Autoimmune Renal Disease Is Exacerbated by S1P-Receptor-1-Dependent Intestinal Th17 Cell Migration to the Kidney.

Th17 cells are most abundant in the gut, where their presence depends on the intestinal microbiota. Here, we examined whether intestinal Th17 cells contribute to extra-intestinal Th17 responses in autoimmune kidney disease. We found high frequencies of Th17 cells in the kidneys of patients with antineutrophil cytoplasmatic antibody (ANCA)-associated glomerulonephritis. We utilized photoconversion of intestinal cells in Kaede mice to track intestinal T cell mobilization upon glomerulonephritis induction, and we found that Th17 cells egress from the gut in a S1P-receptor-1-dependent fashion and subsequently migrate to the kidney via the CCL20/CCR6 axis. Depletion of intestinal Th17 cells in germ-free and antibiotic-treated mice ameliorated renal disease, whereas expansion of these cells upon Citrobacter rodentium infection exacerbated pathology. Thus, in some autoimmune settings, intestinal Th17 cells migrate into target organs, where they contribute to pathology. Targeting the intestinal Th17 cell "reservoir" may present a therapeutic strategy for these autoimmune disorders.

Tr1 Cells Emerge and Suppress Effector Th17 Cells in Glomerulonephritis.

T regulatory type 1 (Tr1) cells, which are defined by their regulatory function, lack of Foxp3, and high expression of IL-10, CD49b, and LAG-3, are known to be able to suppress Th1 and Th17 in the intestine. Th1 and Th17 cells are also the main drivers of crescentic glomerulonephritis (GN), the most severe form of renal autoimmune disease. However, whether Tr1 cells emerge in renal inflammation and, moreover, whether they exhibit regulatory function during GN have not been thoroughly investigated yet. To address these questions, we used a mouse model of experimental crescentic GN and double Foxp3mRFP IL-10eGFP reporter mice. We found that Foxp3neg IL-10-producing CD4+ T cells infiltrate the kidneys during GN progression. Using single-cell RNA sequencing, we could show that these cells express the core transcriptional factors characteristic of Tr1 cells. In line with this, Tr1 cells showed a strong suppressive activity ex vivo and were protective in experimental crescentic GN in vivo. Finally, we could also identify Tr1 cells in the kidneys of patients with antineutrophil cytoplasmic autoantibody-associated GN and define their transcriptional profile. Tr1 cells are currently used in several immune-mediated inflammatory diseases, such as T-cell therapy. Thus, our study provides proof of concept for Tr1 cell-based therapies in experimental GN.

Th17 cell plasticity towards a T-bet-dependent Th1 phenotype is required for bacterial control in Staphylococcus aureus infection.

Staphylococcus aureus is frequently detected in patients with sepsis and thus represents a major health burden worldwide. CD4+ T helper cells are involved in the immune response to S. aureus by supporting antibody production and phagocytosis. In particular, Th1 and Th17 cells secreting IFN-γ and IL-17A, are involved in the control of systemic S. aureus infections in humans and mice. To investigate the role of T cells in severe S. aureus infections, we established a mouse sepsis model in which the kidney was identified to be the organ with the highest bacterial load and abundance of Th17 cells. In this model, IL-17A but not IFN-γ was required for bacterial control. Using Il17aCre × R26YFP mice we could show that Th17 fate cells produce Th17 and Th1 cytokines, indicating a high degree of Th17 cell plasticity. Single cell RNA-sequencing of renal Th17 fate cells uncovered their heterogeneity and identified a cluster with a Th1 expression profile within the Th17 cell population, which was absent in mice with T-bet/Tbx21-deficiency in Th17 cells (Il17aCre x R26eYFP x Tbx21-flox). Blocking Th17 to Th1 transdifferentiation in Th17 fate cells in these mice resulted in increased S. aureus tissue loads. In summary, we highlight the impact of Th17 cells in controlling systemic S. aureus infections and show that T-bet expression by Th17 cells is required for bacterial clearance. While targeting the Th17 cell immune response is an important therapeutic option in autoimmunity, silencing Th17 cells might have detrimental effects in bacterial infections.

