Joachim Velden

CCR6 Recruits Regulatory T Cells and Th17 Cells to the Kidney in Glomerulonephritis

T cells recruited to the kidney contribute to tissue damage in crescentic and proliferative glomerulonephritides. Chemokines and their receptors regulate T cell trafficking, but the expression profile and functional importance of chemokine receptors for renal CD4+ T cell subsets are incompletely understood. In this study, we observed that renal FoxP3+CD4+ regulatory T cells (Tregs) and IL-17-producing CD4+ T (Th17) cells express the chemokine receptor CCR6, whereas IFNgamma-producing Th1 cells are CCR6-. Induction of experimental glomerulonephritis (nephrotoxic nephritis) in mice resulted in upregulation of the only CCR6 ligand, CCL20, followed by T cell recruitment, renal tissue injury, albuminuria, and loss of renal function. CCR6 deficiency aggravated renal injury and increased mortality (from uremia) among nephritic mice. Compared with wild-type (WT) mice, CCR6 deficiency reduced infiltration of Tregs and Th17 cells but did not affect recruitment of Th1 cells in the setting of glomerulonephritis. Adoptive transfer of WT but not CCR6-deficient Tregs attenuated morphologic and functional renal injury in nephritic mice. Furthermore, reconstitution with WT Tregs protected CCR6-/- mice from aggravated nephritis. Taken together, these data suggest that CCR6 mediates renal recruitment of both Tregs and Th17 cells and that the reduction of anti-inflammatory Tregs in the presence of a fully functional Th1 response aggravates experimental glomerulonephritis.

CXCR3 Mediates Renal Th1 and Th17 Immune Response in Murine Lupus Nephritis

Infiltration of T cells into the kidney is a typical feature of human and experimental lupus nephritis that contributes to renal tissue injury. The chemokine receptor CXCR3 is highly expressed on Th1 cells and is supposed to be crucial for their trafficking into inflamed tissues. In this study, we explored the functional role of CXCR3 using the MRL/MpJ-Faslpr (MRL/lpr) mouse model of systemic lupus erythematosus that closely resembles the human disease. CXCR3−/− mice were generated and backcrossed into the MRL/lpr background. Analysis of 20-wk-old CXCR3−/− MRL/lpr mice showed amelioration of nephritis with reduced glomerular tissue damage and decreased albuminuria and T cell recruitment. Most importantly, not only the numbers of renal IFN-γ-producing Th1 cells, but also of IL-17-producing Th17 cells were significantly reduced. Unlike in inflamed kidneys, there was no reduction in the numbers of IFN-γ- or IL-17-producing T cells in spleens, lymph nodes, or the small intestine of MRL/lpr CXCR3−/− mice. This observation suggests impaired trafficking of effector T cells to injured target organs, rather than the inability of CXCR3−/− mice to mount efficient Th1 and Th17 immune responses. These findings show a crucial role for CXCR3 in the development of experimental lupus nephritis by directing pathogenic effector T cells into the kidney. For the first time, we demonstrate a beneficial effect of CXCR3 deficiency through attenuation of both the Th1 and the newly defined Th17 immune response. Our data therefore identify the chemokine receptor CXCR3 as a promising therapeutic target in lupus nephritis.

Analysis and classification of B-cell infiltrates in lupus and ANCA-associated nephritis

Intrarenal B cell infiltrates resembling secondary lymphoid tissue have been found in several forms of inflammatory kidney disease. Their role in renal inflammation is not well defined, perhaps because B cell clusters have been regarded as a single entity while being quite heterogeneous. Therefore we characterized intrarenal lymphoid clusters of 32 patients diagnosed with lupus nephritis and 16 with ANCA associated nephritis. We identified four increasingly organized levels of intrarenal aggregates from scattered B cells to highly compartmentalized B cell clusters with central follicular dendritic cell networks. Most B cells displayed a mature non-antibody producing phenotype with antigen presenting ability. In regions of B cell infiltration, expression of the lymphoid chemokine BCA-1 was found in cells of a dendritic-like morphology and most B cells expressed the corresponding receptor CXCR5. Biopsies containing B cells had significantly higher levels of BCA-1 mRNA expression compared to those without, suggesting a role of BCA-1 and CXCR5 for B cell infiltration into the kidney. Our study proposes a new classification of B cell clusters in lupus and ANCA associated nephritis which might help to study the function of intrarenal B cell clusters in a more differentiated manner.

