PAD-2-mediated citrullination of nucleophosmin provides an effective target for tumor immunotherapy.

BACKGROUND: The enzymatic conversion of arginine to citrulline is involved in gene and protein regulation and in alerting the immune system to stressed cells, including tumor cells. Nucleophosmin (NPM) is a nuclear protein that plays key roles in cellular metabolism including ribosome biogenesis, mRNA processing and chromatin remodeling and is regulated by citrullination. In this study, we explored if the same citrullinated arginines within NPM are involved in gene regulation and immune activation. METHODS: HLA-DP4 and HLA-DR4 transgenic mice were immunized with 22 citrullinated NPM overlapping peptides and immune responses to the peptides were assessed by ex vivo ELISpot assays. Antitumor immunity of NPM targeted vaccination was assessed by challenging transgenic mice with B16F1 HHDII/iDP4, B16F1 HHDII/PAD2KOcDP4, B16F1 HHDII and Lewis lung carcinoma cells/cDP4 cells subcutaneously. Peripheral blood mononuclear cells isolated from healthy donors were stimulated with NPM266-285cit peptides with/without CD45RO+memory cell depletion to assess if the responses in human were naïve or memory. RESULTS: In contrast to NPM regulation, which is mediated by peptidylarginine deiminase (PAD4) citrullination of arginine at position 197, only citrullinated NPM266-285 peptide induced a citrulline-specific CD4 T cell response in transgenic mice models expressing human HLA-DP4 or HLA-DR4. Vaccinations with the NPM266-285cit peptide stimulated antitumor responses that resulted in dramatic tumor therapy, greatly improved survival, and protected against rechallenge without further vaccination. The antitumor response was lost if MHCII expression on the tumor cells was knocked out demonstrating direct presentation of the NPM266-285cit epitope in tumors. This antitumor response was lost in B16 tumors lacking PAD2 enzyme indicating NPM266cit is citrullinated by PAD2 in this model. Assessment of the T cell repertoire in healthy individuals and patients with lung cancer also showed CD4 T cells that respond to NPM266-285cit. The proliferative CD4 responses displayed a Th1 profile as they were accompanied with increased IFNγ and granzyme B expression. Depletion of CD45RO+ memory cells prior to stimulation suggested that responses originated from a naïve population in healthy donors. CONCLUSION: This study indicates PAD2 can citrullinate the nuclear antigen NPM at position 277 which can be targeted by CD4 T cells for antitumor therapy. This is distinct from PAD4 citrullination of arginine 197 within NPM which results in its transport from the nucleoli to the nucleoplasm.

Impact of Posttranslational Modification in Pathogenesis of Rheumatoid Arthritis: Focusing on Citrullination, Carbamylation, and Acetylation.

Rheumatoid arthritis (RA) is caused by prolonged periodic interactions between genetic, environmental, and immunologic factors. Posttranslational modifications (PTMs) such as citrullination, carbamylation, and acetylation are correlated with the pathogenesis of RA. PTM and cell death mechanisms such as apoptosis, autophagy, NETosis, leukotoxic hypercitrullination (LTH), and necrosis are related to each other and induce autoantigenicity. Certain microbial infections, such as those caused by Porphyromonasgingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella copri, can induce autoantigens in RA. Anti-modified protein antibodies (AMPA) containing anti-citrullinated protein/peptide antibodies (ACPAs), anti-carbamylated protein (anti-CarP) antibodies, and anti-acetylated protein antibodies (AAPAs) play a role in pathogenesis as well as in prediction, diagnosis, and prognosis. Interestingly, smoking is correlated with both PTMs and AMPAs in the development of RA. However, there is lack of evidence that smoking induces the generation of AMPAs.

Early and Late Processes Driving NET Formation, and the Autocrine/Paracrine Role of Endogenous RAGE Ligands.