Targeting Monocyte Derived CCL17 Attenuates Murine Crescentic Glomerulonephritis by Affecting Renal CCR4+ Regulatory T-Cell Recruitment.

INTRODUCTION: The chemokine receptor CCR4 is expressed by diverse CD4+ T cell subsets including regulatory T cells (Tregs) but its functional importance for leukocyte recruitment and the relevance of its two corresponding chemokines CCL17 and CCL22 have not been studied in immune-mediated crescentic glomerulonephritis (cGN). METHODS: Utilizing the single-cell RNA sequencing (scRNAseq) data in analyzing leukocytes isolated from both human and murine nephritic kidneys, we identified CCL17 as a potential therapeutic target in immune-mediated renal disease. Using a mouse model of murine cGN, we then delineated the effects of targeting CCL17 by neutralizing antibodies and in Ccl17 gene-deficient mice. RESULTS: Unsupervised scRNAseq analyses identified the CCL17-CCR4 axis as a mechanism potentially involved in renal T-cell migration. Analyses of functional kidney impairment and histopathological kidney damage revealed an attenuation of crescentic GN in anti-CCL17 antibody-treated mice which was corroborated using in Ccl17 gene-deficient mice. Immunohistochemical analyses revealed that these changes were accompanied by an affected renal Treg recruitment in both experimental approaches. CONCLUSION: The chemokine receptor CCR4 and its corresponding chemokine CCL17 are expressed in human and murine cGN and targeting the CCR4-CCL17 axis by neutralizing antibodies as well as Ccl17 gene deficiency led to increased renal Treg recruitment and reduced histological and functional kidney damage in murine cGN.

IKK1 aggravates ischemia-reperfusion kidney injury by promoting the differentiation of effector T cells.

Ischemia-reperfusion injury (IRI) is one of the major causes of acute kidney injury (AKI), and experimental work has revealed detailed insight into the inflammatory response in the kidney. T cells and NFκB pathway play an important role in IRI. Therefore, we examined the regulatory role and mechanisms of IkappaB kinase 1 (IKK1) in CD4+T lymphocytes in an experimental model of IRI. IRI was induced in CD4cre and CD4IKK1Δ mice. Compared to control mice, conditional deficiency of IKK1 in CD4+T lymphocyte significantly decreased serum creatinine, blood urea nitrogen (BUN) level, and renal tubular injury score. Mechanistically, lack in IKK1 in CD4+T lymphocytes reduced the ability of CD4 lymphocytes to differentiate into Th1/Th17 cells. Similar to IKK1 gene ablation, pharmacological inhibition of IKK also protected mice from IRI. Together, lymphocyte IKK1 plays a pivotal role in IRI by promoting T cells differentiation into Th1/Th17 and targeting lymphocyte IKK1 may be a novel therapeutic strategy for IRI.

Transcriptional and Clonal Characterization of Cytotoxic T Cells in Crescentic Glomerulonephritis.

