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Background Myeloperoxidase ANCA-associated vasculitis is a major cause of ESKD. Efficacy of anti-CD20 mAb treatment was tested in a mouse model of the disease. Methods MPO immunization induced anti-MPO autoimmunity, and a subnephritogenic dose of sheep anti-mouse GBM globulin triggered GN. Results Anti-CD20 mAb treatment increased the numbers and immunomodulatory capacity of MPO-specific T regulatory cells (Tregs) and attenuated T cell-mediated and humoral anti-MPO autoimmunity and GN. Disabling of Tregs negated the therapeutic benefit of anti-CD20 treatment. The mechanism of enhancement of Treg activity could be attributed to anti-CD20 mAb effects on inducing B cell apoptosis. Administering anti-CD20 mAb-induced apoptotic splenocytes to mice developing anti-MPO GN was as effective as anti-CD20 mAb treatment in inducing Tregs and attenuating both anti-MPO autoimmunity and GN. A nonredundant role for splenic macrophages in mediating the anti-CD20 mAb-induced immunomodulation was demonstrated by showing that administration of anti-CD20 mAb ex vivo-induced apoptotic splenocytes to unmanipulated mice attenuated autoimmunity and GN, whereas deletion of splenic marginal zone macrophages prevented anti-CD20 mAb-induced immunomodulation and treatment efficacy. Six days after administering anti-CD20 mAb to mice with murine anti-MPO GN, cell-mediated anti-MPO responses and GN were attenuated, and Tregs were enhanced, but ANCA levels were unchanged, suggesting humoral autoimmunity was redundant at this time point. Conclusions Collectively, these data suggest that, as well as reducing humoral autoimmunity, anti-CD20 mAb more rapidly induces protective anti-MPO Treg-mediated immunomodulation by splenic processing of anti-CD20-induced apoptotic B cells.Copyright © 2021 by the American Society of Nephrology

BACKGROUND: Myeloperoxidase ANCA-associated vasculitis is a major cause of ESKD. Efficacy of anti-CD20 mAb treatment was tested in a mouse model of the disease. METHOD(S): MPO immunization induced anti-MPO autoimmunity, and a subnephritogenic dose of sheep anti-mouse GBM globulin triggered GN. RESULT(S): Anti-CD20 mAb treatment increased the numbers and immunomodulatory capacity of MPO-specific T regulatory cells (Tregs) and attenuated T cell-mediated and humoral anti-MPO autoimmunity and GN. Disabling of Tregs negated the therapeutic benefit of anti-CD20 treatment. The mechanism of enhancement of Treg activity could be attributed to anti-CD20 mAb effects on inducing B cell apoptosis. Administering anti-CD20 mAb-induced apoptotic splenocytes to mice developing anti-MPO GN was as effective as anti-CD20 mAb treatment in inducing Tregs and attenuating both anti-MPO autoimmunity and GN. A nonredundant role for splenic macrophages in mediating the anti-CD20 mAb-induced immunomodulation was demonstrated by showing that administration of anti-CD20 mAb ex vivo-induced apoptotic splenocytes to unmanipulated mice attenuated autoimmunity and GN, whereas deletion of splenic marginal zone macrophages prevented anti-CD20 mAb-induced immunomodulation and treatment efficacy. Six days after administering anti-CD20 mAb to mice with murine anti-MPO GN, cell-mediated anti-MPO responses and GN were attenuated, and Tregs were enhanced, but ANCA levels were unchanged, suggesting humoral autoimmunity was redundant at this time point. CONCLUSION(S): Collectively, these data suggest that, as well as reducing humoral autoimmunity, anti-CD20 mAb more rapidly induces protective anti-MPO Treg-mediated immunomodulation by splenic processing of anti-CD20-induced apoptotic B cells.Copyright © 2021 by the American Society of Nephrology.

Epidemiological studies and clinical trials suggest Bacillus Calmette-Guerin (BCG) vaccine has protective effects against coronavirus disease 2019 (COVID-19). There are now over 30 clinical trials evaluating if BCG vaccination can prevent or reduce the severity of COVID-19. However, the mechanism by which BCG vaccination can induce severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses is unknown. Here, we identify 8 novel BCG-derived peptides with significant sequence homology to either SARS-CoV-2 NSP3 or NSP13-derived peptides. Using an in vitro co-culture system, we show that human CD4+ and CD8+ T cells primed with a BCG-derived peptide developed enhanced reactivity to its corresponding homologous SARS-CoV-2-derived peptide. As expected, HLA differences between individuals meant that not all persons developed immunogenic responses to all 8 BCG-derived peptides. Nevertheless, all of the 20 individuals that were primed with BCG-derived peptides developed enhanced T cell reactivity to at least 7 of 8 SARS-CoV-2-derived peptides. These findings provide an in vitro mechanism that may account, in part, for the epidemiologic observation that BCG vaccination confers some protection from COVID-19.© Copyright © 2021 Eggenhuizen, Ng, Chang, Fell, Cheong, Wong, Gan, Holdsworth and Ooi.

