into caspase activation and recruitment domains (CARDs), 2 the p10 and the p20 subunits (4). Two p10 and two p20 proteins subsequently form the active caspase-1 tetramer, which then cleaves and thereby activates IL-1␤ in the so-called canonical inflammasome pathway (5, 6). Furthermore, inflammatory caspases induce pro-inflammatory cell death, referred to as pyroptosis, which involves cell swelling, cell lysis, and the release of cytoplasmic contents (7,8). Pyroptosis depends on the activation of caspase-4, -5, or -11 and caspase-1 (9 -11). Caspase-1 or -11 cleaves gasdermin D, a gasdermin domain-containing protein, at amin...

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.