Rheumatoid factors (RF) and the disease-specific anti-citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients where they probably contribute to ...synovitis. We investigated the influence of IgM and IgA RF on the FcR- and complement-dependent effects of ACPA immune complexes (ACPA-IC). When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions purified from RA serum pools, M-CSF-generated macrophages skewed their cytokine response toward inflammation, with increases in the TNF-α/IL-10 ratio and in IL-6 and IL-8 secretion, and decreases in the IL-1Ra/IL-1β ratio. In the IgM RF-mediated amplification of the inflammatory response of macrophages, the participation of an IgM receptor was excluded, notably by showing that they did not express any established receptor for IgM. Rather, this amplification depended on the IgM RF-mediated recruitment of more IgG into the ACPA-IC. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC, showing its major implication in the effects of RF of the IgA class. LPS further amplified the TNF-α response of macrophages to RF-containing ACPA-IC. Lastly, the presence of IgM or IgA RF increased the capacity of ACPA-IC to activate the complement cascade. Therefore, specifically using autoantibodies from RA patients, the strong FcR-mediated or complement-dependent pathogenic potential of IC including both ACPA and IgM or IgA RF was established. Simultaneous FcR triggering by these RF-containing ACPA-IC and TLR4 ligation possibly makes a major contribution to RA synovitis.
Anticitrullinated protein antibodies (ACPA) are specifically associated with rheumatoid arthritis (RA) and produced in inflamed synovial membranes where citrullinated fibrin, their antigenic target, ...is abundant. We showed that immune complexes containing IgG ACPA (ACPA-IC) induce FcγR-mediated tumour necrosis factor (TNF)-α secretion in macrophages. Since IgM rheumatoid factor (RF), an autoantibody directed to the Fc fragment of IgG, is also produced and concentrated in the rheumatoid synovial tissue, we evaluated its influence on macrophage stimulation by ACPA-IC.
With monocyte-derived macrophages from more than 40 healthy individuals and different human IgM cryoglobulins with RF activity, using a previously developed human in vitro model, we evaluated the effect of the incorporation of IgM RF into ACPA-IC.
IgM RF induced an important amplification of the TNF-α secretion. This effect was not observed in monocytes and depended on an increase in the number of IgG-engaged FcγR. It extended to the secretion of interleukin (IL)-1β and IL-6, was paralleled by IL-8 secretion and was not associated with overwhelming secretion of IL-10 or IL-1Ra. Moreover, the RF-induced increased proinflammatory bioactivity of the cytokine response to ACPA-IC was confirmed by an enhanced, not entirely TNF-dependent, capacity of the secreted cytokine cocktail to prompt IL-6 secretion by RA synoviocytes.
By showing that it can greatly enhance the proinflammatory cytokine response induced in macrophages by the RA-specific ACPA-IC, these results highlight a previously undescribed, FcγR-dependent strong proinflammatory potential of IgM RF. They clarify the pathophysiological link between the presence of ACPA and IgM RF, and RA severity.
Proteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a ...major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin.
Formation of the epitopes recognized by the rheumatoid arthritis (RA)‐specific autoantibodies to citrullinated proteins (ACPA) on filaggrin and on the α‐ and β‐chains of fibrin, their synovial ...target, requires conversion of their arginyl residues into citrullyl residues, but is also affected by their amino‐acyl environment. Using competition with five citrullinated filaggrin‐derived peptides bearing major ACPA epitopes, we confirmed the close cross‐reactivity between filaggrin and citrullinated fibrin. To identify the sequential epitopes recognized on fibrin by ACPA, 71 citrullinated 15‐mer peptides derived from all the sites of the α‐ and β‐chains of fibrin harboring arginyl residues were tested by ELISA using ACPA‐positive RA sera exhibiting different reactivity profiles to the five filaggrin peptides. We identified 18 fibrin‐derived peptides bearing ACPA epitopes. Regarding the ability of fibrinogen arginyl residues to be citrullinated in vitro, 11 of the peptides likely correspond to in vivo targeted epitopes. Two out of them bear major epitopes and are located in the central globular domain of the protein. In the synovial tissue, fibrin citrullination and ACPA binding could impair fibrin degradation by plasmin. The immunological conflict between ACPA and fibrin could therefore sustain synovial inflammation not only via pro‐inflammatory effector mechanisms but also via impairment of fibrinolysis.
