Enzymes like peptidylarginine deiminase 4 (PAD4) generate citrullinated epitopes that are crucial in triggering and driving the anti-citrullinated protein antibody (ACPA) response in rheumatoid arthritis. Here, we assessed whether serum PAD4-potentiating activity is associated with subsequent arthritis development among ACPA-positive individuals during the pre-clinical phase. The study included 267 ACPA-positive individuals from the Karolinska Risk-RA cohort, among whom 101 (37.8%) developed arthritis within the observational period (range 22-60 months). We measured the ability of serum to potentiate PAD4 activity by quantifying the generation of citrullinated products using high-performance HPLC. Study participants were then classified as PAD4act-positive or -negative, and PAD4 status analysed in relation to onset of arthritis, clinical characteristics (ESR, CRP and DAS28) and established risk factors (HLA-DRB genotype, smoking). PAD4act-positivity was identified in 35 individuals, with a significant proportion (21, p = 0.0049) developing arthritis. Kaplan-Meier analysis demonstrated that individuals with PAD4act-positivity had increased rate of progressing to arthritis (p = 0.0022). Additionally, PAD4act-positive individuals were more likely to possess at least one copy of the HLA-DRB shared epitope allele (p = 0.045). Notably, PAD4act-positivity did not associate with pre-clinical CRP, ESR, DAS28, or smoking status. These findings suggest that serum PAD4-potentiating activity may help stratify arthritis risk and time to onset among ACPA-positive at-risk individuals.

Abstract Autoantibodies are hallmarks of many autoimmune diseases, but their potential pathogenic roles, particularly for those targeting intracellular proteins, remain unclear. Anti-MDA5-positive dermatomyositis (anti-MDA5 DM) is characterized by autoantibodies against the intracellular protein MDA5 1,2 , a conserved innate immune receptor that recognizes viral dsRNA by forming filaments 3 . Here, using four patient-derived monoclonal autoantibodies (mAbs), we reconstitute and define the molecular architecture, biogenesis, and immunological activity of pathogenic MDA5 immune complexes. Our cryo-EM analysis revealed that these mAbs bind dsRNA-scaffolded MDA5 filaments in at least two distinct binding modes, each exhibiting striking epitope convergence using germline-encoded residues. Extracellular immune complexes formed between mAbs and filamentous, but not monomeric, MDA5 potently activate multiple innate immune pathways, with the magnitude of activation determined by antibody binding mode and immune complex stoichiometry. Antibody bivalency further crosslinks MDA5 filaments into higher-order aggregates with heightened immunostimulatory activity, demonstrating an active role of autoantibodies in shaping immune complex architecture. Analysis of patient plasma reveals elevated levels of extracellular MDA5 filaments and identifies LINE retroelement-derived dsRNA as a structural scaffold. Notably, MDA5 immune complexes induce endogenous LINE dsRNA expression, likely promoting additional MDA5 filament formation and extracellular release through inflammatory cell death. These data thus support a self-amplifying inflammatory cycle as a pathogenic mechanism for anti-MDA5 DM. Collectively, our study defines a broadly applicable architectural principle, in which higher-order organization and binding modes of autoantibodies––beyond antibody affinity or nucleic acid presence alone––govern innate immune activation.

Microglia survey and regulate central nervous system myelination during embryonic development and adult homeostasis. However, whether microglia–myelin interactions are spatiotemporally regulated remains unexplored. Here, by examining spinal cord white matter tracts in mice, we determined that myelin degeneration was particularly prominent in the dorsal column (DC) during normal aging. This was accompanied by molecular and functional changes in DC microglia as well as an upregulation of transforming growth factor beta (TGF)β signaling. Disrupting TGFβ signaling in microglia led to unrestrained microglial responses and myelin loss in the DC, accompanied by neurological deficits exacerbated with aging. Single-nucleus RNA-sequencing analyses revealed the emergence of a TGFβ signaling-sensitive microglial subset and a disease-associated oligodendrocyte subset, both of which were spatially restricted to the DC. We further discovered that microglia rely on a TGFβ autocrine mechanism to prevent damage of myelin in the DC. These findings demonstrate that TGFβ signaling is crucial for maintaining microglial resilience to myelin degeneration in the DC during aging. This highlights a previously unresolved checkpoint mechanism of TGFβ signaling with regional specificity and spatially restricted microglia–oligodendrocyte interactions. Zhu et al. find that aging causes region-specific myelin damage in the spinal cord, which is counteracted by enhanced TGFβ signaling in microglia, revealing a protective mechanism for healthy aging.

