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ANCA autoantigen gene expression highlights neutrophil heterogeneity where expression in normal-density neutrophils correlates with ANCA-induced activation.

ABSTRACT: ANCA vasculitis is an autoimmune disease with increased expression of the autoantigen genes, myeloperoxidase (MPO) and proteinase 3 (PRTN3), but the origin and significance of expression is less distinct. To clarify this, we measured MPO and PRTN3 messenger RNA in monocytes, normal-density neutrophils, and in enriched leukocytes from peripheral blood mononuclear cells. Increased autoantigen gene expression was detected in normal-density neutrophils and enriched leukocytes from patients during active disease compared to healthy individuals, with the largest difference in enriched leukocytes. RNA-seq of enriched leukocytes comparing active-remission pairs identified a gene signature for low-density neutrophils. Cell sorting revealed low-density neutrophils contained mature and immature neutrophils depending on the presence or absence of CD10. Both populations contributed to autoantigen expression but the frequency of immature cells in low-density neutrophils did not correlate with low-density neutrophil MPO or PRTN3 expression. Low-density neutrophils were refractory to MPO-ANCA induced oxidative burst, suggesting an alternative role for low-density neutrophils in ANCA vasculitis pathogenesis. In contrast, normal-density neutrophils were activated by MPO-ANCA and monoclonal anti-PR3 antibody. Normal-density neutrophil activation correlated with MPO and PRTN3 mRNA. Increased autoantigen gene expression originating from the mature low-density and normal-density neutrophils suggests transcriptional dysregulation is a hallmark of ANCA vasculitis. Thus, the correlation between autoantigen gene expression and antibody-mediated normal-density neutrophil activation connects autoantigen gene expression with disease pathogenesis.

SUBMITTER: Jones BE

PROVIDER: S-EPMC7908903 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundNeutrophils are important for immune surveillance of tumour cells. Neutrophils may also be epigenetically programmed in the tumour microenvironment to promote tumour progression. In addition to the commonly known high-density neutrophils (HDN) based on their separation on density gradient, recent studies have reported the presence of high levels of low-density neutrophils (LDN) in tumour-bearing mice and cancer patients. We reported previously that estrogen promotes the growth of estrogen receptor α-negative mammary tumours in mice undergoing mammary involution through stimulating pro-tumoral activities of neutrophils in the mammary tissue.MethodsFemale BALB/cAnNTac mice at 7-8 weeks old were mated and bilateral ovariectomy was performed 2 days post-partum. At 24 h after forced-weaning of pups to induce mammary involution, post-partum female mice were injected with either E2V, or vehicle control on alternative days for 2-weeks. On 48 h post-weaning, treated female mice were inoculated subcutaneously with 4 T1-Luc2 cells into the 9th abdominal mammary gland. Age-matched nulliparous female was treated similarly. Animals were euthanized on day 14 post-tumour inoculation for analysis. To evaluate the short-term effect of estrogen, post-partum females were treated with only one dose of E2V on day 12 post-tumour inoculation.ResultsEstrogen treatment for 2-weeks reduces the number of blood LDN by more than 10-fold in tumour-bearing nulliparous and involuting mice, whilst it had no significant effect on blood HDN. The effect on tumour-bearing mice is associated with reduced number of mitotic neutrophils in the bone marrow and increased apoptosis in blood neutrophils. Since estrogen enhanced tumour growth in involuting mice, but not in nulliparous mice, we assessed the effect of estrogen on the gene expression associated with pro-tumoral activities of neutrophils. Whilst 48 h treatment with estrogen had no effect, 2-weeks treatment significantly increased the expression of Arg1, Il1b and Tgfb1 in both HDN and LDN of involuting mice. In contrast, estrogen increased the expression of Arg1 and Ccl5 in HDN and LDN of nulliparous mice.ConclusionsProlonged estrogenic stimulation in tumour-bearing mice markedly hampered tumour-associated increase of LDN plausibly by inhibiting their output from the bone marrow and by shortening their life span. Estrogen also alters the gene expression in neutrophils that is not seen in tumour-free mice. The results imply that estrogen may significantly influence the tumour-modulating activity of blood neutrophils.

Project description:The function of oxidatively modified low-density lipoprotein (oxLDL) in the progression of cardiovascular diseases has been extensively investigated and well-characterized with regards to the activation of multiple cellular responses in macrophages and endothelial cells. Although accumulated evidence has revealed the presence of neutrophils in vascular lesions, the effect of oxLDL on neutrophil function has not been properly investigated. In the present decade, neutrophil extracellular traps (NETs) gained immense attention not only as a primary response against pathogenic bacteria but also due to their pathological roles in tissue damage in various diseases, such as atherosclerosis and thrombosis. In this study, we investigated if oxLDL affects NET formation and if it is a risk factor for inflammatory reactions in endothelial cells. HL-60-derived neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA) for 30 min to induce NET formation, followed by incubation with 20 ?g/mL native or oxidized LDL for additional 2 h. Culture media of the stimulated cells containing released NETs components were collected to evaluate NET formation by fluorometric quantitation of released DNA and detection of myeloperoxidase (MPO) by western blot analysis. NET formation of HL-60-derived neutrophils induced by PMA was significantly enhanced by additional incubation with oxLDL but not with native LDL. Treatment of HL-60-derived neutrophils with oxLDL alone in the absence of PMA did not induce NET formation. Furthermore, the culture media of HL-60-derived neutrophils after NET formation were then transferred to human aortic endothelial cell (HAECs) culture. Treatment of HAECs with the culture media containing NETs formed by HL-60-derived neutrophils increased the expression of metalloproteinase-1 protein in HAECs when HL-60-derived neutrophils were incubated with native LDL, and the expression was accelerated in the case of oxLDL. In addition, the culture media from NETs formed by HL-60-derived neutrophils caused the elongation of HAECs, which was immensely enhanced by coincubation with native LDL or oxLDL. These data suggest that oxLDL may act synergistically with neutrophils to form NETs and promote vascular endothelial inflammation.

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ANCA autoantigen gene expression highlights neutrophil heterogeneity where expression in normal-density neutrophils correlates with ANCA-induced activation.

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