SIGNIFICANCE STATEMENT: T-cell infiltration is a hallmark of crescentic GN (cGN), often caused by ANCA-associated vasculitis. Pathogenic T-cell subsets, their clonality, and downstream effector mechanisms leading to kidney injury remain to be fully elucidated. Single-cell RNA sequencing and T-cell receptor sequencing revealed activated, clonally expanded cytotoxic CD4 + and CD8 + T cells in kidneys from patients with ANCA-associated cGN. In experimental cGN, kidney-infiltrating CD8 + T cells expressed the cytotoxic molecule, granzyme B (GzmB), which induced apoptosis in renal tissue cells by activation of procaspase-3, and aggravated disease pathology. These findings describe a pathogenic function of (clonally expanded) cytotoxic T cells in cGN and identify GzmB as a mediator and potential therapeutic target in immune-mediated kidney disease. BACKGROUND: Crescentic GN (cGN) is an aggressive form of immune-mediated kidney disease that is an important cause of end stage renal failure. Antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis is a common cause. T cells infiltrate the kidney in cGN, but their precise role in autoimmunity is not known. METHODS: Combined single-cell RNA sequencing and single-cell T-cell receptor sequencing were conducted on CD3 + T cells isolated from renal biopsies and blood of patients with ANCA-associated cGN and from kidneys of mice with experimental cGN. Functional and histopathological analyses were performed with Cd8a-/- and GzmB-/- mice. RESULTS: Single-cell analyses identified activated, clonally expanded CD8 + and CD4 + T cells with a cytotoxic gene expression profile in the kidneys of patients with ANCA-associated cGN. Clonally expanded CD8 + T cells expressed the cytotoxic molecule, granzyme B (GzmB), in the mouse model of cGN. Deficiency of CD8 + T cells or GzmB ameliorated the course of cGN. CD8 + T cells promoted macrophage infiltration and GzmB activated procaspase-3 in renal tissue cells, thereby increasing kidney injury. CONCLUSIONS: Clonally expanded cytotoxic T cells have a pathogenic function in immune-mediated kidney disease.

The CCR6/CCL20 axis expands RORγt+ Tregs to protect from glomerulonephritis.

Previous studies have identified a unique Treg population, which expresses the Th17 characteristic transcription factor RORγt. These RORγt+ Tregs possess enhanced immunosuppressive capacity, which endows them with great therapeutic potential. However, as a caveat, they are also capable of secreting pro-inflammatory IL-17A. Since the sum function of RORγt+ Tregs in glomerulonephritis (GN) remains unknown, we studied the effects of their absence. Purified CD4+ T cell populations, containing or lacking RORγt+ Tregs, were transferred into immunocompromised RAG1 knockout mice and the nephrotoxic nephritis model of GN was induced. Absence of RORγt+ Tregs significantly aggravated kidney injury, demonstrating overall kidney-protective properties. Analyses of immune responses showed that RORγt+ Tregs were broadly immunosuppressive with no preference for a particular type of T cell response. Further characterization revealed a distinct functional and transcriptional profile, including enhanced production of IL-10. Expression of the chemokine receptor CCR6 marked a particularly potent subset, whose absence significantly worsened GN. As an underlying mechanism, we found that chemokine CCL20 acting through receptor CCR6 signaling mediated expansion and activation of RORγt+ Tregs. Finally, we also detected an increase of CCR6+ Tregs in kidney biopsies, as well as enhanced secretion of chemokine CCL20 in 21 patients with anti-neutrophil cytoplasmic antibody associated GN compared to that of 31 healthy living donors, indicating clinical relevance. Thus, our data characterize RORγt+ Tregs as anti-inflammatory mediators of GN and identify them as promising target for Treg directed therapies.

IL-17 Receptor C Signaling Controls CD4+ TH17 Immune Responses and Tissue Injury in Immune-Mediated Kidney Diseases.

