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

Abstract

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 IFNγ-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.

The currently defined four subsets of CD4⁺ T cells—namely Th1, Th2, Th17, and regulatory T cells (Tregs)—are central players in adaptive immunity.¹ Inappropriate or unbalanced T cell responses underlie several distinct types of autoimmune diseases, some of which target the kidney. In particular, infiltrating effector CD4⁺ T cells of the Th1 type are supposed to initiate and perpetuate glomerular and tubulointerstitial tissue damage in crescentic and proliferative forms of glomerulonephritis either directly by cytotoxic functions or cytokine secretion or indirectly by providing help for induction of autoantibodies and cytokines or immune complexes or by activating macrophages.² Another IL-17–producing CD4⁺ effector T cell subset, termed Th17, has been identified.³ Ongoing studies demonstrate that Th17 cells are involved in driving autoimmune processes previously thought to be exclusively Th1-mediated, such as rheumatoid arthritis,⁴ multiple sclerosis,⁵ and crescentic glomerulonephritis.^6,7 In contrast, the Th2 cell–mediated immune response seems to be of importance in nonproliferative forms of glomerulonephritis, such as minimal-change and membranous nephropathy.² In rodents and humans, a unique subset of CD4⁺CD25⁺FoxP3⁺ Tregs has been shown to control peripheral tolerance. An anti-inflammatory role of Tregs has also been suggested in human and experimental glomerulonephritis^8,9; however, the molecular basis of immunosuppression and the trafficking properties of Tregs are still unknown.

Before T cells can exert their effects on renal damage or repair, they have to reach the site of injury. In recent years, it has become clear that a group of small proteins called chemokines serve as key regulators of directional T cell trafficking under inflammatory conditions.¹⁰ Target cell specificity is achieved by differential expression of corresponding chemokine receptors on the surface of leukocyte subsets. The different CD4⁺ T cell populations in humans and mice display distinct patterns of chemokine receptor expression. Th1-polarized cells preferentially express CXCR3, CCR5, and CXCR6, whereas Th2 cells express higher amounts of CCR3, CCR4, and CCR8.¹⁰ Although CCR6 and CXCR3 have been detected on Th17 cells,^11–13 the chemokine receptor expression profile of this subset has yet to be defined, particularly with respect to functional importance. Tregs with potential anti-inflammatory properties express a wider repertoire of chemokine receptors, many of which they share with proinflammatory T cell subsets, such as CCR4, CCR5, CCR6, CCR8, CXCR3, and CXCR6.^10,13 It remains unclear, however, which of these chemokine receptors (or which receptor combination) is crucial for guiding Tregs to the site of inflammation. In general, the precise molecular basis for the chemokine/chemokine receptor-mediated trafficking of CD4⁺ T cell subsets in glomerulonephritis is not defined.

Several features of CCR6 and its only highly specific ligand CCL20 argue for a critical role of this chemokine–chemokine receptor pair in this context. CCR6 is expressed in human and mouse dendritic cell subpopulations,^14–16 B cells,^17,18 and T cell subsets.^13,19 Recent data suggest that, in particular, autoreactive Th17 cells, which contribute to renal tissue injury in nephritis, are highly CCR6⁺.^11,12 This study was designed to examine the potential role of CCR6 in glomerulonephritis. We therefore induced the T cell–dependent model of nephrotoxic nephritis (NTN) in wild-type (WT) and CCR6^−/− mice to address two major issues: (1) What is the expression pattern of CCR6 on infiltrating renal CD4⁺ T cell subsets? (2) Does CCR6 deficiency influence the clinical course of experimental glomerulonephritis, and, if so, what are the mechanisms?

Results

CCR6 Expression on Renal T Cell Subsets

In a first step, we analyzed CCR6 expression on renal T helper cell subsets in C57BL/6 WT mice with NTN. FACS analysis of isolated renal leukocytes stained for intracellular cytokines, and extracellular markers revealed that approximately 60% of CD4⁺IL-17⁺ T cells (Th17 cells) were CCR6⁺. In contrast, CD4⁺IFNγ⁺ T cells (Th1 cells) were largely CCR6⁻ (Figure 1A). Furthermore, we assessed CCR6 expression on intrarenal Tregs by FACS analysis for the transcription factor FoxP3. Approximately 20% of renal CD4⁺FoxP3⁺ T cells showed CCR6 expression (Figure 1B). The specificity of CCR6 staining was demonstrated by simultaneous analysis of isolated renal leukocytes from nephritic CCR6^−/− mice. In contrast to renal CD4⁺ T helper cells, infiltrating cytotoxic CD8⁺ T cells did not express CCR6 (Figure 1C).

Figure 1: CCR6 is expressed on intrarenal T cell subsets. (A) Representative FACS analysis of isolated renal leukocytes for CCR6 expression on Th1 and Th17 cells at day 7 of NTN. All plots are gated for CD4⁺ T cells. Further gating on IL-17⁺ and IFNγ⁺ cells shows differential expression of CCR6. (B) FACS analysis of intrarenal Tregs for CCR6 expression at day 7 of NTN. Left plot is gated for CD3⁺ events. Further gating on CD4⁺ FoxP3⁺ cells shows CCR6 expression on 20% of the cells of this subset. Analysis of renal leukocytes from CCR6^−/− mice demonstrates specificity of the CCR6 staining. (C) Gating on renal CD8⁺ T cells shows absence of CCR6 expression on this T cell subset. Numbers refer to positive events in percentage of gated events. All experiments were repeated three times.

