RESUMO
Epitope mapping provides critical insight into antibody-antigen interactions. Epitope mapping of autoantibodies from patients with autoimmune diseases can help elucidate disease immunogenesis and guide the development of antigen-specific therapies. Similarly, epitope mapping of commercial antibodies targeting known autoantigens enables the use of those antibodies to test specific hypotheses. Anti-Neutrophil Cytoplasmic Autoantibody (ANCA) vasculitis results from the formation of autoantibodies to multiple autoantigens, including myeloperoxidase (MPO), proteinase-3 (PR3), plasminogen (PLG), and peroxidasin (PXDN). To perform high-resolution epitope mapping of commercial antibodies to these autoantigens, we developed a novel yeast surface display library based on a series of >5000 overlapping peptides derived from their protein sequences. Using both FACS and magnetic bead isolation of reactive yeast, we screened 19 commercially available antibodies to the ANCA autoantigens. This approach to epitope mapping resulted in highly specific, fine epitope mapping, down to single amino acid resolution in many cases. Our study also identified cross-reactivity between some commercial antibodies to MPO and PXDN, which suggests that patients with apparent autoantibodies to both proteins may be the result of cross-reactivity. Together, our data validate yeast surface display using maximally overlapping peptides as an excellent approach to linear epitope mapping.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos , Saccharomyces cerevisiae , Humanos , Mapeamento de Epitopos , Autoanticorpos , Mieloblastina , Autoantígenos , Peroxidase , PeptídeosRESUMO
BACKGROUND: PR3-ANCA vasculitis has a genetic association with HLA-DPB1. We explored immunologic and clinical features related to the interaction of HLA-DPB1*04:01 with a strongly binding PR3 peptide epitope (PR3225-239). METHODS: Patients with ANCA vasculitis with active disease and disease in remission were followed longitudinally. Peripheral blood mononuclear cells from patients and healthy controls with HLA-DPB1*04:01 were tested for HLA-DPB1*04:01 expression and interaction with a PR3 peptide identified via in silico and in vitro assays. Tetramers (HLA/peptide multimers) identified autoreactive T cells in vitro. RESULTS: The HLA-DPB1*04:01 genotype was associated with risk of relapse in PR3-ANCA (HR for relapse 2.06; 95% CI, 1.01 to 4.20) but not in myeloperoxidase (MPO)-ANCA or the combined cohort. In silico predictions of HLA and PR3 peptide interactions demonstrated strong affinity between ATRLFPDFFTRVALY (PR3225-239) and HLA-DPB1*04:01 that was confirmed by in vitro competitive binding studies. The interaction was tested in ex vivo flow cytometry studies of labeled peptide and HLA-DPB1*04:01-expressing cells. We demonstrated PR3225-239 specific autoreactive T cells using synthetic HLA multimers (tetramers). Patients in long-term remission off therapy had autoantigenic peptide and HLA interaction comparable to that of healthy volunteers. CONCLUSIONS: The risk allele HLA-DPB1*04:01 has been associated with PR3-ANCA, but its immunopathologic role was unclear. These studies demonstrate that HLA-DPB1*04:01 and PR3225-239 initiate an immune response. Autoreactive T cells specifically recognized PR3225-239 presented by HLA-DPB1*04:01. Although larger studies should validate these findings, the pathobiology may explain the observed increased risk of relapse in our cohort. Moreover, lack of HLA and autoantigen interaction observed during long-term remission signals immunologic nonresponsiveness.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Vasculite , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , Anticorpos Anticitoplasma de Neutrófilos , Autoantígenos , Cadeias beta de HLA-DP , Humanos , Leucócitos Mononucleares/metabolismo , Mieloblastina/genética , Peroxidase , RecidivaRESUMO
BACKGROUND: Treatment of autoimmune diseases has relied on broad immunosuppression. Knowledge of specific interactions between human leukocyte antigen (HLA), the autoantigen, and effector immune cells, provides the foundation for antigen-specific therapies. These studies investigated the role of HLA, specific myeloperoxidase (MPO) epitopes, CD4+ T cells, and ANCA specificity in shaping the immune response in patients with anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. METHODS: HLA sequence-based typing identified enriched alleles in our patient population (HLA-DPB1*04:01 and HLA-DRB4*01:01), while in silico and in vitro binding studies confirmed binding between HLA and specific MPO epitopes. Class II tetramers with MPO peptides were utilized to detect autoreactive CD4+ T cells. TCR sequencing was performed to determine the clonality of T cell populations. Longitudinal peptide ELISAs assessed the temporal nature of anti-MPO447-461 antibodies. Solvent accessibility combined with chemical modification determined the buried regions of MPO. RESULTS: We identified a restricted region of MPO that was recognized by both CD4+ T cells and ANCA. The autoreactive T cell population contained CD4+CD25intermediateCD45RO+ memory T cells and secreted IL-17A. T cell receptor (TCR) sequencing demonstrated that autoreactive CD4+ T cells had significantly less TCR diversity when compared to naïve and memory T cells, indicating clonal expansion. The anti-MPO447-461 autoantibody response was detectable at onset of disease in some patients and correlated with disease activity in others. This region of MPO that is targeted by both T cells and antibodies is not accessible to solvent or chemical modification, indicating these epitopes are buried. CONCLUSIONS: These observations reveal interactions between restricted MPO epitopes and the adaptive immune system within ANCA vasculitis that may inform new antigen-specific therapies in autoimmune disease while providing insight into immunopathogenesis.