BACKGROUND: IL-17A-producing CD4+ T helper (TH17) cells play a critical role in autoimmune and chronic inflammatory diseases, such as crescentic GN. The proinflammatory effects of IL-17 are mediated by the activation of the IL-17RA/IL-17RC complex. Although the expression of these receptors on epithelial and endothelial cells is well characterized, the IL-17 receptor expression pattern and function on hematopoietic cells, e.g., CD4+ T cell subsets, remains to be elucidated. METHODS: Crescentic GN (nephrotoxic nephritis) was induced in IL-17A, IFNγ, and Foxp3 triple-reporter mice for sorting of renal CD4+ T cell subsets and subsequent single-cell RNA sequencing. Moreover, we generated TH17 cell-specific IL-17RA and IL-17RC gene-deficient mice and studied the functional role of IL-17 signaling in TH17 cells in crescentic GN, imiquimod-induced psoriasis, and in the CD4+CD45RBhigh T cell transfer colitis model. RESULTS: We identified a specific expression of the IL-17 receptor A/C complex on CD4+ TH17 cells. Single-cell RNA sequencing of TH17 cells revealed the activation of the IL-17 receptor signaling pathway in experimental crescentic GN. Disruption of the IL-17RC signaling pathway in CD4+ T cells and, most importantly, specifically in CD4+ TH17 cells, potentiates the IL-17 cytokine response and results in an accelerated course of experimental crescentic GN. Comparable results were observed in experimental models of psoriasis and colitis. CONCLUSIONS: Our findings indicate that IL-17 receptor C signaling has a previously unrecognized function in the regulation of CD4+ TH17 cells and in the control of organ-specific autoimmunity and might provide new insights into the development of more efficient anti-TH17 treatment strategies.

T-lymphocyte-specific knockout of IKK-2 or NEMO induces Th17 cells in an experimental nephrotoxic nephritis mouse model.

Experimental nephrotoxic serum nephritis (NTN) is a model for T-cell-mediated human rapid progressive glomerulonephritis. T-cell receptor stimulation involves intracellular signaling events that ultimately lead to the activation of transcription factors, such as NF-κB. We explored the involvement of the NF-κB components IKK-2 and NEMO in NTN, by using cell-specific knockouts of IKK-2 and NEMO in CD4+ T lymphocytes. Our results demonstrate that although the course of disease was not grossly altered in CD4xIKK2Δ and CD4xNEMOΔ animals, renal regulatory T cells were significantly reduced and T helper (Th)1 and Th17 cells significantly increased in both knockout mouse groups. The expression of the renal cytokines and chemokines IL-1ß, CCL-2, and CCL-20 was also significantly altered in both knockout mice. Lymphocyte transcriptome analysis confirmed the increased expression of Th17-related cytokines in spleen CD4+ T cells. Moreover, our array data demonstrate an interrupted canonical NF-κB pathway and an increased expression of noncanonical NF-κB pathway-related genes in nephritic CD4xNEMOΔ mice, highlighting different downstream effects of deletion of IKK-2 or NEMO in T lymphocytes. We propose that better understanding of the role of IKK-2 and NEMO in nephritis is essential for the clinical application of kinase inhibitors in patients with glomerulonephritis.-Guo, L., Huang, J., Chen, M., Piotrowski, E., Song, N., Zahner, G., Paust, H.-J., Alawi, M., Geffers, R., Thaiss, F. T-lymphocyte-specific knockout of IKK-2 or NEMO induces Th17 cells in an experimental nephrotoxic nephritis mouse model.

Role of regulatory T cells in experimental autoimmune glomerulonephritis.

Anti-glomerular basement membrane (anti-GBM) disease is characterized by antibodies and T cells directed against the Goodpasture antigen, the noncollagenous domain of the α3-chain of type IV collagen [α3(IV)NC1] of the GBM. Consequences are the deposition of autoantibodies along the GBM and the development of crescentic glomerulonephritis (GN) with rapid loss of renal function. Forkhead box protein P3 (Foxp3)+ regulatory T (Treg) cells are crucial for the maintenance of peripheral tolerance to self-antigens and the prevention of immunopathology. Here, we use the mouse model of experimental autoimmune GN to characterize the role of Treg cells in anti-GBM disease. Immunization of DBA/1 mice with α3(IV)NC1 induced the formation of α3(IV)NC1-specific T cells and antibodies and, after 8-10 wk, the development of crescentic GN. Immunization resulted in increased frequencies of peripheral Treg cells and renal accumulation of these cells in the stage of acute GN. Depletion of Treg cells during immunization led to enhanced generation of α3(IV)NC1-specific antibodies and T cells and to aggravated GN. In contrast, depletion or expansion of the Treg cell population in mice with established autoimmunity had only minor consequences for renal inflammation and did not alter the severity of GN. In conclusion, our results indicate that in anti-GBM disease, Treg cells restrict the induction of autoimmunity against α3(IV)NC1. However, Treg cells are inefficient in preventing crescentic GN after autoimmunity has been established.

