Human MHC Class II and Invariant Chain Knock-in Mice Mimic Rheumatoid Arthritis with Allele Restriction in Immune Response and Arthritis Association.

Transgenic mice expressing human major histocompatibility complex class II (MHCII) risk alleles are widely used in autoimmune disease research, but limitations arise due to non-physiologic expression. To address this, physiologically relevant mouse models are established via knock-in technology to explore the role of MHCII in diseases like rheumatoid arthritis. The gene sequences encoding the ectodomains are replaced with the human DRB1*04:01 and 04:02 alleles, DRA, and CD74 (invariant chain) in C57BL/6N mice. The collagen type II (Col2a1) gene is modified to mimic human COL2. Importantly, DRB1*04:01 knock-in mice display physiologic expression of human MHCII also on thymic epithelial cells, in contrast to DRB1*04:01 transgenic mice. Humanization of the invariant chain enhances MHCII expression on thymic epithelial cells, increases mature B cell numbers in spleen, and improves antigen presentation. To validate its functionality, the collagen-induced arthritis (CIA) model is used, where DRB1*04:01 expression led to a higher susceptibility to arthritis, as compared with mice expressing DRB1*04:02. In addition, the humanized T cell epitope on COL2 allows autoreactive T cell-mediated arthritis development. In conclusion, the humanized knock-in mouse faithfully expresses MHCII, confirming the DRB1*04:01 alleles role in rheumatoid arthritis and being also useful for studying MHCII-associated diseases.

Pathogenic antibody response to glucose-6-phosphate isomerase targets a modified epitope uniquely exposed on joint cartilage.

OBJECTIVES: To identify the arthritogenic B cell epitopes of glucose-6-phosphate isomerase (GPI) and their association with rheumatoid arthritis (RA). METHODS: IgG response towards a library of GPI peptides in patients with early RA, pre-symptomatic individuals and population controls, as well as in mice, were tested by bead-based multiplex immunoassays and ELISA. Monoclonal IgG were generated, and the binding specificity and affinity were determined by ELISA, gel size exclusion chromatography, surface plasma resonance and X-ray crystallography. Arthritogenicity was investigated by passive transfer experiments. Antigen-specific B cells were identified by peptide tetramer staining. RESULTS: Peptide GPI293-307 was the dominant B cell epitope in K/BxN and GPI-immunised mice. We could detect B cells and low levels of IgM antibodies binding the GPI293-307 epitopes, and high affinity anti-GPI293-307 IgG antibodies already 7 days after GPI immunisation, immediately before arthritis onset. Transfer of anti-GPI293-307 IgG antibodies induced arthritis in mice. Moreover, anti-GPI293-307 IgG antibodies were more frequent in individuals prior to RA onset (19%) than in controls (7.5%). GPI293-307-specific antibodies were associated with radiographic joint damage. Crystal structures of the Fab-peptide complex revealed that this epitope is not exposed in native GPI but requires conformational change of the protein in inflamed joint for effective recognition by anti-GPI293-307 antibodies. CONCLUSIONS: We have identified the major pathogenic B cell epitope of the RA-associated autoantigen GPI, at position 293-307, exposed only on structurally modified GPI on the cartilage surface. B cells to this neo-epitope escape tolerance and could potentially play a role in the pathogenesis of RA.

Autoantibodies to Disease-Related Proteins in Joints as Novel Biomarkers for the Diagnosis of Rheumatoid Arthritis.

