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Chronic kidney disease (CKD) leads to significant morbidity and mortality worldwide. Glomerulonephritis (GN) is the second leading cause of CKD resulting in end stage renal failure. The most severe and rapidly progressive type of GN is characterized by glomerular crescent formation. The current therapies for crescentic GN, which consist of broad immunosuppressive drugs, are partially effective, non-specific, toxic and cause many serious side effects including infections, cancer, and cardiovascular problems. Therefore, new and safer therapies are needed. Human amniotic epithelial cells (hAECs) are a type of stem cell which are isolated from the placenta after birth. They represent an attractive and novel therapeutic option for the treatment of various inflammatory conditions owing to their unique and selective immunosuppressive ability, as well as their excellent safety profile and clinical applicability. In this review, we will discuss the immunopathogenesis of crescentic GN, issues with currently available treatments and how hAECs offer potential to become a new and harmless treatment option for this condition.© Copyright © 2021 Al Mushafi, Ooi and Odobasic.

Anti-myeloperoxidase vasculitis (MPO-AAV) is a life-threatening autoimmune disease which causes severe inflammation of small blood vessels, mainly in the kidney. As for many other autoimmune diseases, current treatments, which consist of general immunosuppressants, are partially effective, toxic and broadly immunosuppressive, causing significant and serious adverse effects in many patients. Therefore, there is an urgent need for more targeted and less harmful therapies. Tolerogenic dendritic cells, regulatory T cells and stem cells have emerged as attractive, new and safer options for the treatment for various autoimmune diseases due to their unique and selective immunosuppressive capacity. In this review, we will discuss how these cellular therapies offer potential to become novel and safer treatments for MPO-AAV.© Copyright © 2021 Odobasic and Holdsworth.

Background Because of their capacity to induce antigen-specific immunosuppression, tolerogenic dendritic cells are a promising tool for treatment of autoimmune conditions, such as GN caused by autoimmunity against myeloperoxidase (MPO). Methods We sought to generate tolerogenic dendritic cells to suppress anti-MPO GN by culturing bone marrow cells with an NFkB inhibitor (BAY 11-7082) and exposing them to a pulse of MPO. After administering these MPO/BAY dendritic cells or saline to mice with established anti-MPO or anti-methylated BSA (mBSA) immunity, we assessed immune responses and GN. We also examined mechanisms of action of MPO/BAY dendritic cells. Results MPO/BAY dendritic cells decreased anti-MPO immunity and GN without inhibiting immune responses against mBSA; they also induced IL-10-producing regulatory T cells in MPO-immunized mice without affecting IL-10+ CD4+Foxp32 type 1 regulatory T cells or regulatory B cells. MPO/BAY dendritic cells did not inhibit anti-MPO immunity when CD4+Foxp3+ cells were depleted in vivo, showing that regulatory T cells are required for their effects. Coculture experiments with dendritic cells and CD4+Foxp32 or CD4+Foxp3+ cells showed that MPO/BAY dendritic cells generate Foxp3+ regulatory T cells from CD4+Foxp32 cells through several pathways, and induce IL-10+ regulatory T cells via inducible costimulator (ICOS), which was confirmed in vivo. Transfer of MPO/BAY dendritic cell-induced regulatory T cells in vivo, with or without anti-IL-10 receptor antibody, demonstrated that they suppress anti-MPO immunity and GN via IL-10. Conclusions MPO/BAY dendritic cells attenuate established anti-MPO autoimmunity and GN in an antigen-specific manner through ICOS-dependent induction of IL-10-expressing regulatory T cells. This suggests that autoantigen-loaded tolerogenic dendritic cells may represent a novel antigen-specific therapeutic option for anti-MPO GN.Copyright © 2019 by the American Society of Nephrology.

Background. The functional relevance of OX40 ligand (OX40L) in the effector phase of crescentic glomerulonephritis (GN) is unknown. These studies defined the role of endogenous OX40L during the effector stage of murine crescentic GN. Methods. GN was induced by immunization with sheep globulin/adjuvant on Day 0 and injection of sheep anti-mouse glomerular basement membrane immunoglobulin (Ig) on Day 10. Rat IgG or neutralizing anti-OX40L antibody was administered on Days 10-18 and immune responses and renal injury assessed on Day 20. Results. Compared with naive animals, OX40L was upregulated in the lymph nodes (LNs) and on leucocytes and resident non-immune cells in the kidneys of mice with GN. Inhibition of OX40L in GN augmented renal injury, as indicated by increased crescent formation, proteinuria and glomerular leucocyte accumulation. In line with increased injury, anti-OX40L treatment increased proliferation and decreased apoptosis of CD4 T cells in the LNs, without affecting LN CD4 cytokine production and CD8 T-cell responses. Blockade of OX40L decreased LN regulatory T-cell (Treg) proliferation, transforming growth factor beta production and foxp3 expression. OX40L inhibition did not affect B cell expansion or circulating antibody levels. In the kidney, neutralization of OX40L augmented interferon I 3 (IFN I 3) expression by CD4 and CD8 T cells and shifted macrophage polarization towards the pro-inflammatory M1 phenotype. Conclusions. OX40L is protective during the effector phase of murine crescentic GN by reducing the expansion of CD4 T cells and enhancing Treg responses in the LNs, and by locally inhibiting T-cell IFN I 3 production and pro-inflammatory macrophage phenotype in the kidney.Copyright © 2018 The Author(s).

The effects of formyl peptide receptors (FPRs) on effector T cells and inflammation-causing tissue-resident cells are not well known. Here, we explored the effect of FPR activation on efferent T cell responses in models of rheumatoid arthritis (RA) and on the expansion of fibroblast-like synoviocytes (FLS). Compound 43 (Cpd43; FPR1/2 agonist) was administered to mice with collagen-induced arthritis (CIA) or antigen-induced arthritis (AIA) after disease onset. Joint inflammation/damage and immunity were assessed. FLS were cultured with Cpd43 to test its effects on cell apoptosis and proliferation. To explore the effects of endogenous FPR2 ligands on FLS proliferation, FLS FPR2 was blocked or Annexin A1 (AnxA1) expression silenced. Cpd43 reduced arthritis severity in both models. In CIA, Cpd43 decreased CD4 T cell proliferation and survival and increased the production of the protective cytokine, IFNgamma in lymph nodes. In AIA, Cpd43 increased CD4 apoptosis and production of the anti-inflammatory IL-4, while augmenting the proportion of splenic regulatory T cells and their expression of IL-2Ralpha. In both models, Cpd43 increased CD4 IL-17A production, without affecting humoral immunity. FPR2 inhibitors reversed Cpd43-mediated effects on AIA and T cell immunity. Cpd43 decreased TNF-induced FLS proliferation and augmented FLS apoptosis in association with intracellular FPR2 accumulation, while endogenous AnxA1 and FPR2 reduced FLS proliferation via the ERK and NFkappaB pathways. Overall, FPR activation inhibits the expansion of arthritogenic effector CD4 T cells and FLS, and reduces joint injury in experimental arthritis. This suggests the therapeutic potential of FPR ligation for the treatment of RA.Copyright © 2018

The prospects for complement-targeted therapy in ANCA-associated vasculitis have been enhanced by a recent clinical trial in which C5a receptor 1 (C5aR1) inhibition safely replaced glucocorticoids in induction treatment. C5aR1 primes neutrophils for activation by anti-neutrophil cytoplasmic antibody (ANCA) and is therefore required in models of glomerulonephritis induced by anti-myeloperoxidase antibody. Although humoral and cellular autoimmunity play essential roles in ANCA-associated vasculitis, a role for C5aR1 in these responses has not been described. Here, we use murine models to dissect the role of C5aR1 in the generation of anti-myeloperoxidase autoimmunity and the effector responses resulting in renal injury. The genetic absence or pharmacological inhibition of C5aR1 results in reduced autoimmunity to myeloperoxidase with an attenuated Th1 response, increased Foxp3+ regulatory T cells and reduction in generation of myeloperoxidase-ANCA. These changes are mediated by C5aR1 on dendritic cells, which promotes activation, and thus myeloperoxidase autoimmunity and glomerulonephritis. We also use renal intravital microscopy to determine the effect of C5aR1 inhibition on ANCA induced neutrophil dysfunction. We found that myeloperoxidase-ANCA induce neutrophil retention and reactive oxygen species burst within glomerular capillaries. These pathological behaviors are abrogated by C5aR1 inhibition. Thus, C5aR1 inhibition ameliorates both autoimmunity and intra-renal neutrophil activation in ANCA-associated vasculitis.Copyright © 2017 International Society of Nephrology