Induction of CCL20 Expression in NTN

An important prerequisite for a role of CCR6 in renal T cell trafficking is upregulation of the only known CCR6 ligand, CCL20, during renal inflammation. Quantitative reverse transcriptase–PCR (RT-PCR) analysis of renal cortex at various time points after injection of the nephrotoxic sheep serum showed strong induction of renal CCL20 expression in the course of NTN. CCL20 mRNA was already upregulated at day 1 (28-fold of basal expression), showed an expression peak at day 7 (386-fold), and remained elevated until day 14 (252-fold; Figure 2A). To identify the renal compartments contributing to the strong expression of CCL20 mRNA in NTN, we performed in situ hybridization experiments. CCL20 expression localized to tubulointerstitial infiltrates and glomerular cells. Sporadically, CCL20 expression was also detectable in epithelial cells of dilated tubules. CCL20 mRNA was detected only in low levels in non-nephritic control mice (Figure 2B). To characterize the renal distribution of CCL20 expression on the protein level, we performed immunohistochemical staining of paraffin-embedded kidney sections of mice with NTN and controls (Figure 2C). In line with the results from in situ hybridization, CCL20 protein was primarily detected in mononuclear cells of the interstitial and periglomerular infiltrates. Positive staining of intraglomerular cells was restricted to infiltrating cells, whereas podocytes and mesangial cells were largely negative (Figure 2C, middle). Some tubular epithelial cells were also positive for CCL20 protein (Figure 2C, right).

Figure 2: CCL20 expression is induced in NTN. (A) Real-time RT-PCR analysis of renal CCL20 expression in WT mice at various time points after induction of NTN (n = 4 to 5 per time point). mRNA level is expressed as fold of non-nephritic controls. Symbols represent means ± SD. (B) Representative photographs of in situ hybridizations with a specific cRNA probe for CCL20 of nephritic mice and non-nephritic controls. Nephritic WT mice show CCL20 mRNA-expressing cells in the glomeruli (arrows), within the tubulointerstitial infiltrates (arrowheads), and in dilated tubular cells (inset). (C) Representative immunohistochemistry for CCL20. Magnification, ×400.

Renal Phenotype of CCR6^−/− Mice

Because CCR6 deficiency has been shown to influence homeostatic immune cell trafficking and organization of lymphoid tissue in the gut,^14,15 we analyzed 10-week-old C57BL/6 CCR6^−/− mice for structural and functional renal defects. Comparison of periodic acid-Schiff (PAS)-stained kidney sections, blood urea nitrogen (BUN) levels, and albuminuria in healthy WT and CCR6^−/− mice showed no signs of renal alterations in the setting of CCR6 deficiency (data not shown). Comparison of the genetic background of C57BL/6 CCR6^−/− and C57BL/6 WT mice, which served as controls in all experiments by genome-wide analysis of single-nucleotide polymorphisms, demonstrated a >99% match among the two C57BL/6 strains (data not shown).

Aggravated NTN in CCR6^−/− Mice

To gain further insight into the functional role of CCR6 in cell-mediated renal tissue damage, we induced NTN in C57BL/6 WT and C57BL/6 CCR6^−/− mice. Examination of PAS-stained kidney sections in the T cell–mediated autologous phase at day 7 after injection of the nephritogenic sheep serum revealed severe focal glomerular and tubulointerstitial damage. Glomerular alterations included hypercellularity and formation of cellular crescents, capillary aneurysms, and intraglomerular deposition of PAS-positive material. The tubulointerstitial compartment showed abundant leukocyte infiltration and focal destruction of the regular tissue structure with tubular dilation, necrosis of tubular epithelial cells, and intratubular protein casts (Figure 3A).

Figure 3: Nephritis is aggravated in CCR6^−/− mice. (A) Representative photographs of PAS-stained kidney sections of WT and CCR6^−/− mice at day 7 of NTN show glomerular crescent formation (arrows), glomerular deposition of PAS-positive material (arrowheads), and abundant tubulointerstitial leukocyte infiltrates (*). (B) Quantification of glomerular crescent formation in nephritic WT mice (n = 17), nephritic CCR6^−/− (n = 20) mice at day 7 of NTN, and non-nephritic WT controls (n = 14). (C) BUN levels (left) and albumin-to-creatinine ratio (right) of nephritic WT mice (n = 18 and 9, respectively), nephritic CCR6^−/− mice (n = 16 and 13, respectively) at day 7 of NTN, and non-nephritic WT controls (n = 12 and 9, respectively). Symbols represent individual data points, and horizontal lines indicate median values. *P < 0.05; **P < 0.01. (D) Kaplan-Meier survival analysis of WT (n = 23) and CCR6^−/− mice (n = 29) with NTN. Magnification, ×400.

For quantification of glomerular and tubular tissue damage, PAS-stained kidney sections were evaluated as described previously.²⁰ The frequency of glomerular crescent formation at day 7 of NTN was significantly increased in CCR6^−/− mice compared with WT controls (non-nephritic controls 0.2 ± 0.8%; WT 30.8 ± 7.3%; CCR6^−/− 37.8 ± 7.6%; P < 0.01; Figure 3B). Furthermore, nephritic kidneys showed a high percentage of glomerular sclerosis and considerable tubulointerstitial damage. The quantification of glomerular sclerosis and interstitial area, as an indicator of tubulointerstitial injury, showed no significant difference between the two nephritic groups (data not shown).

The impairment of renal function as a result of cell-mediated kidney injury was assessed by measurement of BUN levels and albuminuria (Figure 3C). The BUN level and urinary albumin excretion were significantly increased in nephritic mice compared with non-nephritic controls (P < 0.001). At day 7 of NTN, we found a significant increase in BUN in nephritic CCR6^−/− mice (150 ± 86 mg/dl) compared with their WT counterparts (97 ± 56 mg/dl; P < 0.05). The albumin-to-creatinine ratio was not significantly different between nephritic WT (527 ± 336 g/g) and CCR6^−/− mice (440 ± 293 g/g).

As a consequence of decline in GFR and urinary protein loss, mice with NTN may develop edema, ascites, and moderate to severe clinical impairment. Lethality of WT mice as a result of uremia was 19% (n = 23 in three independent experimental sets). In CCR6^−/− mice, development of severe clinical disease with massive edema and ascites was more common and lethality was increased to 41% (n = 29 in three independent experimental sets; log rank test P = 0.08 [NS] versus WT; Figure 3D).