IL-17C/IL-17 Receptor E Signaling in CD4+ T Cells Promotes TH17 Cell-Driven Glomerular Inflammation.

The IL-17 cytokine family and the cognate receptors thereof have a unique role in organ-specific autoimmunity. Most studies have focused on the founding member of the IL-17 family, IL-17A, as the central mediator of diseases. Indeed, although pathogenic functions have been ascribed to IL-17A and IL-17F in the context of immune-mediated glomerular diseases, the specific functions of the other IL-17 family members in immunity and inflammatory kidney diseases is largely unknown. Here, we report that compared with healthy controls, patients with acute Anti-neutrophil cytoplasmatic antibody (ANCA)-associated crescentic glomerulonephritis (GN) had significantly elevated serum levels of IL-17C (but not IL-17A, F, or E). In mouse models of crescentic GN (nephrotoxic nephritis) and pristane-induced lupus nephritis, deficiency in IL-17C significantly ameliorated the course of GN in terms of renal tissue injury and kidney function. Deficiency of the unique IL-17C receptor IL-17 receptor E (IL-17RE) provided similar protection against crescentic GN. These protective effects associated with a reduced TH17 response. Bone marrow transplantation experiments revealed that IL-17C is produced by tissue-resident cells, but not by lymphocytes. Finally, IL-17RE was highly expressed by CD4+ TH17 cells, and loss of this expression prevented the TH17 responses and subsequent tissue injury in crescentic GN. Our findings indicate that IL-17C promotes TH17 cell responses and immune-mediated kidney disease via IL-17RE expressed on CD4+ TH17 cells. Targeting the IL-17C/IL-17RE pathway may present an intriguing therapeutic strategy for TH17-induced autoimmune disorders.

The chemokine receptor CX3CR1 reduces renal injury in mice with angiotensin II-induced hypertension.

The role of CX3CR1, also known as fractalkine receptor, in hypertension is unknown. The present study determined the role of the fractalkine receptor CX3CR1 in hypertensive renal and cardiac injury. Expression of CX3CR1 was determined using CX3CR1GFP/+ mice that express a green fluorescent protein (GFP) reporter in CX3CR1+ cells. FACS analysis of leukocytes isolated from the kidney showed that 34% of CD45+ cells expressed CX3CR1. Dendritic cells were the majority of positive cells (67%) followed by macrophages (10%), NK cells (6%), and T cells (10%). With the use of confocal microscopy, the receptor was detected in the kidney only on infiltrating cells but not on resident renal cells. To evaluate the role of CX3CR1 in hypertensive end-organ injury, an aggravated model of hypertension was used. Unilateral nephrectomy was performed followed by infusion of angiotensin II (ANG II, 1.5 ng·g-1·min-1) and a high-salt diet in wild-type ( n = 15) and CX3CR1-deficient mice ( n = 18). CX3CR1 deficiency reduced the number of renal dendritic cells and increased the numbers of renal CD11b/F4/80+ macrophages and CD11b/Ly6G+ neutrophils in ANG II-infused mice. Surprisingly, CX3CR1-deficient mice exhibited increased albuminuria, glomerular injury, and reduced podocyte density in spite of similar levels of arterial hypertension. In contrast, cardiac damage as assessed by increased heart weight, cardiac fibrosis, and expression of fetal genes, and matrix components were not different between both genotypes. Our findings suggest that CX3CR1 exerts protective properties by modulating the invasion of inflammatory cells in hypertensive renal injury. CX3CR1 inhibition should be avoided in hypertension because it may promote hypertensive renal injury.

