The CN-index of hnRNP-DL Predicts an Individual “Window of Treatment Success” in RA Patients

Abstract

BackgroundThere is a need for biomarker to identify patients ‘at risk’ for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers.MethodsUsing Protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry. ResultshnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. This CNDL (Citrullinated-Native-hnRNP-DL)-index identified and the bioinformatic value was explored, as a new value for an “individual window of treatment success” in early RA and for the detection of RF-IgM/α-CCP2 seronegative RA patients (24-46%). Negative CNDL-index was found in SLE patients, risk-RA- and early RA-cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CNDL-values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC-analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients.ConclusionCNDL-index defines patients is a possible biomarker for develop RA and the “window of treatment success” thereby potentially closing the sensitivity gap.

Undervalued and novel roles of heterogeneous nuclear ribonucleoproteins in autoimmune diseases: Resurgence as potential biomarkers and targets

“…The recombinant hnRNP DL expressed in Escherichia coli can be recognized by all tested anti-Sm autosera obtained from SLE patients, indicating hnRNP DL as a novel autoantigen for SLE (Chang et al, 2013). Both native and citrullinated hnRNP DL were reactive to sera of SLE patients, and citrullination seemingly brought about novel structural epitopes recognized by SLE sera that were negative for cyclic citrullinated peptides (Marklein et al, 2021). Autoantibodies directed to hnRNP D can also be detected in around 33% of SLE patients and four out of five anti-Sm-positive sera, and the epitope was mapped to the conformational structure within the N-terminal RNA-binding region thought on which the RNA binding activity was unacted (Skriner et al, 2008).…”

“…RF/anti-CCP2 (Marklein et al, 2021). In addition, 20% of sera from RA patients also reacted with AUF1 (Skriner et al, 2008).…”

“…Besides, autoantibodies to hnRNP B1, one of the alternatively spliced variants from hnRNP A2, were associated with the level of C-reactive protein and the erythrocyte sedimentation rate, which reflected the activity of RA (Maslyanskiy et al, 2014). Like RA33, hnRNP DL was also targeted as either native or citrullinated autoantigen in RA patients at dissimilar stages (Marklein et al, 2021). The citrullinated/native index of anti-hnRNP DL autoantibodies seemed to be of value in identifying individuals at risk for RA and predicting the therapeutic response especially in those seronegative for This patient suffered from arthralgias, myalgias, as well as symptoms related to the syndrome of idiopathic chronic urticaria and angioedema with thyroid autoimmunity.…”

“…The alterations in the immunogenicity of self-antigens, such as the apoptosis-induced histone modifications, may also elicit autoimmunity possibly under the condition of dysfunction in removing apoptotic cells (van Bavel et al, 2011). However, although the fact that both unmodified and citrullinated hnRNP A2/B1 and hnRNP DL are targeted as autoantigens in RA challenged the post-transitional modification (i.e., protein citrullination) as the central principle for tolerance breakdown, the transition among distinct forms of autoantigens and their associations with different clinical phenotypes indicated the involvement of epitope spreading (Konig et al, 2016;Marklein et al, 2021).…”

“…Thus, in addition to antigen peptides derived from hnRNPs themselves, it may not exclude the possibility that activation of TLRs on APCs by hnRNPs-associated DNAs or RNAs and the production of inflammatory cytokines such as type I interferons (IFNs) induce bystander activation of autoreactive lymphocytes against hnRNPs (Hoffmann et al, 2011), which applies in the cases of alternative inflammatory adjuvants as well. Parallel to multiple nucleic acids-associated autoantigens in SLE (Han et al, 2015), engagement of both BCR by hnRNPs and TLRs by their associated DNAs or RNAs presumably also augments the autoantibody generation, and knockout mice models testified the dependency of TLR7/9 for producing autoantibodies to hnRNP DL (Marklein et al, 2021). Thus, non-antigen-specific activation of autoreactive lymphocytes against hnRNPs could precipitate the onset or aggravation of corresponding autoimmune diseases.…”