OBJECTIVE: This study was undertaken to develop and characterize a multiplex immunoassay for detection of autoantibodies against peptides derived from proteins known to play a role in development of arthritis and that are also expressed in joints. METHODS: We selected peptides from the human counterpart of proteins expressed in the joints, based on mouse models that showed these to be targeted by pathogenic or regulatory antibodies in vivo. Using bead-based flow immunoassays measuring IgG antibodies, we selected triple helical or cyclic peptides, containing the epitopes, to avoid collinear reactivity. We characterized the analytical performance of the immunoassay and then validated it in 3 independent rheumatoid arthritis (RA) cohorts (n = 2,110), Swedish age- and sex-matched healthy controls, and patients with osteoarthritis (OA), patients with psoriatic arthritis (PsA), and patients with systemic lupus erythematosus (SLE). RESULTS: Screening assays showed 5 peptide antigens that discriminated RA patients from healthy controls with 99% specificity (95% confidence interval [CI] 98-100%). In our validation studies, we reproduced the discriminatory capacity of the autoantibodies in 2 other RA cohorts, showing that the autoantibodies had high discriminatory capacity for RA versus OA, PsA, and SLE. The novel biomarkers identified 22.5% (95% CI 19-26%) of early RA patients seronegative for anti-cyclic citrullinated peptide and rheumatoid factor. The usefulness of the biomarkers in identifying seronegative RA patients was confirmed in validation studies using 2 independent cohorts of RA patients and cohorts of patients with OA, PsA, and SLE. CONCLUSION: A multiplex immunoassay with peptides from disease-related proteins in joints was found to be useful for detection of specific autoantibodies in RA serum. Of note, this immunoassay had high discriminatory capacity for early seronegative RA.

Early Increase of Circulating Transitional B Cells and Autoantibodies to Joint-Related Proteins in Patients With Metastatic Melanoma Developing Checkpoint Inhibitor-Induced Inflammatory Arthritis.

OBJECTIVE: To investigate potential associations between B cell-related immunologic changes and development of inflammatory arthritis (IA) after treatment with immune checkpoint inhibitors (ICIs). METHODS: Patients who developed ICI-induced IA (ICI-IA) and patients who did not develop immune-related adverse events (non-IRAE) after receiving ICIs to treat metastatic melanoma were consecutively recruited. Blood samples were collected at the time of ICI-IA occurrence and at different time points during treatment. Peripheral blood B cell subsets during ICI treatment were analyzed by flow cytometry. Rheumatoid factor, anti-citrullinated protein antibodies, and antibodies against joint-related proteins were measured. RESULTS: Proportions of CD19+ B cells were higher in patients with ICI-IA (n = 7) compared to patients with non-IRAE (n = 15) (median 11.7% [interquartile range (IQR) 9.7-16.2%] versus 8.1% [IQR 5.7-11.0%]; P = 0.03). The proportion and absolute numbers of transitional CD19+CD10+CD24high CD38high B cells were increased in patients with ICI-IA compared to non-IRAE patients (median 8.1% [IQR 4.9-12.1%] versus 3.6% [IQR 1.9-4.9%]; median 10.7 cells/µl [IQR 8.9-19.6] versus 4.4 cells/µl [IQR 2.3-6.6]; P < 0.01 for both). In addition, higher levels of transitional B cells were associated with development of ICI-IA (odds ratio 2.25 [95% confidence interval 1.03-4.9], P = 0.04). Transitional B cells increased before the onset of overt ICI-IA and decreased between the active and quiescent stages of ICI-IA (P = 0.02). Autoantibodies to type II collagen epitopes were detected in up to 43% of ICI-IA patients compared to none of the non-IRAE patients (P = 0.02). CONCLUSION: Development of ICI-IA is accompanied by an increase in transitional B cells and by production of autoantibodies to joint-related proteins. Monitoring of B cell-driven abnormalities upon ICI treatment may help earlier recognition of ICI-IA.

Variants of beta-glucan polysaccharides downregulate autoimmune inflammation.

