Genetic variations in A20 DUB domain provide a genetic link to citrullination and neutrophil extracellular traps in systemic lupus erythematosus

Objectives: Genetic variations in TNFAIP3 (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-κB but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis. Methods: CRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating C103A knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and C103A KI cells. Functional studies were performed in A20 C103A U937 cells and in immune cells from A20 C103A mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 C103A mice and SLE-patients with rs2230926. Results: Genetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-κB signalling. Instead, cells with C103A mutation or rs2230926 polymorphism presented an upregulated expression of PADI4, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 C103A cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes. Conclusions: We propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of PADI4 with resultant protein citrullination and extracellular trap formation

Lack of association between the chemokine receptor 5 polymorphism CCR5delta32 in rheumatoid arthritis and juvenile idiopathic arthritis

<p>Abstract</p> <p>Background</p> <p>The chemokine receptor CCR5 has been detected at elevated levels on synovial T cells, and a 32 bp deletion in the <it>CCR5 </it>gene leads to a non-functional receptor. A negative association between the <it>CCR5Δ32 </it>and rheumatoid arthritis (RA) has been reported, although with conflicting results. In juvenile idiopathic arthritis (JIA), an association with CCR5 was recently reported. The purpose of this study was to investigate if the <it>CCR5Δ32 </it>polymorphism is associated with RA or JIA in Norwegian cohorts.</p> <p>Methods</p> <p>853 RA patients, 524 JIA patients and 658 controls were genotyped for the <it>CCR5Δ32 </it>polymorphism.</p> <p>Results</p> <p>The <it>CCR5Δ32 </it>allele frequency was 11.5% in the controls vs. 10.4% in RA patients (OR = 0.90; <it>P </it>= 0.36) and 9.7% in JIA patients (OR = 0.85; <it>P </it>= 0.20). No decreased homozygosity was observed for <it>CCR5Δ32</it>, as previously suggested.</p> <p>Conclusion</p> <p>Our data do not support an association between the <it>CCR5Δ32 </it>allele and Norwegian RA or JIA patients. Combining our results with those from a recently published meta-analysis still provide evidence for a role for <it>CCR5Δ32 </it>in RA, albeit substantially weaker than the effect first reported.</p

An African-Specific Variant of TP53 Reveals PADI4 as a Regulator of p53-Mediated Tumor Suppression

TP53 is the most frequently mutated gene in cancer, yet key target genes for p53-mediated tumor suppression remain unidentified. Here, we characterize a rare, African-specific germline variant of TP53 in the DNA-binding domain Tyr107His (Y107H). Nuclear magnetic resonance and crystal structures reveal that Y107H is structurally similar to wild-type p53. Consistent with this, we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes; this includes the epigenetic modifier PADI4, which deiminates arginine to the nonnatural amino acid citrulline. Surprisingly, we show that Y107H mice develop spontaneous cancers and metastases and that Y107H shows impaired tumor suppression in two other models. We show that PADI4 is itself tumor suppressive and that it requires an intact immune system for tumor suppression. We identify a p53–PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. Significance: We analyze the African-centric Y107H hypomorphic variant and show that it confers increased cancer risk; we use Y107H in order to identify PADI4 as a key tumor-suppressive p53 target gene that contributes to an immune modulation signature and that is predictive of cancer survival and the success of immunotherapy

Intrinsic function of the peptidylarginine deiminase PADI4 is dispensable for normal haematopoiesis.

Peptidylarginine deiminases (PADIs) are strongly associated with the development of autoimmunity, neurodegeneration and cancer but their physiological roles are ill-defined. The nuclear deiminase PADI4 regulates pluripotency in the mammalian pre-implantation embryo but its function in tissue development is unknown. PADI4 is primarily expressed in the bone marrow, as part of a self-renewal-associated gene signature. It has been shown to regulate the proliferation of multipotent haematopoietic progenitors and proposed to impact on the differentiation of haematopoietic stem cells (HSCs), suggesting that it controls haematopoietic development or regeneration. Using conditional in vivo models of steady state and acute Padi4 ablation, we examined the role of PADI4 in the development and function of the haematopoietic system. We found that PADI4 loss does not significantly affect HSC self-renewal or differentiation potential upon injury or serial transplantation, nor does it lead to HSC exhaustion or premature ageing. Thus PADI4 is dispensable for cell-autonomous HSC maintenance, differentiation and haematopoietic regeneration. This work represents the first study of PADI4 in tissue development and indicates that pharmacological PADI4 inhibition may be tolerated without adverse effects

