Reduced Anti-Histone Antibodies and Increased Risk of Rheumatoid Arthritis Associated with a Single Nucleotide Polymorphism in PADI4 in North Americans.

Autoantibodies against citrullinated proteins are a hallmark of rheumatoid arthritis, a destructive inflammatory arthritis. Peptidylarginine deiminase 4 (PAD4) has been hypothesized to contribute to rheumatoid arthritis by citrullinating histones to induce neutrophil extracellular traps (NETs), which display citrullinated proteins that are targeted by autoantibodies to drive inflammation and arthritis. Consistent with this theory, PAD4-deficient mice have reduced NETs, autoantibodies, and arthritis. However, PAD4's role in human rheumatoid arthritis is less clear. Here, we determine if single nucleotide polymorphism rs2240335 in PADI4, whose G allele is associated with reduced PAD4 in neutrophils, correlates with NETs, anti-histone antibodies, and rheumatoid arthritis susceptibility in North Americans. Control and rheumatoid arthritis subjects, divided into anti-cyclic citrullinated peptide (CCP) antibody positive and negative groups, were genotyped at rs2240335. In homozygotes, in vitro NETosis was quantified in immunofluorescent images and circulating NET and anti-histone antibody levels by enzyme linked immunosorbent assay (ELISA). Results were compared by t-test and correlation of rheumatoid arthritis diagnosis with rs2240335 by Armitage trend test. NET levels did not significantly correlate with genotype. G allele homozygotes in the CCP- rheumatoid arthritis group had reduced anti-native and anti-citrullinated histone antibodies. However, the G allele conferred increased risk for rheumatoid arthritis diagnosis, suggesting a complex role for PAD4 in human rheumatoid arthritis.

DNA Area and NETosis Analysis (DANA): a High-Throughput Method to Quantify Neutrophil Extracellular Traps in Fluorescent Microscope Images.

BACKGROUND: Neutrophil extracellular traps (NETs), extracellular structures composed of decondensed chromatin and antimicrobial molecules, are released in a process called NETosis. NETs, which are part of normal host defense, have also been implicated in multiple human diseases. Unfortunately, methods for quantifying NETs have limitations which constrain the study of NETs in disease. Establishing optimal methods for NET quantification holds the potential to further elucidate the role of NETs in normal and pathologic processes. RESULTS: To better quantify NETs and NET-like structures, we created DNA Area and NETosis Analysis (DANA), a novel ImageJ/Java based program which provides a simple, semi-automated approach to quantify NET-like structures and DNA area. DANA can analyze many fluorescent microscope images at once and provides data on a per cell, per image, and per sample basis. Using fluorescent microscope images of Sytox-stained human neutrophils, DANA quantified a similar frequency of NET-like structures to the frequency determined by two different individuals counting by eye, and in a fraction of the time. As expected, DANA also detected increased DNA area and frequency of NET-like structures in neutrophils from subjects with rheumatoid arthritis as compared to control subjects. Using images of DAPI-stained murine neutrophils, DANA (installed by an individual with no programming background) gave similar frequencies of NET-like structures as the frequency of NETs determined by two individuals counting by eye. Further, DANA quantified more NETs in stimulated murine neutrophils compared to unstimulated, as expected. CONCLUSIONS: DANA provides a means to quantify DNA decondensation and the frequency of NET-like structures using a variety of different fluorescent markers in a rapid, reliable, simple, high-throughput, and cost-effective manner making it optimal to assess NETosis in a variety of conditions.

Peptidylarginine deiminase 2 is required for tumor necrosis factor alpha-induced citrullination and arthritis, but not neutrophil extracellular trap formation.

Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for citrullination in inflamed lungs. PAD2 is important for citrullination in healthy tissues and is present in NETs, but its role in citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis.

Innate nuclear sensor IFI16 translocates into the cytoplasm during the early stage of in vitro human cytomegalovirus infection and is entrapped in the egressing virions during the late stage.

