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Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.Copyright © 2019, The Author(s).

Purpose: The aim of this study was to determine if measurement of B cell protective immunity was associated with susceptibility to sinopulmonary infection in kidney transplant recipients. Methods and Materials: A prospective cohort of 168 patients with stable graft function (median 4.1 years) underwent assessment of B-lymphocyte antigen CD19 (CD19+) cell number, immunoglobulin G concentration, and seroresponses to influenza vaccination upon study entry. Patients received a single dose of a trivalent, seasonal influenza vaccine. Result(s): After 2 years follow-up, 31 patients (18%) developed sinopulmonary infection. CD19+ cell number was strongly associated with future sinopulmonary infection. A higher proportion of patients with CD19+ cell counts below the fifth percentile for controls developed sinopulmonary infections than those above the fifth percentile, 30% (23 of 77 patients) compared with 9% (7 of 79 patients; P =.001). There was a trend toward a higher proportion of patients with reduced immunoglobulin G concentrations developing infections than in the normal range for controls, 29% (14 of 48 patients) compared with 15% (16 of 108 patients; P =.060). Influenza vaccination seroresponses were poor in patients and controls such that they could not be used to identify a subgroup of patients at high risk for the development of severe pulmonary infection. Conclusion(s): Monitoring B-cell numbers represents a simple, inexpensive means of stratifying transplant recipients' risk of sinopulmonary infection.Copyright © 2018 Elsevier Inc.

Aim: To use Goodpasture's disease (GPD), a classical form of HLA-linked autoimmunity, to define the mechanism of HLA-mediated risk, and dominant protection from risk of disease. Background(s): The mechanisms underpinning HLA-mediated susceptibility to, and protection from autoimmune diseases are unknown. GPD is HLAlinked and characterized by an immunodominant CD4+ self-epitope derived from the alpha3 chain of Type IV collagen (alpha3135-145). Method(s): HLA-DR transgenic mice, cells from HLA-typed healthy humans and from patients with GPD were used, with HLA-DR-alpha3135-145 tetramers, Xray crystallography, and in vivo/in vitro models of autoimmunity. Result(s): Autoreactive alpha3135-145-specific T cells clonally expanded in GPD patients. In alpha3135-145-immunized HLA-DR15 transgenic mice with GPD, alpha3135-145-specific T cells infiltrated the kidney. Structurally, HLA-DR15 and HLA-DR1 presented alpha3135-145 in different binding registers, resulting in differential T cell receptor (TCR) usage. HLA-DR15-alpha3135-145 tetramer+ CD4+ T cells in disease susceptible HLA-DR15 transgenic mice exhibited a conventional phenotype (Tconv), secreting pro-inflammatory cytokines. In contrast, HLA-DR1-alpha3135-145 tetramer+ cells in disease resistant HLA-DR1 and HLADR15/ DR1 transgenic mice were largely CD4+Foxp3+ regulatory T cells (Tregs) expressing tolerogenic cytokines. In HLA-DR15/DR1 transgenic mice, HLA-DR1-alpha3135-145 specific Tregs conferred protection to alpha3135-145-specific autoimmunity and protected from GPD (alpha3135-145-immunized, Treg intact vs Treg depleted: segmental necrosis 0+/-0 vs 50+/-13% P<0.01, serum urea 9+/-1 vs 31+/-10 P<0.001). HLA-DR15+ and HLA-DR1+ healthy human donors displayed altered alpha3135-145-specific TCRs, with HLA-DR15-alpha3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-alpha3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes (alpha3135-145 tetramer+ Treg:Tconv ratios: DR15 0.04+/-0.00, DR1 9.61+/-1.79, P<0.001). Conclusio

