Complement C4A Regulates Autoreactive B Cells in Murine Lupus.
ABSTRACT: Systemic lupus erythematosus (SLE) is a severe autoimmune disease mediated by pathogenic autoantibodies. While complement protein C4 is associated with SLE, its isoforms (C4A and C4B) are not equal in their impact. Despite being 99% homologous, genetic studies identified C4A as more protective than C4B. By generating gene-edited mouse strains expressing either human C4A or C4B and crossing these with the 564lgi lupus strain, we show that, overall, C4A-like 564Igi mice develop less humoral autoimmunity than C4B-like 564Igi mice. This includes a decrease in the number of GCs, autoreactive B cells, autoantibodies, and memory B cells. The higher efficiency of C4A in inducing self-antigen clearance is associated with the follicular exclusion of autoreactive B cells. These results explain how the C4A isoform is protective in lupus and suggest C4A as a possible replacement therapy in lupus.
Project description:Human complement C4 is complex, with multiple layers of diversity. The aims of this study were to elucidate the copy number variations (CNVs) of C4A and C4B in relation to disease risk in systemic lupus erythematosus (SLE), and to compare the basis of race-specific C4A deficiency between East Asians and individuals of European descent.The East Asian study population included 999 SLE patients and 1,347 healthy subjects. Variations in gene copy numbers (GCNs) of total C4, C4A, and C4B, as well as C4-Long and C4-Short genes, were determined and validated using independent genotyping technologies. Genomic regions with C4B96 were investigated to determine the basis of the most basic C4B protein occurring concurrently with C4A deficiency.In East Asians, high GCNs of total C4 and C4A were strongly protective against SLE, whereas low and medium GCNs of total C4 and C4A, and the absence of C4-Short genes, were risk factors for SLE. Homozygous C4A deficiency was infrequent in East Asian subjects, but had an odds ratio (OR) of 12.4 (P?=?0.0015) for SLE disease susceptibility. Low serum complement levels were strongly associated with low GCNs of total C4 (OR 3.19, P?=?7.3?×?10(-7) ) and C4B (OR 2.53, P?=?2.5?×?10(-5) ). Patients with low serum complement levels had high frequencies of anti-double-stranded DNA antibodies (OR 4.96, P?=?9.7?×?10(-17) ), hemolytic anemia (OR 3.89, P?=?3.6?×?10(-10) ), and renal disease (OR 2.18, P?=?8.5?×?10(-6) ). The monomodular-Short haplotype found to be prevalent in European Americans with C4A deficiency, which was in linkage disequilibrium with HLA-DRB1*0301, was scarce in East Asians. Instead, most East Asian subjects with C4A deficiency were found to have a recombinant haplotype with bimodular C4-Long and C4-Short genes, encoding C4B1 and C4B96, which was linked to HLA-DRB1*1501. DNA sequencing revealed an E920K polymorphism in C4B96.C4 CNVs and deficiency of C4A both play an important role in the risk and manifestations of SLE in East Asian and European populations.
Project description:Interindividual gene copy-number variation (CNV) of complement component C4 and its associated polymorphisms in gene size (long and short) and protein isotypes (C4A and C4B) probably lead to different susceptibilities to autoimmune disease. We investigated the C4 gene CNV in 1,241 European Americans, including patients with systemic lupus erythematosus (SLE), their first-degree relatives, and unrelated healthy subjects, by definitive genotyping and phenotyping techniques. The gene copy number (GCN) varied from 2 to 6 for total C4, from 0 to 5 for C4A, and from 0 to 4 for C4B. Four copies of total C4, two copies of C4A, and two copies of C4B were the most common GCN counts, but each constituted only between one-half and three-quarters of the study populations. Long C4 genes were strongly correlated with C4A (R=0.695; P<.0001). Short C4 genes were correlated with C4B (R=0.437; P<.0001). In comparison with healthy subjects, patients with SLE clearly had the GCN of total C4 and C4A shifting to the lower side. The risk of SLE disease susceptibility significantly increased among subjects with only two copies of total C4 (patients 9.3%; unrelated controls 1.5%; odds ratio [OR] = 6.514; P=.00002) but decreased in those with > or =5 copies of C4 (patients 5.79%; controls 12%; OR=0.466; P=.016). Both zero copies (OR=5.267; P=.001) and one copy (OR=1.613; P=.022) of C4A were risk factors for SLE, whereas > or =3 copies of C4A appeared to be protective (OR=0.574; P=.012). Family-based association tests suggested that a specific haplotype with a single short C4B in tight linkage disequilibrium with the -308A allele of TNFA was more likely to be transmitted to patients with SLE. This work demonstrates how gene CNV and its related polymorphisms are associated with the susceptibility to a human complex disease.