OBJECTIVES:Autoantibodies targeting melanoma differentiation associated protein 5 (MDA5) are strongly associated with dermatomyositis (DM) and may contribute to its pathogenesis. Here we aimed to investigate MDA5+ B cells, their phenotype and generate MDA5 monoclonal antibodies to assess their epitope specificity. METHODS:MDA5-reactive B cells were captured from peripheral blood of patients with anti-MDA5+ DM (n = 3) using an MDA5-fluorescent probe. B cell receptor (BCR) sequences were analysed from single-sorted B cells (n = 240). Selected clones were re-expressed as IgG1 monoclonal antibodies (mAbs, n = 23). Reactivity was assessed using recombinant MDA5 protein constructs, peptide epitope mapping, ELISA, western blot and a commercial line blot assay. RESULTS:Of 240 anti-MDA5+ sorted B cells, 23 BCRs were re-expressed as mAbs, two of which showed high reactivity and specificity for MDA5. These antibody sequences originated from one CD19+IgD-CD27-CD38+ and one CD19+IgD-CD27+CD38+ IgG+ B cell with low somatic hypermutation (SHM). Both mAbs had nanomolar apparent affinity and bound to sites within the helicase domains of the MDA5 protein but with distinct epitope recognition. Serology screening confirmed targeting of a linear epitope identified in the mAb studies. CONCLUSION:Our results show that anti-MDA5+ B cells recognize the helicase domains, which are the enzymatically active domains of the protein. These results have implications for understanding the etiopathology of anti-MDA5+ DM and development of new antigen-specific therapies.

Evidence suggests that self-tolerance is breached in the lung prior to the clinical onset of rheumatoid arthritis (RA) in the joints. The human leukocyte antigen DR (HLA-DR) shared epitope (SE) represents the strongest genetic risk factor for sero-positive RA. However, to our knowledge, the HLA-DR immunopeptidome of the RA lung and its link to HLA-DR genotype has not been investigated to date. The objective of this study was to optimize the methods for characterizing the HLA-DR immunopeptidome of lung immune cells and apply it to newly diagnosed RA patients versus current-smoker healthy controls, as well as to investigate the connection with the HLA-DR genotype. The HLA-DR immunopeptidome method was improved to facilitate characterization from as few as 6 million bronchoalveolar lavage (BAL) cells per subject, consisting primarily of alveolar macrophages. This method was applied to newly diagnosed RA patients naive to treatment (n=9, LURA cohort), as well as healthy current-smoker controls (n=10, COSMIC cohort). For five of the RA patients, a 6-month follow-up after initiation of the standard-of-care treatment regime was also included. After isolation and purification, peptide samples were separated by nano-flow liquid chromatography coupled to an Orbitrap mass spectrometer equipped with ion mobility device (FAIMS). Mass spectra acquired in data dependent acquisition mode were then searched against a human proteome database. Subsequently, the identified peptides were deconvoluted to their predicted binding HLA-DR allele using MHCMotifDecon based on the sequenced genotype of the individual. An optimized sample preparation and analytic method enabled the detection of over 23,000 peptides from over 3,000 source proteins with between 1,000 and 5,000 peptides identified per sample. Notably, the application of FAIMS with three compensation voltages allowed for efficient transfer of 2+, 3+, and 4+ peptide ions while removing singly charged background ions. Hierarchical clustering revealed that the immunopeptidome was more driven by the HLA-DR genotype than by RA disease or sex. However, since the HLA-DR genotype is a strong risk factor for RA, these results are convoluted. When deconvoluting the peptides to their predicted binding allele, the HLA-DRB1 alleles *01:01, *04:01, *04:04, *04:05, *04:07, and *10:01 were consistently assigned more peptides than other alleles. Except for *04:07 these alleles belong to the SE risk factor alleles, providing a potential explanation between HLA-SE and RA pathogenesis. Native peptides from known citrullinated and non-modified RA autoantigens (such as α-enolase and calreticulin) were detected and validated as binders in prediction algorithms. No significant differences were found between base line and follow-up (post-treatment) samples from RA patients. Taken together, this data characterizes the HLA-DR immunopeptidome in the lung of early RA in an unprecedented manner, which together with future immunogenicity studies will help our understanding of the connection between the lung and the pathogenesis of RA. Finally, more peptides predicted to bind to SE alleles and *04:07 compared to other alleles demands further study on the relative expression of HLA-DR alleles and presentation mechanisms to understand the implications for RA. ### Competing Interest Statement The authors have declared no competing interest.