Renal T Cell Infiltration in CCR6^−/− Mice

To determine whether expression of the CCR6 receptor on T cells has functional relevance for their recruitment to the inflamed kidney, we performed immunohistochemistry of kidney sections for the pan T cell marker CD3 in nephritic WT and CCR6^−/− mice. At day 7 of NTN, nephritic mice showed abundant renal infiltration of CD3⁺ T cells, predominantly in the interstitial and periglomerular area. Also, the frequency of intraglomerular T cells in nephritic mice was markedly increased compared with non-nephritic controls. In comparison with WT mice, interstitial and periglomerular T cell infiltrates in CCR6^−/− mice seemed to be notably reduced (Figure 4A). The quantification of CD3⁺ cells in CCR6^−/− mice with NTN revealed a significant decrease in tubulointerstitial T cell infiltration compared with nephritic WT mice (WT 26.9 ± 7.1/high-power field [hpf]; CCR6^−/− 16.9 ± 3.9/hpf; P < 0.01; Figure 4B). The infiltration of T cells in the glomerular compartment, in contrast, was only slightly reduced in CCR6^−/− mice (WT 0.97 ± 0.42/glomerular cross-section (gcs); CCR6^−/− 0.63 ± 0.37/gcs; Figure 4B).

Figure 4: Recruitment of Th17 cells and Tregs to the kidney is reduced in CCR6^−/− mice. (A) Representative photographs of kidney sections from nephritic WT and CCR6^−/− mice at day 7 of NTN immunohistochemically stained for the pan-T cell marker CD3 (magnification 400x). (B) Quantification of tubulointerstitial and glomerular CD3⁺ T cells in nephritic WT (n = 12) and CCR6^−/− mice (n = 9), as well as non-nephritic WT controls (n = 10). (C) Representative FACS analysis of isolated renal leukocytes stained for intracellular IFNγ and IL-17 from nephritic WT and CCR6^−/− mice at day 7 of NTN (left). Plots are gated for CD4⁺ T cells. The quantification (right) shows a significantly reduced frequency of Th17 cells in CCR6^−/− mice, whereas Th1 cell frequency tends to be increased (n = 6 to 7 per group pooled from two to three mice). (D) Real-time RT-PCR analysis of renal cytokine and chemokine expression in nephritic WT and CCR6^−/− mice, as well as non-nephritic WT controls (n = 9 to 18 per group). mRNA level is expressed as fold of non-nephritic controls. (E) FoxP3 immunohistochemistry of kidney sections from nephritic WT and CCR6^−/− mice at day 7 of NTN. (F) Quantification of tubulointerstitial FoxP3⁺ cells in nephritic WT (n = 13) and CCR6^−/− mice (n = 9), as well as non-nephritic WT controls (n = 9). (G) Real-time RT-PCR of FoxP3 expression in nephritic WT (n = 15) and CCR6^−/− mice (n = 18), as well as non-nephritic WT controls (n = 18). Symbols represent individual data points, and horizontal lines indicate median values. *P < 0.05; **P < 0.01. Magnification, ×400.

Role of CCR6 in Renal Th1, Th17, and Treg Recruitment

Because CCR6 expression was found on pro- and anti-inflammatory renal T helper cell subsets (see Figure 1), a more detailed characterization of the T cell populations contributing to the reduction in overall T cell infiltration in CCR6^−/− mice was indispensable; therefore, in a next step, we aimed to characterize the relative distribution of Th1, Th17, and Tregs in the kidneys of WT and CCR6^−/− mice with NTN. The staining of isolated renal leukocytes for the intracellular marker cytokines IL-17 and IFNγ and extracellular T cell markers (CD3 and CD4) with subsequent FACS analysis showed that the frequency of IL-17–producing CD4⁺ T cells was significantly reduced in nephritic kidneys of CCR6^−/− mice compared with WT controls (WT 8.4 ± 5.4%; CCR6^−/− 2.1 ± 0.3%; P < 0.01; Figure 4C). IFNγ-producing CD4⁺ T cells, in contrast, tended to be increased in CCR6^−/− mice (WT 9.4 ± 5.2%; CCR6^−/− 16.1 ± 9.2%; P > 0.05 [NS]; Figure 4C). Next, we performed real-time RT-PCR analysis of renal cortex from CCR6^−/− and WT mice. Supporting the protein data obtained by FACS analyses, renal mRNA expression of IL-17 was significantly decreased in CCR6^−/− mice compared with their WT counterparts (WT 32.3-fold; CCR6^−/− 2.9-fold; P < 0.01; Figure 4D), whereas IFNγ production was not differentially regulated between the two groups (WT 2.0-fold; CCR6^−/− 2.9-fold; Figure 4D). Furthermore, upregulation of the CCR6 ligand CCL20 was substantially reduced in CCR6^−/− mice (WT 564.9-fold; CCR6^−/− 233.2-fold; P < 0.05; Figure 4D), probably as a result of the reduction of intrarenal Th17 cells, which are a major source of CCL20.¹¹

The renal infiltration of Tregs was assessed by immunohistochemical staining of paraffin-embedded kidney sections for the Treg transcription factor FoxP3 (Figure 4E). FoxP3⁺ cells were predominantly found in the periglomerular and tubulointerstitial compartment, and their number was clearly increased in nephritic kidneys. Quantification of FoxP3⁺ cells in the tubulointerstitium demonstrated a significant reduction of Treg infiltration in CCR6^−/− mice compared with nephritic WT mice (WT 5.9 ± 2.1/low-power field [lpf]; CCR6^−/− 2.7 ± 1.7/lpf; P < 0.01; Figure 4F). Again, these protein-based data were supported by real-time RT-PCR analysis of renal cortex, which showed a similar decrease of FoxP3 mRNA expression in CCR6^−/− mice (WT 35.4-fold; CCR6^−/− 14.4-fold; P < 0.01; Figure 4G). FACS analysis showed a 30 to 40% reduction of the frequency of renal FoxP3⁺ CD4⁺ cells in nephritic CCR6^−/− mice when compared with the nephritic WT group (data not shown).