UNLABELLED: Intrinsic immune mechanisms mediated by constitutively expressed proteins termed "restriction factors" provide frontline antiviral defense. We recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. We show here that at an early time point during the in vitro infection of low-passage-number human embryonic lung fibroblasts, IFI16 binds to HCMV DNA. However, during a later phase following infection, IFI16 is mislocalized to the cytoplasmic virus assembly complex (AC), where it colocalizes with viral structural proteins. Indeed, upon its binding to pUL97, IFI16 undergoes phosphorylation and relocalizes to the cytoplasm of HCMV-infected cells. ESCRT (endosomal sorting complex required for transport) machinery regulates the translocation of IFI16 into the virus AC by sorting and trafficking IFI16 into multivesicular bodies (MVB), as demonstrated by the interaction of IFI16 with two MVB markers: Vps4 and TGN46. Finally, IFI16 becomes incorporated into the newly assembled virions as demonstrated by Western blotting of purified virions and electron microscopy. Together, these results suggest that HCMV has evolved mechanisms to mislocalize and hijack IFI16, trapping it within mature virions. However, the significance of this IFI16 trapping following nuclear mislocalization remains to be established. IMPORTANCE: Intracellular viral DNA sensors and restriction factors are critical components of host defense, which alarm and sensitize immune system against intruding pathogens. We have recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. However, viruses are known to evolve numerous strategies to cope and counteract such restriction factors and neutralize the first line of host defense mechanisms. Our findings describe that during early stages of infection, IFI16 successfully recognizes HCMV DNA. However, in late stages HCMV mislocalizes IFI16 into the cytoplasmic viral assembly complex and finally entraps the protein into mature virions. We clarify here the mechanisms HCMV relies to overcome intracellular viral restriction, which provides new insights about the relevance of DNA sensors during HCMV infection.

Peptidylarginine Deiminase 2 in Murine Antiviral and Autoimmune Antibody Responses.

The peptidylarginine deiminases (PADs) and the citrullinated proteins that they generate have key roles in innate immunity and rheumatoid arthritis, an inflammatory arthritis with antibodies that target citrullinated proteins. However, the importance of PADs, particularly PAD2, in the adaptive immune response, both normal and pathogenic, is newly emerging. In this study, we evaluated a requirement for PAD2 in the antibody response in collagen-induced arthritis (CIA), a T and B cell-driven murine model of rheumatoid arthritis, and in the protective antibody response to murine influenza infection. Using PAD2-/- and PAD2+/+ mice on the DBA/1J background, we found that PAD2 is required for maximal anti-collagen antibody levels, but not collagen-specific plasma cell numbers, T cell activation or polarization, or arthritis severity in CIA. Also, we found that PAD2 is required not just for normal levels of persistent hemagglutination inhibiting antibodies but also for full protection from lethal influenza rechallenge. Together, these data provide evidence for a novel modest requirement for PAD2 in a normal antiviral antibody response and in an abnormal autoantibody response in inflammatory arthritis.

Disordered Antigens and Epitope Overlap Between Anti-Citrullinated Protein Antibodies and Rheumatoid Factor in Rheumatoid Arthritis.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) are commonly present in rheumatoid arthritis (RA) without a clear rationale for their coexistence. Moreover, autoantibodies develop against proteins with different posttranslational modifications and native proteins without obvious unifying characteristics of the antigens. We undertook this study to broadly evaluate autoantibody binding in seronegative and seropositive RA to identify novel features of reactivity. METHODS: An array was created using a total of 172,828 native peptides, citrulline-containing peptides, and homocitrulline-containing peptides derived primarily from proteins citrullinated in the rheumatoid joint. IgG and IgM binding to peptides were compared between cyclic citrullinated peptide (CCP)-positive RF+, CCP+RF-, CCP-RF+, and CCP-RF- serum from RA patients (n = 48) and controls (n = 12). IgG-bound and endogenously citrullinated peptides were analyzed for amino acid patterns and predictors of intrinsic disorder, i.e., unstable 3-dimensional structure. Binding to IgG-derived peptides was specifically evaluated. Enzyme-linked immunosorbent assay confirmed key results. RESULTS: Broadly, CCP+RF+ patients had high citrulline-specific IgG binding to array peptides and CCP+RF- and CCP-RF+ patients had modest citrulline-specific IgG binding (median Z scores 3.02, 1.42, and 0.75, respectively; P < 0.0001). All RA groups had low homocitrulline-specific binding. CCP+RF+ patients had moderate IgG binding to native peptides (median Z score 2.38; P < 0.0001). The highest IgG binding was to citrulline-containing peptides, irrespective of protein identity, especially if citrulline was adjacent to glycine or serine, motifs also seen in endogenous citrullination in the rheumatoid joint. Highly bound peptides had multiple features predictive of disorder. IgG from CCP+RF+ patients targeted citrulline-containing IgG-derived peptides. CONCLUSION: Disordered antigens, which are frequently citrullinated, and common epitopes for ACPAs and RF are potentially unifying features for RA autoantibodies.