Objective Individuals positive for anti-cyclic-peptide-antibodies (anti-CCP) and musculoskeletal complaints (MSK-C) are at risk for developing rheumatoid arthritis (RA). In this study we aimed to investigate factors involved in arthritis progression.Methods Anti-CCP2-positive individuals with MSK-C referred to a rheumatologist were recruited. Individuals lacked arthritis at clinical and ultrasound examination and were followed for >= 3 years or until clinical arthritis diagnosis. Blood samples from inclusion were analysed for nine ACPA reactivities (citrullinated alpha-1-enolase, fibrinogen, filaggrin, histone, vimentin and tenascin peptides); 92 inflammation-associated proteins; and HLA-shared epitope alleles. Cox regression was applied to the data to identify independent predictors in a model.Results Two hundred and sixty-seven individuals were included with median follow-up of 49 months (interquartile range [IQR]: 22-60); 101 (38%) developed arthritis after a median of 14 months (IQR: 6-27). The analysis identified that presence of at least one ACPA reactivity (hazard ratio [HR] 8.0; 95% CI: 2.9, 22), ultrasound-detected tenosynovitis (HR 3.4; 95% CI: 2.0, 6.0), IL-6 levels (HR 1.5; 95% CI: 1.2, 1.8) and IL-15 receptor alpha (IL-15R alpha) levels (HR 0.6; 95% CI: 0.4, 0.9) are significant independent predictors for arthritis progression in a prediction model (Harrell's C 0.76 [s.e. 0.02], AUC 0.82 [95% CI: 0.76, 0.89], cross-validated AUC 0.70 [95% CI: 0.56, 0.85]).Conclusion We propose a high RA risk phase characterized by presence of ACPA reactivity, tenosynovitis, IL-6 and IL-15R alpha and suggest that these factors need to be further investigated for their biological effects and clinical values, to identify individuals at particular low risk and high risk for arthritis progression.

Anti-citrullinated protein autoantibodies (ACPA) are diagnostic for rheumatoid arthritis (RA). The antigens recognized by these autoantibodies are produced by protein arginine deiminases (PADs), particularly PAD4. However, it remains unknown why and how PAD4 causes this aberrant citrullination in RA. Here, we report that poly-perforin pores are present on freshly isolated neutrophils from RA patients, but not on healthy donor neutrophils. Neutrophils with perforin pores also contained intracellular citrullinated proteins in the region adjacent to the pores. This response was replicated in vitro by treating neutrophils with purified perforin, which generated intense dots of anti-perforin immunofluorescence, calcium influx, and intracellular citrullination. Extensive neutrophil killing in Felty's syndrome, an aggressive form of RA, correlated with particularly high ACPA, and PAD4 autoantibodies. In contrast, other forms of death, including NETosis, apoptosis, and pyroptosis, produced minimal citrullination. We conclude that neutrophil targeting by perforin leading to intracellular citrullination takes place in patients with RA.