Background: Accumulating evidence suggests that dysregulation of neutrophil extracellular traps (NETs) could be associated with the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Peptidyl arginine deiminases (PADs) are enzymes that generate citrullinated proteins, which are pro-inflammatory in both innate and adaptive immune responses. Citrullination of histone 3 (H3Cit) by PAD2 and PAD4 facilitates generation of both macrophage extracellular traps (METs) and NETs. BB-Clamidine is a second generation pan (PAD) inhibitor which reduces citrullination of H3Cit and subsequent NET formation. This study investigates the contribution of NETs in the pathogenesis of experimental anti-myeloperoxidase ANCA associated glomerulonephritis (MPO-ANCA GN) and the therapeutic possibility of BB-Cl amidine disrupting NET formation in vivo, and attenuating anti-MPO GN. Method(s): Experimental anti-MPO GN was induced in C57BL/6 mice by myeloperoxidase (MPO) immunisation and GN triggered using a subnephritogenic dose of anti-glomerular basement membrane globulin. BB-Cl-amidine treatment (1mg/kg/daily, n=6 or vehicle, n=7) was administered to disrupt NET formation after the establishment of MPO autoimmunity until termination of the experiment. Result(s): NET accumulation identified by co-localisation of extracellular DNA, H3Cit, PAD4, and MPO, was prominent in glomeruli of untreated (vehicle) mice compared to BB-Cl-amidine treated mice (2.1 +/- 0.4 vs 0.4 +/- 0.1, P=0.004). Histological assessment of kidneys demonstrated prominent glomerular segmental necrosis in the untreated group versus a reduction in mice receiving BB-Cl-amidine (88 +/- 3 vs 41 +/- 3, P=0.004). BBCl-amidine significantly attenuated glomerular leukocyte infiltration; CD4 T cells (1.2 +/- 0.3 vs 0.3 +/- 0.1, P=0.03), macrophages (1 +/- 0.1 vs 0.4 +/- 0.06, P=0.008) and neutrophils (3 +/- 0.5 vs 1.6 +/-0.3, P=0.03), compared to untreated mice. BB-Cl-amidine treated mice had increased numbers of CD

Background: Anti-cytokine monoclonal antibody (mAb) therapies have been effective in many autoimmune diseases but have not been successfully trialled in MPO-ANCA associated vasculitis. This study assessed the efficacy of blocking key CD4+ Th1 subset signature effector cytokines, TNFalpha and IFNgamma in MPO-ANCA GN. Assessing therapeutic efficacy of these anti-cytokine mAbs is now complicated by our recent discovery that Th subset dominance during the development of MPO autoimmunity is biphasic with initial transient Th17 dominance followed by persistent Th1 responses. Method(s): Anti-MPO autoimmunity was induced in C57BL/6 mice by MPO immunization and GN triggered using anti-GBM Ig during early and late development of anti-MPO autoimmunity, and GN assessed 4 days later (days 20 and 32, respectively). mAb treatment began 4hrs post GN triggering. Result(s): Administration of anti-TNFalpha mAb early in anti-MPO development (day 20) had no effect on kidney injury compared with vehicle treated controls: albuminuria [5.7+/-1.7 vs 5.5+/-1.7 mg/24hr, P=0.9]; glomerular segmental necrosis [GSN: 50+/-4 vs 43+/-3, P=0.1]. Similarly, anti-IFNgamma mAb was ineffective in attenuating GN at this timepoint [GSN: 48+/-6 vs 45+/-3, P=0.53]. Failure of these treatments is concordant with our observation that early developing anti-MPO autoimmunity is Th17 dominant. In contrast, anti-TNFalpha therapy during established anti-MPO GN (day 32) markedly attenuated kidney injury [GSN: 27+/-2 vs 50+/-4, P=0.01]. TNFalpha blockade acts locally in the kidney as systemic MPO specific IFNgamma and IL-17 recall responses from lymph nodes draining MPO immunization sites were similar to vehicle treated controls. Neutralizing IFNgamma at this late timepoint induced a phenotypic switch from Th1 responses to a protective Th2 with increased in serum MPO-ANCA [1.19+/-0.25 vs 0.48+/-0.13 OD450nm, P=0.02], increased IL-4 production from MPO challenged LN cells and increased the proportion of activated M2 m

