NETosis markers: Quest for specific, objective, and quantitative markers

AbstractMore than 10years have passed since the discovery of neutrophil extracellular traps (NETs) in 2004. NETs are extracellular web-like DNA decorated with antimicrobial proteins, which are released from activated neutrophils. The state of neutrophils with NET formation is called NETosis. It has been realized that NETosis includes suicidal NETosis and vital NETosis. The former state means cell death of neutrophils, whereas the latter state preserves living neutrophilic functions. Although both suicidal and vital NETosis play essential roles in elimination of microorganisms, excessive formation of NETs, especially the ones derived from suicidal NETosis, can harm the hosts. Therefore, the discovery of NETosis markers and development of evaluation methods are important. In this review, we compare the methods for evaluating NETosis, including immunocytological and immunohistological detection of co-localized neutrophil-derived proteins and extracellular DNA, and citrullinated histones, detection of NET remnants in fluid samples, and flow cytometric detection of cell-appendant NET components, with focus on the specificity, objectivity, and quantitativity. Since the gold standard marker of NETosis or method of NET detection has not been established yet, researchers should choose the most appropriate marker or method in each situation based on the knowledge of the respective virtues and faults

Novel process of intrathymic tumor-immune tolerance through CCR2-mediated recruitment of Sirpα + dendritic cells: A murine model

Immune surveillance system can detect more efficiently secretory tumor-specific antigens, which are superior as a target for cancer immunotherapy. On the contrary, immune tolerance can be induced in the thymus when a tumor antigen is massively secreted into circulation. Thus, the secretion of tumor-specific antigen may have contradictory roles in tumor immunity in a context-dependent manner. However, it remains elusive on the precise cellular mechanism of intrathymic immune tolerance against tumor antigens. We previously demonstrated that a minor thymic conventional dendritic cell (cDC) subset, CD8α -Sirpα + cDCs, but not the major subset, CD8α +Sirpα - cDCs can selectively capture blood-borne antigens and crucially contribute to the self-tolerance. In the present study, we further demonstrated that Sirpα + cDCs can capture a blood-borne antigen leaking inside the interlobular vascular-rich regions (IVRs). Blood-borne antigen selectively captured by Sirpα + cDCs can induce antigen-specific Treg generation or negative selection, depending on the immunogenicity of the presented antigen. Furthermore, CCR2 expression by thymic Sirpα + cDCs and abundant expression of its ligands, particularly, CCL2 by tumor-bearing mice prompted us to examine the function of thymic Sirpα + cDCs in tumor-bearing mice. Interestingly, tumor-bearing mice deposited CCL2 inside IVRs in the thymus. Moreover, tumor formation induced the accumulation of Sirpα + cDCs in IVRs under the control of CCR2-CCL2 axis and enhanced their capacity to take up antigens, resulting in the shift from Treg differentiation to negative selection. Finally, intrathymic negative selection similarly ensued in CCR2-competent mice once the tumor-specific antigen was secreted into bloodstream. Thus, we demonstrated that thymic Sirpα + cDCs crucially contribute to this novel process of intrathymic tumor immune tolerance. © 2012 Baba et al

Possible Implication of Fc γ

Leukocytes can “gnaw away” the plasma membrane of other cells. This phenomenon, called trogocytosis, occurs subsequent to cell-to-cell adhesion. Currently, two mechanisms of trogocytosis, adhesion molecule-mediated trogocytosis and Fcγ receptor-(FcγR-) mediated trogocytosis, have been identified. In our earlier study, we established an in vitro model of FcγR-mediated trogocytosis, namely, CD8 translocation model from T cells to neutrophils. By using this model, we demonstrated that the molecules transferred to neutrophils via FcγR-mediated trogocytosis were taken into the cytoplasm immediately. This result suggests that the chance of molecules transferred via FcγR-mediated trogocytosis to play a role on the cell surface could be time-limited. Thus, we consider the physiological role of FcγR-mediated trogocytosis as a means to remove antibodies (Abs) that bind with self-molecules rather than to extract molecules from other cells. This concept means that FcγR-mediated trogocytosis can be a defense mechanism to Ab-mediated autoimmune response. Moreover, the activity of FcγR-mediated trogocytosis was revealed to be parallel to the endocytotic activity of neutrophils, which was critically related to the susceptibility to systemic autoimmune diseases. The collective findings suggest that FcγR-mediated trogocytosis could physiologically play a role in removal of Abs bound to self-antigens and prevent autoimmune diseases

Severity-based treatment for Japanese patients with MPO-ANCA-associated vasculitis: the JMAAV study

We (JMAAV [Japanese patients with MPO-ANCA-associated vasculitis] Study Group) performed a prospective, open-label, multi-center trial to evaluate the usefulness of severity-based treatment in Japanese patients with myeloperoxidase-anti-neutrophil cytoplasmic antibodies (MPO-ANCA)-associated vasculitis. Patients with MPO-ANCA-associated vasculitis received a severity-based regimen according to the appropriate protocol: low-dose corticosteroid and, if necessary, cyclophosphamide or azathioprine in patients with mild form; high-dose corticosteroid and cyclophosphamide in those with severe form; and the severe-form regimen plus plasmapheresis in those with the most severe form. We followed up the patients for 18 months. The primary end points were the induction of remission, death, and end-stage renal disease (ESRD). Fifty-two patients were registered, and 48 patients were enrolled in this study (mild form, n = 23; severe form, n = 23; most severe form, n = 2). Among the 47 patients who received the predefined therapies, 42 achieved remission within 6 months, 5 died, and 1 developed ESRD. Disease flared up in 8 of the 42 patients with remission during the 18-month follow-up period. The JMAAV trial is the first prospective trial for MPO-ANCA-associated vasculitis to be performed in Japan. The remission and death rates were comparable to those in several previous clinical trials performed in western counties. The regimen employed in this trial was tailor-made based on patients’ disease severity and disease type, and it seems that standardization can be consistent with treatment choices made according to severity