Myeloperoxidase (MPO) is important in intracellular microbial killing by neutrophils but extracellularly causes tissue damage. Its role in adaptive immunity and T-cell2mediated diseases is poorly understood. Here, T-cell responses in lymph nodes (LNs) were enhanced by MPO deletion or in vivo inhibition, causing enhanced skin delayed-type hypersensitivity and antigen (Ag)-induced arthritis. Responses of adoptively transferred OT-II T cells were greater inMPO-deficient than wild-type (WT) recipients. MPO, deposited by neutrophils in LNs after Ag injection interacted with dendritic cells (DCs) in vivo. Culture of murine or human DCs with purified MPO or neutrophil supernatant showed that enzymatically dependent MPO-mediated inhibition of DC activation occurs via MPOgenerated reactive intermediates and involves DC Mac-1. Transfer of DCs cultured with WT, but not MPO-deficient, neutrophil supernatant attenuated Ag-specific immunity in vivo. MPO deficiency or in vivo inhibition increased DC activation in LNs after immunization. Studies with DQ-ovalbumin showed that MPO inhibits Ag uptake/processing by DCs. In vivo DC transfer and in vitro studies showed that MPO inhibits DCmigration to LNs by reducing their expression of CCR7. Therefore, MPO, via its catalytic activity, inhibits the generation of adaptive immunity by suppressing DC activation, Ag uptake/processing, and migration to LNs to limit pathological tissue inflammation.Copyright © 2013 by The American Society of Hematology.

Observations in experimental murine myeloperoxidase (MPO)-ANCA-associated vasculitis (AAV) show mast cells degranulate, thus enhancing injury as well as producing immunomodulatory IL-10. Here we report that, compared with biopsy specimens from control patients, renal biopsy specimens from 44 patients with acute AAV had more mast cells in the interstitium, which correlated with the severity of tubulointerstitial injury. Furthermore, most of the mast cells were degranulated and spindle-shaped in patients with acute AAV, indicating an activated phenotype. We hypothesized that the mast cell stabilizer disodium cromoglycate would attenuate mast cell degranulation without affecting IL-10 production. We induced anti-MPO GN by immunizing mice with MPO and a low dose of anti-glomerular basement membrane antibody. When administered before or after induction of MPO autoimmunity in these mice, disodium cromoglycate attenuated mast cell degranulation, development of autoimmunity, and development of GN, without diminishing IL-10 production. In contrast, administration of disodiumcromoglycate tomast cell-deficientmice had no effect on the development of MPO autoimmunity or GN. MPO-specific CD4+ effector T cell proliferationwas enhanced by co-culture with mast cells, but in the presence of disodiumcromoglycate, proliferation was inhibited and IL-10 production was enhanced. These results indicate that disodium cromoglycate blocks injurious mast cell degranulation specifically without affecting the immunomodulatory role of these cells. Thus as a therapeutic, disodium cromoglycate may substantially enhance the regulatory role of mast cells in MPO-AAV.Copyright © 2016 by the American Society of Nephrology.

Myeloperoxidase (MPO) anti-neutrophil cytoplasmic Ab (ANCA)-associated vasculitis results from autoimmunity to MPO. IL-17A plays a critical role in generating this form of autoimmune injury but its cell of origin is uncertain. We addressed the hypothesis that IL-17A-producing gd T cells are a nonredundant requisite in the development of MPO autoimmunity and glomerulonephritis (GN).We studied MPO-ANCA GN in wild type, ab, or gd T cell-deficient (C57BL/6, bTCR2/2, and dTCR2/2 respectively) mice. Both T cell populations played important roles in the generation of autoimmunity to MPO and GN. Humoral autoimmunity was dependent on intact ab T cells but was unaffected by gd T cell deletion. Following MPO immunization, activated gd T cells migrate to draining lymph nodes. Studies in dTCR2/2 and transfer of gd T cells to dTCR2/2 mice show that gd T cells facilitate the generation of anti-MPO autoimmunity and GN. dTCR2/2 mice that received IL-17A2/2 gd T cells demonstrate that the development of anti-MPO autoimmunity and GN are dependent on gd T cell IL-17A production. Finally, transfer of anti-MPO CD4+ T cell clones to naive dTCR2/2 and wild type mice with planted glomerular MPO shows that gd T cells are also necessary for recruitment of anti-MPO ab CD4+ effector T cells. This study demonstrates that IL-17A produced by gd T cells plays a critical role in the pathogenesis of MPO-ANCA GN by promoting the development of MPO-specific ab T cells.Copyright © 2017 by The American Association of Immunologists, Inc.

Objectives: Systemic lupus erythematosus (SLE) is a serious multisystem autoimmune disease, mediated by disrupted B cell quiescence and typically treated with glucocorticoids. We studied whether B cells in SLE are regulated by the glucocorticoid-induced leucine zipper (GILZ) protein, an endogenous mediator of antiinflammatory effects of glucocorticoids. Methods :We conducted a study of GILZ expression in blood mononuclear cells of patients with SLE, performed in vitro analyses of GILZ function in mouse and human B cells, assessed the contributions of GILZ to autoimmunity in mice, and used the nitrophenol coupled to keyhole limpet haemocyanin model of immunisation in mice. Result(s): Reduced B cell GILZ was observed in patients with SLE and lupus-prone mice, and impaired induction of GILZ in patients with SLE receiving glucocorticoids was associated with increased disease activity. GILZ was downregulated in naive B cells upon stimulation in vitro and in germinal centre B cells, which contained less enrichment of H3K4me3 at the GILZ promoter compared with naive and memory B cells. Mice lacking GILZ spontaneously developed lupus-like autoimmunity, and GILZ deficiency resulted in excessive B cell responses to T-dependent stimulation. Accordingly, loss of GILZ in naive B cells allowed upregulation of multiple genes that promote the germinal centre B cell phenotype, including lupus susceptibility genes and genes involved in cell survival and proliferation. Finally, treatment of human B cells with a cell-permeable GILZ fusion protein potently suppressed their responsiveness to T-dependent stimuli. Conclusion(s): Our findings demonstrated that GILZ is a non-redundant regulator of B cell activity, with important potential clinical implications in SLE.

Neutrophils are no longer seen as leukocytes with a sole function of being the essential first responders in the removal of pathogens at sites of infection. Being armed with numerous pro- and anti-inflammatory mediators, these phagocytes can also contribute to the development of various autoimmune diseases and can positively or negatively regulate the generation of adaptive immune responses. In this review, we will discuss how myeloperoxidase, the most abundant neutrophil granule protein, plays a key role in the various functions of neutrophils in innate and adaptive immunity.Copyright © 2016 Dragana Odobasic et al.