Common infections and polysaccharides, from bacteria and yeasts, could trigger psoriasis and psoriatic arthritis (PsA), and possibly rheumatoid arthritis (RA). The objective of this study was to investigate the effects of ß-glucan polysaccharides in the effector phase of arthritis and as regulators of psoriasis and PsA-like symptoms in mice. Collagen antibody induced arthritis was studied as a model of RA and mannan-induced psoriasis (MIP) was used as model for psoriasis and PsA, using mice with a mutation of Ncf1 on the B10.Q genetic background, making them highly disease susceptible. The mice were exposed to three common variants: 1,6-ß-glucan, 1,3-ß-glucan and 1,3-1,6-ß-glucan. These ß-glucans down-regulated disease in mice if administered simultaneously, before or after mannan. Interestingly, the protection was macrophage mannose receptor (MMR/CD206) dependent with a more pronounced protection long-term than short-term. The number of resident peritoneal macrophages decreased after in vivo challenge with ß-glucan and mannan compared to mannan alone, whereas the numbers of infiltrating cells correspondingly increased, further indicating macrophages as key for ß-glucan mediated regulation. At the doses tested, ß-glucans could not induce arthritis, psoriasis or PsA in wild-type mice. However, ß-glucans could ameliorate the PsA-like symptoms representing a new unforeseen possibility to explore for future clinical treatment.

Chronic Active Arthritis Driven by Macrophages Without Involvement of T Cells: A Novel Experimental Model of Rheumatoid Arthritis.

OBJECTIVE: To develop a new chronic rheumatoid arthritis model that is driven by the innate immune system. METHODS: Injection of a cocktail of 4 monoclonal antibodies against type II collagen, followed on days 5 and 60 by intraperitoneal injections of mannan (from Saccharomyces cerevisiae), was used to induce development of chronic arthritis in B10.Q mice. The role of the innate immune system as compared to the adaptive immune system in this arthritis model was investigated using genetically modified mouse strains. RESULTS: A new model of chronic relapsing arthritis was characterized in B10.Q mice, in which a persistently active, chronic disease was found. This relapsing disease was driven by macrophages lacking the ability to mount a reactive oxygen species response against pathogens, and was associated with the classical/alternative pathway, but not the lectin pathway, of complement activation. The disease was independent of Fcγ receptor type III, and also independent of the activity of adaptive immune cells (B and T cells), indicating that the innate immune system, involving complement activation, could be the sole driver of chronicity. CONCLUSION: Chronic active arthritis can be driven innately by macrophages without the involvement of T and B cells in the adaptive immune system.

The Macrophage Mannose Receptor Regulate Mannan-Induced Psoriasis, Psoriatic Arthritis, and Rheumatoid Arthritis-Like Disease Models.

The injection of mannan into mice can result in the development of psoriasis (Ps) and psoriatic arthritis (PsA), whereas co-injection with antibodies toward collagen type II leads to a chronic rheumatoid-like arthritis. The critical event in all these diseases is mannan-mediated activation of macrophages, causing more severe disease if the macrophages are deficient in neutrophil cytosolic factor 1 (Ncf1), i.e., lack the capacity to make a reactive oxygen species (ROS) burst. In this study, we investigated the role of one of the receptors binding mannan; the macrophage mannose receptor (MR, CD206). MR is a C-type lectin present on myeloid cells and lymphatics. We found that mice deficient in MR expression had more severe mannan-induced Ps, PsA as well as rheumatoid-like arthritis. Interestingly, the MR-mediated protection was partly lost in Ncf1 mutated mice and was associated with an type 2 macrophage expansion. In conclusion, these results show that MR protects against a pathogenic inflammatory macrophage response induced by mannan and is associated with induction of ROS.

Reactive Oxygen Species Regulate Both Priming and Established Arthritis, but with Different Mechanisms.