Prediction of response to remission induction therapy by gene expression profiling of peripheral blood in Japanese patients with microscopic polyangiitis

BackgroundMicroscopic polyangiitis (MPA), which is classified as an anti-neutrophil cytoplasmic antibody (ANCA)-associated small vessel vasculitis, is one of the most frequent primary vasculitides in Japan. We earlier nominated 16 genes (IRF7, IFIT1, IFIT5, OASL, CLC, GBP-1, PSMB9, HERC5, CCR1, CD36, MS4A4A, BIRC4BP, PLSCR1, DEFA1/DEFA3, DEFA4, and COL9A2) as predictors of response to remission induction therapy against MPA. The aim of this study is to determine the accuracy of prediction using these 16 predictors.MethodsThirty-nine MPA patients were selected randomly and retrospectively from the Japanese nationwide RemIT-JAV-RPGN cohort and enrolled in this study. Remission induction therapy was conducted according to the Guidelines of Treatment for ANCA-Associated Vasculitis published by the Ministry of Health, Labour, and Welfare of Japan. Response to remission induction therapy was predicted by profiling the altered expressions of the 16 predictors between the period before and 1 week after the beginning of treatment. Remission is defined as the absence of clinical manifestations of active vasculitis (Birmingham Vasculitis Activity Score 2003: 0 or 1 point). Persistent remission for 18 months is regarded as a “good response,” whereas no remission or relapse after remission is regarded as a “poor response.”Results“Poor” and “good” responses were predicted in 7 and 32 patients, respectively. Five out of 7 patients with “poor” prediction and 1 out of 32 patients with “good” prediction experienced relapse after remission. One out of 7 patients with “poor” prediction was not conducted to remission. Accordingly, the sensitivity and specificity to predict poor response was 85.7% (6/7) and 96.9% (31/32), respectively.ConclusionsResponse to remission induction therapy can be predicted by monitoring the altered expressions of the 16 predictors in the peripheral blood at an early point of treatment in MPA patients

Comparison of severity classification in Japanese patients with antineutrophil cytoplasmic antibody-associated vasculitis in a nationwide, prospective, inception cohort study

OBJECTIVE: To compare disease severity classification systems for six-month outcome prediction in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). METHODS: Patients with newly diagnosed AAV from 53 tertiary institutions were enrolled. Six-month remission, overall survival, and end-stage renal disease (ESRD)-free survival were evaluated. RESULTS: According to the European Vasculitis Study Group (EUVAS)-defined disease severity, the 321 enrolled patients were classified as follows: 14, localized; 71, early systemic; 170, generalized; and 66, severe disease. According to the rapidly progressive glomerulonephritis (RPGN) clinical grading system, the patients were divided as follows: 60, grade I; 178, grade II; 66, grade III; and 12, grade IV. According to the Five-Factor Score (FFS) 2009, 103, 109, and 109 patients had ≤1, 2, and ≥3 points, respectively. No significant difference in remission rates was found in any severity classification. The overall and ESRD-free survival rates significantly differed between grades I/II, III, and IV, regardless of renal involvement. Severe disease was a good predictor of six-month overall and ESRD-free survival. The FFS 2009 was useful to predict six-month ESRD-free survival but not overall survival. CONCLUSIONS: The RPGN grading system was more useful to predict six-month overall and ESRD-free survival than the EUVAS-defined severity or FFS 2009

Comparison of severity classification in Japanese patients with antineutrophil cytoplasmic antibody-associated vasculitis in a nationwide, prospective, inception cohort study

OBJECTIVE: To compare disease severity classification systems for six-month outcome prediction in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). METHODS: Patients with newly diagnosed AAV from 53 tertiary institutions were enrolled. Six-month remission, overall survival, and end-stage renal disease (ESRD)-free survival were evaluated. RESULTS: According to the European Vasculitis Study Group (EUVAS)-defined disease severity, the 321 enrolled patients were classified as follows: 14, localized; 71, early systemic; 170, generalized; and 66, severe disease. According to the rapidly progressive glomerulonephritis (RPGN) clinical grading system, the patients were divided as follows: 60, grade I; 178, grade II; 66, grade III; and 12, grade IV. According to the Five-Factor Score (FFS) 2009, 103, 109, and 109 patients had ≤1, 2, and ≥3 points, respectively. No significant difference in remission rates was found in any severity classification. The overall and ESRD-free survival rates significantly differed between grades I/II, III, and IV, regardless of renal involvement. Severe disease was a good predictor of six-month overall and ESRD-free survival. The FFS 2009 was useful to predict six-month ESRD-free survival but not overall survival. CONCLUSIONS: The RPGN grading system was more useful to predict six-month overall and ESRD-free survival than the EUVAS-defined severity or FFS 2009

Possible implication of disordered neutrophil extracellular traps in the pathogenesis of MPO-ANCA-associated vasculitis

Neutrophil extracellular traps (NETs) are characterized by the presence of extracellular DNA fibers studded with antimicrobial proteins, including myeloperoxidase (MPO). Although NETs play an important role in the innate immune system, the scattered extracellular enzymes, such as MPO, pose risks to the host. Therefore, NETs are strictly regulated by DNase I in the serum, which prevents them from persisting. Recent studies have demonstrated that dysregulation of NETs could be involved in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus. In this review, we interpret the association of disordered NETs with autoimmune diseases, especially propylthiouracil-induced MPO-ANCA-associated vasculitis