Plasticity of Th17 Cells in Autoimmune Kidney Diseases.

The ability of CD4(+) T cells to differentiate into pathogenic Th1 and Th17 or protective T regulatory cells plays a pivotal role in the pathogenesis of autoimmune diseases. Recent data suggest that CD4(+) T cell subsets display a considerable plasticity. This plasticity seems to be a critical factor for their pathogenicity, but also for the potential transition of pathogenic effector T cells toward a more tolerogenic phenotype. The aim of the current study was to analyze the plasticity of Th17 cells in a mouse model of acute crescentic glomerulonephritis and in a mouse chronic model of lupus nephritis. By transferring in vitro generated, highly purified Th17 cells and by using IL-17A fate reporter mice, we demonstrate that Th17 cells fail to acquire substantial expression of the Th1 and Th2 signature cytokines IFN-γ and IL-13, respectively, or the T regulatory transcription factor Foxp3 throughout the course of renal inflammation. In an attempt to therapeutically break the stability of the Th17 phenotype in acute glomerulonephritis, we subjected nephritic mice to CD3-specific Ab treatment. Indeed, this treatment induced an immunoregulatory phenotype in Th17 cells, which was marked by high expression of IL-10 and attenuated renal tissue damage in acute glomerulonephritis. In summary, we show that Th17 cells display a minimum of plasticity in acute and chronic experimental glomerulonephritis and introduce anti-CD3 treatment as a tool to induce a regulatory phenotype in Th17 cells in the kidney that may be therapeutically exploited.

Glomerulopathy induced by immunization with a peptide derived from the goodpasture antigen α3IV-NC1.

Mouse experimental autoimmune glomerulonephritis, a model of human antiglomerular basement membrane disease, depends on both Ab and T cell responses to the Goodpasture Ag noncollagenous domain 1 of the α3-chain of type IV collagen (α3IV-NC1). The aim of our study was to further characterize the T cell-mediated immune response. Repeated immunization with mouse α3IV-NC1 caused fatal glomerulonephritis in DBA/1 mice. Although two immunizations were sufficient to generate high α3IV-NC1-specific IgG titers, Ab and complement deposition along the glomerular basement membranes, and a nephrotic syndrome, two additional immunizations were needed to induce a necrotizing/crescentic glomerulonephritis. Ten days after the first immunization, α3IV-NC1-specific CD4(+) cells producing TNF-α, IFN-γ, or IL-17A were detected in the spleen. With the emergence of necrotizing/crescentic glomerulonephritis, ∼0.15% of renal CD4(+) cells were specific for α3IV-NC1. Using peptides spanning the whole α3IV-NC1 domain, three immunodominant T cell epitopes were identified. Immunization with these peptides did not lead to clinical signs of experimental autoimmune glomerulonephritis or necrotizing/crescentic glomerulonephritis. However, mice immunized with one of the peptides (STVKAGDLEKIISRC) developed circulating Abs against mouse α3IV-NC1 first detected at 8 wk, and 50% of the mice showed mild proteinuria at 18-24 wk due to membranous glomerulopathy. Taken together, our results suggest that autoreactive T cells are able to induce the formation of pathologic autoantibodies. The quality and quantity of α3IV-NC1-specific Ab and T cell responses are critical for the phenotype of the glomerulonephritis.

IL-17F Promotes Tissue Injury in Autoimmune Kidney Diseases.

