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Low Density Granulocytes in ANCA Vasculitis Are Heterogenous and Hypo-Responsive to Anti-Myeloperoxidase Antibodies.

ABSTRACT: Low Density Granulocytes (LDGs), which appear in the peripheral blood mononuclear cell layer of density-separated blood, are seen in cancer, sepsis, autoimmunity, and pregnancy. Their significance in ANCA vasculitis (AAV) is little understood. As these cells bear the autoantigens associated with this condition and have been found to undergo spontaneous NETosis in other diseases, we hypothesized that they were key drivers of vascular inflammation. We found that LDGs comprise a 3-fold higher fraction of total granulocytes in active vs. remission AAV and disease controls. They are heterogeneous, split between cells displaying mature (75%), and immature (25%) phenotypes. Surprisingly, LDGs (unlike normal density granulocytes) are hyporesponsive to anti-myeloperoxidase antibody stimulation, despite expressing myeloperoxidase on their surface. They are characterized by reduced CD16, CD88, and CD10 expression, higher LOX-1 expression and immature nuclear morphology. Reduced CD16 expression is like that observed in the LDG population in umbilical cord blood and in granulocytes of humanized mice treated with G-CSF. LDGs in AAV are thus a mixed population of mature and immature neutrophils. Their poor response to anti-MPO stimulation suggests that, rather than being a primary driver of AAV pathogenesis, LDGs display characteristics consistent with generic emergency granulopoiesis responders in the context of acute inflammation.

SUBMITTER: Ui Mhaonaigh A

PROVIDER: S-EPMC6856659 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Low Density Granulocytes in ANCA Vasculitis Are Heterogenous and Hypo-Responsive to Anti-Myeloperoxidase Antibodies.

Ui Mhaonaigh Aisling A Coughlan Alice M AM Dwivedi Amrita A Hartnett Jack J Cabral Joana J Moran Barry B Brennan Kiva K Doyle Sarah L SL Hughes Katherine K Lucey Rosemary R Floudas Achilleas A Fearon Ursula U McGrath Susan S Cormican Sarah S De Bhailis Aine A Molloy Eleanor J EJ Brady Gareth G Little Mark A MA

Frontiers in immunology 20191107

Low Density Granulocytes (LDGs), which appear in the peripheral blood mononuclear cell layer of density-separated blood, are seen in cancer, sepsis, autoimmunity, and pregnancy. Their significance in ANCA vasculitis (AAV) is little understood. As these cells bear the autoantigens associated with this condition and have been found to undergo spontaneous NETosis in other diseases, we hypothesized that they were key drivers of vascular inflammation. We found that LDGs comprise a 3-fold higher fract ...[more]

PMID: 31781107

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Project description:BackgroundTreatment 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.MethodsHLA 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.ResultsWe 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.ConclusionsThese 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.

Project description:IntroductionIncreasing evidence has suggested that linear epitopes of antineutrophil cytoplasmic antibody (ANCA) directed to myeloperoxidase (MPO) might provide clues to the pathogenesis of propylthiouracil (PTU)-induced ANCA-associated vasculitis (AAV). This study mapped epitopes of MPO-ANCA in sera from patients with PTU-induced MPO-ANCA (with or without vasculitis) and primary AAV, aiming to analyze certain epitopes associated with the development of PTU-induced AAV.MethodsSix recombinant linear fragments, covering the whole amino acid sequence of a single chain of MPO, were produced from Escherichia coli. Sera from 17 patients with PTU-induced AAV, 17 patients with PTU-induced MPO-ANCA but without clinical evidence of vasculitis, and 64 patients with primary AAV were collected at presentation. Of the 17 patients with PTU-induced AAV, 12 also had sera at remission. The epitope specificities were detected by enzyme-linked immunosorbent assay by using the recombinant fragments as solid-phase ligands.ResultsCompared with patients with PTU-induced MPO-ANCA but without clinical vasculitis, sera from PTU-induced AAV patients showed significantly higher reactivity against the H1 fragment of MPO (optical density values: 0.17 (0.10 to 0.35) versus 0.10 (0.04 to 0.21), P?=?0.038) and could recognize a significantly higher number of fragments (two (none to four) versus one (none to two), P?=?0.026). Compared with sera from primary AAV patients, sera from PTU-induced AAV patients had significantly higher reactivity to the P fragment and the H4 fragment (47.1% versus 14.1% P?<?0.001; 41.2% versus 14.1%, P?=?0.034, respectively), and could recognize a significantly higher number of fragments (two (none to four) versus one (none to two), P?=?0.013]. Among the 12 PTU-induced AAV patients with sequential samples, the number of fragments recognized in remission was significantly less than that in initial onset (two (none to four) versus none (none to 0.75), P?<?0.001].ConclusionsLinear epitopes of MPO molecules might be associated closely with PTU-induced AAV. In particular, the P and H4 fragments may be important epitopes in PTU-induced AAV.

Dataset Information

Low Density Granulocytes in ANCA Vasculitis Are Heterogenous and Hypo-Responsive to Anti-Myeloperoxidase Antibodies.

Publications

Low Density Granulocytes in ANCA Vasculitis Are Heterogenous and Hypo-Responsive to Anti-Myeloperoxidase Antibodies.

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