Antifilaggrin autoantibodies (AFA) are a population of IgG autoantibodies associated to rheumatoid arthritis (RA), which includes the so-called "antikeratin" Abs and antiperinuclear factor. AFA are ...the most specific serological markers of RA. We previously showed that they recognize human epidermal filaggrin and other profilaggrin-related proteins of various epithelial tissues. Here, we report further characterization of the protein Ags and epitopes targeted by AFA. All the Ags that exhibit numerous neutral/ acidic isoelectric variants were immunochemically demonstrated to be deiminated proteins. In vitro deimination of a recombinant human filaggrin by a peptidylarginine deiminase generated AFA epitopes on the protein. Moreover, two of three filaggrin-derived synthetic peptides with a citrulline in the central position were specifically and widely recognized by AFA affinity-purified from a series of RA sera. These results indicate that citrulline residues are constitutive of the AFA epitopes, but only in the context of specific amino acid sequences of filaggrin. In competition experiments, the two peptides abolished the AFA reactivity of RA sera, showing that they present major AFA epitopes. These data should help in the identification of a putative deiminated AFA-inducing or cross-reactive articular autoantigen and provide new insights into the pathogenesis of RA. They could also open the way toward specific immunosuppressive and/or preventive therapy of RA.
Peptidylarginine deiminases (PADs) catalyse a post-translational modification of proteins through the conversion of arginine residues into citrullines. The existence of four isoforms of PAD (types I, ...II, III and IV) encoded by four different genes, which are distinct in their substrate specificities and tissue-specific expression, was reported in rodents. In the present study, starting from epidermis polyadenylated RNA, we cloned by reverse transcriptase-PCR a full-length cDNA encoding human PAD type I. The cDNA was 2711 bp in length and encoded a 663-amino-acid sequence. The predicted protein shares 75% identity with the rat PAD type I sequence, but displays only 50-57% identity with the three other known human isoforms. We have described the organization of the human PAD type I gene on chromosome 1p36. A recombinant PAD type I was produced in Escherichia coli and shown to be enzymically active. Human PAD type I mRNAs were detected by reverse transcriptase-PCR not only in the epidermis, but also in various organs, including prostate, testis, placenta, spleen and thymus. In human epidermis extracts analysed by Western blotting, PAD type I was detected as a 70 kDa polypeptide, in agreement with its predicted molecular mass. As shown by immunohistochemistry, the enzyme was expressed in all the living layers of human epidermis, with the labelling being increased in the granular layer. This is the first description of the human PAD type I gene and the first demonstration of its expression in epidermis.
In the inflamed synovium of patients with rheumatoid arthritis (RA), autoantibodies to citrullinated proteins (ACPA) probably form immune complexes (IC) on deposits of citrullinated fibrin. We showed ...that in vitro such ACPA-IC activate a pro-inflammatory cytokine response in M-CSF-differentiated macrophages. Our objective was to evaluate how macrophage polarisation influences this response.
CD14-positive monocytes from healthy donors were cultured in the presence of M-CSF, IFN-γ, interleukin (IL)-4 or IL-10. Expression of markers specific for polarised macrophages was analysed by flow cytometry. Their cytokine secretion was prompted by in vitro generated autoantibodies to citrullinated proteins immune complexes (ACPA-IC) and assayed in the culture supernatants.
IFN-γ-polarised cells exhibited high levels of CD64 and CD80. Low expression of CD14 and high expression of CD206 characterised the IL-4-polarised cells. Exposure to IL-10 or M-CSF raised the expression of CD14, CD32 and CD163. The two cell types lacked CD80 and exhibited similar expression of CD64, CD200R and CD206. In response to ACPA-IC, the secretion of IL-1β, IL-6 and IL-8 was similar among cells exposed to IFN-γ, IL-4 or IL-10. However, the later cells were associated with the highest IL-1Ra:IL-1β ratio and the lowest tumour necrosis factor (TNF)-α:IL-10 ratio. Conversely, M-CSF-exposed cells secreted the highest levels of pro-inflammatory cytokines, exhibited a high TNF-α:IL-10 ratio and the lowest IL-1Ra:IL-1β ratio.
