Objective Anti–citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA). However, the molecular basis for ACPA production is still unclear. The purpose of this study was to determine if circulating plasmablasts from RA patients produce ACPAs and whether Porphyromonas gingivalis facilitates the generation of ACPAs. Methods Using a single-cell antibody cloning approach, we generated 217 and 110 monoclonal recombinant antibodies from circulating plasmablasts from 7 RA patients and 4 healthy controls, respectively. Antibody reactivity with citrullinated antigens was tested by a second-generation anti–cyclic citrullinated peptide (anti-CCP) kit and by enzyme-linked immunosorbent assays (ELISAs) against citrullinated human antigens. Antibody reactivity with P gingivalis was tested by ELISAs against outer membrane antigens (OMAs) and citrullinated enolase from P gingivalis. Results Approximately 19.5% of plasmablast-derived antibodies from anti-CCP–positive RA patients, but none from 1 anti-CCP–negative RA patient or the healthy controls, specifically recognized citrullinated antigens. The immunoglobulin genes encoding these ACPAs were highly mutated, with increased ratios of replacement mutations to silent mutations, suggesting the involvement of active antigen selection in ACPA generation. Interestingly, 63% of the ACPAs cross-reacted with OMAs and/or citrullinated enolase from P gingivalis. The reactivity of ACPAs against citrullinated proteins from P gingivalis was confirmed by immunoblotting and mass spectrometry. Furthermore, some germline-reverted ACPAs retained their reactivity with P gingivalis antigens but completely lost their reactivity with citrullinated human antigens. Conclusion These results suggest that circulating plasmablasts in RA patients produce ACPAs and that this process may be facilitated by anti–P gingivalis immune responses.
Rheumatoid arthritis (RA) is a complex autoimmune disease affecting 1–2% of general worldwide population. The etiopathogenesis of RA involves the interplay of multiple genetic risk factors and environmental triggers. Microbial infections are believed to play an important role in the initiation and perpetuation of RA. Recent clinical studies have shown the association of microbial infections with RA. Accumulated studies using animal models have also found that microbial infections can induce and/or exaggerate the symptoms of experimental arthritis. In this review, we have identified the most common microbial infections associated with RA in the literature and summarized the current evidence supporting their pathogenic role in RA. We also discussed the potential mechanisms whereby infection may promote the development of RA, such as generation of neo-autoantigens, induction of loss of tolerance by molecular mimicry, and bystander activation of the immune system.
Neutrophil extracellular traps (NETs) are web-like structures released by activated neutrophils. Recent studies suggest that NETs play an active role in driving autoimmunity and tissue injury in diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The purpose of this study was to investigate if celastrol, a triterpenoid compound, can inhibit NET formation induced by inflammatory stimuli associated with RA and SLE. We found that celastrol can completely inhibit neutrophil oxidative burst and NET formation induced by tumor necrosis factor alpha (TNFα) with an IC50 of 0.34 µM and by ovalbumin:anti-ovalbumin immune complexes (Ova IC) with an IC50 of 1.53 µM. Celastrol also completely inhibited neutrophil oxidative burst and NET formation induced by immunoglobulin G (IgG) purified from RA and SLE patient sera. Further investigating into the mechanisms, we found that celastrol treatment downregulated the activation of spleen tyrosine kinase (SYK) and the concomitant phosphorylation of mitogen-activated protein kinase kinase (MAPKK/MEK), extracellular-signal-regulated kinase (ERK), and NFκB inhibitor alpha (IκBα), as well as citrullination of histones. Our data reveals that celastrol potently inhibits neutrophil oxidative burst and NET formation induced by different inflammatory stimuli, possibly through downregulating the SYK-MEK-ERK-NFκB signaling cascade. These results suggest that celastrol may have therapeutic potentials for the treatment of inflammatory and autoimmune diseases involving neutrophils and NETs.