AIMS: Neutrophil cytosolic factor 1 (NCF1) is a key regulatory component of the phagocytic NOX2 complex, which produces reactive oxygen species (ROS). Polymorphism of the Ncf1 gene is associated with increased arthritis severity. In this study, we generated targeted Ncf1 knock-in mice with inducible Ncf1 expression and determined the critical time window during which the NOX2-derived ROS protect the mice from arthritis. RESULTS: Targeted Ncf1 knock-in mice lacked NOX2-derived ROS, and in vivo allelic conversion of Ncf1 by the CreERT2 recombinase led to full protein expression and ROS production within 10 days. Mice in which Ncf1 had been activated before immunization with type II collagen (CII) developed only mild clinical symptoms of collagen-induced arthritis (CIA), whereas the ROS-deficient littermates had severe arthritis. The functional Ncf1 restricted the expansion of IL-17A-producing T cells specific for the immunodominant CII peptide. When the Ncf1 gene was activated after the priming phase, Ncf1-dependent protection from autoimmune arthritis was still observed, together with a reduced number of splenic monocytes but it was not associated with alterations in peptide-specific T cell response. The Ncf1-deficient mice expressed pronounced interferon signature, which could be normalized by conditional expression of Ncf1 and was also present in the Ncf1-mutated mouse during arthritis. Innovation and Conclusion: Ncf1 deficiency has been known to predispose to autoimmunity in both humans and rodents. Our in vivo results point to a regulatory role of NOX2-derived ROS not only during priming but also during the effector phase of CIA, most likely via different mechanisms. Antioxid. Redox Signal. 27, 1473-1490.

(18)F-Labeling of Mannan for Inflammation Research with Positron Emission Tomography.

Recently mannan from Saccharomyces cerevisiae has been shown to be able to induce psoriasis and psoriatic arthritis in mice, and the phenotypes resemble the corresponding human diseases. To investigate the pathological processes, we set out to label mannan with fluorine-18 ((18)F) and study the (18)F-labeled mannan in vitro and in vivo with positron emission tomography (PET). Accordingly, mannan has been transformed into (18)F-fluoromannan with (18)F-bicyclo[6.1.0]nonyne. In mouse aorta, the binding of [(18)F]fluoromannan to the atherosclerotic lesions was clearly visualized and was significantly higher compared to blocking assays (P < 0.001) or healthy mouse aorta (P < 0.001). In healthy rats the [(18)F]fluoromannan radioactivity accumulated largely in the macrophage-rich organs such as liver, spleen, and bone marrow and the excess excreted in urine. Furthermore, the corresponding (19)F-labeled mannan has been used to induce psoriasis and psoriatic arthritis in mice, which indicates that the biological function of mannan is preserved after the chemical modifications.

Ncf1 polymorphism reveals oxidative regulation of autoimmune chronic inflammation.

The current review on the function of neutrophil cytosolic factor 1 (NCF1) and induced reactive oxygen species (ROS) is based on a genetic search for the major genes controlling autoimmune inflammatory disorders. Surprisingly, the disease-promoting allele determined a lower ROS response and was therefore in complete contrast to the prevailing dogma. Once cloned, it opened the possibility to dissect this complex field from a new angle and with the possibilities to study the role of ROS in vivo. We found that NCF1 and NADPH oxidase 2 (NOX2) complex-derived ROS is an important regulator of several chronic inflammatory disorders by using models for rheumatoid arthritis, multiple sclerosis, psoriasis and psoriasis arthritis, gout, and lupus. ROS could therefore affect many different types of diseases and the common denominator seems to be that ROS regulate macrophages, which prevents inflammation from going chronic. The role of ROS is currently changing from being seen as toxic agents that will promote inflammation toward a more complex view with ROS as crucial regulators of immune and inflammatory pathways.

Direct Comparison of a Natural Loss-Of-Function Single Nucleotide Polymorphism with a Targeted Deletion in the Ncf1 Gene Reveals Different Phenotypes.

