Anti-myeloperoxidase antibodies attenuate the monocyte response to LPS and shape macrophage development
Reena J. Popat, Seran Hakki, Alpesh Thakker, Alice M. Coughlan, Julie Watson, Mark A. Little, Corinne M. Spickett, Paul Lavender, Behdad Afzali, Claudia Kemper, Michael G. Robson
Reena J. Popat, Seran Hakki, Alpesh Thakker, Alice M. Coughlan, Julie Watson, Mark A. Little, Corinne M. Spickett, Paul Lavender, Behdad Afzali, Claudia Kemper, Michael G. Robson
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Research Article Inflammation

Anti-myeloperoxidase antibodies attenuate the monocyte response to LPS and shape macrophage development

Abstract

Anti-neutrophil cytoplasmic antibody (ANCA) vasculitis is characterized by the presence of autoantibodies to myeloperoxidase and proteinase-3, which bind monocytes in addition to neutrophils. While a pathological effect on neutrophils is acknowledged, the impact of ANCA on monocyte function is less well understood. Using IgG from patients we investigated the effect of these autoantibodies on monocytes and found that anti-myeloperoxidase antibodies (MPO-ANCA) reduced both IL-10 and IL-6 secretion in response to LPS. This reduction in IL-10 and IL-6 depended on Fc receptors and enzymatic myeloperoxidase and was accompanied by a significant reduction in TLR-driven signaling pathways. Aligning with changes in TLR signals, oxidized phospholipids, which function as TLR4 antagonists, were increased in monocytes in the presence of MPO-ANCA. We further observed that MPO-ANCA increased monocyte survival and differentiation to macrophages by stimulating CSF-1 production. However, this was independent of myeloperoxidase enzymatic activity and TLR signaling. Macrophages differentiated in the presence of MPO-ANCA secreted more TGF-β and further promoted the development of IL-10– and TGF-β–secreting CD4+ T cells. Thus, MPO-ANCA may promote inflammation by reducing the secretion of antiinflammatory IL-10 from monocytes, and MPO-ANCA can alter the development of macrophages and T cells to potentially promote fibrosis.

Authors

Reena J. Popat, Seran Hakki, Alpesh Thakker, Alice M. Coughlan, Julie Watson, Mark A. Little, Corinne M. Spickett, Paul Lavender, Behdad Afzali, Claudia Kemper, Michael G. Robson

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Figure 5

MPO-ANCA generates oxidized phospholipids.

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MPO-ANCA generates oxidized phospholipids. (A) The most abundant oxidize...
(A) The most abundant oxidized phospholipids extracted from peripheral blood monocytes incubated for 18 hours with LPS and anti-myeloperoxidase antibodies (MPO-ANCA) (n = 3) or control IgG (n = 3). The data are presented as a percentage of the intensity of the native saturated phospholipid dipalmitoylphosphatidylcholine at m/z 734 in each sample. *P < 0.05 by 2-tailed Student’s t test). (B) Extracted ion chromatograms (XICs) for the control IgG–treated (n = 3) and MPO-ANCA–treated (n = 3) samples, showing the intensity of the long-chain oxidized phospholipid species at m/z 828.8 eluting at ~29.5 minutes, ahead of nonoxidized species of the same m/z ratio. The labels above the peaks indicate their respective area as calculated by Peakview software. Based on these characteristics, the early eluting species at m/z 828.8 was identified (arrow) as 1-palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-3-phosphocholine (PEIPC). The XICs were prepared with a window of 0.7 Da and without smoothing. (C) Surface E06 staining on peripheral blood monocytes incubated for 18 hours with LPS and MPO-ANCA (n = 3) or control IgG (n = 3). (D) Median fluorescence intensity (MFI) data from the experiment shown in C and 2 other monocyte donors to give 3 monocyte donors in total. The effect of adding the MPO inhibitor AZD5904 (4 μM) to MPO-ANCA–treated monocytes is also shown. Control IgG was compared with MPO-ANCA using a 2-tailed Student’s t test. MPO-ANCA was compared with MPO-ANCA + AZD5904 using a 2-tailed paired Student’s t test (with lines indicating paired samples). *P < 0.05. Error bars represent mean ± SEM. For a given monocyte donor, n is the number of IgG preparations from different individuals. They are not technical replicates or repeated measures of the same IgG samples.