Neutrophil extracellular trap (NET) formation has emerged as an important response against various pathogens; it also plays a role in chronic inflammation, autoimmunity, and cancer. Despite a growing understanding of the mechanisms underlying NET formation, much remains to be elucidated. We previously showed that in human neutrophils activated with different classes of physiological stimuli, NET formation features both early and late events that are controlled by discrete signaling pathways. However, the nature of these events has remained elusive. We now report that PAD4 inhibition only affects the early phase of NET generation, as do distinct signaling intermediates (TAK1, MEK, p38 MAPK). Accordingly, the inducible citrullination of residue R2 on histone H3 is an early neutrophil response that is regulated by these kinases; other arginine residues on histones H3 and H4 do not seem to be citrullinated. Conversely, elastase blockade did not affect NET formation by several physiological stimuli, though it did so in PMA-activated cells. Among belated events in NET formation, we found that chromatin decondensation is impaired by the inhibition of signaling pathways controlling both early and late stages of the phenomenon. In addition to chromatin decondensation, other late processes were uncovered. For instance, unstimulated neutrophils can condition themselves to be poised for rapid NET induction. Similarly, activated neutrophils release endogenous proteic factors that promote and largely mediate NET generation. Several such factors are known RAGE ligands and accordingly, RAGE inbibition largely prevents both NET formation and the conditioning of neutrophils to rapidly generate NETs upon stimulation. Our data shed new light on the cellular processes underlying NET formation, and unveil unsuspected facets of the phenomenon that could serve as therapeutic targets. In view of the involvement of NETs in both homeostasis and several pathologies, our findings are of broad relevance.

CD8+ T Cells Variably Recognize Native Versus Citrullinated GRP78 Epitopes in Type 1 Diabetes.

In type 1 diabetes, autoimmune ß-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8+ T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8+ T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8+ T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8+ T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Epitope-Based Chicken-Derived Novel Anti-PAD2 Monoclonal Antibodies Inhibit Citrullination.

The aberrant upregulation of protein arginine deiminase 2- (PAD2-) catalyzed citrullination is reported in various autoimmune diseases (rheumatoid arthritis and multiple sclerosis) and several cancers. Currently, there are no anti-PAD2 monoclonal antibodies (mAbs) that can inhibit the citrullination reaction. Here, an epitope 341YLNRGDRWIQDEIEFGY357 was examined as an antigenic site of PAD2. Chickens were immunized with this epitope, and the generated mAbs were screened for its reactivity against the full-length PAD2. Enzyme-linked immunosorbent assay revealed that six mAbs, which were screened from the phage display library, crossreacted with mouse PAD2. Kinetic analysis revealed that mAbs are bound to PAD2 in the nanomolar range, which indicated a strong binding. Results of the in vitro citrullination inhibition assay revealed that the half-maximal effective concentration values of mAbs for the inhibition of histone or benzoyl-L-arginine ethyl ester citrullination were in the range of 6-75 nM which supports strong inhibition capabilities. Alanine scanning of epitope revealed that the peptide fragment 344RGDRWIQDEIEF355 was responsible for generating strong antibody responses that inhibit the PAD2-catalyzed citrullination reaction. These antibodies can aid in understanding the extracellular PAD2 function and treating diseases associated with aberrant citrullination.

The shared susceptibility epitope of HLA-DR4 binds citrullinated self-antigens and the TCR.

Individuals expressing HLA-DR4 bearing the shared susceptibility epitope (SE) have an increased risk of developing rheumatoid arthritis (RA). Posttranslational modification of self-proteins via citrullination leads to the formation of neoantigens that can be presented by HLA-DR4 SE allomorphs. However, in T cell-mediated autoimmunity, the interplay between the HLA molecule, posttranslationally modified epitope(s), and the responding T cell repertoire remains unclear. In HLA-DR4 transgenic mice, we show that immunization with a Fibß-74cit69-81 peptide led to a population of HLA-DR4Fibß-74cit69-81 tetramer+ T cells that exhibited biased T cell receptor (TCR) ß chain usage, which was attributable to selective clonal expansion from the preimmune repertoire. Crystal structures of pre- and postimmune TCRs showed that the SE of HLA-DR4 represented a main TCR contact zone. Immunization with a double citrullinated epitope (Fibß-72,74cit69-81) altered the responding HLA-DR4 tetramer+ T cell repertoire, which was due to the P2-citrulline residue interacting with the TCR itself. We show that the SE of HLA-DR4 has dual functionality, namely, presentation and a direct TCR recognition determinant. Analogous biased TCR ß chain usage toward the Fibß-74cit69-81 peptide was observed in healthy HLA-DR4+ individuals and patients with HLA-DR4+ RA, thereby suggesting a link to human RA.