The genetic targeting of mouse models has given insight into complex processes. However, phenotypes of genetically targeted mice are susceptible to artifacts due to gene manipulation, which may lead to misinterpretation of the observations. To directly address these issues, we have compared the immunological phenotypes of Ncf1 knockout mice with Ncf1m1J mice possessing a naturally occurring intronic loss-of-function SNP in their Ncf1 gene. Neutrophil cytosolic factor 1 (NCF1) is the key regulatory component of the phagocytic NADPH oxidase 2 (NOX2) complex. Defects in NCF1 lead to lower production of reactive oxygen species (ROS) associated with autoimmune diseases in humans. In mice, collagen induced arthritis (CIA) and psoriatic arthritis are autoimmune disorders known to be regulated by Ncf1, and they were utilized in the present study to compare the Ncf1 knockout with Ncf1m1J mice. Targeted Ncf1 knockout mice were generated on a pure C57BL/6N genetic background, and thereafter crossed with B10.Q.Ncf1m1J mice. The targeting silenced the Ncf1 gene as intended, and both the B6N;B10.Q.Ncf1m1J mice as well as the knockout littermates had reduced ROS production compared to wild type mice. Both also exhibited enhanced STAT1 (signal transducer and activator of transcription 1) protein expression as an indicator of pronounced interferon signature reported recently for Ncf1 deficient mice. Surprisingly, female Ncf1 knockout mice were protected from CIA whereas the Ncf1m1J females developed severe disease. Ovariectomization retrieved the susceptibility of Ncf1 knockout females pointing to a sex hormone regulated protection against CIA in these mice. The data partly explains the discrepancy of the phenotypes reported earlier utilizing the Ncf1m1J mice or Ncf1 knockout mice. These observations indicate that even a targeted knockout mutation may lead to a different biological outcome in comparison to the natural loss-of-function mutation of the same gene.

In vivo imaging of reactive oxygen and nitrogen species in murine colitis.

BACKGROUND: Traditional techniques analyzing mouse colitis are invasive, laborious, or indirect. Development of in vivo imaging techniques for specific colitis processes would be useful for monitoring disease progression and/or treatment effectiveness. The aim was to evaluate the applicability of the chemiluminescent probe L-012, which detects reactive oxygen and nitrogen species, for in vivo colitis imaging. METHODS: Two genetic colitis mouse models were used; K8 knockout (K8(-/-)) mice, which develop early colitis and the nonobese diabetic mice, which develop a transient subclinical colitis. Dextran sulphate sodium was used as a chemical colitis model. Mice were anesthetized, injected intraperitoneally with L-012, imaged, and quantified for chemiluminescent signal in the abdominal region using an IVIS camera system. RESULTS: K8(-/-) and nonobese diabetic mice showed increased L-012-mediated chemiluminescence from the abdominal region compared with control mice. L-012 signals correlated with the colitis phenotype assessed by histology and myeloperoxidase staining. Although L-012 chemiluminescence enabled detection of dextran sulphate sodium-induced colitis at an earlier time point compared with traditional methods, large mouse-to-mouse variations were noted. In situ and ex vivo L-012 imaging as well as [18F]FDG-PET imaging of K8(-/-) mice confirmed that the in vivo signals originated from the distal colon. L-012 in vivo imaging showed a wide variation in reactive oxygen and nitrogen species in young mice, irrespective of K8 genotype. In aging mice L-012 signals were consistently higher in K8(-/-) as compared to K8(+/+) mice. CONCLUSIONS: In vivo imaging using L-012 is a useful, simple, and cost-effective tool to study the level and longitudinal progression of genetic and possibly chemical murine colitis.

Reactive oxygen species deficiency induces autoimmunity with type 1 interferon signature.

AIMS: Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the phagocyte reactive oxygen species (ROS)-producing NOX2 enzyme complex and characterized by recurrent infections associated with hyperinflammatory and autoimmune manifestations. A translational, comparative analysis of CGD patients and the corresponding ROS-deficient Ncf1(m1J) mutated mouse model was performed to reveal the molecular pathways operating in NOX2 complex deficient inflammation. RESULTS: A prominent type I interferon (IFN) response signature that was accompanied by elevated autoantibody levels was identified in both mice and humans lacking functional NOX2 complex. To further underline the systemic lupus erythematosus (SLE)-related autoimmune process, we show that naïve Ncf1(m1J) mutated mice, similar to SLE patients, suffer from inflammatory kidney disease with IgG and C3 deposits in the glomeruli. Expression analysis of germ-free Ncf1(m1J) mutated mice reproduced the type I IFN signature, enabling us to conclude that the upregulated signaling pathway is of endogenous origin. INNOVATION: Our findings link the previously unexplained connection between ROS deficiency and increased susceptibility to autoimmunity by the discovery that activation of IFN signaling is a major pathway downstream of a deficient NOX2 complex in both mice and humans. CONCLUSION: We conclude that the lack of phagocyte-derived oxidative burst is associated with spontaneous autoimmunity and linked with type I IFN signature in both mice and humans.