Renal Cytokine Milieu in Nephritic CCR6^−/− Mice

To address the question of whether the modified trafficking properties of CD4⁺ T cell subsets in CCR6^−/− mice translate to changes in the renal cytokine milieu, we analyzed mRNA expression of pro- and anti-inflammatory mediators (IL-10 and IL-35 versus TNF-α, CCL2, CCL5, and CXCL10). Although there was a tendency for a reduced expression of IL-10 and IL-35 in CCR6^−/− mice, these changes were not significantly different in WT and CCR6^−/− mice (Supplemental Figure 1).

Role of CCR6 in Renal Recruitment of Other Leukocyte Subsets

Because CCR6 is also expressed on macrophages, dendritic cells,^14–16 and B cells,^17,18 we were interested in studying the potential effect of CCR6 deficiency on renal infiltration of these leukocyte subsets; therefore, we performed immunohistochemical staining of kidney sections for F4/80 (interstitial monocytes and dendritic cells), MAC-2 (glomerular monocytes), and B220 (B cells). Quantification of F4/80⁺, MAC-2⁺, and B220⁺ cells showed no significant difference between nephritic WT and CCR6^−/− mice (Supplemental Figure 2).

Systemic Immune Response in CCR6^−/− Mice

To address the question of whether CCR6^−/− mice have an altered systemic cellular immune response, we analyzed splenocytes from nephritic WT and CCR6^−/− mice for production of IFNγ and IL-17. CD4-gated FACS plots of T cells from the spleen showed similar frequencies of Th1 and Th17 cells in both experimental groups (IL-17⁺: WT 0.5 ± 0.1%, CCR6^−/− 0.5 ± 0.1%; IFNγ⁺: WT 4.4 ± 2.6%, CCR6^−/− 5.3 ± 4.2%; Figure 5A). Additional FACS analyses showed that the presence of FoxP3⁺CD4⁺ Tregs within the renal lymph node, where the nephritogenic immune response is assumed to be initiated, was not significantly different between nephritic WT and CCR6^−/− mice (WT 14.4 ± 4.0%; CCR6^−/− 13.9 ± 3.4%; Figure 5B).

Figure 5: Systemic immune response occurs in CCR6^−/− mice. (A and B) Representative FACS analyses for IFNγ- and IL-17–producing T cells in the spleen (A) and FoxP3⁺ T cells in renal lymph nodes (B) of nephritic WT and CCR6^−/− mice at day 7 of NTN. Plots are gated on CD4⁺CD3⁺ events (A) and CD3⁺ events (B), respectively. Numbers refer to positive events in percentage of all gated events. (C and D) Representative photographs of immunohistochemical staining for sheep IgG (C) and total mouse IgG (D) in nephritic WT and CCR6^−/− mice. (E) Semiquantitative scoring of glomerular mouse IgG deposition in nephritic WT (n = 12) and CCR6^−/− mice (n = 9), as well as in non-nephritic WT controls (n = 10). (F) ELISA analysis of serum samples from nephritic WT (n = 13 to 15) and CCR6^−/− mice (n = 12 to 15) at day 7 of NTN for sheep IgG-specific total mouse IgG and IgG subclasses at the indicated dilutions. Symbols represent individual data points, and horizontal lines indicate median values. *P < 0.05; **P < 0.01.

The systemic humoral immune response to sheep IgG is another important premise for initiation of the autologous phase of NTN.² In a first step, we verified by immunohistochemistry of kidney sections for sheep IgG that glomerular deposition of the injected sheep antibody was comparable in WT and CCR6^−/− mice (Figure 5C). Next, we performed immunohistochemical staining of mouse IgG to evaluate the amount of endogenous antibody deposition in injured glomeruli (Figure 5D). Semiquantitative scoring of glomerular mouse IgG showed significantly increased deposition in CCR6^−/− mice (WT 1.25 ± 0.31; CCR6^−/− 1.79 ± 0.70; P < 0.05; Figure 5E). For a more precise description of the IgG antibody response directed against the nephritogenic antigen, we conducted ELISA analyses of serum samples for sheep IgG-specific mouse IgG subclasses (Figure 5F). In line with the results from immunohistochemistry, CCR6^−/− mice displayed significantly increased levels of total IgG, IgG1, and IgG2a 7 days after injection of the nephrotoxic sheep serum (total IgG [1:100]: WT 0.13 ± 0.04, CCR6^−/− 0.19 ± 0.09; IgG1 [1:20]: WT 0.45 ± 0.24, CCR6^−/− 1.06 ± 0.67; IgG2a [1:500]: WT 0.48 ± 0.10, CCR6^−/− 0.68 ± 0.24; P < 0.05 for all; Figure 5F).

Functional Analysis of CCR6^−/− and CCR6-Competent Tregs

One possible explanation for the deleterious phenotype of CCR6^−/− mice in NTN is impaired trafficking of protective Tregs in presence of an unhindered Th1 response. To prove this hypothesis, we performed transfer of WT and CCR6^−/− CD4⁺CD25⁺ Tregs isolated from the spleens of naive mice to WT mice with NTN (Figure 6).

Figure 6: In vivo function of Tregs depends on CCR6. (A) FACS analysis of Tregs isolated from the spleen of naive WT and CCR6^−/− mice for T cell activation markers, the integrin CD103 and CCR6 expression, gated on CD4⁺CD25⁺ T cells. Numbers represent percentage of events from all gated events, representative of three experiments. (B and C) Quantification of glomerular crescent formation and parameters of renal function from nephritic WT mice that received PBS (n = 3 to 4), WT Tregs (n = 3), or CCR6^−/− Tregs (n = 4) both isolated from naive mice and in non-nephritic controls (n = 4). Symbols represent individual data points and the horizontal lines indicate median values. *P < 0.05. (D) ELISA analysis of supernatants from CD3-stimulated T cell co-cultures with CCR6^−/− or CCR6-competent Tregs of nephritic mice and WT CD4⁺ responder T cells of naive mice isolated from the spleen (n = 3 per group). Bars represent means, and error bars indicate SD. **P < 0.01.