Toll-like receptor 9 (TLR9) enhances proinflammatory responses, but whether it can act in a regulatory capacity remains to be established. In experimental murine AKI induced by cisplatin, Tlr92/2 mice developed enhanced renal injury and exhibited fewer intrarenal regulatory T cells (Tregs) compared with genetically intact mice. A series of reconstitution and depletion studies defined a role for TLR9 in maintaining Treg-mediated homeostasis in cisplatin-induced AKI.When Rag12/2 mice were reconstituted with nonregulatory CD252 splenocytes from wild-type (WT) or Tlr92/2 mice, AKI was similarly enhanced. However, when Rag12/2 mice were reconstituted with CD4+CD25+ regulatory cells, WT CD4+CD25+ cells were more renoprotective and localized to the kidney more efficiently than Tlr92/2 CD4+CD25+ cells. In Tregdepleted Foxp3DTR mice, reconstitution with naive WT CD4+CD25+ cells resulted in less severe AKI than did reconstitution with Tlr92/2 Tregs. Tlr92/2 mice were not deficient in CD4+CD25+ cells, and WT and TLR9-deficient Tregs had similar suppressive function ex vivo. However, expression of adhesion molecules important in Treg trafficking was reduced on peripheral CD4+CD25+ cells from Tlr92/2 mice. In conclusion, we identified a pathway by which TLR9 promotes renal Treg accumulation in AKI.

Many forms of glomerulonephritis are mediated by autoimmunity. While autoantibodies are often pathogenic, cell-mediated immunity plays an important role in a number of forms of rapidly progressive glomerulonephritis. This unit describes the induction of cell-mediated autoimmune glomerular disease in mice. One disease model, experimental anti-glomerular basement membrane (GBM) disease, features autoreactivity to a well-defined component of type IV collagen found in the GBM, a3(IV)NC1. The other models the cell-mediated effector response in forms of renal vasculitis, where autoantibodies to myeloperoxidase result in systemic neutrophil activation, resulting in their localization to the glomerulus and the subsequent deposition of myeloperoxidase within glomerular capillaries. There, myeloperoxidase acts as a "planted" autoantigen and is recognized by effector autoreactive myeloperoxidase-specific T cells. These models are useful both in defining mechanisms germane to the development of autoimmunity to a3(IV)NC1 and myeloperoxidase, and in dissecting the role of cell-mediated responses in effecting glomerular injury.Copyright © 2014 by John Wiley & Sons, Inc.

Aim: To determine whether human amniotic epithelial stem cells (hAECs) can attenuate autoimmune anti-myeloperoxidase (MPO) glomerulonephritis. Background(s): hAECs are characteristically pluripotent, with low antigenicity and are highly anti-inflammatory, making them an attractive stem cell based therapeutic. Their use in anti-MPO glomerulonephritis has not been explored. Method(s): Anti-MPO glomerulonephritis was induced by immunisation with MPO/adjuvant (day [d] 0, 7) followed by disease trigger using low dose antiglomerular basement membrane globulin (d16). hAECs or vehicle were administered either 3d before each MPO/adjuvant injection or just before disease trigger (d14) and their effects on anti-MPO autoimmunity and GN assessed. hAECs were also co-cultured with splenocytes from MPO-immunised animals and their effect on MPO-specific T cell responses measured. Result(s): hAECs suppressed MPO-specific T cell responses when co-cultured with splenocytes in vitro (2.7 +/- 0.6 vs 7.5 +/- 0.8% bromodeoxyuridine+CD4+ cells, p < 0.01). In vivo, when used as a preventative, hAECs attenuated MPO-specific dermal delayed-type hypersensitivity (DTH; 0.1 +/- 0.03 vs 0.5 +/- 0.DELTAmm, p < 0.05), while enhancing proliferation of foxp3+CD25+ regulatory T cells (1.20 +/- 0.09 vs 0.84 +/- 0.13%, p < 0.05), CD4+ IL-10 producing cells (0.05 +/- 0.01 vs 0.26 +/- 0.09% p < 0.05) and surface expression of indoleamine 2,3 dioxygenase+ (0.027 +/- 0.004 vs 0.28 +/- 0.08%, p < 0.05), without affecting dendritic cells. Therapeutic administration of hAECs attenuated the development of glomerulonephritis (segmental necrosis: 15.6 +/- 2.2 vs 40.6 +/- 2.2%, p < 0.05), correlating with reduced accumulation of macrophages (0.2 +/- 0.04 vs 0.4 +/- 0.06 cells/ glomerular cross section [c/gcs], p < 0.05), CD4+ cells (0.3 +/- 0.03 vs 0.4 +/- 0.05 c/ gcs, p < 0.05) and neutrophils (0.2 +/- 0.03 vs 0.4 +/- 0.03 c/gcs, p < 0.01). Systemic anti-MPO autoimmunity was also reduced (DTH: 0.01 +/- 0.01 vs 0.4

The glomerulonephritides are diseases characterized by immune-mediated glomerular inflammation. Most severe and rapidly progressive forms of glomerulonephritis feature the participation of injurious leukocytes that localize to glomeruli. This unit describes classical models of rapidly progressive glomerulonephritis in mice, induced by injecting heterologous globulin (raised in sheep) that binds to the glomerular basement membrane. These models have been particularly useful in defining the participation of effector leukocytes in severe glomerular disease. In these models, injury typically occurs in two phases. In the initial, heterologous phase, injury is mediated by the globulin bound within the glomerulus acting as an antibody. The later, autologous phase of injury is mediated by the host's adaptive immunity to the heterologous globulin now functioning as a planted foreign antigen within glomeruli. As autologous phase injury is driven by immunity to sheep globulin, assessment of antigen-specific systemic immunity to sheep globulin is critical when using this model.Copyright © 2014 by John Wiley & Sons, Inc.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis involves innate and adaptive immune cells in the induction of autoimmunity and in autoimmune effector responses. Most Fcgamma receptors (FcgammaRs) activate immune cells, but FcgammaRIIB, found in humans and mice on B cells and innate cells, is an inhibitory receptor. Here we tested whether endogenous FcgammaRIIB negatively regulates autoreactivity and effector responses in experimental anti-myeloperoxidase (MPO) glomerulonephritis, using wild-type and FcgammaRIIB -/- mice. After MPO immunization, FcgammaRIIB -/- mice developed higher MPO-ANCA titers and increased anti-MPO T-cell responses. Transfer of FcgammaRIIB-deficient dendritic cells loaded with a nephritogenic MPO peptide (MPO 409-428) into wild-type mice induced stronger autoimmunity than dendritic cells derived from wild-type mice. Transferring anti-MPO antibodies into lipopolysaccharide-primed mice resulted in increased glomerular neutrophil accumulation and injury in FcgammaRIIB -/- mice, showing a role for FcgammaRIIB in suppressing neutrophil activation. Inducing active autoimmunity to MPO followed by triggering T cell-mediated glomerular injury by transfer of sub-nephritogenic doses of lipopolysaccharide and anti-MPO antibodies resulted in more disease in FcgammaRIIB -/- mice. Thus, endogenous FcgammaRIIB negatively regulates anti-MPO autoimmunity and glomerulonephritis by dendritic cells, B cells, and neutrophils to limit MPO-ANCA production, T-cell responses, and neutrophil activation.Copyright © 2014 International Society of Nephrology.

Objective Myeloperoxidase (MPO) locally contributes to organ damage in various chronic inflammatory conditions by generating reactive intermediates. The contribution of MPO in the development of experimental lupus is unknown. The aim of this study was to define the role of MPO in murine lupus nephritis (LN). Methods LN was induced in C57BL/6 wild-type (WT) and MPO knockout (MPO-/-) mice by intraperitoneal injection of pristane. Autoimmunity and glomerulonephritis were assessed 20 and 40 weeks after pristane administration. Cell apoptosis, leukocyte accumulation, and cytokine levels in the peritoneal cavity of WT and MPO-/- mice were assessed 3 or 6 days after pristane injection. Results MPO-/- mice developed more severe nephritis than did WT mice 20 and 40 weeks after pristane injection, despite having reduced glomerular deposition of antibody and complement and diminished levels of markers of oxidative stress (oxidized DNA and glutathione sulfonamide). Enhancement of renal disease in MPO-deficient mice correlated with increased accumulation of CD4+ T cells and macrophages in glomeruli, which, in turn, was associated with augmented generation of CD4+ T cell responses and increased activation and migration of dendritic cells in secondary lymphoid organs. In addition, the enhanced renal injury in MPO-/- mice was associated with increased glomerular accumulation of neutrophils and deposition of neutrophil extracellular traps. MPO deficiency also increased early cell apoptosis, leukocyte accumulation, and proinflammatory cytokine expression in the peritoneum. Conclusion MPO attenuates pristane-induced LN by inhibiting early inflammatory responses in the peritoneum and limiting the generation of CD4+ T cell autoimmunity in secondary lymphoid organs.Copyright © 2015, American College of Rheumatology.

