CD103 Regulates Dermal Regulatory T Cell Motility and Interactions with CD11c-Expressing Leukocytes to Control Skin Inflammation.

Dermal regulatory T cells (Tregs) are essential for maintenance of skin homeostasis and control of skin inflammatory responses. In mice, Tregs in the skin are characterized by high expression of CD103, the αE integrin. Evidence indicates that CD103 promotes Treg retention within the skin, although the mechanism underlying this effect is unknown. The main ligand of CD103, E-cadherin, is predominantly expressed by cells in the epidermis. However, because Tregs are predominantly located within the dermis, the nature of the interactions between E-cadherin and CD103-expressing Tregs is unclear. In this study, we used multiphoton intravital microscopy to examine the contribution of CD103 to Treg behavior in resting and inflamed skin of mice undergoing oxazolone-induced contact hypersensitivity. Inhibition of CD103 in uninflamed skin did not alter Treg behavior, whereas 48 h after inducing contact hypersensitivity by oxazolone challenge, CD103 inhibition increased Treg migration. This coincided with E-cadherin upregulation on infiltrating myeloid leukocytes in the dermis. Using CD11c-enhanced yellow fluorescent protein (EYFP) × Foxp3-GFP dual-reporter mice, inhibition of CD103 was found to reduce Treg interactions with dermal dendritic cells. CD103 inhibition also resulted in increased recruitment of effector CD4+ T cells and IFN-γ expression in challenged skin and resulted in reduced glucocorticoid-induced TNFR-related protein expression on Tregs. These results demonstrate that CD103 controls intradermal Treg migration, but only at later stages in the inflammatory response, when E-cadherin expression in the dermis is increased, and provide evidence that CD103-mediated interactions between Tregs and dermal dendritic cells support regulation of skin inflammation.

Reimagining possibilities: the journey of a PhD parent.

In this commentary, I discuss my non-traditional pathway to a PhD, a late-bloomer's account of balancing a young family and PhD studies, a guide for those contemplating the same path.

Unravelling the Link between the Gut Microbiome and Autoimmune Kidney Diseases: A Potential New Therapeutic Approach.

The gut microbiota and short chain fatty acids (SCFA) have been associated with immune regulation and autoimmune diseases. Autoimmune kidney diseases arise from a loss of tolerance to antigens, often with unclear triggers. In this review, we explore the role of the gut microbiome and how disease, diet, and therapy can alter the gut microbiota consortium. Perturbations in the gut microbiota may systemically induce the translocation of microbiota-derived inflammatory molecules such as liposaccharide (LPS) and other toxins by penetrating the gut epithelial barrier. Once in the blood stream, these pro-inflammatory mediators activate immune cells, which release pro-inflammatory molecules, many of which are antigens in autoimmune diseases. The ratio of gut bacteria Bacteroidetes/Firmicutes is associated with worse outcomes in multiple autoimmune kidney diseases including lupus nephritis, MPO-ANCA vasculitis, and Goodpasture's syndrome. Therapies that enhance SCFA-producing bacteria in the gut have powerful therapeutic potential. Dietary fiber is fermented by gut bacteria which in turn release SCFAs that protect the gut barrier, as well as modulating immune responses towards a tolerogenic anti-inflammatory state. Herein, we describe where the current field of research is and the strategies to harness the gut microbiome as potential therapy.

Toll-like Receptor 9 Induced Dendritic Cell Activation Promotes Anti-Myeloperoxidase Autoimmunity and Glomerulonephritis.

ANCA-associated vasculitis (AAV) is intricately linked with infections. Toll-like receptors (TLR) provide a potential link between infection and anti-myeloperoxidase (MPO) autoimmunity. TLR9 ligation has been shown to promote anti-MPO autoimmunity and glomerular vasculitis in murine MPO-AAV. This study investigates dendritic cell TLR9 ligation in murine experimental anti-MPO glomerulonephritis. We analyzed autoimmune responses to MPO following transfer of TLR9 stimulated, MPO pulsed dendritic cells and kidney injury following a sub-nephritogenic dose of sheep anti-mouse glomerular basement membrane globulin. TLR9 ligation enhanced dendritic cell activation upregulating CD40 and CD80 expression, promoting systemic anti-MPO autoimmunity and T cell recall responses and exacerbating kidney injury. CD40 upregulation by TLR9 was critical for the induction of nephritogenic autoimmunity. The presence of DEC205, which transports the TLR9 ligand to TLR9 located in the endosome, also promoted kidney injury. This confirms TLR9 mediated dendritic cell activation as a mechanism of anti-MPO autoimmunity in AAV and further defines the link between infection and the generation of MPO specific autoimmune inflammation.