Assuntos
Imunidade Adaptativa/imunologia , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Epitopos/imunologia , Peroxidase/imunologia , Vasculite/imunologia , Sequência de Aminoácidos , Animais , Autoanticorpos/imunologia , Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Humanos , Leucócitos Mononucleares/imunologia , Estudos Longitudinais , Camundongos , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
OBJECTIVE: There is accumulating evidence that complement activation is important in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) pathogenesis. This study was undertaken to investigate complement activation in AAV with myeloperoxidase (MPO) positivity and AAV with proteinase 3 (PR3) positivity after determining optimal methods for measuring activated complement factors in circulation. METHODS: Participants included 98 patients with AAV (45 MPO-ANCA positive, 53 PR3-ANCA positive) and 35 healthy controls. Plasma was obtained from blood collected using EDTA tubes, with or without 100 µg/ml Futhan. Levels of Bb, C3a, C5a, soluble C5b-9 (sC5b-9), properdin, and C4d were measured by enzyme-linked immunosorbent assay. Group comparisons were made using Wilcoxon's 2-sample test. Paired data were analyzed using a matched pairs signed rank test. RESULTS: Compared to healthy controls, certain complement analyte levels were high in patients with active AAV with MPO positivity, including C3a (P < 0.0001), C5a (P = 0.0004), and sC5b-9 (P = 0.0007). During remission, levels of Bb (P = 0.001), C3a (P < 0.0001), and sC5b-9 (P = 0.003) were higher. Compared to healthy controls, C3a (P < 0.0001), C5a (P = 0.002), sC5b-9 (P = 0.0001), and C4d (P = 0.005) levels were higher in patients with active AAV with PR3 positivity; levels of C3a (P < 0.0001) and C4d (P = 0.007) were also higher duriing remission. There were no significant differences in any complement analyte for either ANCA serotype between patients with active disease and those with disease in remission. Among patients with paired samples, sC5-9 levels were significantly lower during disease remission compared to active disease. C5a was significantly lower among patients with disease in long-term remission who were not receiving therapy. For Bb, C5a, and sC5b-9, median levels and individual values were considerably higher in control and patient samples processed without Futhan compared to those processed with Futhan. CONCLUSION: Complement activation occurs in both MPO-positive AAV and PR3-positive AAV. The complement activation profile differs according to disease activity and possibly ANCA serotype. Futhan reduces in vitro complement activation and provides a more accurate measurement.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Complemento C3a/imunologia , Complemento C5a/imunologia , Fator B do Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Mieloblastina/imunologia , Peroxidase/imunologia , Adulto , Idoso , Estudos de Casos e Controles , Complemento C4/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Properdina/imunologia , Índice de Gravidade de DoençaRESUMO
Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.
Assuntos
Rim/imunologia , Nefrite Lúpica/imunologia , Biomarcadores , Biópsia , Análise por Conglomerados , Biologia Computacional/métodos , Células Epiteliais/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Interferons/metabolismo , Rim/metabolismo , Rim/patologia , Leucócitos/imunologia , Leucócitos/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Anotação de Sequência Molecular , Células Mieloides/imunologia , Células Mieloides/metabolismo , Análise de Célula Única , TranscriptomaRESUMO
BACKGROUND: Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease characterized by destructive vascular inflammation. Two prominent ANCA autoantigens are myeloperoxidase (MPO) and proteinase 3 (PR3), and transcription of MPO and PRTN3, the genes encoding the autoantigens, is associated with disease activity. We investigated whether patients with AAV have alterations in histone modifications, particularly those associated with transcriptional activation, at MPO and PRTN3. RESULTS: We identified a network of genes regulating histone modifications that were differentially expressed in AAV patients compared to healthy controls. We focused on four genes (EHMT1 and EHMT2, ING4, and MSL1) and found their expression correlated with expression of MPO and PRTN3. Methylation of histone H3K9, catalyzed by EHMT1 and EHMT2 and associated with gene silencing, was most depleted at MPO and PRTN3 in patients with active disease and the highest MPO and PRTN3 expression. Acetylation of histone H4K16, modified by complexes containing ING4 and MSL1 and associated with gene activation, was most enriched at MPO and PRTN3 in patients with active disease and the highest MPO and PRTN3 expression. Methylation at H3K4, a mark of transcriptional activation, was enriched at MPO and PRTN3 in patients and healthy controls. CONCLUSIONS: MPO and PRTN3 in neutrophils of AAV patients with active disease have a distinct pattern of histone modifications, which implicates epigenetic mechanisms in regulating expression of autoantigen genes and suggests that the epigenome may be involved in AAV pathogenesis.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , Redes Reguladoras de Genes , Histonas/metabolismo , Mieloblastina/genética , Peroxidase/genética , Acilação , Epigênese Genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Metilação , Análise de Sequência com Séries de Oligonucleotídeos/métodosRESUMO