Project description:BACKGROUND: The fourth component of human complement (C4), an essential factor of the innate immunity, is represented as two isoforms (C4A and C4B) in the genome. Although these genes differ only in 5 nucleotides, the encoded C4A and C4B proteins are functionally different. Based on phenotypic determination, unbalanced production of C4A and C4B is associated with several diseases, such as systemic lupus erythematosus, type 1 diabetes, several autoimmune diseases, moreover with higher morbidity and mortality of myocardial infarction and increased susceptibility for bacterial infections. Despite of this major clinical relevance, only low throughput, time and labor intensive methods have been used so far for the quantification of C4A and C4B genes. RESULTS: A novel quantitative real-time PCR (qPCR) technique was developed for rapid and accurate quantification of the C4A and C4B genes applying a duplex, TaqMan based methodology. The reliable, single-step analysis provides the determination of the copy number of the C4A and C4B genes applying a wide range of DNA template concentration (0.3-300 ng genomic DNA). The developed qPCR was applied to determine C4A and C4B gene dosages in a healthy Hungarian population (N = 118). The obtained data were compared to the results of an earlier study of the same population. Moreover a set of 33 samples were analyzed by two independent methods. No significant difference was observed between the gene dosages determined by the employed techniques demonstrating the reliability of the novel qPCR methodology. A Microsoft Excel worksheet and a DOS executable are also provided for simple and automated evaluation of the measured data. CONCLUSION: This report describes a novel real-time PCR method for single-step quantification of C4A and C4B genes. The developed technique could facilitate studies investigating disease association of different C4 isotypes.
Project description:Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of pathogenic IgG antinuclear antibodies. Pathogenic IgG autoantibody production requires B-cell activation, leading to the production of activation-induced deaminase (AID) and class switching of IgM genes to IgG. To understand how and when B cells are activated to produce these IgG autoantibodies, we studied cells from 564Igi, a mouse model of SLE. 564Igi mice develop a disease profile closely resembling that found in human SLE patients, including the presence of IgG antinucleic acid Abs. We have generated 564Igi mice that conditionally express an activation-induced cytidine deaminase transgene (Aicda(tg) ), either in all B cells or only in mature B cells. Here, we show that class-switched pathogenic IgG autoantibodies were produced only in 564Igi mice in which AID was functional in early-developing B cells, resulting in loss of tolerance. Furthermore, we show that the absence of AID in early-developing B cells also results in increased production of self-reactive IgM, indicating that AID, through somatic hypermutation, contributes to tolerance. Our results suggest that the pathophysiology of clinical SLE might also be dependent on AID expression in early-developing B cells.
Project description:The presence of autoantibodies in systemic lupus erythematosus, particularly those of the IgG subclass, have long been associated with disease onset and activity. Here we explored the prevalence of autoreactive IgE in SLE and its relevance to disease in French (n = 79) and United States (US) (n = 117) cohorts with a mean age of 41.5 ± 12.7 and 43.6 ± 15.3 years and disease duration of 13.5 ± 8.5 and 16.6 ± 11.9 years, respectively. Our findings show that approximately 65% of all SLE subjects studied produced IgE antibodies to the seven autoantigens tested. This positivity was increased to almost 83% when only those subjects with active disease were considered. SLE subjects who were positive for anti-dsDNA, -Sm, and -SSB/La -specific IgE showed a highly significant association in the levels of these antibodies with disease activity similar to that of the corresponding IgG's. A strong association of IgE autoantibodies with active nephritis was also found in the combined cohort analysis. A test of the predictive value of autoreactive IgE's and IgGs for disease activity (SLE Disease Activity Index (SLEDAI) ? 4) revealed that the best predictors were dsDNA-specific IgE and IgG, and that the age of an SLE subject influenced this predictive model. The finding argue that the overall levels of IgE autoantibodies, independently or in combination with IgG autoantibodies, may serve as indicators of active disease.