Equity and diversity in the nephrology workforce in Australia and New Zealand.

BACKGROUND: Despite diversity initiatives, inequities persist in medicine with negative implications for the workforce and patients. Little is known about workplace inequity in nephrology. AIM: To describe perceptions and experiences of bias by health professionals in the Australian and New Zealand Society of Nephrology (ANZSN), focussing on gender and race. METHODS: A web-based survey of ANZSN members recorded degree of perceived inequity on a Likert scale, ranging from 1 (none) to 5 (complete). Groups were compared using Mann-Whitney U-test and logistic regression. Comments were synthesised using qualitative methods to explore themes of inequity and pathways to an inclusive future. RESULTS: Of the 620 members of the ANZSN, there were 134 (22%) respondents, of whom 57% were women and 67% were White. The majority (88%) perceived inequities in the workforce. Perceived drivers of inequity were gender (84/113; 75%), carer responsibilities (74/113; 65%) and race (64/113; 56%). Half (74/131) had personally experienced inequity, based on gender in 70% (52/74) and race in 39% (29/75) with perceived discrimination coming from doctors, patients, academics and health administrators. White males were least likely (odds ratio 0.39; 95% confidence interval 0.18-0.90) to experience inequity. Dominant themes from qualitative analysis indicated that the major impacts of inequity were limited opportunities for advancement and lack of formal assistance for those experiencing inequities. Proposed solutions to reduce inequity included normalising the discourse on inequity at an organisational level, with policy changes to ensure diverse representation on committees and in executive leadership positions. CONCLUSIONS: Inequity, particularly driven by gender and race, is common for nephrology health professionals in Australia and New Zealand and impacts career progression.

Anti-CD20 mAb-Induced B Cell Apoptosis Generates T Cell Regulation of Experimental Myeloperoxidase ANCA-Associated Vasculitis.

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

The Expanding Role of Extracellular Traps in Inflammation and Autoimmunity: The New Players in Casting Dark Webs.

The first description of a new form of neutrophil cell death distinct from that of apoptosis or necrosis was discovered in 2004 and coined neutrophil extracellular traps "(NETs)" or "NETosis". Different stimuli for NET formation, and pathways that drive neutrophils to commit to NETosis have been elucidated in the years that followed. Critical enzymes required for NET formation have been discovered and targeted therapeutically. NET formation is no longer restricted to neutrophils but has been discovered in other innate cells: macrophages/monocytes, mast Cells, basophils, dendritic cells, and eosinophils. Furthermore, extracellular DNA can also be extruded from both B and T cells. It has become clear that although this mechanism is thought to enhance host defense by ensnaring bacteria within large webs of DNA to increase bactericidal killing capacity, it is also injurious to innocent bystander tissue. Proteases and enzymes released from extracellular traps (ETs), injure epithelial and endothelial cells perpetuating inflammation. In the context of autoimmunity, ETs release over 70 well-known autoantigens. ETs are associated with pathology in multiple diseases: lung diseases, vasculitis, autoimmune kidney diseases, atherosclerosis, rheumatoid arthritis, cancer, and psoriasis. Defining these pathways that drive ET release will provide insight into mechanisms of pathological insult and provide potential therapeutic targets.

Apoptotic Cell-Induced, Antigen-Specific Immunoregulation to Treat Experimental Antimyeloperoxidase GN.

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

Biologicals targeting T helper cell subset differentiating cytokines are effective in the treatment of murine anti-myeloperoxidase glomerulonephritis.