Aim: To test the hypothesis that endogenous FcgammaRIIB negatively regulates autoimmune anti-myeloperoxidase (MPO) glomerulonephritis, using wild type (WT) and FcgammaRIIB-/- mice. Background(s): Anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis involves innate and adaptive immune cells both in the induction of autoimmunity and in autoimmune effector responses. Most Fcgamma receptors activate immune cells, but FcgammaRIIB, found in humans and mice on B cells and innate cells, is an inhibitory receptor. Method(s): The effects of endogenous FcgammaRIIB were determined by comparing: anti-MPO antibody titres; neutrophil activation ex vivo; glomerular neutrophil recruitment in vivo; anti-MPO T cell responses; dendritic cell activation of T cells and disease severity. Result(s): After MPO immunisations, FcgammaRIIB-/- mice developed higher anti- MPO IgG titres (WT: 0.35 +/- 0.06 vs. FcgammaRIIB-/-: 0.64 +/- 0.05 OD450, P < 0.01). FcgammaRIIB negatively regulated ANCA-directed neutrophil activation as FcgammaRIIB-/- neutrophils had increased ROS production after stimulation with anti-MPO IgG and LPS ex vivo (WT: 4.7 +/- 0.1% vs. FcgammaRIIB-/-: 6.0 +/- 0.3% R123+ neutrophils, P < 0.05). Furthermore, the passive transfer of anti-MPO IgG and LPS in vivo resulted in increased glomerular neutrophil recruitment in FcgammaRIIB-/- mice (WT: 0.5 +/- 0.1 vs. FcgammaRIIB-/-: 1.0 +/- 0.1 neutrophils/glomerular cross section, P < 0.001) with enhanced glomerular injury. FcgammaRIIB-/- mice also developed enhanced anti-MPO T cells responses after MPO immunisation. FcgammaRIIB on dendritic cells suppressed T cell priming as the transfer of MPOpulsed FcgammaRIIB-/- dendritic cells into WT mice enhanced T cell priming (WT: 0.6 +/- 0.0% vs. FcgammaRIIB-/-: 1.4 +/- 0.1% IFN-gamma+CD4+ cells, P < 0.001). In an active T cell mediated disease model, FcgammaRIIB-/- mice developed more severe glomerulonephritis compared with WT mice evidenced by increased glomerular segmen

In acute kidney injury, which is a significant cause of morbidity and mortality, cytokines and leukocytes promote inflammation and injury. We examined the pathogenic role of IL-17A in cisplatin-induced acute kidney injury. Intrarenal IL-17A mRNA transcription and protein expression were increased in wild-type mice after cisplatin-induced renal injury. An important role for IL-17A in the nephrotoxicity of cisplatin was demonstrated by observing protection from cisplatin-induced functional and histological renal injury in Il17a-/- and Rorgammat-/- mice, as well as in mice treated pre-emptively with anti-IL-17A antibodies. Both renal injury and renal IL-1beta and IL-17A production were attenuated in Asc-/- and Tlr2-/- mice, suggesting that cisplatin induces endogenous TLR2 ligand production and activates the ASC-dependent inflammasome complex, resulting in IL-1beta and injurious IL-17A production. Neutrophils and natural killer cells are the likely targets of these pathways, because combined depletion of these cells was strongly protective; anti-IL-17A antibodies had no additional effect in this setting. Although IL-17A can also be produced by CD4+ and gammadelta T cells, IL-17A from those cells does not contribute to renal injury. Cisplatin-induced injury was unchanged in gammadelta T-cell-deficient mice, whereas Il17a-/- CD4 + T cells induced similar injury as did wild-type CD4+ T cells on transfer to cisplatin-injected Rag1-/- mice. These studies demonstrate an important role for TLR2, the ASC inflammasome, and IL-17A in innate leukocytes in cisplatin-induced renal injury. © 2014 American Society for Investigative Pathology.

Aim: To define the role of interleukin (IL)-17A in systemic lupus erythematosus (SLE) induced by pristane. Background(s): SLE and lupus nephritis are significant causes of morbidity and mortality. Clinical studies have demonstrated that serum IL-17A levels are increased in disease, although direct evidence of pathogenicity is lacking. Method(s): We injected pristane (500 ml) intraperitoneally into C57BL/6 wild type (WT) mice and assessed IL-17A production after 6 days, using flow cytometry. Subsequently we treated WT and IL-17A-/- mice with pristane and assessed cellular immunity (8 weeks), humoral autoimmunity and renal injury, both functionally and histologically, after 7 months. Result(s): After 6 days, IL-17A production was readily detected in pristane treated WT mice, but not in control (WT) mice. The majority of IL-17A producing cells were innate cells (macrophages 42.3 +/- 5.2%, neutrophils 23.8 +/- 2.7%, natural killer T cells 11.2 +/- 1.3%, gammadelta T cells11.2 +/- 1.3%) only 14.1 +/- 1.5% were CD4+Tcells. After 8 weeks systemic IFNY (WT 591.3 +/- 188.6 vs. IL-17A-/- 176.0 +/- 15.8 ng/ml, P < 0.05) and TNF (WT 294.7 +/- 11.6 vs. IL-17A-/- 196.6 +/- 35.7 ng/ml, P < 0.05) production were decreased in IL-17A-/- mice, IL-17A was still readily detected in WT mice. After 7 months humoral autoimmunity was diminished in IL-17A-/- mice, with decreased levels of total IgG and anti-dsDNA, at serial dilutions. Glomerular IgG (WT 1.8 +/- 0.1 vs. IL-17A-/- 1.1 +/- 0.1 intensity score, P < 0.0001) and complement (WT 2.0 +/- 0.1 vs. IL-17A-/- 1.3 +/- 0.1 intensity score, P < 0.0001) deposition was decreased in the absence of IL-17A. Compared to WT mice, glomerular injury assessed by albuminuria (WT 271.5 +/- 55.8 vs. IL-17A-/- 87.6 +/- 15.9 mg/24 hours, P < 0.05) and histological injury (WT 32.0 +/- 2.1 vs. IL-17A-/- 22.9 +/- 3.1 abnormal glomeruli P < 0.05) were diminished in IL-17A-/- mice. Conclusion(s): These results demonstrate that innate immune cells produce IL-17

Interleukin (IL)-17A is increased both in serum and in kidney biopsies from patients with lupus nephritis, but direct evidence of pathogenicity is less well established. Administration of pristane to genetically intact mice results in the production of autoantibodies and proliferative glomerulonephritis, resembling human lupus nephritis. These studies sought to define the role of IL-17A in experimental lupus induced by pristane administration. Pristane was administered to wild-type (WT) and IL-17A-/- mice. Local and systemic immune responses were assessed after 6days and 8weeks, and autoimmunity, glomerular inflammation and renal injury were measured at 7months. IL-17A production increased significantly 6days after pristane injection, with innate immune cells, neutrophils (Ly6G+) and macrophages (F4/80+) being the predominant source of IL-17A. After 8weeks, while systemic IL-17A was still readily detected in WT mice, the levels of proinflammatory cytokines, interferon (IFN)-gamma and tumour necrosis factor (TNF) were diminished in the absence of endogenous IL-17A. Seven months after pristane treatment humoral autoimmunity was diminished in the absence of IL-17A, with decreased levels of immunoglobulin (Ig)G and anti-dsDNA antibodies. Renal inflammation and injury was less in the absence of IL-17A. Compared to WT mice, glomerular IgG, complement deposition, glomerular CD4+ T cells and intrarenal expression of T helper type 1 (Th1)-associated proinflammatory mediators were decreased in IL-17A-/- mice. WT mice developed progressive proteinuria, but functional and histological renal injury was attenuated in the absence of IL-17A. Therefore, IL-17A is required for the full development of autoimmunity and lupus nephritis in experimental SLE, and early in the development of autoimmunity, innate immune cells produce IL-17A. © 2014 British Society for Immunology.