Neutrophil-Associated Inflammatory Changes in the Pre-Diabetic Pancreas of Early-Age NOD Mice.

A growing body of evidence indicates that neutrophils are the first major leukocyte population accumulating inside the pancreas even before the onset of a lymphocytic-driven impairment of functional beta cells in type 1 diabetes mellitus (T1D). In humans, pancreata from T1D deceased donors exhibit significant neutrophil accumulation. We present a time course of previously unknown inflammatory changes that accompany neutrophil and neutrophil elastase accumulation in the pancreas of the non-obese diabetic (NOD) mouse strain as early as 2 weeks of age. We confirm earlier findings in NOD mice that neutrophils accumulate as early as 2 weeks of age. We also observe a concurrent increase in the expression of neutrophil elastase in this time period. We also detect components of neutrophil extracellular traps (NET) mainly in the exocrine tissue of the pancreas during this time as well as markers of vascular pathology as early as 2 weeks of age. Age- and sex-matched C57BL/6 mice do not exhibit these features inside the pancreas. When we treated NOD mice with inhibitors of myeloperoxidase and neutrophil elastase, two key effectors of activated neutrophil activity, alone or in combination, we were unable to prevent the progression to hyperglycemia in any manner different from untreated control mice. Our data confirm and add to the body of evidence demonstrating neutrophil accumulation inside the pancreas of mice genetically susceptible to T1D and also offer novel insights into additional pathologic mechanisms involving the pancreatic vasculature that have, until now, not been discovered inside the pancreata of these mice. However, inhibition of key neutrophil enzymes expressed in activated neutrophils could not prevent diabetes. These findings add to the body of data supporting a role for neutrophils in the establishment of early pathology inside the pancreas, independently of, and earlier from the time at onset of lymphocytic infiltration. However, they also suggest that inhibition of neutrophils alone, acting via myeloperoxidase and neutrophil elastase only, in the absence of other other effector cells, is insufficient to alter the natural course of autoimmune diabetes, at least in the NOD model of the disease.

Preparation and characterization of cyclic citrullinated peptide-immobilized latex beads for measurement of anti-citrillinated protein antibody through latex particle-enhanced turbidimetric immunoassay.

The anti-citrillinated protein antibody (ACPA) plays an important role in early diagnosis of rheumatoid arthritis (RA), and is usually detected by using cyclic citrullinated peptide (CCP) as antigen. The ACPA against CCP test is usually performed utilizing enzyme-linked immunosorbent assay (ELISA), but the ELISA is expensive and time-consuming. Here, latex particle-enhanced turbidimetric immunoassay (LTIA) based on CCP-immobilized latex bead was proposed for fast measurements of ACPA of RA patients. CCP-immobilized latex bead was fabricated through three methods, including direct coupling, overall coupling and layer by layer coupling. According to the optimized experiments, layer-by-layer coupling was the best method with advantages of time-saving, simple operation and good repeatability. In addition, a spacer arm of appropriate length between latex beads and CCP could avoid stereoscopic obstacles and make ACPA closer to CCP. The CCP-immobilized latex bead based on layer by layer coupling (CCP-LB-LLC) was used for assembling the homemade kit, which was applied in fast measurements of ACPA through LTIA. The homemade kit possessed a low limit of detection (0.2 U/mL) and an acceptable the batch-to-batch reproducibility. In addition, the homemade kit can be stored at 4 °C for at least one month. When used to detect 20 clinical samples, the results of homemade kit were consistent with commercial ELISA. Furthermore, LTIA based on the homemade kit was simpler and cheaper than ELISA. These results demonstrated that the homemade kit could be useful for diagnosis of RA patients.

Complementary Effects of Carbamylated and Citrullinated LL37 in Autoimmunity and Inflammation in Systemic Lupus Erythematosus.