Despite their phenotypic similarity, IL-10-polarised and M-CSF-polarised macrophages clearly differ in their cytokine response to ACPA-IC. M-CSF-polarised cells exhibit the highest pro-inflammatory potential. Since M-CSF is abundant in the RA synovium, therein it probably drives macrophages towards a strong pro-inflammatory cytokine response to the locally formed ACPA-IC.
Deimination, the conversion of protein-bound arginines into citrullines, is a post-translational modification catalyzed by a peptidylarginine deiminase (Pad). In the epidermis, three Pads are ...expressed, namely Pad1, 2 and 3, and the major deiminated protein is filaggrin. Deimination of fibrin has been observed in various pathological inflammatory conditions. Here, we analyzed the expression of Pads and citrullination of proteins during cutaneous wound healing, i.e. in a physiological inflammatory condition. Full-thickness punches were performed on adult mouse back skin, and wound recovery was analyzed over 10 days by immunohistology and western blotting. Pad1 was immunodetected in all the neo-epidermis. Pad3, normally expressed in the stratum granulosum, was not detected in the hyperproliferative tongue of the neo-epidermis, but was shown to be co-localized with (pro)filaggrin in a large number of keratinocyte layers in its differentiating part. Deiminated proteins were detected in the stratum corneum of the neo-epidermis in the late phase of re-epithelialization, and in the clot and the clot-derived scab. In the clot where we only detected Pad4, one of the deiminated proteins was shown to be fibrin. Deimination of the clot proteins, and more generally wound healing and keratinocyte differentiation, seemed to be Pad2-independent, as shown using Padi2(-/-) mice.
Neutrophil extracellular traps (NET), produced by activated polymorphonuclear neutrophils (PMN), are supposed to play a role in the pathogenesis of rheumatoid arthritis (RA), a chronic inflammatory ...autoimmune disease characterized by anti-citrullinated protein antibodies (ACPA). Indeed, NET contain citrullinated autoantigens and some RA autoantibodies recognize NET. However, the mechanisms by which NET trigger or perpetuate the inflammatory process in RA are hitherto not elucidated. We hypothesized that, in addition to citrullination, NET might also contain stimulatory proteins and directly activate inflammatory target cells, as PMN and macrophages.
NET antigenic and inflammatory properties were analyzed in 157 healthy donors (HD) and RA patients, the largest analysis reported so far. Primary PMN and monocyte-derived macrophages were isolated and immunoglobulin G (IgG) purified. NET were induced (NETosis), isolated and quantified. NET antigenicity was analyzed by fluorescence microscopy. PMN and macrophages were stimulated with NET with/without ACPA, C1q, LL-37 or lipopolysaccharide (LPS) and cell activation was estimated by flow cytometry and ELISA.
PMN from RA patients produced more NET than HD PMN. We next dissected how NET mechanistically affect inflammatory cells. Particularly, we show for the first time that RA and HD NET activated both resting macrophages and PMN, but importantly RA NET were more stimulatory, leading to secretion of inflammatory cytokines and up-regulation of HLA/CD86/CD11b. IgG from ACPA-positive RA patients specifically recognized RA and even HD NET. Nevertheless, NET-induced cell activation occurs independently of immune complex formation with ACPA. Likewise, endosomal acidification was not required. Notably, we also report that complement C1q increased the NET stimulatory activity on macrophages only, due to higher expression of C1q receptors, which was further supported by the LL-37 antimicrobial peptide. In contrast, NET specifically inhibited interleukin (IL)-6 secretion by LPS-activated macrophages and not PMN, especially with C1q/LL-37. This inhibition was not mediated by NET-derived proteases or LPS neutralization and was associated with the simultaneous induction of IL-10 secretion.