FACS analysis of the isolated CD4⁺CD25⁺ Tregs after intracellular FoxP3 staining revealed that >95% of the isolated cells were FoxP3⁺ (data not shown). To address the question of whether CCR6 deficiency alters the baseline activation of Tregs, we performed FACS analysis for a set of T cell activation markers. The analysis revealed that the isolated T cell populations from CCR6^−/− and WT mice showed similar percentages of CD62L⁻ cells, CD69⁺ cells, and CD44^high cells (Figure 6A). Furthermore, expression of the integrin CD103, a Treg marker for the capacity to access peripheral tissues during inflammation, was not different between the groups. Approximately 12% of isolated Tregs were CCR6⁺.

Transfer of 2 × 10⁶ naive WT Tregs to WT mice 24 hours before injection of the nephritogenic sheep serum substantially ameliorated the course of NTN, as indicated by reduced histopathologic kidney damage and decreased impairment of renal function (Figure 6, B and C). A similar number of CCR6^−/− Tregs, however, were unable to reduce crescent formation or the increase of BUN and albuminuria (Figure 6, B and C). Evaluation of PAS-stained kidney sections for the frequency of crescentic glomeruli showed decreased numbers in mice that received WT Tregs (WT+PBS 40.0 ± 7.2%; WT+WT Tregs 17.8 ± 8.4%), whereas WT mice that received CCR6^−/− Tregs showed no amelioration of tissue damage (WT+CCR6^−/− Tregs 44.2 ± 6.3%; P < 0.01 versus WT+WT Tregs; Figure 6B). In line with the results from histopathology, transfer of WT Tregs ameliorated the increase of BUN (WT+PBS 155 ± 96 mg/dl; WT+WT Tregs 52 ± 17 mg/dl) and urinary protein excretion (WT+PBS 131 ± 57 g/g; WT+WT Tregs 34 ± 32 g/g), whereas transfer of CCR6^−/− Tregs did not improve parameters of renal function (BUN: WT+CCR6^−/− Tregs 112 ± 39 mg/dl, P = 0.05 [NS] versus WT+WT Tregs; albumin-to-creatinine ratio: WT+CCR6^−/− Tregs 119 ± 126 g/g; Figure 6C).

To evaluate the immunosuppressive capacity of CCR6^−/− Tregs from nephritic mice compared with their nephritic WT counterparts, we performed T cell co-culture experiments (Figure 6D). ELISA analysis of supernatants from co-cultures of splenic Tregs and CD4⁺ responder T cells showed that CCR6^−/− Tregs were able to suppress production of proinflammatory IL-2 by responder cells to a similar degree as CCR6-competent Tregs (WT responder cells 1939 ± 108 pg/ml; WT responder cells + WT Tregs 383 ± 68 pg/ml; WT responder cells + CCR6^−/− Tregs 550 ± 128 pg/ml; P > 0.05 [NS]; Figure 6D). Furthermore, the production of anti-inflammatory IL-10 by responder cells was comparably stimulated by CCR6^−/− and WT Tregs (WT responder cells 221 ± 16 pg/ml; WT responder cells + WT Tregs 1685 ± 276 pg/ml; WT responder cells + CCR6^−/− Tregs 1581 ± 295 pg/ml; P > 0.05 [NS]; Figure 6D).

Attenuation of Nephritis in CCR6^−/− Mice by Reconstitution with WT Tregs

To provide further evidence that the aggravated phenotype of CCR6^−/− mice in NTN is a result of a defect in Treg function, we reconstituted CCR6^−/− mice with 2 × 10⁶ WT Tregs isolated from naive mice 24 hours before induction of NTN. The transfer of WT Tregs partially protected CCR6^−/− mice from crescent formation (CCR6^−/− + PBS 26.7 ± 6.1%; CCR6^−/− + WT Tregs 11.1 ± 7.7%; P < 0.05; Figure 7), loss of renal function (BUN: CCR6^−/− + PBS 50 ± 10 mg/dl, CCR6^−/− + WT Tregs 27 ± 5 mg/dl; P < 0.05; Figure 7), and development of albuminuria (CCR6^−/− + PBS 107 ± 83 g/g; CCR6^−/− + WT Tregs 1.4 ± 1.8 g/g; P > 0.05 [NS]; Figure 7). Interestingly, this amelioration of nephritis was independent of the humoral immune response against the nephritogenic antigen. The augmented production of sheep IgG-specific antibodies and their enhanced deposition in the glomeruli, which was found in CCR6^−/− mice with NTN (see Figure 5), was not significantly reduced by reconstitution with WT Tregs (Supplemental Figure 3).

Figure 7: WT Tregs are transferred to CCR6^−/− mice. Quantification of glomerular crescent formation (left), BUN levels (middle), and albumin-to-creatinine ratio (right) of CCR6^−/− mice that received either PBS (n = 4) or WT Tregs (n = 3) analyzed at day 7 after induction of NTN. Symbols represent individual data points, and horizontal lines indicate median values. *P < 0.05.

Discussion

In the face of an expanding array of functionally diverse T helper cell subsets, it is of great importance to identify the differential factors that are responsible for recruitment of proinflammatory and potentially pro-resolving T cell populations to the target organ in autoimmune disease. Preventing renal infiltration of harmful T cells while keeping migration of protective T cells intact seems to be a potential approach for treatment of human glomerulonephritis. Such a selective control of T cell migration under inflammatory conditions could possibly be achieved by targeting of T helper cell lineage–specific chemokine receptors.

In this study, we investigated the function of the chemokine receptor CCR6, which has been found to be highly expressed by human and rodent Th17 cells.^11,12,21 Because Th17 cells have been shown to contribute significantly to renal tissue injury in experimental glomerulonephritis,^6,7 CCR6 might be a promising target for interference with renal infiltration of this effector T cell population.