Project description:Germinal centers (GCs) are the primary sites of clonal B cell expansion and affinity maturation, directing the production of high-affinity antibodies. This response is a central driver of pathogenesis in autoimmune diseases, such as systemic lupus erythematosus (SLE), but the natural history of autoreactive GCs remains unclear. Here, we present a novel mouse model where the presence of a single autoreactive B cell clone drives the TLR7-dependent activation, expansion, and differentiation of other autoreactive B cells in spontaneous GCs. Once tolerance was broken for one self-antigen, autoreactive GCs generated B cells targeting other self-antigens. GCs became independent of the initial clone and evolved toward dominance of individual clonal lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease.
Project description:APS is the association of antiphospholipid antibodies (aPL) with thromboses and/or recurrent pregnancy loss (RPL). Among patients with SLE, one-third have aPL and 10-15% have a manifestation of secondary APS. Animal studies suggested that complement activation plays an important role in the pathogenesis of thrombosis and pregnancy loss in APS. We performed a cross-sectional study on complement proteins and genes in 525 patients with aPL. Among them, 237 experienced thromboses and 293 had SLE; 111 had both SLE and thromboses, and 106 had neither SLE nor thrombosis. Complement protein levels were determined by radial immunodiffusion for C4, C3 and factor H; and by functional ELISA for mannan binding lectin (MBL). Total C4, C4A and C4B gene copy numbers (GCN) were measured by TaqMan-based realtime PCR. Two to six copies of C4 genes are frequently present in a diploid genome, and each copy may code for an acidic C4A or a basic C4B protein. We observed significantly (a) higher protein levels of total C4, C4A, C4B, C3, and anticardiolipin (ACLA) IgG, (b) increased frequencies of lupus anticoagulant and males, and (c) decreased levels of complement factor H, MBL and ACLA-IgM among patients with thrombosis than those without thrombosis (N = 288). We also observed significantly lower GCNs of total C4 and C4A among aPL-positive patients with both SLE and thrombosis than others. By contrast, aPL-positive subjects with SLE had significantly reduced protein levels of C3, total C4, C4A, C4B and ACLA-IgG, and higher frequency of females than those without SLE. Patients with thrombosis but without SLE (N = 126), and patients with SLE but without thrombosis (N = 182) had the greatest differences in mean protein levels of C3 (p = 2.6 × 10-6), C4 (p = 2.2 × 10-9) and ACLA-IgG (p = 1.2 × 10-5). RPL occurred in 23.7% of female patients and thrombotic SLE patients had the highest frequency of RPL (41.0%; p = 3.8 × 10-10). Compared with non-RPL females, RPL had significantly higher frequency of thrombosis and elevated C4 protein levels. Female patients with homozygous C4A deficiency all experienced RPL (p = 0.0001) but the opposite was true for patients with homozygous C4B deficiency (p = 0.017). These results provide new insights and biomarkers for diagnosis and management of APS and SLE.