Identification of a region in p47phox/NCF1 crucial for phagocytic NADPH oxidase (NOX2) activation.

A point mutation in the mouse Ncf1(m1J) gene decreases production of ROS by the phagocytic NOX2 complex. Three mRNA splice variants are expressed, but only one is expressed as a protein, although at lower levels than the WT NCF1 (also known as p47phox). Our aim was to investigate whether the mutant p47phox, lacking 8 aa, is active, but as a result of its low expression, ROS production is decreased in Ncf1(m1J) mice, or whether the mutant p47phox completely lacks the capability to activate the NOX2 complex. The p47phox mutant (Δ228-235), which was equal to the protein in Ncf1(m1J) mice, failed to activate the NOX2 complex. When the deleted region was narrowed down to 2 aa, the p47phox protein remained inactive and failed to translocate to the membrane upon activation. Single amino acid substitutions revealed Thr233 to be vital for ROS production. Residues Tyr231 and Val232 also seemed to be important for p47phox function, as p47phox_Y231G and p47phox_V232G resulted in a >50% decrease in ROS production by the NOX2 complex. In addition, we identified the epitope of the D-10 anti-p47phox mAb. In conclusion, the p47phox protein variant expressed in Ncf1(m1J) mice is completely defective in activating the NOX2 complex to produce ROS, and the effect is dependent on SH3 region amino acids at positions 231-233, which are vital for the proper assembly of the NOX2 complex.

Hydrogen peroxide as an immunological transmitter regulating autoreactive T cells.

SIGNIFICANCE: An unexpected finding, revealed by positional cloning of genetic polymorphisms controlling models for rheumatoid arthritis, exposed a new function of Ncf1 and NADPH oxidase (NOX) 2 controlled oxidative burst. RECENT ADVANCES: A decreased capacity to produce ROS due to a natural polymorphism was found to be the major factor leading to more severe arthritis and increased T cell-dependent autoimmunity. CRITICAL ISSUES: In the vein of this finding, we here review a possible new role of ROS in regulating inflammatory cell and autoreactive T cell activity. It is postulated that peroxide is an immunologic transmitter secreted by antigen-presenting cells that downregulate the responses by autoreactive T cells. FUTURE DIRECTIONS: This may operate at different levels of T cell selection and activation: during negative selection in the thymus, priming of T cells in draining lymph nodes, and while interacting with macrophages in peripheral target tissues.

Superoxide dismutase 3 limits collagen-induced arthritis in the absence of phagocyte oxidative burst.

Extracellular superoxide dismutase (SOD3), an enzyme mediating dismutation of superoxide into hydrogen peroxide, has been shown to reduce inflammation by inhibiting macrophage migration into injured tissues. In inflamed tissues, superoxide is produced by the phagocytic NOX2 complex, which consists of the catalytic subunit NOX2 and several regulatory subunits (e.g., NCF1). To analyze whether SOD3 can regulate inflammation in the absence of functional NOX2 complex, we injected an adenoviral vector overexpressing SOD3 directly into the arthritic paws of Ncf1(∗/∗) mice with collagen-induced arthritis. SOD3 reduced arthritis severity in both oxidative burst-deficient Ncf1(∗/∗) mice and also in wild-type mice. The NOX2 complex independent anti-inflammatory effect of SOD3 was further characterized in peritonitis, and SOD3 was found to reduce macrophage infiltration independently of NOX2 complex functionality. We conclude that the SOD3-mediated anti-inflammatory effect on arthritis and peritonitis operates independently of NOX2 complex derived oxidative burst.

Synthesis and anti-inflammatory effects of a series of novel 7-hydroxycoumarin derivatives.