PAD4 is not essential for disease in the K/BxN murine autoantibody-mediated model of arthritis

INTRODUCTION: Both murine and human genome-wide association studies have implicated peptidyl arginine deiminase (PAD4) as a susceptibility gene in rheumatoid arthritis (RA). In addition, patients with RA commonly have autoantibodies which recognize PAD4 or and/or citrullinated peptides. This study aims to evaluate the role of PAD4 in the effector phase of arthritis. METHODS: PAD4 knock out (KO) and wild type (WT) C57BL/6J mice were injected with K/BxN sera to induce disease. Progression of disease was monitored by measuring paw and ankle swelling and clinical indexes of disease, and pathogenesis was assessed by indexing of clinical progression on paws collected from WT and PAD4 KO mice injected with K/BxN serum. PAD4 activity was determined by visualization of neutrophil extracellular traps (NETs) and immunohistological analysis of histone citrullination. RESULTS: PAD4 activity is readily detectable in the inflamed synovium of WT but not PAD4 deficient animals, as demonstrated by histone citrullination and NET formation. However, PAD4 WT and KO animals develop K/BxN serum transfer disease with comparable severity and kinetics, with no statistically significant differences noted in clinical scores, swelling, joint erosion or joint invasion. CONCLUSIONS: PAD4 WT and KO mice develop disease in the K/BxN serum transfer model of arthritis with similar severity and kinetics, indicating that PAD4 is dispensable in this effector phase model of disease

The Role of NADPH Oxidase and Neutrophil Extracellular Traps in the Pathogenesis of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by loss of tolerance to nuclear antigens and tissue destruction. While the sources of autoantigens in SLE are unknown, release of contents from dying cells and/or inadequate clearance of resulting debris are likely possibilities. Prior reports suggest that neutrophil extracellular traps (NETs) and its associated death pathway, NETosis, are sources of autoantigen in SLE. However, we showed that inhibition of NETs by targeting the NADPH oxidase complex, via Cybb-deletion, exacerbated disease in the MRL/lpr model of SLE. To clarify the contribution of NETs in SLE pathogenesis, we employed a genetic approach to delete peptidyl arginine deiminase IV (Padi4) and neutrophil elastase (Elane), two distal mediators of NET formation, in MRL/lpr mice. Both Padi4 and Elane deficiency did alter disease course, directly challenging the concept that NETs promote autoimmunity. Therefore, it remains unknown how NADPH oxidase regulates autoimmunity. NADPH oxidase can constrain inflammation by neutrophil, macrophage, and lymphocyte dependent mechanisms. To identify the cell lineage in which Cybb deficiency drives SLE, we employed bone marrow chimera and conditional knockout approaches to delete Cybb in the myeloid compartment of MRL/lpr mice. Myeloid Cybb deficiency is sufficient to worsen glomerular and interstitial nephritis, suggesting that Cybb is protective in SLE due to a fundamental regulatory activity of Cybb within the myeloid compartment. NADPH oxidase is a critical mediator of LC3-associated phagocytosis (LAP), which is important for the clearance of dead cells by macrophages. As Cybb modulates SLE pathogenesis by its function in myeloid cells, we assessed LAP in lupus. To test the hypothesis that exacerbated SLE in Cybb-deficient mice is due to defective LAP, we genetically deleted another critical LAP mediator, Rubicon, in Cybb-sufficient and Cybb-deficient SLE prone mice. Unexpectedly, Rubicon deficiency increased the lifespan and ameliorated renal disease in Cybb-deficient MRL/lpr mice. Strikingly, Rubicon deficiency reduced the autoantibody responses to RNA associated autoantigens. These data suggest that a defect in LAP is not a major driver of SLE and highlights RUBICON as a novel mediator of lupus pathogenesis. The mechanism by which myeloid Cybb regulates SLE remains enigmatic and requires further investigation

PAD4-Mediated Neutrophil Extracellular Trap Formation Is Not Required for Immunity against Influenza Infection

