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ObjectiveNeutrophil infiltration into the synovial joint is a hallmark of rheumatoid arthritis (RA), a disease characterised by progressive bone erosion. However, the mechanisms by which neutrophils participate in bone destruction remain unclear. Carbamylation is a posttranslational modification linked to increased bone erosion in RA and we previously showed that carbamylation is present in RA neutrophil extracellular traps (NETs). However, it remains unclear whether NETs and their carbamylated protein cargo directly promote bone destruction and alter osteoclast biology.MethodsNETs and carbamylated NETs (cNETs) were assessed for their capacity to induce osteoclast formation in CD14+ monocytes. Chemical inhibitors and neutralising antibodies were used to elucidate the pathway by which NETs induce osteoclastogenesis. HLA-DRB1*04:01 mice received intra-articular injection of cNETs for 4 weeks. Joints were isolated and assessed for osteoclast formation. Plasma and synovial fluid samples from patients with RA (n=32) were assessed for the presence of carbamylated histone, and correlations to disease specific outcomes were performed.ResultsWe found that NETs, when cNETs, instruct monocytes to undergo rapid osteoclast formation. NET-mediated osteoclastogenesis appears to depend on Toll-like receptor 4 signalling and NET-associated proteins including histones and neutrophil elastase. In vivo, we identified that the number of osteoclasts increased following immunisation with cNETs in HLA-DRB1*04:01 transgenic mice. Furthermore, carbamylated histones are increased in plasma and synovial fluid from patients with RA and correlate with active bone resorption and inflammatory markers.ConclusionsOur results suggest that NETs have a direct role in RA-associated bone erosion by promoting osteoclast formation.

BACKGROUND: Measurement of hemoglobin A1c (HbA1c) in the blood is integral to and essential for the treatment of patients with diabetes mellitus. HbA1c reflects the mean blood glucose concentration over the preceding 8 to 12 weeks. Although the clinical value of HbA1c was initially limited by large differences in results among various methods, the investment of considerable effort to implement standardization has brought about a marked improvement in analysis. CONTENT: The focus of this review is on the substantial progress that has been achieved in enhancing the accuracy and, therefore, the clinical value of HbA1c assays. SUMMARY: The interactions between the National Glycohemoglobin Standardization Program and manufacturers of HbA1c methods have been instrumental in standardizing HbA1c. Proficiency testing using whole blood has allowed accuracy-based assessment of methods in individual clinical laboratories that has made an important contribution to improving the HbA1c measurement in patient samples. These initiatives, supported by the efforts of the IFCC network, have led to a continuing enhancement of HbA1c methods. Many of the factors that previously influenced HbA1c results independently of blood glucose have been eliminated from most modern methods. These include carbamylation, labile intermediates, and common hemoglobin variants. Nevertheless, some factors (e.g., race and aging) may alter HbA1c interpretation, but whether these differences have clinical implications remains contentious. HbA1c has a fundamental role in the diagnosis and management of diabetes. Ongoing improvements in HbA1c measurement and quality will further enhance the clinical value of this analyte.

[This corrects the article DOI: 10.3389/fimmu.2025.1532250.].

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis associated with psoriasis. A third of psoriatic patients develop PsA unknown mechanisms. No reliable diagnostic markers are available for PsA, or prognostic biomarkers for PsA development in psoriasis. We previously uncovered a pro-inflammatory role for cathelicidin LL37 in lesional psoriasis skin. LL37 binds nucleic acids and stimulates plasmacytoid/myeloid dendritic cells (pDC, mDCs) to secrete type I interferon (IFN-I) and pro-inflammatory factors. LL37 becomes an autoantigen for psoriatic Th1-Th17/CD8 T cells. Anti-LL37 antibodies were detected in systemic lupus erythematosus, an autoimmune disease characterized by neutrophil-extracellular-traps release (NETosis) in target organs. LL37 can be substrate of irreversible post-translational modifications, citrullination or carbamylation, linked to neutrophil activity. Here we analyzed inflammatory factors, included LL37, in PsA and psoriasis plasma and PsA synovial fluids (SF)/biopsies. We show that LL37 (as a product of infiltrating neutrophils) and autoantibodies to LL37 are elevated in PsA, but not OA SF. Anti-LL37 antibodies correlate with clinical inflammatory markers. Anti-carbamylated/citrullinated-LL37 antibodies are present in PsA SF/plasma and, at lower extent, in psoriasis plasma, but not in controls. Plasma anti-carbamylated-LL37 antibodies correlate with PsA (DAS44) but not psoriasis (PASI) disease activity. Ectopic lymphoid structures, and deposition of immunoglobulin-(Ig)G-complexes (IC) co-localizing with infiltrating neutrophils, are observed in PsA and not OA synovial tissues (ST). Activated complement (C5a, C9), GM-CSF and IFN-I are up-regulated in PsA and not OA synovia and in PsA and psoriasis plasma but not in HD. C9 and GM-CSF levels in PsA SF correlate with clinical inflammatory markers and DAS44 (C9) and with anti-carbamylated/citrullinated-LL37 antibodies (GM-CSF and IFN-I). Thus, we uncover a role for LL37 as a novel PsA autoantibody target and correlation studies suggest participation of anti-LL37 antibodies to PsA pathogenesis. Notably, plasma antibodies to carbamylated-LL37, which correlate with DAS44, suggest their use as new disease activity markers. GM-CSF and complement C5a and C9 elevation may be responsible for autoantigens release by neutrophils and their modification, fueling inflammation and autoreactivity establishment. Finally, targeting GM-CSF, C5a, C9 can be beneficial in PsA.