Objectives: Anti-glomerular basement membrane (GBM) disease is a small vessel vasculitis caused by anti-a3(IV)NC1 T and B cell autoreactivity. An immunodominant T cell epitope, a3135-145, has been defined. HLA-DR15 confers an increased disease risk (OR 8.5) while HLA-DR1 is dominantly protective from this risk. These studies aimed to define the mechanisms by which different HLA-DR alleles lead to susceptibility or protection from disease. Method(s): a3135-145-specific CD4+ T cells were enumerated in anti-GBM patients, HLA-typed healthy humans and HLA-DR transgenic mice using HLA-DR-a3135-145 tetramers. The crystal structures of HLADR15-a3135-145 and HLA-DR1-a3135-145 were solved. a3135-145-specific T cell receptor (TCR) usage was determined by single cell sequencing. T cell phenotype was determined by flow cytometry. T cell responses were determined in vivo, ex vivo and by culturing naive T cells with a3135-145 in vitro. Experimental autoimmune anti-GBM disease was induced by a3135-145 immunization of HLA-DR transgenic mice (Treg intact or Treg depleted). Result(s): a3135-145-specific CD4+ T cells were expanded in anti-GBM patients and infiltrated kidneys of a3135-145-immunized HLA-DR15 transgenic mice. Mice transgenic for both HLA-DR15 and HLA-DR1 showed similar reactivity to most a3 peptides, but lacked proinflammatory responses to a3135-145. Structurally, HLA-DR15 and HLA-DR1 presented a3135-145 in different binding registers. Compared to the conformation of a3135-145 in HLA-DR15, in HLA-DR1 a3135-145 adopted a more pronounced conformation with a flipped backbone. These different HLA-peptide conformations altered a3135-145-specific TCR usage and T cell phenotypes. HLA-DR15-a3135-145-specific T cells exhibited a Foxp3-conventional T cell phenotype resulting in proinflammatory T cell autoreactivity, anti-GBM antibody production and disease. In contrast, HLA-DR1-a3135-145-specific T cells were predominantly anti-inflammatory Foxp3+ Tregs that proliferated and respon

Aim: To test the hypothesis that biological neutralization of the dominant T helper (TH) cell subset inducing cytokine attenuates anti-MPO GN. Background(s): Both CD4+ TH17 and TH1 cells, have been demonstrated to be pathogenic in anti-MPO GN. Definition of the dominant TH subset would allow targeted biological treatment. Method(s): Anti-MPO autoimmunity was induced by MPO immunization and GN triggered using anti-GBM Ig. TH subsets in anti-MPO GN was defined by their cytokine profile in draining lymph nodes (dLN) and glomerular effector responses. TH subsets was assessed early after induction of anti-MPO GN on day 20 (d20) and when disease was fully established (d32). Result(s): In WT mice, anti-MPO GN progressively intensifies from d20 to d32 with increasing frequency of segmental necrosis (10+/-1vs17+/-2%, P<0.01) and albuminuria (1.22+/-0.2vs2.0+/-0.2mg/24hr, P<0.05). Analysis of TH cytokines in dLN showed key TH17 cytokines, IL-17A (2.8+/-0.1vs1.6+/-0.3ng/ml, P<0.05) and IL-6 were maximal at d20 and subsides by d32 while TH1 dominant cytokines, IFNgamma (19.9+/-8.1vs52.9+/-12.3pg/ml, P<0.05) and TNF-alpha were maximum at d32. Gene analysis of isolated kidney CD4+ T cells demonstrated that expression of TH17 genes (IL-17A, CCR6, TGF-beta) are more upregulated early while upregulation of TH1 genes (IFNgamma, CXCR3, STAT4) increases by d32. This suggests TH17 dominates GN early and TH1 late. Treatment with monoclonal antibodies (mAb) to the TH17 inducing cytokine, IL-23p19, attenuated GN (albuminuria; 105.3+/-28.2vsIgG2b controls; 325.4+/-60.5 mug/24hr, P<0.01) on d20 but not on d32. Whereas mAb to TH1 inducing cytokine, IL-12p35, was protective at d32 (albuminuria, 788.2+/-323.3vs IgG2a controls; 1797+/-229.9 mug/24hr, P<0.05). Conclusion(s): In anti-MPO GN, TH subset dominance is biphasic, TH17 early then TH1 late. Anti-cytokine biological treatment is effective only when it targets the dominant TH cytokines inducing anti-MPO GN.