Autoantibodies to malondialdehyde (MDA) proteins constitute a subset of anti-modified protein autoantibodies in rheumatoid arthritis (RA), which is distinct from citrulline reactivity. Serum anti-MDA IgG levels are commonly elevated in RA and correlate with disease activity, CRP, IL6, and TNF-alpha. MDA is an oxidation-associated reactive aldehyde that together with acetaldehyde mediates formation of various immunogenic amino acid adducts including linear MDA-lysine, fluorescent malondialdehyde acetaldehyde (MAA)-lysine, and intramolecular cross-linking. We used single-cell cloning, generation of recombinant antibodies (n = 356 from 25 donors), and antigen-screening to investigate the presence of class-switched MDA/MAA+ B cells in RA synovium, bone marrow, and bronchoalveolar lavage. Anti-MDA/MAA+ B cells were found in bone marrow plasma cells of late disease and in the lung of both early disease and risk-individuals and in different B cell subsets (memory, double negative B cells). These were compared with previously identified anti-MDA/ MAA from synovial memory and plasma cells. Seven out of eight clones carried somatic hypermutations and all bound MDA/MAA-lysine independently of protein backbone. How-ever, clones with somatic hypermutations targeted MAA cross-linked structures rather than MDA-or MAA-hapten, while the germline-encoded synovial clone instead bound linear MDA-lysine in proteins and peptides. Binding patterns were maintained in germline converted clones. Affinity purification of polyclonal anti-MDA/MAA from patient serum revealed higher proportion of anti-MAA versus anti-MDA compared to healthy controls. In conclusion, IgG anti-MDA/MAA show distinct targeting of different molecular structures. Anti-MAA IgG has been shown to promote bone loss and osteoclastogenesis in vivo and may contribute to RA pathogenesis.

ObjectiveThe lung is implicated as a site for breach of tolerance prior to onset of seropositive rheumatoid arthritis (RA). To substantiate this, we investigated lung‐resident B cells in bronchoalveolar lavage (BAL) samples from untreated early RA patients and anti–citrullinated protein antibody (ACPA)–positive individuals at risk for developing RA.MethodsSingle B cells (n = 7,680) were phenotyped and isolated from BAL samples from individuals at risk of RA (n = 3) and at RA diagnosis (n = 9). The immunoglobulin variable region transcripts were sequenced and selected for expression as monoclonal antibodies (n = 141). Monoclonal ACPAs were tested for reactivity patterns and binding to neutrophils.ResultsUsing our single‐cell approach, we found significantly increased proportions of B lymphocytes in ACPA+ compared to ACPA– individuals. Memory and double‐negative B cells were prominent in all subgroups. Upon antibody re‐expression, 7 highly mutated citrulline‐autoreactive clones originating from different memory B cell subsets were identified, both in individuals at risk of RA and early RA patients. Lung IgG variable gene transcripts from ACPA+ individuals carried frequent mutation‐induced N‐linked Fab glycosylation sites (P < 0.001), often in the framework 3 of the variable region. Two of the lung ACPAs bound to activated neutrophils, 1 from an individual at risk of RA and 1 from an early RA patient.ConclusionT cell–driven B cell differentiation resulting in local class switching and somatic hypermutation are evident in lungs before as well as in early stages of ACPA+ RA. Our findings add to the notion of lung mucosa being a site for initiation of citrulline autoimmunity preceding seropositive RA.image

Objective The objective of this study was to discover autoantibodies to non-modified proteins associated with the presence/absence of ACPAs in RA .Methods The autoantibody repertoire of 80 ACPA-negative and 80 ACPA-positive RA subjects from the Swedish population-based Epidemiological Investigation of RA (EIRA) cohort was screened using a suspension bead array built on protein fragments earlier described as autoimmunity targets. Four autoantibodies positive in the initial screening were validated in another set of EIRA samples containing 317 ACPA-positive, 302 ACPA-negative and 372 age- and sex-matched controls. The relationship between the four autoantibodies and lung abnormalities on high-resolution CT (HRCT) was examined in 93 early-RA patients from the LURA cohort. Association between the autoantibodies, smoking and MHC class II alleles was assessed by logistic regression analysis. Results Anti-ANOS1 and anti-MURC IgG levels were associated with ACPA-positive status [odds ratio (OR) = 3.02; 95% CI 1.87-4.89; and OR = 1.86; 95% CI 1.16-2.97, respectively] and increased in ACPA-positive patients compared with controls. Anti-ANOS1 IgG was associated with smoking habit (OR = 2.11; 95% CI 1.22-3.69) and anti-MURC IgG with the presence of the MHC class II 'shared-epitope' genes (OR = 1.95; 95% CI 1.11-3.46). Anti-TSPYL4 IgG was associated with being ACPA negative (OR = 0.41; 95% CI 0.19-0.89). Anti-TSPYL4 IgG and anti-MAP2K6 IgG levels were increased in the ACPA-negative patients compared with controls. Presence of anti-MAP2K6 IgG and anti-TSPYL4 IgG correlated negatively with HRCT-defined lung abnormalities. Conclusion These four autoantibodies may be useful in diagnostics and in predicting clinical phenotypes of RA.