Anti-myeloperoxidase nephritogenic autoimmunity induces severe glomerulonephritis. To assess the therapeutic potential of monoclonal antibodies targeting T helper (Th) subset differentiation determining cytokines, we studied a murine model of anti-myeloperoxidase glomerulonephritis. The temporal participation of T helper subsets was determined by quantitating gene expression of CD4+ T-cells isolated from nephritic kidneys and cytokine production by lymphocytes from nodes draining myeloperoxidase immunization sites. Th17 cytokines (IL-17A and IL-6) rose rapidly but declined as autoimmunity matured when Th1 cytokines (IL-12 and TNF) predominated. Therefore, T helper subset participation in anti-myeloperoxidase autoimmunity is biphasic, with Th17 early and Th1 late. To confirm the functional relevance of this biphasic pattern, we compared systemic anti-myeloperoxidase autoimmunity in wild type, Th17 deficient and Th1 deficient mice. Early, Th1 deficient mice developed similar autoimmunity and glomerulonephritis to wild type mice. However, Th17 deficient mice had significantly reduced anti-myeloperoxidase autoimmunity. In late autoimmunity, Th1 deficient mice developed reduced autoimmunity and were protected from anti-myeloperoxidase glomerulonephritis. The therapeutic potential of these findings were demonstrated by neutralizing monoclonal antibodies. Targeting IL-23p19 attenuated early Th17 dominated anti-myeloperoxidase autoimmunity and glomerulonephritis but not late phase disease. Targeting IL-12p35 attenuated late phase Th1 dominated anti-myeloperoxidase autoimmunity and glomerulonephritis but not early autoimmunity or glomerulonephritis. Targeting both T helper subsets with an anti-IL-12p40 monoclonal antibody was effective during both early and late phases of anti-myeloperoxidase glomerulonephritis. Thus, definition of dominant T helper differentiating subsets in anti-myeloperoxidase glomerulonephritis by renal CD4+ T-cell cytokine gene expression allows effective proper phase monoclonal antibody treatment of anti-myeloperoxidase glomerulonephritis.

Pathogenic Role for γδ T Cells in Autoimmune Anti-Myeloperoxidase Glomerulonephritis.

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 γδ T cells are a nonredundant requisite in the development of MPO autoimmunity and glomerulonephritis (GN). We studied MPO-ANCA GN in wild type, αß, or γδ T cell-deficient (C57BL/6, ßTCR-/- , and δTCR-/- respectively) mice. Both T cell populations played important roles in the generation of autoimmunity to MPO and GN. Humoral autoimmunity was dependent on intact αß T cells but was unaffected by γδ T cell deletion. Following MPO immunization, activated γδ T cells migrate to draining lymph nodes. Studies in δTCR-/- and transfer of γδ T cells to δTCR-/- mice show that γδ T cells facilitate the generation of anti-MPO autoimmunity and GN. δTCR-/- mice that received IL-17A-/- γδ T cells demonstrate that the development of anti-MPO autoimmunity and GN are dependent on γδ T cell IL-17A production. Finally, transfer of anti-MPO CD4+ T cell clones to naive δTCR-/- and wild type mice with planted glomerular MPO shows that γδ T cells are also necessary for recruitment of anti-MPO αß CD4+ effector T cells. This study demonstrates that IL-17A produced by γδ T cells plays a critical role in the pathogenesis of MPO-ANCA GN by promoting the development of MPO-specific αß T cells.

Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis.

In antineutrophil cytoplasmic antibody-associated vasculitis (AAV), Toll-like receptors (TLRs) may be engaged by infection-associated patterns and by endogenous danger signals, linking infection and innate inflammation with this autoimmune disease. This study examined intrarenal TLR2, TLR4, and TLR9 expression and renal injury in AAV, testing the hypothesis that increased TLR expression correlates with renal injury. Patients with AAV exhibited both glomerular and tubulointerstitial expression of TLR2, TLR4, and TLR9, with TLR4 being the most prominent in both compartments. Glomerular TLR4 expression correlated with glomerular segmental necrosis and cellular crescents, with TLR2 expression correlating with glomerular segmental necrosis. The extent and intensity of glomerular and tubulointerstitial TLR4 expression and the intensity of glomerular TLR2 expression inversely correlated with the presenting estimated glomerular filtration rate. Although myeloid cells within the kidney expressed TLR2, TLR4, and TLR9, TLR2 and TLR4 colocalized with endothelial cells and podocytes, whereas TLR9 was expressed predominantly by podocytes. The functional relevance of intrarenal TLR expression was further supported by the colocalization of TLRs with their endogenous ligands high-mobility group box 1 and fibrinogen. Therefore, in AAV, the extent of intrarenal TLR4 and TLR2 expression and their correlation with renal injury indicates that TLR4, and to a lesser degree TLR2, may be potential therapeutic targets in this disease.

