Objective. Systemic juvenile idiopathic arthritis (JIA) is an immunoinflammatory disease characterized by arthritis and systemic manifestations. The role of natural killer (NK) cells in the pathogenesis of systemic JIA remains unclear. The purpose of this study was to perform a comprehensive analysis of NK cell phenotype and functionality in patients with systemic JIA.Methods. Transcriptional alterations specific to NK cells were investigated by RNA sequencing of highly purified NK cells from 6 patients with active systemic JIA and 6 age-matched healthy controls. Cytokines (NK cellstimulating and others) were quantified in plasma samples (n 5 18). NK cell phenotype and cytotoxic activity against tumor cells were determined (n 5 10), together with their interferon-g (IFNg)-producing function (n 5 8).Results. NK cells from the systemic JIA patients showed an altered gene expression profile compared to cells from the healthy controls, with enrichment of immunoinflammatory pathways, increased expression of innate genes including TLR4 and S100A9, and decreased expression of immune-regulating genes such as IL10RA and GZMK. In the patients' plasma, interleukin-18 (IL-18) levels were increased, and a decreased ratio of IFNg to IL-18 was observed. NK cells from the patients exhibited specific alterations in the balance of inhibitory and activating receptors, with decreased killer cell lectin-like receptor G1 and increased NKp44 expression. Although NK cells from the patients showed increased granzyme B expression, consistent with intact cytotoxicity and degranulation against a tumor cell line, decreased granzyme K expression in CD56 bright NK cells and defective IL-18-induced IFNg production and signaling were demonstrated.Conclusion. NK cells are active players in the inflammatory environment typical of systemic JIA. Although their cytotoxic function is globally intact, subtle defects in NK-related pathways, such as granzyme K expression and IL-18-driven IFNg production, may contribute to the immunoinflammatory dysregulation in this disease.Systemic juvenile idiopathic arthritis (JIA) is a chronic immunoinflammatory childhood disorder of unknown etiology that is characterized by arthritis and systemic features such as quotidian fever, rash, lymphadenopathy, and serositis (1,2). An interplay of environmental factors and genetic predisposition is considered to underlie the pathogenesis (1). About 10% of systemic JIA patients develop

Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. While many cancer drugs target genetic vulnerabilities, loss-of-function screens fail to identify essential genes in drug mechanism of action. Here, we report CRISPRres, a CRISPR-Cas-based genetic screening approach to rapidly derive and identify drug resistance mutations in essential genes. It exploits the local genetic variation created by CRISPR-Cas-induced non-homologous end-joining (NHEJ) repair to generate a wide variety of functional in-frame mutations. Using large sgRNA tiling libraries and known drug–target pairs, we validate it as a target identification approach. We apply CRISPRres to the anticancer agent KPT-9274 and identify nicotinamide phosphoribosyltransferase (NAMPT) as its main target. These results present a powerful and simple genetic approach to create many protein variants that, in combination with positive selection, can be applied to reveal the cellular target of small-molecule inhibitors.

IFN-stimulated gene 15 (ISG15) deficiency in humans leads to severe IFNopathies and mycobacterial disease, the latter being previously attributed to its extracellular cytokine-like activity. In this study, we demonstrate a novel role for secreted ISG15 as an IL-10 inducer, unique to primary human monocytes. A balanced ISG15-induced monocyte/IL-10 versus lymphoid/IFN-γ expression, correlating with p38 MAPK and PI3K signaling, was found using targeted in vitro and ex vivo systems analysis of human transcriptomic datasets. The specificity and MAPK/PI3K-dependence of ISG15-induced monocyte IL-10 production was confirmed in vitro using CRISPR/Cas9 knockout and pharmacological inhibitors. Moreover, this ISG15/IL-10 axis was amplified in leprosy but disrupted in human active tuberculosis (TB) patients. Importantly, ISG15 strongly correlated with inflammation and disease severity during active TB, suggesting its potential use as a biomarker, awaiting clinical validation. In conclusion, this study identifies a novel anti-inflammatory ISG15/IL-10 myeloid axis that is disrupted in active TB.