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular deoxyribonucleic acid (ecDNA) is released during different forms of cell death including apoptosis, necrosis, necroptosis, neutrophil extracellular traps (NETs) and pyroptosis. Measurement of this cell death is time consuming with several different biomarkers required to identify the different biochemical forms of cell death. Measurement of ecDNA is generally conducted in serum and urine as a surrogate for renal damage, not in the actual target organ where the pathological injury occurs. The current difficulty in investigating ecDNA in the kidney is the lack of methods for formalin fixed paraffin embedded tissue (FFPE) both experimentally and in archived human kidney biopsies. This protocol provides a summary of the steps required to stain for ecDNA in FFPE tissue (both human and murine), quench autofluorescence and measure the ecDNA in the resulting images using a machine learning tool from the publicly available open source ImageJ plugin trainable Weka segmentation. Trainable Weka segmentation is applied to ecDNA within the glomeruli where the program learns to classify ecDNA. This classifier is applied to subsequent acquired kidney images, reducing the need for manual annotations of each individual image. The adaptability of the trainable Weka segmentation is demonstrated further in kidney tissue from experimental murine anti-MPO glomerulonephritis (GN), to identify NETs and ecMPO, common pathological contributors to anti-MPO GN. This method provides objective analysis of ecDNA in kidney tissue that demonstrates clearly the efficacy in which the trainable Weka segmentation program can distinguish ecDNA between healthy normal kidney tissue and diseased kidney tissue. This protocol can easily be adapted to identify ecDNA, NETs and ecMPO in other organs.

Background: Neutrophil extracellular trap (NETs) are implicated in the pathogenesis of myeloperoxidase anti neutrophil cytoplasmic antibody vasculitis (MPO-AAV). Neutrophil elastase (NE) is implicated in initiating NET formation through proteolytic degradation of the nuclear envelope, histone degradation and chromatin decondensation in concert with other neutrophil enzymes to facilitate DNA release. NETs are decorated with multiple proinflammatory enzymes including MPO (the autoantigen in this disease) and NE. NE released through degranulation is degraded by endogenous NE inhibitors, however NE bound to NET derived DNA is protected providing a reservoir of proteolytic NE enabling tissue destruction, microvascular injury and further recruitment of neutrophils. This study investigates the pathogenic contribution of NE release through NET formation in experimental MPO-AAV using animals deficient in neutrophil elastase. Method(s): A 20 day experimental model of anti-MPO GN was induced in B6.129X1-Elanetm1Sds/J mice (neutrophil elastase knock out mice) and wild-type (WT) litter mate controls by myeloperoxidase (MPO) immunisation and GN triggered using a subnephritogenic dose of anti-glomerular basement membrane globulin (anti GBM Ig), [n=10 (WT) and n=10 (Elane-/-)]. Result(s): Elane-/-animals were protected from excessive NET production and glomerular injury with significantly reduced numbers of glomerular neutrophils, extracellular MPO deposition, CD4 T cells and macrophages influx (all, P <0.05 compared to WT). Histological assessment of kidneys demonstrated a reduction in segmental necrosis in the Elane-/-group compared to the WT group (P<0.05). Functional injury measured by creatinine/albuminuria ratio was significantly reduced in the Elane -/-group compared to the WT group (P=0.04). Systemic autoimmunity to MPO was significantly reduced in Elane-/-mice with reduced frequency of MPO specific CD4 effector T cells from the draining lymph nodes and a significant reduct

A dysregulation of neutrophil extracellular trap (NETs) may be associated with the pathogenesis of myeloperoxidase (MPO) anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Peptidyl-argininedeiminase- 4 (PAD4) is an enzyme that facilitates generation of NETs. This study investigates the contribution of NETs in the pathogenesis of experimental MPO-ANCA associated glomerulonephritis (GN) by using PAD4 KO mice in an experimental murine model of MPO-ANCA GN. PAD4 KO mice had significant NET abrogation and were protected from glomerular injury and recruitment of inflammatory leukocytes: CD4 T cells, neutrophils and macrophages, and glomerular MPO (the autoantigen in this disease) (all, P < 0.05 compared to WT). We investigated the therapeutic possibility of 2 inhibitors; BB-Cl amidine (BB-CLA) a second generation pan PAD inhibitor and GSK484 a specific PAD4 inhibitor, both which inhibit the enzymatic activity of PAD4 and subsequent NET formation. BBCLA treatment reduced glomeruli NET accumulation, segmental necrosis (marker of glomerular injury) and leukocyte infiltration with CD4 T Cells, macrophages and neutrophils significantly reduced (all, P< 0.05 compared to the vehicle treated group). BB-Cl-amidine treated mice had increased numbers of CD4+ FoxP3+ T regulatory cells from lymph nodes compared to the vehicle treated group (P= 0.0012). PAD4 inhibition with GSK484 resulted in a significant reduction in the number of glomeruli with NETs and MPO deposition was significantly reduced (P< 0.05). Segmental necrosis and leukocyte recruitment to the glomeruli was significantly reduced (CD4 T cells, Neutrophils and macrophages all P< 0.05 compared to vehicle). In conclusion, PAD4 inhibition may be of potential therapeutic benefit in MPO-ANCA vasculitis.