Susceptibility and protection against human autoimmune diseases, including type I diabetes, multiple sclerosis, and Goodpasture disease, is associated with particular human leukocyte antigen (HLA) alleles. However, the mechanisms underpinning such HLA-mediated effects on self-tolerance remain unclear. Here we investigate the molecular mechanism of Goodpasture disease, an HLA-linked autoimmune renal disorder characterized by an immunodominant CD4+ T-cell selfepitope derived from the a3 chain of type IV collagen (a3135-145)1-4. While HLA-DR15 confers a markedly increased disease risk, the protective HLA-DR1 allele is dominantly protective in trans with HLA-DR15 (ref. 2). We show that autoreactive a3135-145-specific T cells expand in patients with Goodpasture disease and, in a3135-145- immunized HLA-DR15 transgenic mice, a3135-145-specific T cells infiltrate the kidney and mice develop Goodpasture disease. HLA-DR15 and HLA-DR1 exhibit distinct peptide repertoires and binding preferences and present the a3135-145 epitope in different binding registers. HLA-DR15-a3135-145 tetramer+ T cells in HLA-DR15 transgenic mice exhibit a conventional T-cell phenotype (Tconv) that secretes pro-inflammatory cytokines. In contrast, HLADR1- a3135-145 tetramer+ T cells in HLA-DR1 and HLA-DR15/DR1 transgenic mice are predominantly CD4+Foxp3+ regulatory T cells (Treg cells) expressing tolerogenic cytokines. HLA-DR1-induced Treg cells confer resistance to disease in HLA-DR15/DR1 transgenic mice. HLA-DR15+ and HLA-DR1+ healthy human donors display altered a3135-145-specific T-cell antigen receptor usage, HLADR15- a3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-a3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes. Moreover, patients with Goodpasture disease display a clonally expanded a3135-145-specific CD4+ T-cell repertoire. Accordingly, we provide a mechanistic basis for the dominantly protective effect of HLA in autoimmune disease, whereby HLA polymorphism shapes the relative ab

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.

Aim: To determine the therapeutic efficacy and mechanisms of action of anti- CD20 B cell monoclonal antibodies (mAb) in the treatment of murine autoimmune MPO-ANCA glomerulonephritis (anti-MPO GN). Background(s): Anti-CD20 treatment (with rituximab) is effective treatment for human anti-MPO GN. Its efficacy is attributed to B cell depletion. However, B cells also potentially play roles in immunoregulation and the initiation and maintenance of CD4 T cell autoimmunity. Method(s): Experimental anti-MPO autoimmunity was induced by MPO immunisation and GN triggered using a subnephritogenic dose of anti-GBM globulin. B cell depletion using mouse anti-CD20 mAb (or control mouse anti-IgG2a mAb) commenced after the induction of anti-MPO autoimmunity (day 14) and continued through the development of GN. Result(s): Administration of anti-CD20 mAb protected mice from the development of renal injury, compared to controls (segmental glomerular necrosis: 16 +/- 4 vs 44 +/- 4%, P < 0.01 and albuminuria: 221.0 +/- 81.6 vs 898.8 +/- 330.3 mug/24 hrs, P < 0.05). Attenuation of GN was associated with reduced systemic anti-MPO autoimmunity; serum anti-MPO IgG (ANCA) titres (0.21 +/- 0.02 vs 0.34 +/- 0.07 OD450nm, P < 0.05) and dermal MPO induced DTH swelling (0.07 +/- 0.02 vs 0.2 +/- 0.03DELTAmm, P < 0.01). Anti-CD20 mAb treatment decreased MPO specific recall proliferation in draining lymph node cells (104.5 +/- 12.7 vs 217.7 +/- 55.3 counts per minute, P < 0.05) with reduced frequency of IFN-gamma and IL-17A producing cells (ELISPOT; 22 +/- 6 vs 115 +/- 27 cells, P < 0.01 and 6 +/- 3 vs 40 +/- 18 cells, P < 0.05, respectively). Furthermore anti-CD20 treatment increased the proportion of proliferating Foxp3+ T regulatory cells (2.9 +/- 0.1 vs 1.7 +/- 0.1%, P < 0.001; 15% of proliferating Foxp3+ T cells in both groups produced IL-10). Conclusion(s): Anti-CD20 B cell directed immunotherapy suppresses anti-MPO autoimmunity and the development of GN by reducing the development of T and B c