A number of 7-hydroxycoumarins have been synthesised by Pechmann cyclisation using differently substituted resorcinols employing perchloric acid as the condensing agent. All the compounds have been characterised by analytical and spectroscopic methods. The anti-inflammatory properties were tested with LPS-induced inflammation in J774 macrophages. Expression of iNOS and COX-2 was determined by Western blot, NO by nitrite assay and IL-6 by ELISA analyses. Fifteen of the tested 7-hydroxycoumarins also inhibited IL-6 production but none of them had any major inhibitory effect on COX-2 expression.

Dual specificity phosphatase 1 regulates human inducible nitric oxide synthase expression by p38 MAP kinase.

The role of dual specificity phosphatase 1 (DUSP1) in inducible nitric oxide synthase (iNOS) expression in A549 human pulmonary epithelial cells, J774 mouse macrophages and primary mouse bone marrow-derived macrophages (BMMs) was investigated. iNOS expression was induced by a cytokine mixture (TNF, IFNγ and IL-1ß) in A549 cells and by LPS in J774 cells, and it was inhibited by p38 MAPK inhibitors SB202190 and BIRB 796. Stimulation with cytokine mixture or LPS enhanced also DUSP1 expression. Down-regulation of DUSP1 by siRNA increased p38 MAPK phosphorylation and iNOS expression in A549 and J774 cells. In addition, LPS-induced iNOS expression was enhanced in BMMs from DUSP1((-/-)) mice as compared to that in BMMs from wild-type mice. The results indicate that DUSP1 suppresses iNOS expression by limiting p38 MAPK activity in human and mouse cells. Compounds that enhance DUSP1 expression or modulate its function may be beneficial in diseases complicated with increased iNOS-mediated NO production.

Positioning of a polymorphic quantitative trait nucleotide in the Ncf1 gene controlling oxidative burst response and arthritis severity in rats.

The Ncf1 gene, encoding the P47(PHOX) protein that regulates production of reactive oxygen species (ROS) by the phagocyte NADPH oxidase (NOX2) complex, is associated with autoimmunity and arthritis severity in rats. We have now identified that the single-nucleotide polymorphism (SNP) resulting in an M153T amino acid substitution mediates arthritis resistance and thus explains the molecular polymorphism underlying the earlier identified Ncf1 gene effect. We identified the SNP in position 153 to regulate ROS production using COS(PHOX) cells transfected with mutated Ncf1. To determine the role of this SNP for control of arthritis, we used the Wistar strain, identified to carry only the postulated arthritis resistant SNP in position 153. When this Ncf1 allele was backcrossed to the arthritis susceptible DA strain, both granulocyte ROS production and arthritis resistance were restored. Position 153 is located in the hinge region between the PX and SH3 domains of P47(PHOX). Mutational analysis of this position revealed a need for an -OH group in the side chain but we found no evidence for phosphorylation. The polymorphism did not affect assembly of the P47(PHOX)/P67(PHOX) complex in the cytosol or membrane localization, but is likely to operate downstream of assembly, affecting activity of the membrane NOX2 complex.

NOX2 complex-derived ROS as immune regulators.

Reactive oxygen species (ROS) are a heterogeneous group of highly reactive molecules that oxidize targets in a biologic system. During steady-state conditions, ROS are constantly produced in the electron-transport chain during cellular respiration and by various constitutively active oxidases. ROS production can also be induced by activation of the phagocyte NADPH oxidase 2 (NOX2) complex in a process generally referred to as an oxidative burst. The induced ROS have long been considered proinflammatory, causing cell and tissue destruction. Recent findings have challenged this inflammatory role of ROS, and today, ROS are also known to regulate immune responses and cell proliferation and to determine T-cell autoreactivity. NOX2-derived ROS have been shown to suppress antigen-dependent T-cell reactivity and remarkably to reduce the severity of experimental arthritis in both rats and mice. In this review, we discuss the role of ROS and the NOX2 complex as suppressors of autoimmunity, inflammation, and arthritis.