Reciprocal regulation of Th2 and Th17 cells by PAD2-mediated citrullination.

Dysregulated citrullination, a unique form of posttranslational modification catalyzed by the peptidylarginine deiminases (PADs), has been observed in several human diseases, including rheumatoid arthritis. However, the physiological roles of PADs in the immune system are still poorly understood. Here, we report that global inhibition of citrullination enhances the differentiation of type 2 helper T (Th2) cells but attenuates the differentiation of Th17 cells, thereby increasing the susceptibility to allergic airway inflammation. This effect on Th cells is due to inhibition of PAD2 but not PAD4. Mechanistically, PAD2 directly citrullinates GATA3 and RORγt, 2 key transcription factors determining the fate of differentiating Th cells. Citrullination of R330 of GATA3 weakens its DNA binding ability, whereas citrullination of 4 arginine residues of RORγt strengthens its DNA binding. Finally, PAD2-deficient mice also display altered Th2/Th17 immune response and heightened sensitivity to allergic airway inflammation. Thus, our data highlight the potential and caveat of PAD2 as a therapeutic target of Th cell-mediated diseases.

Relative efficiencies of peptidylarginine deiminase 2 and 4 in generating target sites for anti-citrullinated protein antibodies in fibrinogen, alpha-enolase and histone H3.

OBJECTIVE: Peptidylarginine deiminase 2 (PAD2) and PAD4 are expressed in the synovium of rheumatoid arthritis (RA) patients and catalyze citrullination of arginine residues in proteins targeted by anti-citrullinated protein antibodies (ACPAs). Little is known about the relative importance of PAD2 and PAD4 in generating citrullinated self-antigens. Here we investigate the ability of PAD2 and PAD4 to generate citrullinated targets for ACPAs in four human proteins. METHODS: Synovial fluid (SF) and plasma were collected from 42 RA patients. Human fibrinogen, human alpha-enolase (ENO1), human histone H3, and human serum albumin (HSA) were citrullinated in vitro by PAD2 or PAD4. The total degree of citrullination was determined using the anti-modified citrulline approach. Antibody binding to native and citrullinated proteins was measured by ELISA. RESULTS: ACPAs within pooled SF from multiple RA patients reacted equally well with, and cross-reacted with, PAD2- and PAD4-citrullinated fibrinogen. ACPAs from most individual patient SF and plasma samples bound equally well to PAD2- and PAD4-citrullinated fibrinogen or ENO1. When histone H3 was used as target, PAD4 was generally superior in generating epitopes recognized by ACPAs. No binding to citrullinated HSA was observed. CONCLUSION: In most patients, PAD2 and PAD4 are equally efficient in generating citrullinated target sites for ACPAs in fibrinogen and ENO1. The binding of autoantibodies to histone H3 was generally higher after citrullination with PAD4 than with PAD2. Citrullinated HSA is not a target for ACPAs.

Citrullination of NF-κB p65 promotes its nuclear localization and TLR-induced expression of IL-1ß and TNFα.