During an inflammatory response, neutrophils migrate to the site of infection where they can kill invading pathogens by phagocytosis, secretion of anti-microbicidal mediators or the release of neutrophil extracellular traps (NETs). NETs are specialized anti-microbial structures comprised of decondensed chromatin decorated with microbicidal agents. Increased amount of NETs have been found in patients suffering from the chronic lung inflammatory disease cystic fibrosis, correlating with increased severity of pulmonary obstruction. Furthermore, acute lung inflammation during influenza A infection is characterized by a massive influx of neutrophils into the lung. The role of NETs during virus-mediated lung inflammation is unknown. Peptidylarginine deiminase 4 (PAD4)-mediated deimination of histone H3 and H4 is required for NET formation. Therefore, we generated a PAD4-deficient mouse strain that has a striking inability to form NETs. These mice were infected with influenza A/WSN, and the disease was monitored at the level of leukocytic lung infiltration, lung pathology, viral replication, weight loss and mortality. PAD4 KO fared comparable to WT mice in all the parameters tested, but they displayed slight but statistically different weight loss kinetics during infection that was not reflected in enhanced survival. Overall, we conclude that PAD4-mediated NET formation is dispensable in a mouse model of influenza A infection

Genetics of rheumatic disease

Many of the chronic inflammatory and degenerative disorders that present to clinical rheumatologists have a complex genetic aetiology. Over the past decade a dramatic improvement in technology and methodology has accelerated the pace of gene discovery in complex disorders in an exponential fashion. In this review, we focus on rheumatoid arthritis, systemic lupus erythematosus and ankylosing spondylitis and describe some of the recently described genes that underlie these conditions and the extent to which they overlap. The next decade will witness a full account of the main disease susceptibility genes in these diseases and progress in establishing the molecular basis by which genetic variation contributes to pathogenesis

What epidemiology has told us about risk factors and aetiopathogenesis in rheumatic diseases

This article will review how epidemiological studies have advanced our knowledge of both genetic and environmental risk factors for rheumatic diseases over the past decade. The major rheumatic diseases, including rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus, scleroderma, osteoarthritis, gout, and fibromyalgia, and chronic widespread pain, will be covered. Advances discussed will include how a number of large prospective studies have improved our knowledge of risk factors, including diet, obesity, hormones, and smoking. The change from small-scale association studies to genome-wide association studies using gene chips to reveal new genetic risk factors will also be reviewed

Novel Roles for Arginine Modifying Enzymes in Immune Regulation

Arginine modifying enzymes are important transcriptional regulators involved in many cellular processes. Cytokine expression by immune cells is a highly regulated process, since cytokine imbalance is associated with severe pathological consequences including autoimmunity, autoinflammation and asthma. Th2 mediated IL-4 expression is a tightly regulated process. The NFAT interacting protein NIP45 is an essential regulator of IL-4 expression by Th2 cells. Furthermore, NIP45-mediated IL-4 expression is enhanced by arginine methylation of NIP45. Here, I demonstrate that arginine deimination of NIP45 by PAD4 negatively regulates IL-4 expression. PAD4 can deiminate NIP45 both in vitro and in vivo. Overexpression of PAD4 can suppress NIP45-mediated IL-4 expression. Interestingly, the suppressive activity of PAD4 is independent of its catalytic activity. Furthermore, Th2 cells from PAD4-deficient mice that I generated for this thesis display elevated IL-4 secretion. Taken together, I have established that PAD4 is a negative regulator of IL-4 expression. Although PAD4 can deiminate NIP45, the catalytic activity of PAD4 is not required for suppression of NIP45-mediated IL-4 secretion. CARM1 coactivates NFkB-mediated transcription of several proinflammatory genes including IL-6, TNFa, and IP-10 in MEFs. In contrast, I demonstrate that CARM1 is a negative regulator of NFkB-mediated proinflammatory cytokine secretion in macrophages. CARM1-deficient BMDMs secrete significantly more IL-6 and TNFa upon LPS stimulation than CARM1-sufficient BMDMs, without exhibiting a difference in IL-10 secretion. In addition, CARM1 is reported to coactivate glucocorticoid-receptor mediated transcription. Here, I show that glucocorticoid mediated trans-repression of NFkB in BMDMs is not regulated by CARM1. In conclusion, the results from this thesis reveal novel roles for arginine modifying enzymes in cytokine expression by Th2 cells and macrophages. PAD4 is a negative regulator of NIP45-mediated IL-4 secretion in Th2 cells. In addition, the generation of the PAD4 conditional knockout strain will allow a more detailed analysis of PAD4 in immune regulation. Furthermore, CARM1 is a negative regulator of NFkB-mediated proinflammatory cytokine secretion in macrophages, revealing cell-type specific differences for CARM1 function in macrophages and MEFs