Carbamylated proteins and dysregulated neutrophils are implicated in rheumatoid arthritis (RA) pathogenesis. Herein, we characterized the neutrophils present in RA synovial fluid (SF) using proteomic techniques and evaluated their contribution to protein carbamylation. RA-SF neutrophil proteomic profile and SF proteome signature were investigated using high-resolution mass spectrometry. Carbamylated proteins and the degree of protein carbamylation were evaluated by mass spectrometric analysis. ELISA and chemiluminescence kits were used to examine myeloperoxidase (MPO) activity, and hydrogen peroxide (H O ) generation. SF neutrophils exhibited a shift in proteomic cargo with up-regulated proteins involved in defense responses, neutrophil degranulation, and reactive oxygen species metabolic processes, while proteins down-regulated were associated with megakaryocyte differentiation, leukocyte migration, and integrin-mediated signaling pathway. Elevated levels of neutrophil-derived proteins were detected in RA-SF. In addition, we specifically identified many carbamylated proteins and observed an increased frequency of protein carbamylation in RA-SF samples. Functionally, neutrophils from RA-SF showed a significantly increased level of MPO release and HH O generation. Moreover, MPO activity was higher in RA-SF than in autologous blood samples, which correlated well with the degree of protein carbamylation in RA-SF. Synovial neutrophils were found to be activated and increased releasing protein cargo, including MPO and ROS, into the synovial fluid. Presence of many carbamylated proteins in RA-SF and an increased MPO activity showed a strong correlation to the degree of protein carbamylation, suggesting neutrophil-derived MPO in promoting generation of aberrantly carbamylated proteins.

Because of their long lifespan, matrix proteins of the vascular wall, such as elastin, are subjected to molecular aging characterized by non-enzymatic post-translational modifications, like carbamylation which results from the binding of cyanate (mainly derived from the dissociation of urea) to protein amino groups. While several studies have demonstrated a relationship between increased plasma concentrations of carbamylated proteins and the development of cardiovascular diseases, molecular mechanisms explaining the involvement of protein carbamylation in these pathological contexts remain to be fully elucidated. The aim of this work was to determine whether vascular elastic fibers could be carbamylated, and if so, what impact this phenomenon would have on the mechanical properties of the vascular wall. Our experiments showed that vascular elastin was carbamylated in vivo. Fiber morphology was unchanged after in vitro carbamylation, as well as its sensitivity to elastase degradation. In mice fed with cyanate-supplemented water in order to increase protein carbamylation within the aortic wall, an increased stiffness in elastic fibers was evidenced by atomic force microscopy, whereas no fragmentation of elastic fiber was observed. In addition, this increased stiffness was also associated with an increase in aortic pulse wave velocity in ApoE −/− mice. These results provide evidence for the carbamylation of elastic fibers which results in an increase in their stiffness at the molecular level. These alterations of vessel wall mechanical properties may contribute to aortic stiffness, suggesting a new role for carbamylation in cardiovascular diseases.