Chemokines play a critical role in the cross-regulation of Th1 and Th17 immune responses in murine crescentic glomerulonephritis

Th1 and Th17 subtype effector CD4(+) T cells are thought to play a critical role in the pathogenesis of human and experimental crescentic glomerulonephritis. The time course, mechanism, and functions of Th1 and Th17 cell recruitment, and their potential interaction in glomerulonephritis, however, remain to be elucidated. We performed interventional studies using IL-17- and IFN-γ-gene-deficient mice, as well as neutralizing antibodies that demonstrated the importance of the Th17-mediated immune response during the early phase of the disease. At a later stage, we found that Th1 cells were critical mediators of renal tissue injury. Early recruitment of IL-17-producing Th17 cells triggered expression of the chemokine CXCL9 in the kidney that drove the infiltration of Th1 cells bearing its receptor CXCR3. At a later stage, Th1 cell-derived IFN-γ was found to inhibit local chemokine CCL20 expression, acting through its receptor CCR6 on Th17 cells, thereby limiting the renal Th17 immune response. Thus, our findings provide mechanistic evidence for a cytokine-chemokine-driven feedback loop that orchestrates the observed differential Th1 and Th17 cell infiltration into the inflamed kidney. This contributes to the observed time-dependent function of these two major pathogenic effector CD4(+) T cell subsets in crescentic glomerulonephritis.

Renal IL-17 expression in human ANCA-associated glomerulonephritis

Interleukin-17A (IL-17) promotes inflammatory renal tissue damage in mouse models of crescentic glomerulonephritis, including murine experimental autoimmune anti-myeloperoxidase glomerulonephritis, which most likely depends on IL-17-producing Th17 cells. In human anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, however, the cellular sources of IL-17 remain to be elucidated. Therefore, we analyzed human kidney biopsies of active necrotizing and crescentic ANCA-associated glomerulonephritis by immunohistochemistry using an IL-17-specific antibody and by immunofluorescent colocalization with cell type markers. We detected numerous IL-17-expressing (IL-17(+)) cells in the glomeruli and in the tubulointerstitium. Unexpectedly, most of these IL-17(+) cells were polymorphonuclear neutrophilic granulocytes, while IL-17(+) T cells and IL-17(+) mast cells were present at significantly lower frequencies. IL-17 was not detected in other infiltrating or resident kidney cells. In those patients who had not received immunosuppressive treatment before biopsy, serum creatinine levels were positively correlated with tubulointerstitial IL-17(+) neutrophils as well as IL-17(+) T cells. Furthermore, we could demonstrate that purified human blood neutrophils expressed IL-17 protein and released it upon stimulation in vitro. In conclusion, these results support a pathogenic role for IL-17 in human ANCA-associated glomerulonephritis. Our data suggest that in the acute stage of the disease neutrophils may act as an important immediate-early innate source of IL-17 and may thereby initiate and promote ongoing renal inflammation. IL-17 may thus be a target for treating acute ANCA-associated glomerulonephritis.

Deficiency of the Interleukin 17/23 Axis Accelerates Renal Injury in Mice With Deoxycorticosterone Acetate+Angiotensin II–Induced Hypertension

T cells participate in angiotensin II (Ang II)–induced hypertension. However, the specific subsets of T cells that are important in the end-organ damage are unknown. T-helper 17 cells are a recently identified subset that produces interleukin 17 (IL-17) and requires interleukin 23 (IL-23) for expansion. To evaluate the role of the T-helper 17 immune response in hypertensive renal and cardiac end-organ damage, hypertension was induced with deoxycorticosterone acetate (DOCA)+Ang II in wild-type (n=39) and IL-17–deficient (n=31) mice. The injury was evaluated at day 4 and day 14. To inactivate the IL-17/IL-23 axis at a different point, DOCA+Ang II hypertension was also induced in IL-23p19–deficient mice. Renal infiltration by T-helper 17 cells was increased in hypertensive wild-type mice. Systolic blood pressure did not differ between hypertensive IL-17–deficient and wild-type mice. Three days after induction of hypertension, a significantly higher albuminuria was found in IL-17–deficient than in wild-type mice (196±64 versus 58±16 mg/mg albumin/creatinine). Histology revealed significantly more glomerular injury (1.04±0.06 versus 0.67±0.05) and renal infiltration of &ggr;&dgr; T cells in IL-17–deficient than in wild-type mice after 14 days. Similarly, significantly higher albuminuria, glomerular injury, and &ggr;&dgr; T cell infiltration were found in IL-23p19–deficient mice with DOCA+Ang II–induced hypertension. DOCA+Ang II also induced cardiac damage as assessed by heart weight, cardiac fibrosis, as well as expression of fetal genes and matrix components, but no significant differences were found among IL-17–/–, IL-23p19–/–, and wild-type mice. IL-17/IL-23 deficiency accelerates DOCA+Ang II–induced albuminuria and hypertensive renal but not cardiac end-organ damage.

Function of the Th17/Interleukin‐17A Immune Response in Murine Lupus Nephritis

The CD4+ T cell immune response plays a pivotal role in the immunopathogenesis of human and experimental lupus nephritis, but the contribution of the Th17/interleukin‐17 (IL‐17) immune pathway to renal tissue injury in systemic lupus erythematosus (SLE) remains to be elucidated. The aim of this study was to characterize the function of the Th17/IL‐17A immune response in 2 murine models of lupus nephritis.