LL37 acts as T-cell/B-cell autoantigen in Systemic lupus erythematosus (SLE) and psoriatic disease. Moreover, when bound to "self" nucleic acids, LL37 acts as "danger signal," leading to type I interferon (IFN-I)/pro-inflammatory factors production. T-cell epitopes derived from citrullinated-LL37 act as better antigens than unmodified LL37 epitopes in SLE, at least in selected HLA-backgrounds, included the SLE-associated HLA-DRB1*1501/HLA-DRB5*0101 backgrounds. Remarkably, while "fully-citrullinated" LL37 acts as better T-cell-stimulator, it loses DNA-binding ability and the associated "adjuvant-like" properties. Since LL37 undergoes a further irreversible post-translational modification, carbamylation and antibodies to carbamylated self-proteins other than LL37 are present in SLE, here we addressed the involvement of carbamylated-LL37 in autoimmunity and inflammation in SLE. We detected carbamylated-LL37 in SLE-affected tissues. Most importantly, carbamylated-LL37-specific antibodies and CD4 T-cells circulate in SLE and both correlate with disease activity. In contrast to "fully citrullinated-LL37," "fully carbamylated-LL37" maintains both innate and adaptive immune-cells' stimulatory abilities: in complex with DNA, carbamylated-LL37 stimulates plasmacytoid dendritic cell IFN-α production and B-cell maturation into plasma cells. Thus, we report a further example of how different post-translational modifications of a self-antigen exert complementary effects that sustain autoimmunity and inflammation, respectively. These data also show that T/B-cell responses to carbamylated-LL37 represent novel SLE disease biomarkers.

The Role of Citrullination in Inflammatory Bowel Disease: A Neglected Player in Triggering Inflammation and Fibrosis?

Citrullination is a posttranslational modification of proteins mediated by a specific family of enzymes called peptidylarginine deiminases (PAD). Dysregulation of these enzymes is involved in the etiology of various diseases, from cancer to autoimmune disorders. In inflammatory bowel disease (IBD), data for a role of citrullination in the disease process are starting to accumulate at different experimental levels including gene expression analyses, RNA, and protein quantifications. Most data have been generated in ulcerative colitis, but data in Crohn disease are lacking so far. In addition, the citrullination of histones is the fundamental process promoting inflammation through the formation of neutrophil extracellular traps (NETs). Interestingly, NETs have also been shown to activate fibroblasts into myofibroblasts in fibrotic interstitial lung disease. Therefore, citrullination merits more thorough study in the bowel to determine its role in driving disease complications such as fibrosis. In this review we describe the process of citrullination and the different players in this pathway, the role of citrullination in autoimmunity with a special focus on IBD, the emerging role for citrullination and NETs in triggering fibrosis, and, finally, how this process could be therapeutically targeted.

Do RA associated HLA-DR molecules bind citrullinated peptides or peptides from PAD4 to help the development of RA specific antibodies to citrullinated proteins?

PURPOSE: Rheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding a five amino acid basic motive, the shared epitope SE). Each HLA-DRB1 genotype defines a genotype specific risk of developing RA. RA is preceded by the emergence of anti citrullinated protein antibodies (ACPAs). Citrullin is a neutral version of arginin, a basic amino acid, formed after post translational modification by Peptidyl Arginyl Deiminases (PADs). HLA-DRB1 genes associated with RA are also associated with ACPAs. Two models might explain this association. Here we tested both models for prediction of HLA-DRB1 genotypic risks of developing RA. METHODS: We calculated the likelihoods for the 2 HLA-DR molecules encoded by 12 common HLA-DRB1 genotypes to bind at least one randomly chosen peptide from PAD4 or fibrinogen(native or citrullinatd) and compared them with the 12 respective HLA-DRB1genotypic risks of developing RA. RESULTS: HLA-DRB1 Genotypic risks of developing RA correlate with likelihoods of binding PAD4 peptides, not citrullinated Fibrinogen peptides. Thus, the molecular basis for the association of HLA-DR and ACPA positive RA is most likely the capability for RA associated HLA-DR molecules to bind peptides(s) from PAD4.

Extracellular vesicles and post-translational protein deimination signatures in haemolymph of the American lobster (Homarus americanus).