The TH17 immune response has a central role in the pathogenesis of autoimmune diseases, implicating the TH17 master cytokine, IL-17A, as the critical mediator of diseases such as human and experimental crescentic GN. However, the relative importance of additional TH17 effector cytokines, including IL-17F, in immune-mediated tissue injury remains to be fully elucidated. Here, using a mouse model of acute crescentic GN (nephrotoxic nephritis), we identified CD4+ T cells and γδ T cells as the major cellular source of IL-17F in the inflamed kidney. Interventional studies using IL-17F gene-deficient mice, IL-17F-neutralizing antibodies, and adoptive transfer experiments into Rag1-/- mice demonstrated that CD4+ T cell-derived IL-17F drives renal tissue injury in acute crescentic GN. Notably, IL-17F-deficient nephritic mice had fewer renal infiltrating neutrophils than wild-type nephritic mice, and neutrophil depletion did not affect the course of GN in IL-17F-deficient mice. Moreover, in the chronic model of pristane-induced SLE, IL-17F-deficient mice developed less severe disease than wild-type mice, with respect to survival and renal injury. Finally, we show that IL-17F induced expression of the neutrophil-attracting chemokines CXCL1 and CXCL5 in kidney cells. The finding that IL-17F has a nonredundant function in the development of renal tissue injury in experimental GN might be of great importance for the development of anti-IL-17 cytokine therapies in TH17-mediated human autoimmune diseases.

CXCR3+ Regulatory T Cells Control TH1 Responses in Crescentic GN.

Chemokines and chemokine receptors are implicated in regulatory T cell (Treg) trafficking to sites of inflammation and suppression of excessive immune responses in inflammatory and autoimmune diseases; however, the specific requirements for Treg migration into the inflamed organs and the positioning of these cells within the tissue are incompletely understood. Here, we report that Tregs expressing the TH1-associated chemokine receptor CXCR3 are enriched in the kidneys of patients with ANCA-associated crescentic GN and colocalize with CXCR3(+) effector T cells. To investigate the functional role of CXCR3(+) Tregs, we generated mice that lack CXCR3 in Tregs specifically (Foxp3(eGFP-Cre) × Cxcr3(fl/fl)) and induced experimental crescentic GN. Treg-specific deletion of CXCR3 resulted in reduced Treg recruitment to the kidney and an overwhelming TH1 immune response, with an aggravated course of the nephritis that was reversible on anti-IFNγ treatment. Together, these findings show that a subset of Tregs expresses CXCR3 and thereby, acquires trafficking properties of pathogenic CXCR3(+) TH1 cells, allowing Treg localization and control of excessive TH1 responses at sites of inflammation.

TH1 and TH17 cells promote crescent formation in experimental autoimmune glomerulonephritis.

Autoimmunity against the Goodpasture antigen α3IV-NC1 results in crescentic glomerulonephritis (GN). Both antibodies and T cells directed against α3IV-NC1 have been implicated in disease development and progression. Using the model of experimental autoimmune glomerulonephritis (EAG) in DBA/1 mice, we aimed to characterize the frequency and function of α3IV-NC1-specific CD4(+) T cells in the kidneys. DBA/1 mice repeatedly immunized with human α3IV-NC1 developed necrotizing/crescentic GN. Kidneys with crescentic GN contained CD4(+) cells responding to α3IV-NC1 with the production of IFN-γ or IL-17A, demonstrating the accumulation of both α3IV-NC1-specific TH1 and TH17 cells. To test the functional relevance of TH1 and TH17 cells, EAG was induced in DBA/1 mice deficient in IFN-γR, IL-17A or IL-23p19. Mice of all knockout groups mounted α3IV-NC1 IgG, developed nephrotic range proteinuria, and IgG deposition to the glomerular basement membranes at levels similar to immunized wild-type mice. However, all knockout groups showed significantly fewer glomerular crescents and attenuated tubulointerstitial damage. Our results suggest that both α3IV-NC1-specific TH1 and TH17 cells accumulate in the kidneys and are crucial for the development of necrotizing/crescentic GN.

CXCL5 drives neutrophil recruitment in TH17-mediated GN.