Carbamylation is a non-enzymatic post-translational protein modification common in patients with uremia that causes pro-atherogenic alterations in plasma proteins. It is abundantly present in late-stage atherosclerotic plaques; however, the pathogenic relevance and functional consequences of this accumulation are not known. Human atherosclerotic plaque tissue samples were stratified by plaques' stage and kidney function. Immunohistochemistry revealed a significantly higher carbamylated lysine (carb-lys) abundance in latestage hemorrhaged plaques of chronic kidney disease patients compared to early-stage plaques, and a significant negative correlation to glomerular filtration rate for the advanced plaques. While we saw the difference in the total levels of carbamylation between early and advanced plaques, cellular carbamylation signal, studied in a parallel cohort of stable vs unstable plaques, did not differ between plaque stages but significantly correlated to CD68, PLIN2, and LGALS3 signals. Functional effects of carbamylated LDL (carbLDL) uptake on macrophages were studied on an in-house developed confocal-based microscale multi-assay platform to screen multiple cellular functions and demonstrated similar foam cell formation compared to the uptake of oxidized LDL (oxLDL). However, in contrast to oxLDL, carbLDL did not induce PPARg reporter gene expression, suggesting differential capacity to induce lipogenic pathways. Moreover, unlike oxLDL, carbLDL did not induce apoptosis or ROS production. Taken together, our findings demonstrate an accumulation of carbamylated protein during plaque progression in patients with reduced kidney function. This can be, at least partially, explained by uptake of carbLDL particles by the macrophages. CarbLDL uptake, in turn, can induce foam cell formation but seems less cytotoxic than oxLDL.

Tissue aging is a complex phenomenon involving molecular aging of matrix proteins, which mainly results from their progressive alteration by nonenzymatic post-translational modifications (NEPTMs) such as glycation and carbamylation. These two reactions, which correspond to the binding of reactive metabolites ( i.e . reducing sugars and urea-derived cyanate, respectively) on amino groups of proteins, occur during aging and are amplified in various chronic diseases such as diabetes mellitus or chronic renal disease (CKD). Since these reactions target the same functional groups, they can reciprocally compete for protein modification. Determining which NEPTM is predominant in tissues is necessary to better understand their role in the development of long-term complications of chronic diseases. For that purpose, two different murine models were used for reproducing such a competitive context: a CKD-diabetic mice model and a cyanate-consuming mice model. The competition has been evaluated by quantifying glycation and carbamylation products by LC-MS/MS in skin and aorta total extracts as well as in skin type I collagen. The results showed that the simultaneous enhancement of glycation and carbamylation reactions resulted in a decrease of the formation of glycation products (especially Amadori products) whereas the concentrations of homocitrulline, a carbamylation product, remained similar. These results, which have been obtained in both tissues and in purified skin type I collagen, suggest that carbamylation takes precedence over glycation for the modification of tissue proteins, but only in pathological conditions favouring these two NEPTMs. While glycation has been considered for a long time the predominant NEPTM of matrix proteins, carbamylation seems to also play an important role in tissue aging. The existence of competition between these NEPTMs must be taken into account to better understand the consequences of molecular aging of matrix proteins in tissue aging.

Background Uremic impairment of wound healing is a well-established phenomenon, however the etiology of this condition continues to be a medical enigma. Carbamylation, posttranslational modification (PTM) occurring with high frequency in uremic milieu, is known to have impact on structural and functional properties of proteins and peptides. Herein we show that carbamylation of the members of kinin-kallikrein system, that play an essential role in wound healing process, results in its aberrant functionality and impedes the complex process of tissue regeneration in uremic patients. Results Through enzymatic assays we demonstrate that carbamylation of kininogen results in aberrant bradykinin generation. We confirmed that bradykinin is efficiently carbamylated in uremic conditions and, alternatively, by activated neutrophiles. Moreover, this modification affects proteolytic cleavage of the peptide, potentially leading to the accumulation of the carbamylated form. Modified peptide demonstrated lower affinity toward its receptors. Carbamylation diminished bradykinin’s ability to stimulate expression of the B 1 receptor and cytokines essential in wound healing process. Carbamylated bradykinin was significantly less potent in promoting angiogenesis and keratinocyte motility as compared to the native form. In the in vivo murine model of wound healing, we observed impaired collagen fiber production and delayed re-epithelialisation in the presence of carbamylated form. Conclusions Carbamylation-driven impairment of wound healing is a mechanistic link to wound persistence in uremia. Importantly, production of carbamylated bradykinin in localized inflammatory milieus could be a significant contributor to delayed wound healing and formation of chronic wounds in diabetes or psoriasis.

The presence of toxins is believed to be a major factor in the development of uremia in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD). Uremic toxins have been divided into 3 groups: small substances dissolved in water, medium molecules: peptides and low molecular weight proteins, and protein-bound toxins. One of the earliest known toxins is urea, the concentration of which was considered negligible in CKD patients. However, subsequent studies have shown that it can lead to increased production of reactive oxygen species (ROS), and induce insulin resistance in vitro and in vivo, as well as cause carbamylation of proteins, peptides, and amino acids. Other uremic toxins and their participation in the damage caused by oxidative stress to biological material are also presented. Macromolecules and molecules modified as a result of carbamylation, oxidative stress, and their adducts with uremic toxins, may lead to cardiovascular diseases, and increased risk of mortality in patients with CKD.