In Vivo Imaging of Inflamed Glomeruli Reveals Dynamics of Neutrophil Extracellular Trap Formation in Glomerular Capillaries.

Neutrophil extracellular traps (NETs) have been documented in glomeruli of patients with glomerulonephritis. However, the dynamics of NET formation in the glomerulus and their functional contribution to acute glomerular injury are poorly understood. Herein, we used in vivo multiphoton microscopy to investigate NET formation in the acutely inflamed glomerulus. Glomerular inflammation was induced using an antibody against the glomerular basement membrane. After induction of inflammation, multiphoton microscopy revealed that approximately 20% of glomeruli contained structures composed of extracellular DNA within the capillaries. These structures were not seen in mice depleted of neutrophils, consistent with them being NETs. Most contained myeloperoxidase, as seen in NETs in other tissues, whereas intraglomerular NETs did not contain significant levels of the histone H2Ax or neutrophil elastase. In vivo imaging revealed that intraglomerular NETs were present only transiently, suggesting that NETs were susceptible to disruption under the high shear conditions in glomerular capillaries. Investigation of NETs under flow conditions in vitro supported this concept. Dissolution of NETs via DNase I did not alter anti-glomerular basement membrane antibody-induced glomerular injury, as assessed via albuminuria, although the degree of microscopic hematuria was reduced by this intervention. These data indicate that in this model of acute, neutrophil-dependent glomerulonephritis, NETs are generated in the glomerular capillaries, where they are short lived and make a modest contribution to glomerular injury.

CD8+ T Cells Effect Glomerular Injury in Experimental Anti-Myeloperoxidase GN.

Observations in patients with ANCA-associated vasculitis suggest that CD8+ T cells participate in disease, but there is no experimental functional evidence of pathologic involvement for these cells. Myeloperoxidase (MPO) is a well defined autoantigen in ANCA-associated vasculitis. Studies in experimental models of anti-MPO GN suggest that, after ANCA-induced neutrophil localization, deposited MPO within glomeruli is recognized by autoreactive T cells that contribute to injury. We tested the hypothesis that CD8+ T cells mediate disease in experimental ANCA-associated vasculitis. CD8+ T cell depletion in the effector phase of disease attenuated injury in murine anti-MPO GN. This protection associated with decreased levels of intrarenal IFN-γ, TNF, and inflammatory chemokines and fewer glomerular macrophages. Moreover, we identified a pathogenic CD8+ T cell MPO epitope (MPO431-439) and found that cotransfer of MPO431-439-specific CD8+ T cell clones exacerbated disease mediated by MPO-specific CD4+ cells in Rag1-/- mice. Transfer of MPO431-439-specific CD8+ cells without CD4+ cells mediated glomerular injury when MPO was planted in glomeruli. These results show a pathogenic role for MPO-specific CD8+ T cells, provide evidence that CD8+ cells are a therapeutic target in ANCA-associated vasculitis, and suggest that a molecular hotspot within the MPO molecule contains important CD8+, CD4+, and B cell epitopes.

Epidemiology of long-stay patients in a university teaching hospital.

BACKGROUND: Patients admitted to acute care hospitals may have multiple comorbidities, and a small proportion may stay for a protracted period. AIMS: To assess the proportion of hospital patients who are long stay (≥14 days) and evaluate associations with baseline variables and subsequent inpatient morbidity and mortality. METHODS: This is a retrospective observational study of patients aged ≥18 years staying in hospital for at least 24 h between 1 July 2013 and 30 June 2014. RESULTS: There were 22 094 admissions in 15 623 patients. The median (interquartile range (IQR)) length of stay (LOS) was 4 (2-8) days, and 10% had a LOS >16 days. Long-stay admissions comprised 13.1% of admissions but used 49.1% of bed days. Long-stay admissions were more likely to be associated with intensive care unit admission (21.2 vs 6.0%), medical emergency team review (20.5 vs 4.3%) and a longer duration of mechanical ventilation (P < 0.0001 all comparisons). Long-stay patients were more likely to develop in-hospital complications, were more likely to die in hospital (8.2 vs 3.1%) and were less likely to be discharged home (P < 0.001 all comparisons). Multiple variable analysis revealed several associations with prolonged stay, including multiple admissions in the study period, the nature of the admitting unit, the Charlson comorbidity index at admission, admission from another hospital and any history of smoking. CONCLUSIONS: Patients staying at least 14 days comprised one seventh of hospital admissions but used half of bed days and suffered increased in-hospital morbidity and mortality. Several pre-admission associations with prolonged stay were identified.