Proteinase 3 (PR3) and myeloperoxidase (MPO) are two major autoantigens in patients with vasculitis with ANCA. The genes encoding these autoantigens are abnormally expressed in peripheral granulocytes of patients with active ANCA-associated vasculitis. This study provides evidence that this transcriptional dysregulation results in a variety of mRNA processing events from the PRTN3 gene locus. In addition to elevated levels of PR3 message, leukocyte RNA from patients contained PR3 transcripts with an alternative 3' untranslated region. Furthermore, we detected usage of an alternative transcription start site within intron 1 of the PRTN3 gene locus that coincided with active disease (odds ratio, 3.3; 95% confidence interval, 1.3 to 8.4; P=0.01). This promoter may be developmentally regulated, because it was active in normal human bone marrow, multiple leukemia cell lines, MCF-7 cells, and subjects after GM-CSF treatment but not subjects with a neutrophil left shift. This transcript, which lacks exon 1 of PRTN3, encodes a 24-kD protein (p24(PR3/MBN)) with a sequence similar to that previously described for myeloblastin. Notably, PR3, p24(PR3/MBN), and MPO were synthesized in cultured neutrophils from patients with active ANCA-associated vasculitis, indicating that increased transcription results in newly synthesized autoantigens in peripheral neutrophils of patients. The synthesis of p24(PR3/MBN) seems to expand the autoantigen repertoire, because immunoblots showed that sera from patients recognized p24(PR3/MBN). These findings emphasize the importance of transcriptional dysregulation of the autoantigen in autoimmune disease.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , Autoantígenos/genética , Biossíntese de Proteínas/genética , Transcrição Gênica/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/metabolismo , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/fisiopatologia , Autoantígenos/fisiologia , Sequência de Bases , Estudos de Casos e Controles , Células Cultivadas , Éxons/genética , Éxons/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mieloblastina/genética , Mieloblastina/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patologia , Peroxidase/genética , Peroxidase/metabolismo , Biossíntese de Proteínas/fisiologia , Transcrição Gênica/fisiologiaRESUMO
To protect infants from infection, human milk contains high levels of the enzyme lysozyme, unlike the milk of dairy animals. We have genetically engineered goats to express human lysozyme (hLZ milk) in their milk at 68% the amount found in human milk to help extend this protection. This study looked at the effect of hLZ on bacteria in raw milk over time. As the microbial diversity of goats' milk has yet to be investigated in depth using next-generation sequencing (NGS) technologies, we applied NGS and clone library sequencing (CLS) to determine the microbiota of raw goat milk (WT milk) and hLZ milk at early, mid and late lactation. Overall, in WT milk, the bacterial populations in milk at early and mid lactation were similar to each other with a shift occurring at late lactation. Both methods found Proteobacteria as the dominant bacteria at early and mid lactation, while Actinobacteria surged at late lactation. These changes were related to decreases in Pseudomonas and increases in Micrococcus. The bacterial populations in hLZ milk were similar to WT milk at early and mid lactation with the only significant differences occurring at late lactation with the elevation of Bacillaceae, Alicyclobacillaceae, Clostridiaceae and Halomonadaceae.
Assuntos
Animais Geneticamente Modificados/fisiologia , Bactérias/isolamento & purificação , Biodiversidade , Cabras/fisiologia , Microbiota , Leite/enzimologia , Leite/microbiologia , Muramidase/análise , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/microbiologia , Bactérias/classificação , Bactérias/genética , Feminino , Cabras/genética , Cabras/microbiologia , Humanos , Lactação , Leite/química , Muramidase/genética , Muramidase/metabolismoRESUMO
In many modern teleost fish, chorion (egg envelope) glycoproteins are synthesized in the liver of females, and the expression of those genes is controlled by endogenous estrogen released from the ovary during maturation. However, among the classical teleosts, such as salmonid, carp, and zebrafish, the chorion glycoproteins are synthesized in the oocyte, as in higher vertebrates. Sturgeon, which are members of the subclass Chondrostei, represent an ancient lineage of ray-finned fishes that differ from other teleosts in that their sperm possess acrosomes, their eggs have numerous micropyles, and early embryo development is similar to that of amphibians. In order to understand the molecular mechanisms of chorion formation and the phylogenetic relationship between sturgeon and other teleosts, we used specific antibodies directed against the primary components of sturgeon chorion glycoproteins, using immunoblotting and immunocytochemistry approaches. The origin of each chorion glycoprotein was determined through analyses of both liver and ovary, and their localization during ovarian development was investigated. Our data indicate that the origin of the major chorion glycoproteins of sturgeon, ChG1, ChG2, and ChG4, derive not only from the oocyte itself but also from follicle cells in the ovary, as well as from hepatocytes. In the follicle cell layer, granulosa cells were found to be the primary source of ChGs during oogenesis in white sturgeon. The unique origins of chorion glycoproteins in sturgeon suggest that sturgeons are an intermediate form in the evolution of the teleost lineage.