Background Individuals testing positive for anti-cyclic-citrullinated-peptide-antibodies (Anti-CCP) and musculoskeletal (MSK) complaints are at risk for developing rheumatoid arthritis (RA). Objectives We aim to identify factors involved in arthritis progression in a population considered at risk for RA. Methods Anti-CCP-positive individuals with MSK complaints referred to rheumatologist in the Region Stockholm were recruited. Individuals lacked arthritis at clinical and ultrasound examination and were followed for ≥3 years or until arthritis diagnosis was made. Blood samples from inclusion were analyzed for 9 selected anti-citrullinated-protein-antibody (ACPA) reactivities (citrullinated α-1-enolase, fibrinogen, filaggrin, histone, vimentin and tenascin peptides); as well as a panel of 92 inflammation-associated proteins and HLA-SE alleles. Cox regression was applied to the data and a predictive multivariate model was identified. Results are shown with a confidence interval (CI) of 95 percent. Results 267 individuals were recruited. 101 (38%) developed arthritis in median after 14 months (IQR: 6-27). In the multivariate analysis: ACPA reactivity (HR 8.0, CI 2.9-22, p<0.0001), IL15R-α levels (HR 0.6, CI 0.4-0.9, p 0.006), IL6 levels (HR 1.5, CI 1.2-1.8, p<0.0001) and the presence of tenosynovitis as detected by ultrasound (HR 3.4, CI 2.0-6.0, p<0.0001) were significantly associated with arthritis. Diagnostic accuracy for ACPA reactivity test had a sensitivity of 96% (CI 92-99.8), a specificity of 30% (CI 30-46), a positive predictive value of 51% (CI 43-58) and a negative predictive value of 94% (CI 87-99.7). Diagnostic accuracy for ultrasound assessed tenosynovitis had a sensitivity of 17% (CI 9-24), a specificity of 99% (CI 97-100), a positive predictive value of 89% (CI 74-100) and a negative predictive value of 64% (CI 57-70). Conclusion We propose a high-risk RA phase characterized by the presence of certain ACPA reactivities, IL15-Rα, IL6, and tenosynovitis, parameters that could be used to identify individuals at particular low risk and high risk for arthritis progression. Acknowledgements None. Disclosure of Interests Alexandra Cîrciumaru: None declared, Yogan Kisten: None declared, Monika Hansson: None declared, Linda Mathsson-Alm Employee of: ThermoFisher Scientific, Vijay Joshua: None declared, Heidi Wähämaa: None declared, Malena Loberg Haarhaus: None declared, Joakim Lindqvist: None declared, Fei Guozhong: None declared, Nancy Vivar Pomiano: None declared, Hamed Rezaei: None declared, Erik af Klint: None declared, Aleksandra Antovic: None declared, Bence Réthi: None declared, Anca Catrina: None declared, Aase Hensvold: None declared.