IL-17A is increased in serum and in kidney biopsies from patients with lupus nephritis, but direct evidence of pathogenicity is less well established. These studies sought to define the role of IL-17A in experimental lupus induced by the administration of pristane to WT and IL-17A-/- mice. Local and systemic immune responses were assessed after 6 days and 8 weeks, and autoimmunity, glomerular inflammation and renal injury were determined at 7 months. Peritoneal IL-17A production increased significantly 6 days after pristane injection, with innate cells (neutrophils and macrophages) being the predominant source of IL-17A. After 8 weeks, while systemic IL-17A was still readily detected in WT mice, IFN-gamma and TNF were diminished in IL-17A-/- mice. Seven months after pristane, humoral autoimmunity was diminished in IL-17A-/- mice, with decreased serum lgG and anti-dsDNA antibody titers. Renal inflammation and injury was less in the absence of IL-17A. Compared to WT mice, glomerular IgG, complement deposition, glomerular CD4+ T cells and intrarenal pro-inflammatory mediator expression were decreased in IL-17A-/- mice. WT mice developed progressive albuminuria, but albuminuria (7 months: WT 271+/-56, IL-17A 88+/-16mug/day; P<0.05) and histological abnormalities were attenuated in IL-17A-/- mice. Therefore, IL-17A is required for the full development of systemic autoimmunity and experimental lupus nephritis. Early in the development of autoimmunity, innate immune cells produce IL-17A.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis involves innate and adaptive immune cells in the induction of autoimmunity and in autoimmune effector responses. Most Fcgamma receptors (FcgammaRs) activate immune cells, but FcgammaRIIB, found in humans and mice on B cells and innate cells, is an inhibitory receptor. These studies tested the hypothesis that endogenous FcgammaRIIB negatively regulates autoreactivity and effector responses in experimental anti-myeloperoxidase (MPO) glomerulonephritis, using wild type and FcgammaRIIB-/- mice. After MPO immunization, FcgammaRIIB-/- mice developed higher MPO-ANCA titers and increased anti-MPO T cell responses. Transfer of FcgammaRIIB deficient dendritic cells loaded with a nephritogenic MPO peptide (MPO409-428) into wild type mice induced stronger autoimmunity than dendritic cells derived from wild type mice. Transferring anti-MPO antibodies into LPS primed mice resulted in increased glomerular neutrophil accumulation and injury in FcgammaRIIB-/- mice, showing a role for FcgammaRIIB in suppressing neutrophil activation. Inducing active autoimmunity to MPO followed by triggering T cell mediated glomerular injury by transfer of subnephritogenic doses of LPS and anti-MPO antibodies resulted in more disease in FcgammaRIIB-/- mice. Therefore endogenous FcgammaRIIB negatively regulates anti-MPO autoimmunity and glomerulonephritis via dendritic cells, B cells and neutrophils to limit MPO-ANCA production, T cell responses and neutrophil activation.

Objective Myeloperoxidase (MPO) is implicated as a local mediator of tissue damage when released extracellularly in many chronic inflammatory diseases. The purpose of this study was to explore the role of endogenous MPO in experimental rheumatoid arthritis (RA). Methods K/BxN serum-transfer arthritis was induced in C57BL/6 wild-type (WT) and MPO knockout (MPO-/-) mice, and disease development was assessed. MPO activity was measured in joint tissues from mice with or without K/BxN arthritis. Collagen-induced arthritis (CIA) was induced in WT and MPO-/- mice, and disease development and immune responses were examined. MPO expression was assessed in synovial biopsy samples from patients with active RA, and the effect of MPO on synovial fibroblasts was tested in vitro. Results MPO was up-regulated in the joints of mice with K/BxN arthritis, and MPO deficiency attenuated the severity of the disease without affecting circulating cytokine levels. In CIA, MPO-/- mice had enhanced CD4+ T cell responses and reduced frequency of regulatory T cells in the lymph nodes and spleen, as well as augmented interleukin-17A and diminished interferon-gamma secretion by collagen-stimulated splenocytes, without an effect on circulating anticollagen antibody levels. Despite enhanced adaptive immunity in secondary lymphoid organs, CIA development was attenuated in MPO-/- mice. Intracellular and extracellular MPO was detected in the synovium of patients with active RA, and human MPO enhanced the proliferation and decreased the apoptosis of synovial fibroblasts in vitro. Conclusion MPO contributes to the development of arthritis despite suppressing adaptive immunity in secondary lymphoid organs. This suggests distinct effects of local MPO on arthritogenic effector responses. Copyright © 2014 by the American College of Rheumatology.

Myeloperoxidase (MPO) is important in intracellular microbial killing by neutrophils but extracellularly causes tissue damage. Its role in adaptive immunity and T-cell-mediated diseases is poorly understood. Here, T-cell responses in lymph nodes (LNs) were enhanced by MPO deletion or in vivo inhibition, causing enhanced skin delayed-type hypersensitivity and antigen (Ag)-induced arthritis. Responses of adoptively transferred OT-II T cells were greater in MPO-deficient than wild-type (WT) recipients. MPO, deposited by neutrophils in LNs after Ag injection, interacted with dendritic cells (DCs) in vivo. Culture of murine or human DCs with purified MPO or neutrophil supernatant showed that enzymatically dependent MPO-mediated inhibition of DC activation occurs via MPO-generated reactive intermediates and involves DC Mac-1. Transfer of DCs cultured with WT, but not MPO-deficient, neutrophil supernatant attenuated Ag-specific immunity in vivo. MPO deficiency or in vivo inhibition increased DC activation in LNs after immunization. Studies with DQ-ovalbumin showed that MPO inhibits Ag uptake/processing by DCs. In vivo DC transfer and in vitro studies showed that MPO inhibits DC migration to LNs by reducing their expression of CCR7. Therefore, MPO, via its catalytic activity, inhibits the generation of adaptive immunity by suppressing DC activation, Ag uptake/processing, and migration to LNs to limit pathological tissue inflammation.

CD80 and CD86 play a critical role in the initiation of T-cell responses. However, their role in the in vivo effector CD4+ T-cell responses has been less extensively investigated. The current studies have examined the functional relevance of CD80 and CD86 in the effector CD4+ T-cell responses inducing antigen-induced arthritis. Arthritis was induced in C57BL/6 mice by sensitization to methylated bovine serum albumin (mBSA) on day 0, booster immunization (day 7) and intra-articular injection of mBSA (day 21). Control or anti-CD80 and/or anti-CD86 monoclonal antibodies were administered from day 21 to day 28. Arthritis severity and immune responses were assessed on day 28. The development of arthritis was significantly suppressed by inhibition of CD80 or CD86. Blockade of both CD80 and CD86 caused a trend towards reduced disease severity compared to control antibody-treated mice. Neutralization of CD80 attenuated accumulation of CD4+ T cells in joints and enhanced splenocyte production and circulating levels of interleukin-4. Inhibition of CD86 or both CD80 and CD86 reduced T-cell accumulation in joints without affecting T helper type 1/type 2 (Th1/Th2) differentiation or antibody levels. Blockade of CD86, and not CD80, significantly suppressed splenocyte interleukin-17 (IL-17) production. These results provide further in vivo evidence that CD80 and CD86 play important pathogenic roles in effector T-cell responses. CD80 exacerbates arthritis by downregulating systemic levels of IL-4 and increasing T-cell accumulation in joints without affecting IL-17 production. CD86 enhances disease severity by upregulating IL-17 production and increasing the accumulation of effector T cells in joints without affecting Th1/Th2 development. © 2008 Blackwell Publishing Ltd.