The American lobster (Homarus americanus) is a commercially important crustacean with an unusual long life span up to 100 years and a comparative animal model of longevity. Therefore, research into its immune system and physiology is of considerable importance both for industry and comparative immunology studies. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family that catalyses post-translational protein deimination via the conversion of arginine to citrulline. This can lead to structural and functional protein changes, sometimes contributing to protein moonlighting, in health and disease. PADs also regulate the cellular release of extracellular vesicles (EVs), which is an important part of cellular communication, both in normal physiology and in immune responses. Hitherto, studies on EVs in Crustacea are limited and neither PADs nor associated protein deimination have been studied in a Crustacean species. The current study assessed EV and deimination signatures in haemolymph of the American lobster. Lobster EVs were found to be a poly-dispersed population in the 10-500 nm size range, with the majority of smaller EVs, which fell within 22-115 nm. In lobster haemolymph, 9 key immune and metabolic proteins were identified to be post-translationally deiminated, while further 41 deiminated protein hits were identified when searching against a Crustacean database. KEGG (Kyoto encyclopedia of genes and genomes) and GO (gene ontology) enrichment analysis of these deiminated proteins revealed KEGG and GO pathways relating to a number of immune, including anti-pathogenic (viral, bacterial, fungal) and host-pathogen interactions, as well as metabolic pathways, regulation of vesicle and exosome release, mitochondrial function, ATP generation, gene regulation, telomerase homeostasis and developmental processes. The characterisation of EVs, and post-translational deimination signatures, reported in lobster in the current study, and the first time in Crustacea, provides insights into protein moonlighting functions of both species-specific and phylogenetically conserved proteins and EV-mediated communication in this long-lived crustacean. The current study furthermore lays foundation for novel biomarker discovery for lobster aquaculture.

Uncovering a Shared Epitope-Activated Protein Citrullination Pathway.

Rheumatoid arthritis (RA) is closely associated with shared epitope (SE)-coding HLA-DRB1 alleles and circulating anticitrullinated protein Abs (ACPA), but neither the respective pathogenic roles of SE and ACPA in RA nor the mechanisms underlying their coassociation are known. It was recently shown that the SE functions as a signal transduction ligand that activates a cell surface calreticulin-mediated, proarthritogenic, bone erosive pathway in an experimental model of RA. In this study, we demonstrate that stimulation of murine macrophages with LPS or DTT facilitated cell surface translocation of calreticulin, which in turn enabled increased SE-activated calcium signaling and activation of peptidylarginine deiminase with the resultant increased cellular abundance of citrullinated proteins. The i.p. administration of LPS to transgenic mice carrying a human SE-coding HLA-DRB1 allele lead to increased serum levels of TNF-α and anticitrullinated cyclic peptide Abs, along with terminal phalanx bone destruction. These data uncover a previously unknown signal transduction pathway by which the SE facilitates protein citrullination, ACPA production, and bone destruction.

Combination vaccine based on citrullinated vimentin and enolase peptides induces potent CD4-mediated anti-tumor responses.