Neutrophil trafficking to sites of inflammation is essential for the defense against bacterial and fungal infections, but also contributes to tissue damage in TH17-mediated autoimmunity. This process is regulated by chemokines, which often show an overlapping expression pattern and function in pathogen- and autoimmune-induced inflammatory reactions. Using a murine model of crescentic GN, we show that the pathogenic TH17/IL-17 immune response induces chemokine (C-X-C motif) ligand 5 (CXCL5) expression in kidney tubular cells, which recruits destructive neutrophils that contribute to renal tissue injury. By contrast, CXCL5 was dispensable for neutrophil recruitment and effective bacterial clearance in a murine model of acute bacterial pyelonephritis. In line with these findings, CXCL5 expression was highly upregulated in the kidneys of patients with ANCA-associated crescentic GN as opposed to patients with acute bacterial pyelonephritis. Our data therefore identify CXCL5 as a potential therapeutic target for the restriction of pathogenic neutrophil infiltration in TH17-mediated autoimmune diseases while leaving intact the neutrophil function in protective immunity against invading pathogens.

B-cell-derived IL-10 does not vitally contribute to the clinical course of glomerulonephritis.

IL-10-secreting regulatory B cells have been postulated as negative mediators of inflammation. However, their impact on immune-mediated diseases requires further investigation. We recently found that IL-10-secreting B cells infiltrate the kidney during crescentic glomerulonephritis (GN). We therefore studied the function of B-cell-derived IL-10 in light of the potential risks associated with increasingly used B-cell depleting therapies. Lack of IL-10 production by B cells, however, did not influence acute or adaptively mediated progressive renal injury in terms of renal function and histological damage in the nephrotoxic nephritis model of GN. Renal leukocyte infiltration and cytokine expression were similar apart from increased macrophages in mice lacking B-cell-derived IL-10. Systemic immune responses as assessed by cytokine production, leukocyte composition, proliferation, and activation were indistinguishable, while production and renal deposition of Ag-specific IgG were mildly impaired in the absence of B-cell-produced IL-10. Importantly, detailed analysis of systemic and renal regulatory T cells did not show any differences between nephritic mice bearing IL-10-deficient B cells and WT controls. Finally, studies in reporter mice revealed that B cells are only a minor source of systemic IL-10. In summary, our data reveal that endogenous B-cell-derived IL-10 does not play a major role in the nephrotoxic nephritis model of crescentic GN.

Stat3 programs Th17-specific regulatory T cells to control GN.

A pathogenic role for Th17 cells in inflammatory renal disease is well established. The mechanisms underlying their counter-regulation are, however, largely unknown. Recently, Th17 lineage-specific regulatory T cells (Treg17) that depend on activation of the transcription factor Stat3 were identified. We studied the function of Treg17 in the nephrotoxic nephritis (NTN) model of crescentic GN. The absence of Treg17 cells in Foxp3(Cre)×Stat3(fl/fl) mice resulted in the aggravation of NTN and skewing of renal and systemic immune responses toward Th17. Detailed analysis of Stat3-deficient Tregs revealed that the survival, activation, proliferation, and suppressive function of these cells remained intact. However, Tregs from Foxp3(Cre)×Stat3(fl/fl) mice lacked surface expression of the chemokine receptor CCR6, which resulted in impaired renal trafficking. Furthermore, aggravation of NTN was reversible in the absence of Th17 responses, as shown in CD4(Cre)×Stat3(fl/fl) mice lacking both Treg17 and Th17 cells, suggesting that Th17 cells are indeed the major target of Treg17 cells. Notably, immunohistochemistry revealed CCR6-bearing Treg17 cells in kidney biopsy specimens of patients with GN. CCR6 expression on human Treg17 cells also appears dependent on STAT3, as shown by analysis of Tregs from patients with dominant-negative STAT3 mutations. Our data indicate the presence and involvement of Stat3/STAT3-dependent Treg17 cells that specifically target Th17 cells in murine and human crescentic GN, and suggest the kidney-specific action of these Treg17 cells is regulated by CCR6-directed migration into areas of Th17 inflammation.