Myeloperoxidase (MPO) is an enzyme that is found in neutrophils and monocytes/macrophages. Intracellularly, it plays a major role in microbial killing, but extracellularly, it may cause host tissue damage. The role of endogenous MPO was studied during neuhophil-mediated (heterologous) and T helper 1 (Th1)/macrophage-mediated (autologous) phases of crescentic glomerulonephritis. Glomerulonephritis was induced in C57BL/6 wild-type (WT) and MPO-deficient (MPO-/-) mice by intravenous injection of sheep anti-mouse glomerular basement membrane globulin. MPO activity was increased in kidneys of WT mice during both the heterologous and autologous phases of glomerulonephritis. During the heterologous phase of glomerulonephritis, proteinuria was decreased, whereas glomerular neutrophil accumulation and F-selectin expression were enhanced in MPO-/- mice. In the autologous, crescentic phase of glomerulonephritis, MPO-/- mice had increased accumulation of CD4+ cells and macrophages in glomeruli compared with WT mice. However, no difference in renal injury (crescent formation, proteinuria, and serum creatinine levels) was observed. Neutrophils and macrophages from MPO-/- mice exhibited reduced production of reactive oxygen species. Assessment of systemic immunity to sheep globulin showed that MPO-/- mice had increased splenic CD4+ cell proliferation, cytokine production, and dermal delayed-type hypersensitivity, as well as enhanced levels of circulating IgG, IgG1, and IgG3. MPO-/- mice also had an augmented Th1:Th2 ratio compared with WT mice (IFN-gamma:IL-4 and IgG3:IgG1 ratios). These results suggest that endogenous MPO locally contributes to glomerular damage during neutrophil-mediated glomerulonephritis, whereas it attenuates initiation of the adaptive immune response inducing crescentic, autologous-phase glomerulonephritis by suppressing T cell proliferation, cytokine production, and Th1:Th2 ratio. Copyright © 2007 by the American Society of Nephrology.

The expression of recombinant, biologically active mouse myeloperoxidase (MPD) and the recombinant non-collagenous (NC1) domain of mouse collagen alpha 3 Type IV was achieved for the first time in Sf21 cells (Spodoptera frugiperda ovarian insect cells) using a baculovirus expression system. Following purification, the proteins were identified by reducing and non-reducing SDS-PAGE electrophoresis. Recombinant mouse MPO has a molecular weight of approximately 90 kDa and mouse alpha3(IV)NC1 approximately 32 kDa. In addition, milligram quantities of native mouse myeloperoxidase were purified from 32Dcl3 cells. Both native and recombinant myeloperoxidase were biologically active. This study also demonstrated that the immunization of myeloperoxidase deficient (Mpo -/-) mice with purified recombinant mouse myeloperoxidase induced a significant antibody response to native myeloperoxidase. © 2005 Elsevier B.V. All rights reserved.

Experimental crescentic glomerulonephritis is driven by systemic cellular immune responses. A pathogenic role for T helper type 1 (Th1) and Th17 cells is well established. T-bet, a key transcription factor required for Th1 lineage commitment, and retinoic acid-related orphan receptor-gammat (Rorgammat), a key Th17 transcription factor, are required for full expression of disease. Similarly, several Th1- and Th17-associated cytokines have been implicated in disease augmentation. The role of Th2 cells in the disease is less clear, although Th2-associated cytokines, interleukin (IL)-4 and IL-10, are protective. We sought to determine the role of signal transducer and activation of transcription 6 (STAT6), a key regulator of Th2 responses, in experimental crescentic glomerulonephritis. Compared to wild-type mice, histological and functional renal injury was enhanced significantly in STAT6-/- mice 21 days after administration of sheep anti-mouse glomerular basement membrane globulin. Consistent with the enhanced renal injury, both Th1 and Th17 nephritogenic immune responses were increased in STAT6-/- mice. Conversely, production of IL-5, a key Th2-associated cytokine, was decreased significantly in STAT6-/- mice. Early in the disease process systemic mRNA expression of T-bet and Rorgamma was increased in STAT6-/- mice. We conclude that STAT6 is required for attenuation of Th1 and Th17 nephritogenic immune responses and protection from crescentic glomerulonephritis. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

The inducible co-stimulatory molecule (ICOS)/ICOS ligand (ICOSL) co-stimulatory pathway is critical in T cell activation, differentiation, and effector function. Its role was investigated in a model of Th1-driven crescentic glomerulonephritis (GN). GN was induced by sensitizing mice to sheep globulin (day 0) and challenging them with sheep anti-mouse glomerular basement membrane antibody (Ab; day 10). Disease and immune responses were assessed on day 20. For testing the role of ICOSL in the induction of GN, control or anti-ICOSL mAb were administered from days 0 to 8. For examining the role of ICOSL in the effector phase of GN, treatment lasted from days 10 to 18. Blockade of ICOSL during the induction of GN increased glomerular accumulation of CD4+ T cells and macrophages and augmented renal injury. These results correlated with attenuated splenocyte production of protective Th2 cytokines IL-4 and IL-10 and decreased apoptosis of splenic CD4+ T cells. ICOSL was upregulated within glomeruli of mice with GN. Inhibition of ICOSL during the effector phase of GN enhanced glomerular T cell and macrophage accumulation and augmented disease, without affecting the systemic immune response (cytokine production, T cell apoptosis/proliferation, Ab levels). Increased presence of leukocytes in glomeruli of mice that received anti-ICOSL mAb was associated with enhanced cellular proliferation and upregulation of P-selectin and intercellular adhesion molecule-1 within glomeruli. These studies demonstrate that ICOSL is protective during the induction of GN by augmenting Th2 responses and CD4+ T cell apoptosis. They also show that ICOSL is upregulated in nephritic glomeruli, where it locally reduces accumulation of T cells and macrophages and attenuates renal injury. Copyright © 2006 by the American Society of Nephrology.

Most humans with microscopic polyarteritis and anti-myeloperoxidase (anti-MPO), anti-neutrophil cytoplasmic antibodies (ANCA) develop "pauci-immune" crescentic glomerulonephritis. For dissection of the roles of ANCA and cell-mediated effectors in microscopic polyarteritis, experimental autoimmune anti-MPO glomerulonephritis was induced by immunizing C57BL/6 mice with human MPO. Autoimmunity to mouse MPO (ANCA and CD4+ cell reactivity) was induced. Challenge with anti-glomerular basement membrane globulin resulted in accumulation of neutrophils, CD4+ cells and macrophages, and significant numbers of crescentic glomeruli compared with similarly challenged control-immunized mice. MPO-deficient (Mpo-/-) mice immunized with MPO developed similar immune responses to MPO but failed to recruit effector cells to glomeruli or develop significant crescent formation, suggesting that MPO is acting as a planted glomerular autoantigen. Effector CD4+ cell depletion in this model attenuated crescentic glomerulonephritis and effector cell influx without altering ANCA titers. However, B cell-deficient mice, with no ANCA, still developed severe crescentic glomerulonephritis with accumulation of effector cells. Intravital microscopy studies demonstrated that passive transfer of sera from MPO-immunized Mpo -/- mice to LPS-primed mice rapidly induced glomerular neutrophil accumulation and release of MPO. These studies provide in vivo evidence in a relevant vascular bed for both humoral and cellular anti-MPO responses as key inducers of injury. ANCA induces glomerular neutrophil infiltration and MPO deposition. Subsequently, anti-MPO CD4+ cells recognize MPO as a planted glomerular antigen and act with macrophages to amplify severe glomerular injury. Copyright © 2006 by the American Society of Nephrology.