As hypothesized, CCR6 was abundantly expressed on intrarenal Th17 cells in an experimental model of crescentic glomerulonephritis (NTN), whereas IFNγ-producing Th1 cells were CCR6⁻. Interestingly, CCR6 was also detectable on renal Tregs. Further supporting a putative role for CCR6 in renal T cell trafficking, we found profoundly upregulated renal mRNA and protein levels of the only CCR6 ligand CCL20 in NTN. To provide functional evidence, we induced this T cell–dependent model of nephritis in C57BL/6 WT and C57BL/6 CCR6^−/− mice. In contrast to our expectations, CCR6^−/− mice developed aggravated glomerulonephritis in terms of glomerular crescent formation, BUN level, and mortality as a result of uremia compared with WT controls. The quantification of renal T cell infiltration by immunohistochemical CD3 staining showed a reduced overall T cell infiltration. This observation suggests that in addition to the numeric amount of infiltrating T cells, the balance of pro- and anti-inflammatory CD4⁺ T cell subsets might be of central importance for renal injury. The detailed characterization of renal T helper cell subsets in CCR6^−/− mice showed a substantial reduction in both potentially pathogenic Th17 cells and potentially protective Tregs, whereas proinflammatory Th1 cells remained unaltered. We hypothesized that the decreased presence of anti-inflammatory Tregs in the inflamed kidney in the presence of a functionally intact Th1 response might be causative for the deleterious phenotype of CCR6^−/− mice in NTN even though the numbers of renal Th17 cells were also reduced.

Evidence of a potential role of Tregs in renal inflammation comes from the observation that numbers of circulating Tregs and kidney disease activity are correlated in patients with anti–glomerular basement membrane glomerulonephritis.⁹ Furthermore, the potential of Tregs to suppress the pathogenic immune responses in kidney disease was proved in a model of crescentic glomerulonephritis in mice. The adoptive transfer of Tregs was shown to inhibit the generation of a systemic nephritogenic Th1 immune response in secondary lymphoid organs, including renal lymph nodes.⁸ Very recently, it was shown by the same group of investigators²² that the chemokine receptor CCR7 is crucial for homing of Tregs to renal lymph nodes, where they downregulate the nephritogenic immune response. Whereas intrarenal Tregs could not be detected in the aforementioned study,⁸ here we could clearly demonstrate the presence of Tregs in the kidney of nephritic animals by FACS analysis for FoxP3⁺CD4⁺ T cells and immunohistochemical FoxP3 staining. These findings suggest that, in addition to the downregulation of the nephritogenic immune response in secondary lymphoid organs, Tregs migrate to the kidney and might locally suppress immune-mediated renal inflammation.²³ Aggravation of renal disease in CCR6^−/− mice might thus be caused by impaired trafficking of Tregs either to the place of T cell priming in lymphoid tissue or to the inflamed target organ. In our experiments, however, the frequency of Tregs in renal lymph nodes was unchanged in CCR6^−/− mice, arguing against a predominant role of CCR6 for trafficking of Tregs to the secondary lymphoid organs.

Alternatively, lack of CCR6 could directly cause defects of Treg activation or immunosuppressive capacity; however, our in vitro studies demonstrated that CCR6^−/− Tregs showed similar activation status and were equipotent with their WT counterparts in inhibiting proinflammatory cytokine production by anti-CD3–stimulated T cells. To test further whether lack of CCR6 impairs Treg trafficking in vivo, we performed adoptive transfer experiments of WT and CCR6^−/− Tregs to nephritic animals. In line with our hypothesis, these experiments showed significant improvement of renal disease only in animals that were administered an injection of WT but not in those supplemented with CCR6^−/− Tregs. Furthermore, reconstitution of nephritic CCR6^−/− mice with WT Tregs reduced renal tissue injury, underscoring the importance of Treg dysfunction in CCR6^−/− mice.

Further evidence for a critical role of CCR6 in Treg trafficking comes from another study that was published independently during preparation of this manuscript. Villares et al.²⁴ showed that CCR6^−/− mice develop aggravated chronic experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis, as a result of the impaired infiltration of CCR6^−/− Tregs into the inflamed central nervous system (CNS). Another group of investigators²⁵ demonstrated that a defective function of CCR6^−/− dendritic cells with a regulatory phenotype also contributes to the increased susceptibility of CCR6^−/− mice to severe chronic EAE. In that study, despite severe neuroinflammation, Th17 cells in the CNS showed a tendency to reduction, whereas Th1 cells were slightly increased.²⁵ In another experimental model of intestinal inflammation induced by transfer of T helper cells to mice with severe combined immunodeficiency, CCR6^−/− Th17 cells were able to induce excessive intestinal inflammation.²⁶ The inflamed intestinal tissue contained increased numbers of Th1 cells with simultaneously reduced Treg and Th17 cell infiltration.²⁶ Taken together, these data support the hypothesis that the balance between CD4⁺ effector T cell populations is decisive for the control or escalation of autoimmune responses in various target organs. Another level of complexity was added to this issue by several recent studies that suggested a previously unrecognized flexibility in T cell lineage commitment.²⁷ It has become increasingly clear that FoxP3-expressing Tregs can locally convert into IL-17– or IFNγ-producing pathogenic effector T cells.²⁸

Whereas CCR6^−/− mice developed more severe EAE in the aforementioned studies, three other recently published studies found opposite effects of CCR6 deficiency on the clinical outcome in models of encephalitis. In two of those reports,^29,30 the authors were able to demonstrate that CCR6 expression on Th17 cells is critical for development of EAE. Whereas the initial site-specific entry of Th17 cells via the choroids plexus into the uninflamed CNS was CCR6 dependent, leukocyte infiltration was CCR6 independent at later stages of disease.³⁰ Furthermore, Liston et al.³¹ demonstrated that CCR6 drives the priming phase of EAE by mediating the influx of dendritic cells into and the egress of activated lymphocytes from the draining lymph nodes of the CNS. Both of these proposed mechanism for CCR6 function, however, are less important in renal disease. First, there is no equivalent of the blood-brain barrier, which limits lymphocyte influx to certain routes. Second, priming of the nephritogenic immune response in CCR6^−/− mice was not impaired in our experiments. Production of anti-sheep Ig as a major premise for initiation of the autologous T cell–dependent phase of NTN was not reduced but on the contrary even augmented in CCR6^−/− animals. The mechanisms underlying increased antibody production in CCR6^−/− mice remain unclear. Because some B cell subsets strongly express CCR6,^17,18 one reason for increased antibody production may lie in defective control of autoantibody response as a result of impaired co-localization of CCR6⁺ Tregs and CCR6⁺ B cells in secondary lymphoid organs.³²