Purpose: Systemic lupus erythematosus (SLE) is a complex autoimmune disease with marked disparities in prevalence and disease severity among different ethnic groups. The purpose of this study is to characterize a Latin American cohort and identify genetic risk factors for developing SLE and its end-organ manifestations in this Latin Hispanic cohort. Methods: In this cohort, SNP rs9271366 (tag SNP for HLA-DRB1*15:01) confers the highest risk for SLE among the 13 MHC gene alleles that display association with SLE (p = 8.748E-10; OR = 3.5). Among the 26 non-MHC gene alleles analyzed, SNP rs2476601 in PTPN22 gene confers the highest risk for SLE (p = 0.0001; OR = 5.6). ITGAM, TNFSF4, TNIP1, STAT4, CARD11, BLK, and TNXB gene alleles were confirmed as SLE-susceptible alleles in the DR cohort. However, IRF5 and TNFAIP3 gene alleles, established risk factors for SLE in populations of European and Asian ancestry, are not significantly associated with SLE in this cohort. We also defined a novel HLA-DRA haplotype that confers an increased risk for lupus nephritis (LN) and alleles in HLA-DRA2 and TNFSF4 genes as genetic risk factors for developing neuropsychiatric (NP) SLE. Results: In this cohort, SNP rs9271366 (tag SNP for HLA-DRB1*15:01) confers the highest risk for SLE among the 13 MHC gene alleles that display association with SLE (p = 8.748E-10; OR = 3.5). Among the 26 non-MHC gene alleles analyzed, SNP rs2476601 in PTPN22 gene confers the highest risk for SLE (p = 0.0001; OR = 5.6). ITGAM, TNFSF4, TNIP1, STAT4, CARD11, BLK, and TNXB gene alleles were confirmed as SLE-susceptible alleles in the DR cohort. However, IRF5 and TNFAIP3 gene alleles, established risk factors for SLE in populations of European and Asian ancestry, are not significantly associated with SLE in this cohort. We also defined a novel HLA-DRA haplotype that confers an increased risk for lupus nephritis (LN) and alleles in HLA-DRA2 and TNFSF4 genes as genetic risk factors for developing neuropsychiatric (NP) SLE. Conclusion: Our data suggest that the Latin American population shares some common genetic risk factors for SLE as other populations, but also has distinct risk gene alleles that contribute to SLE susceptibility and development of LN and NPSLE. This is the first study focusing on genetic risk factors for SLE in the DR, a Latin American population that has never been characterized before.
Understanding the B-cell response during chronic human immunodeficiency virus (HIV) infection is essential for eliciting broad and potent neutralizing antibodies (Abs). In this study, we analyzed the plasmablast repertoire of chronically HIV-infected individuals in combination with antiretroviral therapy (ART). Among the obtained 72 recombinant monoclonal antibodies (mAbs), 27.8% weakly bound to HIV gp140 and were non-neutralizing. Remarkably, 56.9% were polyreactive and 55.6% were autoreactive. The prominent feature of being polyreactive/autoreactive is not limited to anti-gp140 Abs. Furthermore, these polyreactive/autoreactive Abs displayed striking cross-reactivity with DWEYS in the N-methyl-d-aspartate receptor (NMDAR), and this binding induced SH-SY5Y cell apoptosis. We also found higher frequencies of VH4-34 utilization and VH replacement in the plasmablast repertoire of chronically HIV-infected individuals, which may contribute to the generation of poly/autoreactive Abs. Taken together, these data demonstrate that circulating plasmablasts in chronically HIV-infected individuals experienced with ART predominantly produce poly/autoreactive Abs with minimal anti-HIV neutralizing capacity and potential cross-reactivity with autoantigens. This may represent another dysfunction of B cells during chronic HIV infection.