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

Aim: To determine whether administration of antigen-coupled ethylenecarbodiimide (ECDI)-fixed splenocytes (Ag-Sp) can suppress autoimmune anti-myeloperoxidase (MPO) glomerulonephritis. Background(s): In some autoimmune conditions, treatment with apoptotic, ECDIinduced Ag-SP can induce peripheral T cell tolerance by facilitating host APCs to reprocess and present the Ag in a non-immunogenic manner. Recent discovery of immunodominant MPO peptides makes this approach relevant to anti-MPO glomerulonephritis. Method(s): We compared disease between C57BL/6 (WT) mice that received either no cells, MPO409-428-Sp or OVA323-339-Sp. A standard model of anti-MPO glomerulonephritis was used. Anti-MPO autoimmunity was induced by MPO409-428 immunisation and glomerulonephritis triggered using low dose antiglomerular basement membrane globulin. Result(s): Compared to mice with disease not receiving Ag-SP, glomerulonephritis was less severe in mice treated with MPO409-428-Sp prior to the induction of autoimmunity (glomerular segmental necrosis 23 +/- 3 vs 8 +/- 1% P < 0.05) and decreased glomerular leukocytes (macrophages; 1.7 +/- 0.03 vs 1.1 +/- 0.01 cells per glomerular cross section [c/gcs], P < 0.05; CD4+ cells 1.1 +/- 0.06 vs 0.54 +/- 0.06c/ gcs, P < 0.05). Systemic anti-MPO autoimmunity, assessed by MPO-induced dermal delayed type hypersensitivity (DTH) was also reduced in MPO409-428-Sp treated mice (0.14 +/- 0.04 vs 0.26 +/- 0.03DELTAmm, P < 0.05). To determine if the disease suppression by Ag-Sp is antigen specific, mice were either treated with OVA323-339-Sp or MPO409-428-Sp. Compared with administering MPO409-428-Sp, glomerular injury was more severe when mice were injected with OVA323-339-Sp before MPO immunization (segmental necrosis 43 +/- 5 vs 19 +/- 1%, P < 0.05). Administering MPO409-428-Sp to WT mice with established anti-MPO autoimmunity resulted in less glomerular disease (segmental necrosis 44 +/- 4 vs 31 +/- 4%, P < 0.05) and limited systemic anti-MPO responses (