Many citrullinated proteins are known autoantigens in rheumatoid arthritis, a disease mediated by inflammatory cytokines, such as tumor necrosis factor-α (TNFα). Citrullinated proteins are generated by converting peptidylarginine to peptidylcitrulline, a process catalyzed by the peptidylarginine deiminases (PADs), including PAD1 to PAD4 and PAD6. Several major risk factors for rheumatoid arthritis are associated with heightened citrullination. However, the physiological role of citrullination in immune cells is poorly understood. We report that suppression of PAD activity attenuates Toll-like receptor-induced expression of interleukin-1ß (IL-1ß) and TNFα by neutrophils in vivo and in vitro but not their global transcription activity. Mechanistically, PAD4 directly citrullinates nuclear factor κB (NF-κB) p65 and enhances the interaction of p65 with importin α3, which brings p65 into the nucleus. The citrullination-enhanced interaction of p65 with importin α3 and its nuclear translocation and transcriptional activity can be attributed to citrullination of four arginine residues located in the Rel homology domain of p65. Furthermore, a rheumatoid arthritis-prone variant of PAD4, carrying three missense mutations, is more efficient in interacting with p65 and enhancing NF-κB activity. Together, these data not only demonstrate a critical role of citrullination in an NF-κB-dependent expression of IL-1ß and TNFα but also provide a molecular mechanism by which heightened citrullination propagates inflammation in rheumatoid arthritis. Accordingly, attenuating p65-mediated production of IL-1ß and TNFα by blocking the citrullination of p65 has great therapeutic potential in rheumatoid arthritis.

Tumor necrosis factor alpha, citrullination, and peptidylarginine deiminase 4 in lung and joint inflammation.

BACKGROUND: The relationship between lung and joint inflammation in rheumatoid arthritis is poorly understood. Lung inflammation with resultant protein citrullination may trigger anti-citrullinated protein antibodies, inflammation, and arthritis. Alternatively, lung and joint inflammation may be two manifestations of a single underlying pathology. The lung has increased citrullination and TNF-α levels are high in rheumatoid arthritis; however, it is unknown if TNF-α can induce lung protein citrullination. The citrullinating enzyme peptidylarginine deiminase 4 (PAD4) exacerbates TNF-α-induced arthritis, but a role for PAD4 in lung citrullination and TNF-α-induced lung inflammation has not been explored. Our aim was to use TNF-α-overexpressing mice to clarify the intersection of TNF-α, citrullination, PAD4, arthritis, and lung inflammation. METHODS: Lung protein citrullination in wild-type mice, mice that overexpress TNF-α systemically (TNF(+)), TNF(+)PAD4(+/+), and TNF(+)PAD4(-/-) mice was quantified by both gel electrophoresis using a citrulline probe and western blot. Hematoxylin and eosin (H&E)-stained lung sections from TNF(+)PAD4(+/+) and TNF(+)PAD4(-/-) mice were scored for lung inflammation. H&E-stained ankle joint sections from mice that overexpress TNF-α only in the lungs were assessed for arthritis. RESULTS: TNF(+) mice have increased lung protein citrullination. TNF(+)PAD4(-/-) mice do not have significantly reduced lung protein citrullination, but do have decreased lung inflammation compared to TNF(+)PAD4(+/+) mice. Mice that overexpress TNF-α only in the lungs do not develop arthritis. CONCLUSIONS: PAD4 exacerbates lung inflammation downstream of TNF-α without having a major role in generalized protein citrullination in inflamed lungs. Also, TNF-α-induced lung inflammation is not sufficient to drive murine arthritis.

Mislocalization of the interferon inducible protein IFI16 by environmental insults: implications in autoimmunity.