Systemic lupus erythematosus is a common autoimmune disease, with kidney involvement a serious complication associated with poor prognosis. Humoral immune responses constitute the hallmark of disease, however T helper cells are required for the generation of autoantibodies, as well as the induction and progression of renal injury. Administration of pristane to genetically intact mice results in the development of hypergammaglobulinaemia with the production of lupus like autoantibodies and proliferative glomerulonephritis, with similarities to human lupus nephritis. TLRs are intricately linked to the development of autoimmunity and are involved in the development of lupus nephritis. We injected wild type, TLR9-/- and TLR4-/- mice with pristane and assessed cellular and humoral autoimmunity and renal injury, 8 months later. TLR9-/- mice demonstrated a predominant decrease in Th1 cytokine production which resulted in decreased anti-RNP antibody levels, while anti-dsDNA levels remained intact. Compared to wild type mice treated with pristane, functional and histological renal injury and glomerular immunoglobulin and complement deposition was decreased in TLR9-/- mice. TLR4-/- mice demonstrated a global decrease in both Th1, IFNgamma, and Th17 associated IL-17A and IL-6 cytokine production. Autoantibody levels of anti-dsDNA and anti-RNP were both decreased. Renal injury was attenuated in TLR4-/- mice which demonstrated less glomerular immunoglobulin and complement deposition. These results demonstrate that both TLR9 and TLR4 are required for 'full-blown' autoimmunity and organ injury in experimental lupus induced by pristane. © 2010 Elsevier Ltd.

T-bet is a transcription factor that is essential for T helper (Th)1 lineage commitment and optimal IFN-gamma production by CD4+ T cells. We examined the role of T-bet in the development of experimental crescentic glomerulonephritis, which is induced by Th1-predominant, delayed-type hypersensitivity-like responses directed against a nephritogenic antigen. Anti-glomerular basement membrane (GBM) glomerulonephritis was induced in T-bet-/- and wild-type C57BL/6 mice. Compared with wild-type controls, renal injury was attenuated in T-bet-/- mice with glomerulonephritis, evidenced by less proteinuria, glomerular crescents, and tubulointerstitial inflammation. Accumulation of glomerular CD4+ T cells and macrophages was decreased, and was associated with reduced intrarenal expression of the potent Th1 chemoattractants CCL5/RANTES and CXCL9/Mig. Supporting the pro-inflammatory nature of T-bet signaling, assessment of systemic immunity confirmed that T-bet-/- mice had a reduction in Th1 immunity. The kinetic profile of T-bet mRNA in wild-type mice supported the hypothesis that T-bet deficiency attenuates renal injury in part by shifting the Th1/Th2 balance away from a Th1 phenotype. Expression of renal and splenic IL-17A, characteristically expressed by the Th17 subset of effector T cells, which have been implicated in the pathogenesis of autoimmune disease, was increased in T-bet-/- mice. We conclude that T-bet directs Th1 responses that induce renal injury in experimental crescentic glomerulonephritis. Copyright © 2008 by the American Society of Nephrology.

Objective: Autoantibodies constitute the hallmark of antineutrophil cytoplasmic antibody-associated vasculitis (AAV); however, CD4+ T cells play an essential role in the development of autoimmunity. Infection is associated with vasculitis, with Toll-like receptors (TLRs) a potential link between infection and autoimmunity. This study was undertaken to investigate the role of TLR ligation on cellular and humoral autoimmunity and glomerular injury in experimental myeloperoxidase (MPO)-induced AAV. Method(s): We analyzed autoimmune responses in wild-type mice immunized with MPO alone or coimmunized with MPO and a TLR-2 or TLR-9 ligand. The major vascular injury found in human disease, glomerulonephritis with focal necrosis, was triggered by administering a subnephritogenic dose of nephrotoxic serum. Result(s): MPO alone induced low-titer antineutrophil cytoplasmic antibodies (ANCAs) without delayedtype hypersensitivity or CD4 cytokine responses. However, when MPO was given with either TLR ligand, cellular and humoral autoimmunity was enhanced, but with distinctly different CD4 subsets and IgG ANCA isotypes. TLR-2 ligand induced Th17 autoimmunity, with retinoic acid receptor-related orphan nuclear receptor kappat-dependent interleukin-17A (IL-17A) production. TLR-9 ligand promoted Th1 autoimmunity, with enhanced production of interferon-kappa (IFNkappa) and Th1- associated IgG subclasses. Glomerular vasculitis developed only after the administration of nephrotoxic serum in mice immunized with either TLR ligand and MPO. Glomerulonephritis directed by MPO and TLR-2 ligation was attenuated when IL-17A was neutralized, while glomerulonephritis induced by MPO and TLR-9 ligation was attenuated when IFNkappa was neutralized. Conclusion(s): Our findings indicate a pathogenic role of TLRs in initiating autoimmune AAV. TLR-2 induces Th17 CD4 cells while TLR-9 can also direct vasculitis, by directing Th1 autoimmunity. © 2011, American College of Rheumatology.

Mast cells contribute to the modulation of the immune response, but their role in autoimmune renal disease is not well understood. Here, we induced autoimmunity resulting in focal necrotizing GN by immunizing wild-type or mast cell-deficient (KitW-sh/W-sh) mice with myeloperoxidase. Mast cell-deficient mice exhibited more antimyeloperoxidase CD4+ T cells, enhanced dermal delayed-type hypersensitivity responses to myeloperoxidase, and more severe focal necrotizing GN. Furthermore, the lymph nodes draining the sites of immunization had fewer Tregs and reduced production of IL-10 in mice lacking mast cells. Reconstituting these mice with mast cells significantly increased the numbers of Tregs in the lymph nodes and attenuated both autoimmunity and severity of disease. After immunization with myeloperoxidase, mast cellsmigrated fromthe skin to the lymph nodes to contact Tregs. In an ex vivo assay, mast cells enhanced Treg suppression through IL-10. Reconstitution of mast cell-deficient mice with IL-10-deficient mast cells led to enhanced autoimmunity to myeloperoxidase and greater disease severity compared with reconstitution with IL-10-intact mast cells. Taken together, these studies establish a role for mast cells in mediating peripheral tolerance to myeloperoxidase, protecting them from the development of focal necrotizing GN in ANCA-associated vasculitis. Copyright © 2012 by the American Society of Nephrology.

Aim: To demonstrate a role for mast cells (MC) in immunomodulating autoimmune anti-Myeloperoxidase (MPO) glomerulonephritis (AIMPOGN). Background(s): Upon degranulation, MCs release cytokines involved in effector and regulatory immune responses. Preliminary studies suggest that MCs interact with regulatory T cells (Tregs) modulating autoimmunity. Method(s): We compared autoimmunity and GN between WT, MC deficient KitWsh/Wsh and KitWsh/Wsh mice reconstituted with WT or IL-10-/- MCs. AIMPOGN was induced by immunizing mice with MPO and GN triggered using low dose anti-glomerular basement membrane antibody. Four days later, renal injury and systemic immune responses were assessed. Result(s): Compared with WT mice, KitWsh/Wsh mice developed enhanced autoimmunity (MPO induced lymph node [LN] CD4 IL-17A recall responses (1403 +/- 420.6 vs 520.7 +/- 92.2 pg/ml P < 0.05) and reduced immunoregulatory IL-10 (15.5 +/- 1.2 vs 49.3 +/- 11.9 pg/ml P < 0.05). GN was more severe in KitWsh/Wsh mice with increased proteinuria (7.4 +/- 1.9 vs 2.5 +/- 0.6 mg/24 hr P < 0.05), glomerular CD4 cells (0.6 +/- 0.1 vs 0.2 +/- 0.1 cell/glomerular cross section [c/gcs] P < 0.05), macrophages influx (1.7 +/- 0.2 vs 0.8 +/- 0.2 c/gcs P < 0.05) and histological injury. The percentage of Tregs in LNs draining immunization sites was reduced (17.1 +/- 0.3 vs 15.9 +/- 0.4 P < 0.05). MC reconstituted KitWsh/Wsh mice showed attenuation of increased autoimmunity and GN seen in KitWsh/Wsh mice. However reconstitution with IL-10-/- MCs did not significantly attenuate autoimmunity (IL-17A: 5.5 +/- 1.1 vs 1.9 +/- 0.7 ng/ml P < 0.05) and GN severity (proteinuria: 11.5 +/- 1.8 vs 6.8 +/- 0.4 mg/24 hr P < 0.05). Ex vivo culture of isolated anti-MPO CD4 effectors with combinations of MCs, IL-10-/- MCs and Foxp3+ Tregs demonstrated that MCs modulate Teffector function by IL-10 production to enhanced Treg functional capacities. Conclusion(s): In AIMPOGN MCs play an important role in limiting anti-MPO autoimmunity a