In summary, we showed that CCR6^−/− mice develop more severe glomerulonephritis compared with WT controls. Characterization of renal T cell subsets indicates that CCR6 has an essential function in the course of nephritis by controlling the migration of Tregs and Th17 cells to the inflamed kidney. The defective in vivo function of Tregs in CCR6^−/− mice disrupts the CD4⁺ effector T cell balance and might thereby explain the aggravated course of the disease in these animals. Our study emphasizes the potential pitfalls that lie in pharmacologic interventions with the chemokine receptor system to treat renal autoimmune disease. Because many chemokine receptors show promiscuous expression patterns, blockade of a chemokine receptor that is expressed by a proinflammatory leukocyte subset could potentially interfere with trafficking of another leukocyte subset, which might be needed to control the inflammatory reaction. It is therefore inevitable to evaluate such a therapeutic approach by carrying out detailed studies in experimental models of glomerulonephritis and to validate these findings in human disease.

Concise Methods

Animals

CCR6^−/− mice (C57BL/6 background; strain B6.129P2-CCR6^−/−J) were purchased from The Jackson Laboratory (Bar Harbor, ME), and CCR6^−/− genotype was confirmed by PCR analysis in each animal. Knockout mice underwent embryo transfer to meet the general standards of our institution. Age-matched C57BL/6 WT controls (8 to 10 weeks old) also derived from the strain bred in our animal facility. All animals were raised in specific pathogen-free conditions. Animal experiments were performed according to national and institutional animal care and ethical guidelines and were approved by local committees.

Induction of NTN and Functional Studies

NTN was induced in 8- to 10-week-old male C57BL/6 CCR6^−/− and C57BL/6 WT mice by intraperitoneal injection of 2.5 mg of nephrotoxic sheep serum per gram of mouse body weight as described previously.²⁰ Controls were administered an intraperitoneal injection of an equal amount of nonspecific sheep IgG. For urine sample collection, mice were housed in metabolic cages for 6 hours. Urinary albumin excretion was determined by standard ELISA analysis (Mice-Albumin Kit; Bethyl, Montgomery, TX). Blood samples for BUN measurement and assessment of systemic antibody response were obtained at the time of killing. Urinary creatinine was measured by standard laboratory methods.

Real-Time RT-PCR Analysis

Total RNA of renal cortex was prepared according to standard laboratory methods. Real-time PCR was performed for 40 cycles (initial denaturation at 95°C for 10 minutes, denaturation at 95°C for 15 seconds, and primer annealing and elongation at 60°C for 1 minute) with 1.5 μl of cDNA samples in the presence of 2.5 μl (0.9 μM) of specific murine primers (primer sequences are available upon request) and 12.5 μl of 2× Platinum SYBR Green qPCR Supermix (Invitrogen, Karlsruhe, Germany) in a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA). All samples were run in duplicate and normalized to 18S rRNA to account for small RNA and cDNA variability.

In Situ Hybridization

In situ hybridization procedures were performed as described previously.²⁰ In brief, the CCL20 cRNA probe was labeled by in vitro transcription (Maxiscript; Ambion) with α[³⁵S]UTP (1250 Ci/mmol; PerkinElmer) of subcloned cDNA corresponding to nucleotides 74 through 349 of cDNA sequence NM_016960. In situ hybridization was performed on 16-μm cryosections of renal tissue using 1 × 10⁶ cpm per slide of the ³⁵S-labeled antisense and sense RNA probes, respectively. After washing, sections were dipped into Kodak NTB nuclear track emulsion and exposed for 3 weeks; after development, sections were stained with Mayer's Hemalaun.

Morphologic Examinations

Light microscopy and immunohistochemistry were performed by routine procedures. Crescent formation and glomerular sclerosis (deposition of PAS-positive material) were assessed in 30 glomeruli per mouse in a blinded manner in PAS-stained paraffin sections. As a measure for tubulointerstitial injury, the interstitial area was estimated by point counting four independent areas of renal cortex per mouse in low-magnification fields (×200) as described previously.²⁰ Paraffin-embedded sections (2 μm) were stained with an antibody directed against the pan-T cell marker CD3 (A0452; Dako, Hamburg, Germany), the regulatory T cell transcription factor FoxP3 (FJK-16s; eBiosciences, San Diego, CA), the monocyte/dendritic cell–specific markers F4/80 (BM8; BMA, Hiddenhausen, Germany) and MAC-2 (M3/38; Cedarlane, Burlington, Ontario, Canada), the B cell–specific marker B220 (RA3-6B2; R&D Systems, Wiesbaden, Germany), sheep IgG, mouse IgG (both Jackson Immunoresearch Laboratories), and CCL20 (AB9829; Abcam, Cambridge, UK). Tissue sections were developed with the Vectastain ABC-AP kit (Vector Laboratories, Burlingame, CA) or the ZytoChem Plus (AP) Polymer Kit (Zytomed Systems, Berlin, Germany) for CCL20 staining. For FoxP3 staining, tissue sections were incubated with a polyclonal rabbit anti-rat secondary antibody (Dako) and developed with the ZytoChem Plus (AP) Polymer Kit (Zytomed). MAC-2⁺ and CD3⁺ cells in 30 gcs and F4/80⁺ and CD3⁺ cells in 30 tubulointerstitial hpf (magnification ×400) per kidney were counted by light microscopy in a blinded manner. For quantification of FoxP3⁺ and B220⁺ cells, at least 10 lpf (magnification ×200) were counted. Glomerular mouse IgG deposition was scored from 0 to 3 in 30 glomeruli per mouse.