We show that NET possess both pro- and anti-inflammatory properties depending on target cells, their activation levels and C1q/LL-37. Thus, independently of ACPA, NET modulate RA chronic inflammation via this new dual activity we identified. In addition, NET may trigger autoimmunity in RA as ACPA recognize NET antigens but not non-activated PMN. Therefore, we conclude that excess of NETosis together with enhanced NET activity participate to RA pathogenesis at different levels.
Dual activity of NET. Activated PMN enter NETosis in response to some stimuli. In RA, PMN are significantly more sensitive to NETosis triggering. These NET directly activate (pink arrows) steady state PMN (left) and macrophages (MΦ, right), leading to strong IL-8, TNF and IL-6 secretion (three important cytokines in RA) but low IL-10 induction, i.e. a pro-inflammatory profile. In addition, RA NET are significantly more stimulatory than normal NET. Although NET are recognized by ACPA, the resulting immune complexes do not clearly enhance PMN and MΦ response to NET. Likewise, endosomal TLR are probably not involved in NET sensing. Regarding MΦ, NET-induced activation is specifically increased in the presence of DNA-binding molecules like C1q and LL-37 (blue arrows). C1q in combination with LL-37 carry NET to PMN and MΦ which express surface C1q and LL-37 receptors (C1qR, LL-37-R). Because MΦ express much more C1qR than PMN, MΦ (and not PMN) activation by NET is enhanced by C1q or C1q/LL-37. On the contrary, NET induce an anti-inflammatory profile in strongly-activated MΦ but not PMN (green arrows). Indeed, NET specifically (partly) inhibit secretion of the pro-inflammatory cytokine IL-6 by MΦ in response to LPS, especially in the presence of both C1q and LL-37, and inversely enhance IL-10 secretion by those MΦ. Display omitted
•NET are pro-inflammatory and directly activate RESTING neutrophils and macrophages.•NET from RA patients are more stimulatory than NET from healthy donors.•On the contrary, NET are anti-inflammatory on strongly ACTIVATED macrophages only.•C1q enhances NET regulatory activities on macrophages, due to high C1q receptors expression, especially with LL-37.•NET have a dual immunoregulatory activity depending on target cells, without requirement of ACPA.
Rheumatoid arthritis (RA) is the most frequent human autoimmune disease, affecting about 1% of the adult population worldwide. A better knowledge of the autoimmune mechanisms involved is essential. ...We identified the epithelial targets of various autoantibodies specifically associated to RA, as variants of (pro)filaggrin. We also showed that these targets correspond to deiminated (“citrullinated”) proteins, of which arginyl residues have been posttranslationally transformed into citrullyl residues by a peptidylarginine deiminase (PAD). Moreover, we and others established that citrullyl residues are indispensable elements of the epitopes recognized by these autoantibodies but only in the context of specific aminoacid sequences. We also demonstrated that these autoantibodies to citrullinated proteins (ACPA) are secreted by plasma cells of the synovial tissue and that their major targets correspond to citrullinated forms of the α- and β-chains of fibrin, abundant in the tissue. These results have allowed the development of new efficient immunochemical methods for the detection of ACPA. Some of them are already commercially available. These new methods have permitted the high diagnostic value of ACPA which are present very early in the course of the disease, and also their prognostic value, to be confirmed. ACPA detection should therefore prove to be also a very valuable tool to guide the choice of therapeutic strategies, from the earliest stages of the disease. The synthesis of ACPA in the rheumatoid synovial tissue and the existence therein of a specific antigenic target constitute a strong argument for the involvement of this specific immunological conflict in the pathophysiology of RA. Indeed, it could lead to activation of effector mechanisms with pro-inflammatory effects, thus to formation in the tissue of new fibrin deposits, secondarily citrullinated. We therefore, propose a new pathophysiological model accounting for the self-maintenance and chronicity of rheumatoid inflammation. Numerous questions about the pathophysiological significance of the autoimmune response to deiminated proteins in RA remain to be answered to confirm this model.
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