Immature Renal Dendritic Cells Recruit Regulatory CXCR6+ Invariant Natural Killer T Cells to Attenuate Crescentic GN

Immature renal dendritic cells (DCs) are protective early in murine crescentic GN, but the mechanisms underlying this protection are unknown. Here, depletion of DCs reduced the recruitment of invariant natural killer T (iNKT) cells, which attenuate GN, into the kidney in the early stage of experimental crescentic GN. More than 90% of renal iNKT cells expressed the chemokine receptor CXCR6, and renal DCs produced high amounts of the cognate ligand CXCL16 early after induction of nephritis, suggesting that renal DC-derived CXCL16 might attract protective CXCR6(+) iNKT cells. Consistent with this finding, CXCR6-deficient mice exhibited less iNKT cell recruitment and developed nephritis that was more severe, similar to the aggravated nephritis observed in mice depleted of immature DCs. Finally, adoptive transfer of CXCR6-competent NKT cells ameliorated nephritis. Taken together, these results suggest an immunoprotective mechanism involving immature DCs, CXCL16, CXCR6, and regulatory iNKT cells, which might stimulate the development of new therapeutic strategies for GN.

Early-Onset and Robust Amyloid Pathology in a New Homozygous Mouse Model of Alzheimer's Disease

Background Transgenic mice expressing mutated amyloid precursor protein (APP) and presenilin (PS)-1 or -2 have been successfully used to model cerebral β-amyloidosis, one of the characteristic hallmarks of Alzheimers disease (AD) pathology. However, the use of many transgenic lines is limited by premature death, low breeding efficiencies and late onset and high inter-animal variability of the pathology, creating a need for improved animal models. Here we describe the detailed characterization of a new homozygous double-transgenic mouse line that addresses most of these issues. Methodology/Principal Findings The transgenic mouse line (ARTE10) was generated by co-integration of two transgenes carrying the K670N/M671L mutated amyloid precursor protein (APPswe) and the M146V mutated presenilin 1 (PS1) both under control of a neuron-specific promoter. Mice, hemi- as well as homozygous for both transgenes, are viable and fertile with good breeding capabilities and a low rate of premature death. They develop robust AD-like cerebral β-amyloid plaque pathology with glial inflammation, signs of neuritic dystrophy and cerebral amyloid angiopathy. Using our novel image analysis algorithm for semi-automatic quantification of plaque burden, we demonstrate an early onset and progressive plaque deposition starting at 3 months of age in homozygous mice with low inter-animal variability and 100%-penetrance of the phenotype. The plaques are readily detected in vivo by PiB, the standard human PET tracer for AD. In addition, ARTE10 mice display early loss of synaptic markers and age-related cognitive deficits. By applying a γ-secretase inhibitor we show a dose dependent reduction of soluble amyloid β levels in the brain. Conclusions ARTE10 mice develop a cerebral β-amyloidosis closely resembling the β-amyloid-related aspects of human AD neuropathology. Unifying several advantages of previous transgenic models, this line particularly qualifies for the use in target validation and for evaluating potential diagnostic or therapeutic agents targeting the amyloid pathology of AD.

AT1 antagonism and renin inhibition in mice: pivotal role of targeting angiotensin II in chronic kidney disease

The role of the renin-angiotensin system in chronic kidney disease involves multiple peptides and receptors. Exerting antipodal pathophysiological mechanisms, renin inhibition and AT(1) antagonism ameliorate renal damage. However, it is unclear which mechanism exerts better nephroprotection. We compared the renin inhibitor aliskiren with the AT(1) antagonist losartan in mice with chronic kidney disease due to renal ablation. Doses were adjusted to equipotent inhibition of the renin-angiotensin system, determined via a dose-response quantifying plasma and renal renin expression. Six-week treatment with either 500 mg/l drinking water losartan or 50 mg·kg(-1)·day(-1) aliskiren significantly decreased albuminuria, glomerular damage, and transcription rates of renal injury markers to a similar extent. An array analysis comparing renal gene expression of losartan- and aliskiren-treated mice evaluating >34,000 transcripts demonstrated regulation for 14 genes only, with small differences. No superior nephroprotection was found by combining losartan and aliskiren. Compared with plasma concentrations, aliskiren accumulated ∼7- to 29-fold in the heart, liver, lung, and spleen and ∼156-fold in the kidney. After withdrawal, plasma concentrations dropped to zero within 24 h, whereas renal tissue concentrations declined slowly over days. Withdrawal of aliskiren in mice with chronic kidney disease revealed a significantly delayed re-increase in albuminuria compared with withdrawal of losartan. This study demonstrates equieffective nephroprotection of renin inhibition and AT(1) antagonism in mice with chronic kidney disease without additional benefit of combination therapy. These observations underscore the pivotal role of targeting ANG II to reduce renal injury.