Characterization of the diversified immunoglobulin (Ig) repertoire may provide insight into pathways that shape an efficient antibody (Ab) repertoire for immune response against human immunodeficiency virus (HIV) infection. This study aimed to profile characteristics of the plasmablast repertoire during chronic HIV infection. Ig variable regions of plasmablasts from both chronically HIV-infected donors (HIVDs) previously treated with antiretroviral therapy (ART) and healthy donors (HDs) were amplified by single-cell PCR to establish the basis for further repertoire analysis. We compared the plasmablast repertoires expressed in multiple chronically HIVDs after ART treatment cessation and HDs. We also examined the non-productive repertoire to identify the indication of the immediate products of the rearrangement machinery without an impact of selection during HIV infection. We found multiple differences between the productive repertoires of HIVD and HD subjects, including biased usages of VH3-49, VH1-2, VH3-33, VH3-74, and VH5-51 in VH and D1-7, D1-14, D1-20, and D5-5/18 in D segments in the HIVD group, as well as shorter and preferential glycine usages in CDRH3 regions. Gene selections were also detected in light chains. Notably, differences between productive rearrangements of HIVDs and HDs outnumbered those between productive and non-productive rearrangements within HIVDs. HIV infection may exert a dominant impact on the development of the plasmablast repertoire. The impact of selection is of limited significance in shaping the plasmablast repertoire. Overall, the data indicate that the environment in which the plasmablasts live can affect the distribution of the VH and VL genes in the repertoire and the amino acid compositions of the expressed Abs.
Systemic lupus erythematosus (SLE) is characterized by the over-production of high affinity autoantibodies. However, it is not clear how these autoantibodies are generated and selected. VH replacement is originally considered as a receptor editing process to change unwanted non-functional immunoglobulin heavy chain (IgH) genes or IgH genes encoding self reactive BCRs. Our sequence analysis shows that VH replacement products are highly enriched in IgH genes derived from different autoimmune diseases, including SLE. In this study, we performed single cell RT-PCR to study the functional antibody repertoires in active SLE patients. Analysis of 180 recombinant antibodies derived from SLE patients showed that 19.0% of the antibodies derived from naïve B cells and 56.9% of the antibodies derived from plasmablasts are autoreactive. In contrast, only 7.7% and 17.4% of the antibodies derived from naïve B cells and plasmablast B cells are autoreactive in healthy donors. The VH replacement products are significantly increased in IgH genes obtained from SLE patients (20%) compared to that in healthy controls (10%). Importantly, the accumulated VH replacement products in SLE patients have long CDR3 regions enriched with positively charged amino acids; 74% of them directly encode anti-nuclear antigen or polyreactive antibodies. Based on these results, we conclude that the accumulated VH replacement products in SLE patients contribute to the generation of auto/polyreactive antibodies in SLE.
In the post-HAART stage, a substantial percent of HIV patients develop HIV-Associated Neurocognitive Dysfunction and HIV-Associated Dementia. Finding early biomarkers is essential to diagnose and prevent these diseases. HIV infection causes B cell dysfunctions. To evaluate the functional antibody repertoire during HIV infection, we cloned the paired IgH and IgL genes from single circulating plasmablasts of 5 HIV patients and expressed 80 recombinant antibodies to determine their functions. We also expressed 80 antibodies from 5 donors without HIV infection as control. Analyses of antibodies from HIV-infected individuals showed that 30% of them weakly bind to gp120. Surprisingly, about 72% of the antibodies cloned from HIV-infected individuals were polyreactive, which is sharp contrast to the 16% of polyreactive antibodies from control donors. Up to 60% of them cross-reacted with the DWDYS peptide present in NMDA receptor. We found 17% of antibodies bound to SH-SY5Y or U251 cell surface antigens. Detailed analyses of one such antibody HIV201P5B2 derived from HIV patients showed that it induced NMDAR clustering and internalization and eventually caused neuronal cell apoptosis. Taken together, the dominant production of polyreactive antibodies in the plasmablasts of HIV patients revealed an abnormal alteration of B cell function during HIV infection. Anti-NDMAR antibodies generated in HIV patients may provide a new diagnostic biomarker for HAND and HAD in AIDS patient.
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