Project description:Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of antinucleic acid autoantibodies, high levels of circulating type I interferon (IFN-I), and an IFN-I-dependent elevated expression of activating Fc?R. Increases in neutrophils and monocytes are often observed in clinical SLE, but how these contribute to autoantibody and IFN-I production is poorly understood. Here, we analyzed SLE pathogenesis in 564Igi mice, an SLE-model strain carrying gene-targeted heavy and light chain antibody genes encoding an anti-RNA autoantibody in a C57BL/6 background. Similar to human SLE patients, 564Igi mice produce anti-RNA autoantibodies and expanded neutrophil and monocyte populations. These myeloid cells produced IFN-I and exhibit increased Fc?RIV expression induced via an IFN-I autocrine loop. A direct effect of IFN-I on 56 Igi BM B cells and neutrophils was supported by their upregulation of "IFN-I signature genes". In addition, 564Igi developing B cells showed upregulated TLR7 resulting in IgG2a/2b class switch recombination and autoantibody production. Our results indicate that the production of anti-RNA autoantibody is sufficient to induce an increase of BM, blood, and spleen IFN-I-producing neutrophils, and suggest a mechanism by which autoantibody and IFN-I contribute to SLE by activating B lymphocytes, neutrophils, and monocyte effector cells in vivo.
Project description:Complete deficiency of complement C4 is among the strongest genetic risk factors for human systemic lupus erythematosus (SLE). C4 is a constituent of the RP-C4-CYP21-TNX (RCCX) module in the human leukocyte antigen (HLA) that exhibits inter-individual copy-number and gene-size variations. Here, we studied two North-African families with complete C4 deficiency and SLE. The first included a Moroccan male SLE patient (1P) and a sibling, who were both homozygous for HLA-A*02 B*17 DRB1*07. The second had an Algerian female SLE patient (2P) homozygous for HLA-A*01 B*17 DRB1*13. Early SLE disease onset, the presence of photosensitive rashes, anti-Ro/SSA, renal disease and high titers of antinuclear antibodies were the common features of complete C4 deficiency. Southern blot analyses showed that 1P had monomodular RCCX with a long C4A, whereas 2P had bimodular RCCX with one long C4A and one short C4B. Genomic DNA fragments for these mutant genes were amplified and sequenced. A C>T transition that created the R540X nonsense mutation in C4A was found in 1P. An identical 4-bp insertion that generated the Y1537X nonsense mutation was discovered in both C4A and C4B of 2P. The high concordance of SLE and C4 deficiency among patients with non-DR3 and non-DR2 haplotypes underscores the importance of C4 proteins in the protection against SLE.
Project description:INTRODUCTION:Homozygous deficiencies of complement C4A or C4B are detected in 1-10% of populations. In genome-wide association studies C4 deficiencies are missed because the genetic variation of C4 is complex. There are no studies where the clinical presentation of these patients is analyzed. This study was aimed to characterize the clinical features of patients with homozygous C4A or C4B deficiency. MATERIAL AND METHODS:Thirty-two patients with no functional C4A, 87 patients with no C4B and 120 with normal amount of C4 genes were included. C4A and C4B numbers were assessed with genomic quantitative real-time PCR. Medical history was studied retrospectively from patients' files. RESULTS:Novel associations between homozygous C4A deficiency and lymphoma, coeliac disease and sarcoidosis were detected. These conditions were present in 12.5%, (4/32 in patients vs. 0.8%, 1/120, in controls, OR = 17.00, 95%CI = 1.83-158.04, p = 0.007), 12.5% (4/32 in patients vs. 0%, 0/120 in controls, OR = 1.14, 95%CI = 1.00-1.30, p = 0.002) and 12.5%, respectively (4/32 in patients vs. 2.5%, 3/120 in controls, OR = 5.571, 95%CI = 1.79-2.32, p = 0.036). In addition, C4A and C4B deficiencies were both associated with adverse drug reactions leading to drug discontinuation (34.4%, 11/32 in C4A-deficient patients vs. 14.2%, 17/120 in controls, OR = 3.174, 95%CI = 1.30-7.74, p = 0.009 and 28.7%, 25/87 in C4B-deficient patients, OR = 2.44, 95%CI = 1.22-4.88, p = 0.010). CONCLUSION:This reported cohort of homozygous deficiencies of C4A or C4B suggests that C4 deficiencies may have various unrecorded disease associations. C4 gene should be considered as a candidate gene in studying these selected disease associations.