Background Autoantibodies to malondialdehyde (MDA) proteins constitute a subset of anti-modified protein autoantibodies (AMPA) in rheumatoid arthritis (RA), which is not directly overlapping with citrulline reactivity. We have previously shown that IgM anti-MDA is prominent in the natural antibody repertoire at birth and may have homeostatic roles. Yet, IgG anti-MDA is elevated in RA and correlates with DAS28, CRP, IL6 and TNF-α [1]. Moreover, RA-derived anti-MDA monoclonal antibodies induce bone erosion and osteoclastogenesis by modulating cell metabolism [2]. MDA is an oxidation-associated reactive aldehyde that together with acetaldehyde (AA) mediates formation of a range of amino acid adducts including linear MDA-lysine, fluorescent MAA-lysine, and intramolecular cross-linking. MDA/MAA proteins carry multiple adduct-variants, and we here propose that distinct subsets of anti-MDA/MAA antibodies, defined based on the MDA-induced molecular structures recognized, have different pathogenic properties. Moreover, we have investigated the presence of class-switched MDA+ B cells in RA synovium, bone marrow, and bronchoalveolar lavage (BAL). Objectives Identification of RA MDA+ B cells from different compartments and distinct MDA/MAA reactivity patterns. Methods Human recombinant mAbs were generated from single-cell sorted B cells from BAL and bone marrow (n=148) and screened for reactivity to MDA and MAA modified antigens. MDA-cyanoborohydride and MDA/MAA-hapten proteins without cross-linking were used for characterization. Serology screening of IgG reactivity to MAA-BSA was performed as previously described [3]. Results IgG MAA-reactivity is elevated in 49% of ACPA+ RA patients (n=278) compared to 14% of population controls (n=437). We did not detect any significant increase in IgG anti-MAA in ACPA+ RA-risk individuals at baseline,14% had high anti-MAA (n=263), compared to controls. But for risk-individuals that developed arthritis, anti-MAA levels increased at onset of arthritis with 26% displaying elevated levels (n=53). We identified IgG+ MDA+ cells among RA bone marrow plasma cells and BAL B cells from both early RA and ACPA+ RA-risk, which could be compared to three previously identified RA synovial clones. Seven out of eight investigated clones carried somatic hypermutations (SHM). Interestingly, all clones with SHM were found to specifically target MAA cross-linked structures rather than MDA- or MAA-hapten, while the germline-encoded synovial clone F04 bound strongly to linear MDA-lysine in proteins and peptides. Germline converting resulted in reduced MDA/MAA binding, suggesting different origin of the clones compared to F04. All binding was independent of protein backbone. Yet, only a subset of MAA-selective clones with SHM induced robust TNF-α expression in monocyte-derived macrophages and enhanced osteoclastogenesis. Conclusion MDA/MAA positive B cells can be identified in bone marrow and synovium in established RA and in the lungs of early and RA-risk individuals, but serum levels are only increased in conjunction with onset of arthritis and with inflammation. SHM and class-switching suggest affinity maturation and T-cell dependence of these responses. Distinct targeting of different MDA/MAA molecular structures was observed. Future studies will elucidate how MDA recognition patters are related to the anti-MDA inflammatory and bone-modulating properties and disease stages. References [1]Grönwall, C., et al J Autoimmun. 2017 84:29-45 [2]Sakuraba, K., et al J Autoimmun. 2022 133:102903 [3]Grönwall, C., et al Front Immunol. 202112:627986 Acknowledgements: NIL. Disclosure of Interests None Declared.

Transforming growth factor-β (TGF-β) signaling is critical for microglial maturation during development and the maintenance of microglial homeostasis in adulthood. It remains unclear whether regional susceptibilities to the loss of TGF-β signaling in microglia also exist, and the contributing factors have yet to be identified. We find that deletion of Tgfbr2 on microglia leads to microglial activation and demyelination in mouse spinal cords, primarily in the dorsal column (DC). Tgfbr2 -deficient microglia exhibit distinct transcriptomic changes, and those sorted from the DC display a more proinflammatory profile compared to those from the ventral column (VC) and grey matter (GM). Single nucleus RNA sequencing (snRNA-seq) of the spinal cord uncovers a microglial subtype that emerges exclusively following Tgfbr2 deletion (termed TGFβ signaling-suppressed microglia, TSM), exhibiting high expression of Mmp12, Gpnmb, Lgals3, Mgll, and Alcam, predominantly located in the DC. Phenotypically, disruption of microglial TGF-β signaling results in behavioral deficits that are more severe in female and older mice, whereas young male mice are less affected. Mechanistically, we reveal a significantly higher level of TGF-β1/TGFBR2 in the spinal cords of normal older mice compared to the young mice, with the DC region richer in genes of the TGF-β signaling pathway than the VC and GM regions. This indicates that older mice and the DC region require more TGFβ1 to maintain tissue homeostasis and, reciprocally, are more responsive and sensitive to the disruption of TGF-β signaling in microglia. Herein, we report a demyelinating disease with region-specificity and its susceptibility to the loss of microglial TGF-β signaling with gender and age differences. Our findings contribute valuable information to our understanding of the importance of microglia in regulating myelin health, especially during the aging process.