Background Myeloperoxidase (MPO)-ANCA-associated GN is a significant cause of renal failure. Manipulating autoimmunity by inducing regulatory T cells is potentially a more specific and safer therapeutic option than conventional immunosuppression. Methods To generate MPO-specific regulatory T cells, we used a modified protein-conjugating compound, 1-ethyl-3-(3'dimethylaminopropyl)-carbodiimide (ECDI), to couple the immunodominant MPO peptide (MPO409-428) or a control ovalbumin peptide (OVA323-339) to splenocytes and induced apoptosis in the conjugated cells. We then administered MPO-and OVA-conjugated apoptotic splenocytes (MPO-Sps and OVA-Sps, respectively) to mice and compared their effects on development and severity of anti-MPO GN. We induced autoimmunity to MPO by immunizing mice with MPO in adjuvant; to trigger GN, we used low-dose antiglomerular basement membrane globulin, which transiently recruits neutro-phils that deposit MPO in glomeruli. We also compared the effects of transferring CD4+ T cells from mice treated with MPO-Sp or OVA-Sp to recipient mice with established anti-MPO autoimmunity. Results MPO-Sp but not OVA-Sp administration increased MPO-specific, peripherally derived CD4+ Foxp3- type 1 regulatory T cells and reduced anti-MPO autoimmunity and GN. However, in mice depleted of regulatory T cells, MPO-Sp administration did not protect from anti-MPO autoimmunity or GN. Mice with established anti-MPO autoimmunity that received CD4+ T cells transferred from mice treated with MPO-Sp (but not CD4+ T cells transferred from mice treated with OVA-Sp) were protected from anti-MPO autoimmunity and GN, confirming the induction of therapeutic antigen-specific regulatory T cells. Conclusions These findings in a mouse model indicate that administering apoptotic splenocytes conjugated with the immunodominant MPO peptide suppresses anti-MPO GN by inducing antigen-specific tolerance.Copyright © 2019 by the American Society of Nephrology.

Background: Myeloperoxidase (MPO)-ANCA-associated GN is a significant cause of renal failure. Manipulating autoimmunity by inducing regulatory T cells is potentially a more specific and safer therapeutic option than conventional immunosuppression. Method(s): To generate MPO-specific regulatory T cells, we used a modified protein-conjugating compound, 1-ethyl-3-(3'dimethylaminopropyl)-carbodiimide (ECDI), to couple the immunodominant MPO peptide (MPO409-428) or a control ovalbumin peptide (OVA323-339) to splenocytes and induced apoptosis in the conjugated cells. We then administered MPO- and OVA-conjugated apoptotic splenocytes (MPO-Sps and OVA-Sps, respectively) to mice and compared their effects on development and severity of anti-MPO GN. We induced autoimmunity to MPO by immunizing mice with MPO in adjuvant; to trigger GN, we used low-dose antiglomerular basement membrane globulin, which transiently recruits neutrophils that deposit MPO in glomeruli. We also compared the effects of transferring CD4+ T cells from mice treated with MPO-Sp or OVA-Sp to recipient mice with established anti-MPO autoimmunity. Result(s): MPO-Sp but not OVA-Sp administration increased MPO-specific, peripherally derived CD4+Foxp3- type 1 regulatory T cells and reduced anti-MPO autoimmunity and GN. However, in mice depleted of regulatory T cells, MPO-Sp administration did not protect from anti-MPO autoimmunity or GN. Mice with established anti-MPO autoimmunity that received CD4+ T cells transferred from mice treated with MPO-Sp (but not CD4+ T cells transferred from mice treated with OVA-Sp) were protected from anti-MPO autoimmunity and GN, confirming the induction of therapeutic antigen-specific regulatory T cells. Conclusion(s): These findings in a mouse model indicate that administering apoptotic splenocytes conjugated with the immunodominant MPO peptide suppresses anti-MPO GN by inducing antigen-specific tolerance.Copyright © 2019 by the American Society of Nephrology.