Mast Cell Stabilization Ameliorates Autoimmune Anti-Myeloperoxidase Glomerulonephritis.

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 disodium cromoglycate to mast cell-deficient mice had no effect on the development of MPO autoimmunity or GN. MPO-specific CD4(+) effector T cell proliferation was enhanced by co-culture with mast cells, but in the presence of disodium cromoglycate, 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.

Renal participation of myeloperoxidase in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis.

Myeloperoxidase (MPO) is an important neutrophil lysosomal enzyme, a major autoantigen, and a potential mediator of tissue injury in MPO-ANCA-associated vasculitis (MPO-AAV) and glomerulonephritis. Here we examined MPO deposition in kidney biopsies from 47 patients with MPO-AAV. Leukocyte accumulation and fibrin deposition consistent with cell-mediated immunity was a major feature. Tubulointerstitial macrophage, CD4+ and CD8+ T-cell, and neutrophil numbers correlated with low presenting eGFR. MPO was not detected in kidneys from patients with minimal change or thin basement membrane disease, but was prominent in glomerular, periglomerular, and tubulointerstitial regions in MPO-AAV. Extracellular MPO released from leukocytes was pronounced in all MPO-AAV patients. Similar numbers of neutrophils and macrophages expressed MPO in the kidneys, but colocalization studies identified neutrophils as the major source of extracellular MPO. Extraleukocyte MPO was prominent in neutrophil extracellular traps in the majority of patients; most of which had traps in half or more glomeruli. These traps were associated with more neutrophils and more MPO within glomeruli. Glomerular MPO-containing macrophages generated extracellular trap-like structures. MPO also localized to endothelial cells and podocytes. The presence of the most active glomerular lesions (both segmental necrosis and cellular crescents) correlated with intraglomerular CD4+ cells and MPO+ macrophages. Thus, cellular and extracellular MPO may cause glomerular and interstitial injury.

Neutrophil myeloperoxidase regulates T-cell-driven tissue inflammation in mice by inhibiting dendritic cell function.

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.

The HLA-DRB1*15:01-restricted Goodpasture's T cell epitope induces GN.

Human anti-glomerular basement membrane (GBM) disease strongly associates with HLA-DRB1*15:01. The target autoantigen in this disease is the noncollagenous domain of the α3 chain of type IV collagen, α3(IV)NC1, but critical early T cell epitopes presented by this human MHC class II molecule are unknown. Here, by immunizing HLA-DRB1*15:01 transgenic mice with whole recombinant α3(IV)NC1 and with overlapping α3(IV)NC1 peptides, we defined a HLA-DRB1*15:01-restricted α3(IV)NC1 T cell epitope (α3136-146) with four critical residues. This peptide was not immunogenic in HLA-DRB1*01:01 transgenic or C57BL/6 mice. The T cell epitope is naturally processed from α3(IV)NC1. CD4(+) T cell clones, generated from HLA-DRB1*15:01 transgenic mice and specific for α3136-146, transferred disease into naive HLA-DRB1*15:01 transgenic mice, evidenced by the development of necrotizing crescentic GN, albuminuria, renal impairment, and accumulation of CD4(+) T cells and macrophages in glomeruli. Because Fcγ receptors are implicated in disease susceptibility, we crossed HLA transgenic mice onto an FcγRIIb-deficient background. Immunization with either α3136-146 or α3(IV)NC1 induced GN in HLA-DRB1*15:01 transgenic FcγRIIb-deficient mice, but HLA-DRB1*01:01 transgenic FcγRIIb-deficient mice were unaffected. Taken together, these results demonstrate that the HLA-DRB1*15:01-restricted T cell epitope α3136-146 can induce T cell responses and injury in anti-GBM GN.

Thymic deletion and regulatory T cells prevent antimyeloperoxidase GN.

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 to MPO and protecting from GN. 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 thymic MPO 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 had more 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.

Mast cells contribute to peripheral tolerance and attenuate autoimmune vasculitis.

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 (Kit(W-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 cells migrated from the 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.