Background and ObjectiveStudies of autoimmunity and anti-citrullinated protein antibodies (ACPA) in idiopathic pulmonary fibrosis (IPF) have been confined to investigations of anti-cyclic citrullinated peptide (anti-CCP) antibodies which utilize synthetic peptides as surrogate markers for in vivo citrullinated antigens. We studied immune activation by analysing the prevalence of in vivo anti-modified protein antibodies (AMPA) in IPF. MethodsWe included patients with incident and prevalent IPF (N = 120), sex and smoking-matched healthy controls (HC) (N = 120) and patients with RA (N = 104). Serum (median time: 11 months [Q1-Q3: 1-28 months] from diagnosis) was analysed for presence of antibodies towards native and posttranslational modified (citrullinated [Cit, N = 25]; acetylated [Acet, N = 4] and homocitrullinated [Carb, N = 1]) peptides derived from tenascin (TNC, N = 9), fibrinogen (Fib, N = 11), filaggrin (Fil, N = 5), histone (N = 8), cathelicidin (LL37, N = 4) and vimentin (N = 5) using a custom-made peptide microarray. ResultsAMPA were more frequent and in increased levels in IPF than in HC (44% vs. 27%, p < 0.01), but less than in RA (44% vs. 79%, p < 0.01). We specifically observed AMPA in IPF towards certain citrullinated, acetylated and carbamylated peptides versus HC: tenascin (Cit((2033))-TNC2025-2040; Cit((2197))-TNC2177-2200; Cit((2198))-TNC2177-2200)(,) fibrinogen (Cit((38,42))-Fib & alpha;(36-50); Cit((72))-Fib & beta;(60-74)) and filaggrin (Acet-Fil(307-324), Carb-Fil(307-324)). No differences in survival (p = 0.13) or disease progression (p = 0.19) between individuals with or without AMPA was observed in IPF. However, patients with incident IPF had better survival if AMPA were present (p = 0.009). ConclusionA significant proportion of IPF patients present with specific AMPA in serum. Our results suggest autoimmunity as a possible characteristic for a subgroup of IPF that may affect disease outcome.

Seropositive rheumatoid arthritis (RA) is characterized by the presence of rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA) with different fine-specificities. Yet, other serum anti-modified protein autoantibodies (AMPA), e.g. anti-carbamylated (Carb), -acetylated (KAc), and malondialdehyde acetaldehyde (MAA) modified protein antibodies, have been described. In this comprehensive study, we analyze 30 different IgG and IgA AMPA reactivities to Cit, Carb, KAc, and MAA antigens detected by ELISA and autoantigen arrays in N=1985 newly diagnosed RA patients. Association with patient characteristics such as smoking and disease activity were explored. Carb and KAc reactivities by different assays were primarily seen in patients also positive for anti-citrulline reactivity. Modified vimentin (mod-Vim) peptides were used for direct comparison of different AMPA reactivities, revealing that IgA AMPA recognizing mod-Vim was mainly detected in subsets of patients with high IgG anti-Cit-Vim levels and a history of smoking. IgG reactivity to acetylation was mainly detected in a subset of patients with Cit and Carb reactivity. Anti-acetylated histone reactivity was RA-specific and associated with high anti-CCP2 IgG levels, multiple ACPA fine-specificities, and smoking status. This reactivity was also found to be present in CCP2+ RA-risk individuals without arthritis. Our data further demonstrate that IgG autoreactivity to MAA was increased in RA compared to controls with highest levels in CCP2+ RA, but was not RA-specific, and showed low correlation with other AMPA. Anti-MAA was instead associated with disease activity and was not significantly increased in CCP2+ individuals at risk of RA. Notably, RA patients could be subdivided into four different subsets based on their AMPA IgG and IgA reactivity profiles. Our serology results were complemented by screening of monoclonal antibodies derived from single B cells from RA patients for the same antigens as the RA cohort. Certain CCP2+ clones had Carb or Carb+KAc+ multireactivity, while such reactivities were not found in CCP2- clones. We conclude that autoantibodies exhibiting different patterns of ACPA fine-specificities as well as Carb and KAc reactivity are present in RA and may be derived from multireactive B-cell clones. Carb and KAc could be considered reactivities within the “Cit-umbrella” similar to ACPA fine-specificities, while MAA reactivity is distinctly different.