Aim: To define the presence of intrarenal degranulated mast cells (MCs) in humans with MPO-ANCA glomerulonephritis and determine whether a MC stabiliser, disodium chromoglycate (DSCG) limits experimental murine anti- MPO glomerulonephritis. Background(s): MCs are tuneable cells that potentially have pro- or antiinflammatory roles. However, MC degranulation is a proinflammatory event. Method(s): Human renal biopsies MPO-ANCA glomerulonephritis (n = 24), controls (thin basement membrane or minimal change disease n = 10) were examined for the presence of MCs and their degranulation. Experimental anti-MPO glomerulonephritis was induced using a standard protocol, by MPO immunisation and glomerulonephritis triggered using a subnephritogenic dose of anti-glomerular basement membrane globulin. Daily administration of DSCG (or saline control) to prevent MC degranulation commenced either 2 days prior to MPO immunisation or 10 days after establishment of MPO autoimmunity. Result(s): MPO-ANCA glomerulonephritis patients had significant interstitial MC infiltration (MPO-ANCA: 0.8 +/- 0.1 vs ctrl: 0.2 +/- 0.1 cells per high power field, P = 0.16) and increased MC numbers correlated with low presenting eGFR (r = -0.49, P = 0.15). A high proportion of MCs were degranulated (MPOANCA: 58 +/- 8 vs ctrl: 1 +/- 1%, P = 0.001) and were associated with severe tubulointerstitial injury (TI) (mild TI injury score <30%: 0.2+/-.06 vs severe TI injury score >30%TI; 0.9 +/- 0.2, P = 0.17). In experimental anti-MPO glomerulonephritis, DSCG treatment prior to initial MPO immunisation resulted in diminished anti-MPO autoimmunity (IFN-gamma; Ctrl: 13.5 +/- 3.7 vs DSCG: 3.6 +/- 1.3 ng/mL, P < 0.05), enhanced IL-10 (Ctrl: 124 +/- 10 vs DSCG: 198 +/- 16pg/mL, P = 0.01) and attenuated glomerulonephritis (segmental glomerular necrosis; Ctrl: 62 +/- 6 vs DSCG: 34 +/- 4%, P = 0.01). When treatment commenced in established MPO autoimmunity, DSCG limited the development of glomerulonephritis (segmental glom

Aim: To define the role of Toll like receptor (TLR)9 in anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Background(s): Neutrophils are early effector cells in ANCA vasculitis. Clinical studies suggest infections promote vasculitis, with TLRs a potential link between infection and glomerulonephritis. Method(s): We used myeloperoxidase (MPO) deficient mice to generate MPOANCA. TLR9 was assessed in human and murine MPO-ANCA glomerulonephritis by immunofluorescence. C57BL/6 wild type (WT) mice were injected with: 1) TLR9 ligand (CpG-ODN) alone, 2) MPO-ANCA alone or 3) CpGODN+ MPO-ANCA. Neutrophil recruitment was assessed after five hours. TLR9-/- mice were injected with CpG-ODN+MPO-ANCA. WT and TLR9-/- mice were irradiated and reconstituted with WT or TLR9-/- bone marrow cells (BM) to generate bone marrow chimeras. WT BM-> WT mice (BM + Tissue Cell [TC]+), TLR9-/-BM->WT mice (BM-TC+) and WT BM->TLR9-/- mice (BM+TC-) all received CpG-ODN+MPO-ANCA. Result(s): TLR9 immunostaining is increased in murine and human kidney biopsies with active MPO-ANCA glomerular injury TLR9 ligation enhances MPOANCA induced glomerular neutrophil recruitment. Compared to WT mice receiving CpG-ODN or MPO-ANCA alone, neutrophil recruitment synergistically increased after CpG-ODN+MPO-ANCA (CpG-ODN alone 0.3 +/- 0.1, MPO-ANCA alone 0.9 +/- 0.1, CpG-ODN+MPO-ANCA 1.5 +/- 0.1 neutrophils/ glomerular cross section (n/gcs), P < 0.01). Enhanced neutrophil recruitment corresponded with increased kidney mRNA CXCL1, CXCL2 and TNF expression. Control oligonucleotides (GpC-DNA) did not affect recruitment. In TLR9-/- mice receiving CpG-ODN+MPO-ANCA, neutrophil recruitment (WT 1.4 +/- 0.1 vs. TLR9-/- 0.6 +/- 0.1 n/gcs, P < 0.001) and kidney mRNA expression of CXCL1, CXCL2 and TNF decreased. In chimeric mice, neutrophil recruitment was similarly decreased in BM-TC+ and BM+TC- mice (BM+TC+ 1.7 +/- 0.2, BM-TC+ 0.8 +/- 0.1, BM+TC- 1.0 +/- 0.1 n/gcs, P < 0.001). In additional experiments functional and hist