Objective: We have defined an immunodominant and nephritogenic T cell epitope from MPO [20 amino acids, known as peptide 52 (pep52)]. This work examines whether aberrant presentation of MPO could attenuate autoimmunity and anti-MPO GN (induced by immunizing mice, then inducing glomerular neutrophil influx and MPO deposition with low-dose anti-GBM globulin). Method(s): Aberrant presentation of MPO was performed by intranasal insufflation of pep52 or immunization with MPO-pulsed BM-derived CD40-/- DCs. Result(s): Nasal insufflation of pep52 over 3 consecutive days protected mice from the development of autoimmunity when immunized subsequently with 200 mug pep52, compared with control mice given nasal insufflation with an irrelevant peptide. The frequency of CD4+ cells responding to MPO producing IFN-gamma was 457 +/- 107 (per 106, control mice) versus 289 +/- 90 (tolerized mice) and IL-17A-producing cells was 231 +/- 154 versus 49 +/- 32; P < 0.001. Anti-MPO IgG was also reduced. When pep52 immunized mice were challenged with anti-GBM globulin, GN was reduced in tolerized mice with reduced glomerular segmental necrosis, glomerular neutrophils, macrophages, CD4+ cells and proteinuria. Immunization of C57BL/6 mice with MPO-pulsed CD40+/+ DCs induced strong anti-MPO autoimmunity, which induced GN when triggered with anti-GBM globulin. However, immunization with CD40-/- DCs protected from the development of autoimmunity and GN. Draining lymph node cells isolated from CD40-/- DC-immunized mice showed higher levels of IL-10, suggesting the induction of Tr1 Tregs. Conclusion(s): Aberrant presentation of immunodominant peptide or MPO autoantigen tolerizes and protects from autoimmune anti-MPO GN.

Experimental crescentic glomerulonephritis (GN) is characterized by T helper 1 (Th1) directed nephritogenic immune responses and cell-mediated glomerular injury. IL-12p40, the common cytokine chain for both IL-12 and IL-23, is important in the generation and potentially the maintenance of Th1 responses, whereas IL-18 is a co-factor for Th1 responses that may have systemic and local proinflammatory effects. For testing the hypothesis that both endogenous IL-12p40 and endogenous IL-18 play pathogenetic roles in crescentic GN, accelerated anti-glomerular basement membrane GN was induced in mice genetically deficient in IL-12p40 (IL-12p40-/-), IL-18 (IL-18 -/-), or both IL-12p40 and IL-18 (IL-12p4-/-IL-18 -/-). Compared with wild-type C57BL/6 mice, IL-12p40-/- mice failed to make a nephritogenic Th1 response and developed markedly reduced crescent formation and renal leukocytic infiltration, despite renal production of chemoattractants and adhesion molecules. IL-18-/- mice developed an intact antigen-specific systemic Th1 response, a similar degree of crescent formation, but fewer glomeruli affected by other severe histologic changes and fewer leukocytes in glomeruli and interstitium. IL-18 was expressed within diseased kidneys. Local production of TNF, IL-1beta, IFN-gamma, CCL3 (MIP-1alpha), and CCL4 (MIP-1beta) was reduced in IL-18-/- mice, demonstrating a local proinflammatory role for IL-18. Combined deletion of IL-12p40 and IL-18 did not result in synergistic effects. Consistent with the hypothesis that inflammation leads to fibrosis, all three groups of deficient mice expressed lower levels of intrarenal TGF-beta1 and/or alpha1(I) procollagen mRNA. These studies demonstrate that in severe experimental crescentic GN, IL-12p40 is the key Th1-defining cytokine chain, whereas IL-18 has local proinflammatory roles. Copyright © 2005 by the American Society of Nephrology.

The participation of renal expression of CD80 and CD86 in the immunopathogenesis of crescentic Th1-mediated antiglomerular basement membrane (anti-GBM) glomerulonephritis (GN) has not been assessed. Immunohistochemical staining demonstrated prominent upregulation of both molecules in glomeruli of mice with anti-GBM GN, suggesting a potential role for the local expression of CD80 and CD86 in nephritogenic effector T cell responses. For testing this hypothesis, control or inhibitory anti-CD80 and/or anti-CD86 mAb were administered to mice during the effector phase of the disease but after the establishment of a systemic immune response. Anti-CD80 or anti-CD86 mAb treatment had no effect on the development of GN or infiltration of leukocytes into glomeruli; however, administration of anti-CD80/86 mAb attenuated glomerular accumulation of CD4+ T cells and macrophages, crescent formation, and proteinuria, correlating with reduced antigen-specific skin delayed-type hypersensitivity. Attenuated glomerular infiltration of leukocytes in mice that were treated with anti-CD80/86 mAb was associated with decreased intraglomerular expression of adhesion molecules P-selectin and intercellular adhesion molecule-1, as well as attenuated renal mRNA levels of proinflammatory cytokines IFN-gamma and migration inhibitory factor, without reducing chemokine and chemokine receptor expression in the kidney or intraglomerular apoptosis and proliferation. The systemic Th1/Th2 balance (assessed by splenocyte production of IFN-gamma and IL-4 and circulating levels of IgG1 and IgG2a) was not affected by the inhibition of CD80 and CD86. These studies show that CD80 and CD86 are expressed in glomeruli of mice with crescentic anti-GBM GN, in which they play a critical role in facilitating accumulation of Th1 effectors and macrophages, thus exacerbating renal injury. Copyright © 2005 by the American Society of Nephrology.

BACKGROUND: CD80 and CD86 costimulatory molecules have been shown to affect the induction of Th1-mediated crescentic antiglomerular basement membrane (GBM) antibody-initiated glomerulonephritis (GN). The aim of the current studies was to define the mechanisms by which CD80 and CD86 regulate the development of this disease. METHOD(S): Anti-GBM GN was induced in CD80-/-, CD86-/-, and CD80/86-/- mice, as well as in C57BL/6 controls. Renal injury and immune responses were assessed after 21 days. To examine whether costimulation by OX40-ligand compensates for the absence of CD80 and CD86 in inducing GN, OX40-ligand was blocked in wild-type and CD80/86-/- mice. RESULT(S): Crescentic GN and glomerular accumulation of CD4+ T cells and macrophages were attenuated in CD80-/- mice, correlating with significantly enhanced apoptosis and decreased proliferation of spleen CD4+ T cells. GN was exacerbated in CD86-/- mice, which was associated with attenuated IL-4 and enhanced IFN-gamma levels. In contrast, CD80/86-/- mice developed crescentic GN similar to that in controls. Inhibition of OX40-ligand exacerbated GN in wild-type mice by enhancing IFN-gamma production, and attenuated disease in CD80/86-/- mice by reducing glomerular CD4+ T-cell and macrophage accumulation. CONCLUSION(S): CD80 is pathogenic in crescentic GN by enhancing survival and proliferation of CD4+ T cells, whereas CD86 is protective by enhancing Th2 and attenuating Th1 responses. Furthermore, in the presence of CD80 and CD86, OX40-ligand attenuates, whereas in their absence it enhances GN, suggesting that, in the absence of CD80 and CD86, the OX40/OX40-ligand pathway is an alternative costimulatory pathway in inducing crescentic GN.