BACKGROUND: Stress-induced post-translational modifications occur during autophagy and can result in generation of new epitopes and immune recognition. One such modification is the conversion of arginine to citrulline by peptidylarginine deiminase enzymes. METHODS: We used Human leukocyte antigen (HLA) transgenic mouse models to assess the immunogenicity of citrullinated peptide vaccine by cytokine Enzyme linked immunosorbant spot (ELISpot) assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma and ID8 ovarian models expressing either constitutive or interferon-gamma (IFNγ) inducible Major Histocompatibility Complex (MHC) class II (MHC-II) as represented by most human tumors. To determine the importance of CD4 T cells in tumor therapy, we analyzed the immune cell infiltrate into murine tumors using flow cytometry and performed therapy studies in the presence of CD4 and CD8 T cell depletion. We assessed the T cell repertoire to citrullinated peptides in ovarian cancer patients and healthy donors using flow cytometry. RESULTS: The combination of citrullinated vimentin and enolase peptides (Modi-1) stimulated strong CD4 T cell responses in mice. Responses resulted in a potent anti-tumor therapy against established tumors and generated immunological memory which protected against tumor rechallenge. Depletion of CD4, but not CD8 T cells, abrogated the primary anti-tumor response as well as the memory response to tumor rechallenge. This was further reinforced by successful tumor regression being associated with an increase in tumor-infiltrating CD4 T cells and a reduction in tumor-associated myeloid suppressor cells. The anti-tumor response also relied on direct CD4 T cell recognition as only tumors expressing MHC-II were rejected. A comparison of different Toll-like receptor (TLR)-stimulating adjuvants showed that Modi-1 induced strong Th1 responses when combined with granulocyte-macrophage colony-stimulating factor (GMCSF), TLR9/TLR4, TLR9, TLR3, TLR1/2 and TLR7 agonists. Direct linkage of the TLR1/2 agonist to the peptides allowed the vaccine dose to be reduced by 10-fold to 100-fold without loss of anti-tumor activity. Furthermore, a CD4 Th1 response to the citrullinated peptides was seen in ovarian cancer patients. CONCLUSIONS: Modi-1 citrullinated peptide vaccine induces potent CD4-mediated anti-tumor responses in mouse models and a CD4 T cell repertoire is present in ovarian cancer patients to the citrullinated peptides suggesting that Modi-1 could be an effective vaccine for ovarian cancer patients.

Bacterial citrullinated epitopes generated by Porphyromonas gingivalis infection-a missing link for ACPA production.

OBJECTIVES: Porphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA. METHODS: Recombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated. RESULTS: RACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57-71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018). CONCLUSIONS: RACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.

Immunogenic and inflammatory responses to citrullinated proteins are enhanced following modification with malondialdehyde-acetaldehyde adducts.

BACKGROUND/OBJECTIVE: Malondialdehyde-acetaldehyde adducts (MAA) act as potent immune adjuvants and co-localize with citrullinated antigens in tissues effected by rheumatoid arthritis (RA). We sought to examine the role of MAA-adducts in promoting RA-related autoimmunity and inflammation. METHODS: DBA/J1 mice were immunized with human serum albumin (HSA), HSA-MAA, citrullinated HSA (HSA-Cit), or HSA-MAA-Cit with subsequent measurement of serum anti-citrullinated protein antibody (ACPA) and anti-Cit T cell responses. Cellular binding of the same antigens was examined using THP-1 monocytes and Chinese Hamster Ovary (CHO) cells transfected with specific scavenger receptors (SRs: TLR4, SR-B2, SREC-1). The effects of these antigens on THP-1 activation were then examined by quantifying plate adherence, pro-inflammatory (TNFα, IL-1ß, IL-10) cytokine release, and SR (CD14, SR-B2)/co-stimulatory molecule (CD80, HLA-DR) expression. Comparisons were completed using one-way ANOVA with Tukey's post-hoc test. RESULTS: Mice immunized with co-modified HSA produced significantly higher ACPA concentrations than all other groups whereas T cell responses to citrullinated proteins were highest following immunization with HSA-MAA. Both transfected CHO and THP-1 cells demonstrated significantly higher binding of HSA-MAA-Cit vs. HSA or HSA-Cit. THP-1 cells exposed to HSA-MAA-Cit expressed significantly higher concentrations of TNFα, IL-1ß, and IL-10 vs. all other groups. Furthermore, THP-1 cells demonstrated significantly increased plate adherence and higher expression of CD14, SR-B2, and HLA-DR following incubation with HSA-MAA-Cit vs. HSA or HSA-Cit. CONCLUSION: These studies demonstrate that MAA-adduction of citrullinated antigen greatly enhances immune and cellular responses, potentially acting as a key co-factor in RA pathogenesis.

Antibodies and B cells recognising citrullinated proteins display a broad cross-reactivity towards other post-translational modifications.

OBJECTIVE: Autoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs. METHODS: BCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated. RESULTS: Most mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens. CONCLUSIONS: Our study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis.

Gingimaps: Protein Localization in the Oral Pathogen Porphyromonas gingivalis.

