The role of redox regulation in immune-mediated arthritis has been previously described. However, the relationship between innate immune cells, including innate lymphoid cells (ILCs) and phagocyte-derived ROS, in this process remains unclear. Here, we characterize ILCs and measure the IL-1 family cytokines along with other cytokines relevant to ILC functions and development in serum-induced arthritic joints in wild type and phagocytic NADPH oxidase (NOX2)-deficient Ncf1 −/− mice. We found more severe serum-induced joint inflammation and increased NCR + ILC3s in inflamed joints of Ncf1 −/− mice. Furthermore, in vitro stimulation with IL-1β on Tbet + ILC1s from joints facilitated their differentiation into ROR-γt + ILC3s. Moreover, treatment with IL-1 antagonists effectively lowered the proportions of NCR + ILC3s and IL-17A producing ILC3s in Ncf1 −/− arthritic mice and ameliorated the joint inflammation. These results suggest that NOX2 is an essential reg- ulator of ILC transdifferentiation and may mediate this process in a redox-dependent manner through IL-1β production in the inflammatory joint. Our findings shed important light on the role of ILCs in the initiation and progression in tissue inflammation and delineate a novel innate immune cell-mediated pathogenic mechanism through which redox regulation may determine the direction of immune responses in joints.Keywords: chronic granulomatous disease r innate lymphoid cells r NADPH oxidase r reactive oxygen species r serum-induced arthritis Additional supporting information may be found online in the Supporting Information section at the end of the article.
Immune-mediated arthritis is an important chronic inflammatory disease of joints causing debilitating morbidity in affected patients. The mechanisms underlying immune-mediated arthritis have been intensively investigated, however the cellular and molecular factors contributing to the joint inflammation in different redox conditions have not been clearly elucidated. Previous research showed that phagocyte-produced reactive oxygen species (ROS) plays an anti-inflammatory role in K/BxN serum-transfer arthritis and NOX2-deficient mice tend to have more severe arthritis. Although many leukocytes play critical roles in the development of immune-mediated arthritis, the role of neutrophils, which are the main producers of ROS in inflammation, is still controversial. We hence assessed the immunomodulatory function of neutrophils from arthritic joints of NOX2-deficient and wild type mice in this study. We found more neutrophils accumulation in NOX2-deficient inflamed joints. RNA-sequencing and quantitative PCR revealed significantly increased expression of acute inflammation genes including IL1b, Cxcl2, Cxcl3, Cxcl10 and Mmp3 in activated neutrophils from the inflamed joints of NOX2-deficient mice. Moreover, gene set enrichment analysis (GSEA) showed enriched gene signatures in type I and II IFN responses, IL-6-JAK-STAT3 signaling pathway and TNF-α signaling pathway via NF-κB in NOX2-deficient neutrophils. In addition, we found that NOX2-deficient neutrophils expressed lower levels of PD-L1 and were less suppressive than WT neutrophils. Moreover, treatment of PD-L1-Fc decreased cytokine expression and ameliorated the severity of inflammatory arthritis. Our results suggest that NOX2-derived ROS is critical for regulating the function and gene expression in arthritic neutrophils. Both the strong pro-inflammatory and weakened anti-inflammatory functions of neutrophils due to abnormal redox regulation may be targets of treatment for immune-mediated arthritis.
Reactive oxygen species (ROS) are produced by cells to play important roles in the effector and regulatory functions of immune cells. Our previous studies revealed that ROS act as negative regulators in IL-1β-mediated inflammation. Enhanced severity of inflammation in immune-mediated arthritis was noted when leukocyte NADPH oxidase (NOX2), which mediates the production of ROS, was defective. We hence hypothesized that drugs that modulate the leukocyte ROS production may regulate the pathogenicity of immune-mediated arthritis. Here, we devised a high-content image assay using the dye H2DCF-DA for identifying LOPAC 1280 library compounds that may facilitate or suppress superoxide production in differentiated HL-60 (dHL-60) cells. ROS levels were measured after the cells were treated with phorbol 12-myristate 13-acetate (PMA) or menadione which may induce ROS production from NOX2 or mitochondria. From a screening of 640 compounds in a 3-point interpolate titration in cell-based imaging analysis, we identified 74 drugs that may enhance the ROS production and 72 compounds that may inhibit ROS production by PMA-stimulated-dHL-60 cells. The drugs that we identified included the agonist/antagonist of neurotransmitters and their receptors, ion channel blockers, serotonin antagonists, cell stress modulators and cell cycle inhibitors. To further clarified our hypothesis, we treated NOX2-deficient mice with menadione, which tended to increase the oxidant stress in the joint tissue. The severity of the immune-mediated arthritic joint was significantly suppressed after the treatment. This novel screening method hence may be used to identify potential drugs for treating redox-related inflammatory diseases.
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