Antigen-Specific Humoral Immune Response

Mouse anti-sheep IgG antibody titers were measured by ELISA using sera collected 7 days after induction of the nephritis as described previously.²⁰ In brief, ELISA microtiter plates were coated with 100 μl of 100 μg/ml sheep IgG (Sigma, St. Louis, MO) in carbonate-bicarbonate buffer overnight at 4°C. After blocking with 1% BSA in Tris-buffered saline (Sigma), the plates were incubated with serial dilutions of mouse serum (1:100 to 1:12,500) for 1 hour at room temperature. Bound mouse IgG was detected using peroxidase-conjugated goat anti-mouse IgG (Biozol, Eching, Germany) at 1:1.000, TMB peroxidase substrate, and absorbance readings (at 450 nm) on a spectrophotometer. Lack of cross-reactivity of the secondary antibody with sheep IgG was demonstrated by omitting the primary antibody. Ig isotypes (IgG1 and IgG2a) were measured using the ELISA technique already described. The bound mouse Ig isotypes were detected using peroxidase-conjugated rabbit anti-mouse IgG1 and IgG2a antibodies (Zymed-Invitrogen, Karlsruhe, Germany) at a dilution of 1:1.000.

Leukocyte Isolation from Various Tissues

Previously described methods for leukocyte isolation from murine kidneys were used.²⁰ In brief, kidneys were finely minced and digested for 45 minutes at 37°C with 0.4 mg/ml collagenase D (Roche, Mannheim, Germany) and 0.01 mg/ml DNAse I in DMEM medium (Roche) supplemented with 10% heat-inactivated FCS (Invitrogen). Cell suspensions were sequentially filtered through 70- and 40-μm nylon meshes and washed with HBSS without Ca²⁺ and Mg²⁺ (Invitrogen). Single-cell suspensions were separated using Percoll density gradient (70 and 40%) centrifugation. The leukocyte-enriched cell suspension was aspirated from the Percoll interface. Single-cell suspension of spleen and lymph nodes was prepared according to standard laboratory procedures. In brief, tissues were minced and sequentially passed through 70- and 40-μm nylon meshes. In case of spleen single-cell suspension, erythrocytes were lysed with ammonium chloride. Subsequently, cells were washed several times with HBSS and resuspended in RPMI 1640 with 10% FCS (Invitrogen). Viability of the cells was assessed by trypan blue staining before flow cytometry.

Flow Cytometry

For T cell differentiation, isolated cells were stained for 25 minutes at 4°C with fluorochrome-labeled antibodies specific for CD3 (APC [R&D Systems] or Pacific Blue [eBiosciences]) and CD4 (PerCP [Miltenyi Biotec, Bergisch Gladbach, Germany] or APC-Alexa Fluor750). For analysis of CCR6 expression and T cell activation, status antibodies against CCR6, CD69, CD44, CD62L, and CD103 were used (all BD Biosciences, Franklin Lakes, NJ). Before antibody incubation, unspecific staining was blocked with normal mouse serum (Sigma). Staining of intracellular IFNγ and IL-17 was performed as described previously.⁶ In brief, splenocytes or isolated renal leukocytes were activated by incubation at 37°C in 5% CO₂ for 5 hours with phorbol 12-myristate 13-acetate (50 ng/ml; Sigma) and ionomycin (1 μg/ml; Calbiochem-Merck, Darmstadt, Germany) in RPMI 1640 with 10% FCS. After 30 minutes of incubation, Brefeldin A (10 μg/ml; Sigma) was added. After several washing steps and staining of cell surface markers, cells were incubated for 20 minutes at 4°C in Cytofix/Cytoperm (BD Biosciences) to permeabilize cell membranes. Then, intracellular IFNγ and IL-17 were stained using a rat anti-mouse IFNγ antibody (FITC; BD Biosciences) and an anti-mouse IL-17 antibody (PE; BD Biosciences). Intracellular FoxP3 staining was performed using the anti-mouse FoxP3 staining kit according to the manufacturer's instructions (eBiosciences). Analyses were performed on a Becton Dickinson FACScanto or FACScalibur System.

Isolation and Culture of Splenic CD4⁺CD25⁺ Tregs and Responder T Cells

Sorting procedures from the single-cell suspension of spleens were carried out by MACS according to the manufacturer's instructions (MACS CD4⁺ T-Cell-Isolation Kit; Miltenyi Biotec). Briefly, CD4⁺ T cells were enriched using a biotinylated antibody cocktail depleting all other blood cell types and anti-biotin microbeads. CD4⁺CD25⁺ T cells were isolated by positive selection using PE-labeled anti-CD25 mAb and anti-PE microbeads. Intracellular FoxP3 expression and activation status of isolated CD4⁺CD25⁺ cells was assessed by flowcytometry. CD4⁺CD25⁺ Tregs (2 × 10⁶) isolated from spleens of naive CCR6^−/− or WT mice were transferred to recipient mice by intravenous injection. In vitro experiments were performed as described previously.³³ In brief, 1 × 10⁵ WT responder T cells (CD4⁺CD25⁻) isolated from healthy mice were cultured alone or with 1 × 10⁵ CD4⁺CD25⁺ Tregs from nephritic WT or CCR6^−/− mice for 72 hours in 96-well round-bottom plates precoated with anti-CD3 mAb (5 μg/ml; clone 145-2C11; BD Bioscience, Heidelberg, Germany). Cytokine concentrations were measured in supernatants by ELISA.

Statistical Analysis

Results in the text are expressed as means ± SD. In figures, symbols represent individual data points, and the horizontal lines represent the median values. Differences between individual experimental groups were compared by t test. In case of multiple comparisons, one-way ANOVA with post hoc analysis by Tukey-Kramer test was used. For survival analysis, the Kaplan-Meier plot with a log-rank test was used. Experiments that did not yield enough independent data for statistical analysis because of the experimental setup were repeated at least three times.

Disclosures

None.

This work was supported by grants from the Deutsche Forschungsgemeinschaft (KFO 0228 TP01 to U.P. and J.E.T., TP03 to H.W.M., TP04 to G.T., and PA754/6-3 to U.P.) and the Universitätsklinikum Hamburg-Eppendorf (FFM 10/09 to J.E.T.).

We thank S. Schröder for excellent technical assistance.