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 determine whether nasal insufflation of MPO409-428 can prevent or treat autoimmune anti-MPO ANCA associated glomerulonephritis. Background(s): We have defined the immunodominant MPO T cell epitope (MPO409-428). Nasal insufflation of dominant auto-antigens can induce T regulatory cell (Treg) mediated protective tolerance. Method(s): We compared autoimmunity and glomerulonephritis in C57BL/6 (WT) mice nasally insufflated with either MPO409-428 or an irrelevant peptide, OVA323-339. Autoimmunity was induced by MPO immunisation and glomerulonephritis triggered using a subnephritogenic dose of anti-GBM globulin. Result(s): Compared to OVA323-339, nasal administration of MPO409-428 reduced the development of anti-MPO autoimmunity and triggered glomerulonephritis. Renal injury [segmental glomerular necrosis; 23 +/- 13 vs 4 +/- 1%, P = 0.01], serum anti-MPO IgG [0.47 +/- 0.04 vs 0.08 +/- 0.01 OD450nm, P = 0.001] and anti-MPO T cell responses to MPO [IFN-gamma ELISPOT; 222.30 +/- 18.67 vs 143.7 cells, P = 0.05 and IL-17A ELISPOT; 124 +/- 48 vs 14 +/- 0.4 cells, P = 0.05]. Nasal administration of MPO409-428 to WT mice with established MPO autoimmunity attenuated glomerulonephritis [segmental glomerular necrosis; 22 +/- 3vs 9 +/- 2%, P = 0.01] and reduced glomerular T cell and macrophage accumulation. To determine the mechanism of these effects, we assessed Treg function. Anti-MPO CD4+Foxp3- T cell effectors were co-cultured with CD4+Foxp3+ Tregs. Tregs from MPO nasally insufflated mice induced significantly greater T effector cell suppression [[3H]-Thymidine proliferation; Teff alone:834 +/- 136 vs Teff and Tregs: 117 +/- 22 cpm, P = 0.01]. Functionally, adoptive transfer of CD4+ T cells from mice nasally insufflated with MPO409-428 to mice with established anti-MPO autoimmunity significantly limited the development of triggered glomerulonephritis (compared to mice that did not receive CD4 T cells) [segmental glomerular necrosis; 23 +/- 2 vs 9 +/- 1%, P = 0.01]. Conclu

Loss of tolerance to neutrophil myeloperoxidase (MPO) underlies the development of ANCA-associated vasculitis and GN, but the mechanisms underlying this loss of tolerance are poorly understood. Here, we assessed the role of the thymus in deletion of autoreactive anti-MPO T cells and the importance of peripheral regulatory T cells in maintaining tolerance toMPO and protecting fromGN. Thymic expression of MPO mRNA predominantly localized to medullary thymic epithelial cells. To assess the role of MPO in forming the T cell repertoire and the role of the autoimmune regulator Aire in thymicMPO expression, we compared the effects of immunizing Mpo-/- mice, Aire-/- mice, and control littermates with MPO. Immunized Mpo -/- and Aire-/- mice developed significantly more proinflammatory cytokine-producing anti-MPO T cells and higher ANCA titers than control mice. When we triggered GN with a subnephritogenic dose of anti-glomerular basement membrane antibody, Aire-/- mice hadmore severe renal disease than Aire+/+ mice, consistent with a role for Aire-dependent central deletion in establishing tolerance to MPO. Furthermore, depleting peripheral regulatory T cells in wild-type mice also led to more anti-MPO T cells, higher ANCA titers, and more severe GN after immunization with MPO. Taken together, these results suggest that Aire-dependent central deletion and regulatory T cell-mediated peripheral tolerance both play major roles in establishing and maintaining tolerance to MPO, thereby protecting against the development of anti-MPO GN. Copyright © 2013 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.

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