The nuclear DNA sensor IFI16, a member of PYHIN family of proteins, was previously studied for its role in cell cycle regulation, tumor suppression, apoptosis and DNA damage signaling. Autoantibodies against IFI16 are prevalent in the sera of patients with systemic autoimmunity, thus depicting physiological significance as an autoantigen. At present, the nuclear IFI16 protein has been thoroughly investigated for its role as an innate immune sensor involved in inflammasome signaling and viral restriction. While the sub-cellular localization of IFI16 during such events has been known, very little knowledge about its presence and significance in the extracellular space is available. Recently our group has discovered the presence of circulating IFI16 in the sera from systemic autoimmune patients indicating that in this setting it may be mislocalized form its nuclear site and secreted in the extracellular milieu. In this review, we will discuss the leakage of endogenous IFI16 that has been experimentally proved using in vivo and in vitro models. Also we will comment on the significance of mislocalized inflammasome components in the extracellular space and how it can be responsible for chronic inflammation.

The Extracellular IFI16 Protein Propagates Inflammation in Endothelial Cells Via p38 MAPK and NF-κB p65 Activation.

The nuclear interferon-inducible-16 (IFI16) protein acts as DNA sensor in inflammasome signaling and as viral restriction factor. Following Herpesvirus infection or UV-B treatment, IFI16 delocalizes from the nucleus to the cytoplasm and is eventually released into the extracellular milieu. Recently, our group has demonstrated the occurrence of IFI16 in sera of systemic-autoimmune patients that hampers biological activity of endothelia through high-affinity membrane binding. As a continuation, we studied the activity of endotoxin-free recombinant IFI16 (rIFI16) protein on primary endothelial cells. rIFI16 caused dose/time-dependent upregulation of IL-6, IL-8, CCL2, CCL5, CCL20, ICAM1, VCAM1, and TLR4, while secretion of IL-6 and IL-8 was amplified with lipopolysaccharide synergy. Overall, cytokine secretion was completely inhibited in MyD88-silenced cells and partially by TLR4-neutralizing antibodies. By screening downstream signaling pathways, we found that IFI16 activates p38, p44/42 MAP kinases, and NF-kB. In particular, activation of p38 is an early event required for subsequent p44/42 MAP kinases activity and cytokine induction indicating a key role of this kinase in IFI16 signaling. Altogether, our data conclude that extracellular IFI16 protein alone or by synergy with lipopolysaccharide acts like Damage-associated molecular patterns propagating "Danger Signal" through MyD88-dependent TLR-pathway.

Nuclear DNA sensor IFI16 as circulating protein in autoimmune diseases is a signal of damage that impairs endothelial cells through high-affinity membrane binding.

IFI16, a nuclear pathogenic DNA sensor induced by several pro-inflammatory cytokines, is a multifaceted protein with various functions. It is also a target for autoantibodies as specific antibodies have been demonstrated in the sera of patients affected by systemic autoimmune diseases. Following transfection of virus-derived DNA, or treatment with UVB, IFI16 delocalizes from the nucleus to the cytoplasm and is then eventually released into the extracellular milieu. In this study, using an in-house capture enzyme-linked immunsorbent assay we demonstrate that significant levels of IFI16 protein can also exist as circulating form in the sera of autoimmune patients. We also show that the rIFI16 protein, when added in-vitro to endothelial cells, does not affect cell viability, but severely limits their tubulogenesis and transwell migration activities. These inhibitory effects are fully reversed in the presence of anti-IFI16 N-terminal antibodies, indicating that its extracellular activity resides within the N-terminus. It was further demonstrated that endogenous IFI16 released by apoptotic cells bind neighboring cells in a co-culture. Immunofluorescence assays revealed existence of high-affinity binding sites on the plasma membrane of endothelial cells. Free recombinant IFI16 binds these sites on HUVEC with dissociation constant of 2.7 nM, radioiodinated and unlabeled IFI16 compete for binding sites, with inhibition constant (Ki) of 14.43 nM and half maximal inhibitory concentration (IC50) of 67.88 nM; these data allow us to estimate the presence of 250,000 to 450,000 specific binding sites per cell. Corroborating the results from functional assays, this binding could be completely inhibited using anti-IFI16 N-terminal antibody, but not with an antibody raised against the IFI16 C-terminal. Altogether, these data demonstrate that IFI16 may exist as circulating protein in the sera of autoimmune patients which binds endothelial cells causing damage, suggesting a new pathogenic and alarmin function through which this protein triggers the development of autoimmunity.