Porphyromonas gingivalis is an oral pathogen involved in the widespread disease periodontitis. In recent years, however, this bacterium has been implicated in the etiology of another common disorder, the autoimmune disease rheumatoid arthritis. Periodontitis and rheumatoid arthritis were known to correlate for decades, but only recently a possible molecular connection underlying this association has been unveiled. P. gingivalis possesses an enzyme that citrullinates certain host proteins and, potentially, elicits autoimmune antibodies against such citrullinated proteins. These autoantibodies are highly specific for rheumatoid arthritis and have been purported both as a symptom and a potential cause of the disease. The citrullinating enzyme and other major virulence factors of P. gingivalis, including some that were implicated in the etiology of rheumatoid arthritis, are targeted to the host tissue as secreted or outer-membrane-bound proteins. These targeting events play pivotal roles in the interactions between the pathogen and its human host. Accordingly, the overall protein sorting and secretion events in P. gingivalis are of prime relevance for understanding its full disease-causing potential and for developing preventive and therapeutic approaches. The aim of this review is therefore to offer a comprehensive overview of the subcellular and extracellular localization of all proteins in three reference strains and four clinical isolates of P. gingivalis, as well as the mechanisms employed to reach these destinations.

Generation of Distinct Patterns of Rheumatoid Arthritis Autoantigens by Peptidylarginine Deiminase Types 2 and 4 During Perforin-Induced Cell Damage.

OBJECTIVE: To address the independent roles of peptidylarginine deiminase type 2 (PAD2) and PAD4 in generating rheumatoid arthritis (RA) autoantigens by using a system that mimics intracellular citrullination in the RA joint. METHODS: PAD2- or PAD4-expressing 293T cells and mock-transfected cells were used as targets in cytotoxic assays using lymphokine-activated killer cells, cytotoxic YT cell granule contents, or purified human perforin. Protein citrullination and autoantigen production were determined by immunoblotting using the anti-modified citrulline-Senshu method and RA sera (n = 30), respectively. RESULTS: RA sera recognized at least 3 categories of autoantigens in PAD-expressing target cells killed by the cytotoxic lymphocyte granule-induced death pathway. These included: 1) autoantigens targeted in their native form, 2) citrullinated antigens, and 3) antigens cleaved by cytotoxic proteases (e.g., granzymes). Interestingly, although target cells expressing PAD2 or PAD4 showed prominent hypercitrullination of a broad range of proteins during cytotoxic granule-induced cell damage, autoantibodies in RA sera targeted only a very limited number of antigens in hypercitrullinated cells. Furthermore, RA sera showed distinct reactivities to autoantigens generated by PAD2 or PAD4. CONCLUSION: The cytotoxic granule-induced death pathway has the capacity to modify antigens by inducing hypercitrullination and antigen cleavage in target cells. Interestingly, among a large number of citrullinated proteins generated by PAD2 and PAD4 in cells, only a few are likely involved in the production of autoantibodies in RA.

Deiminated proteins in extracellular vesicles and serum of llama (Lama glama)-Novel insights into camelid immunity.

Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, causing functional and structural changes in target proteins. Protein deimination causes generation of neo-epitopes, affects gene regulation and also allows for protein moonlighting. Furthermore, PADs have been found to be a phylogenetically conserved regulator for extracellular vesicle (EVs) release. EVs are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins in serum and serum-EVs are described for the first time in camelids, using the llama (Lama glama L. 1758) as a model animal. We report a poly-dispersed population of llama serum EVs, positive for phylogenetically conserved EV-specific markers and characterised by TEM. In serum, 103 deiminated proteins were overall identified, including key immune and metabolic mediators including complement components, immunoglobulin-based nanobodies, adiponectin and heat shock proteins. In serum, 60 deiminated proteins were identified that were not in EVs, and 25 deiminated proteins were found to be unique to EVs, with 43 shared deiminated protein hits between both serum and EVs. Deiminated histone H3, a marker of neutrophil extracellular trap formation, was also detected in llama serum. PAD homologues were identified in llama serum by Western blotting, via cross reaction with human PAD antibodies, and detected at an expected 70 kDa size. This is the first report of deiminated proteins in serum and EVs of a camelid species, highlighting a hitherto unrecognized post-translational modification in key immune and metabolic proteins in camelids, which may be translatable to and inform a range of human metabolic and inflammatory pathologies.