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CD4⁺CD25⁺ Regulatory T Cells Protect against Injury in an Innate Murine Model of Chronic Kidney Disease

Deepika Mahajan^*, Yiping Wang^*, Xiahong Qin^*, Ying Wang^*, Guoping Zheng^*, Yuan Min Wang, Stephen I. Alexander and David C.H. Harris^*

^* Centre for Transplantation and Renal Research, The University of Sydney at Westmead Millennium Institute, and Centre for Kidney Research, Children’s Hospital at Westmead, Westmead, Sydney, Australia

Address correspondence to: Dr. Deepika Mahajan, Centre for Transplantation and Renal Research, The University of Sydney at Westmead Millennium Institute, Westmead NSW 2145, Australia. Phone: +61-2-9845-8906; Fax: +61-2-9633-9351; E-mail: deepika_mahajan{at}wmi.usyd.edu.au

Received for publication August 10, 2005. Accepted for publication August 1, 2006.

	Abstract

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Studies of mechanisms of disease regulation by CD4⁺CD25⁺ regulatory T cells (Treg) have been focused on their interaction with effector T cells; however, the possibility that regulation might involve noncognate cells has not been explored in detail. This study investigated the effect of CD4⁺CD25⁺ Treg on macrophage proinflammatory properties and phenotype in vitro and found that they modulate macrophages by inhibiting their activation, leading to reduced proinflammatory cytokine production and a downregulated effector phenotype. For testing the in vivo significance of this effect, CD4⁺CD25⁺ T cells that expressed high levels of Foxp3 were reconstituted into SCID mice after induction of Adriamycin nephropathy, a noncognate model of chronic renal disease. CD4⁺CD25⁺ T cells significantly reduced glomerular and interstitial injury. In addition, there was a significant fall in the number of macrophages in both the glomeruli and interstitium of SCID mice that were reconstituted with Treg as compared with the Adriamycin alone group. Blockade of TGF- using neutralizing antibodies significantly impaired the protective effect of Treg. These findings delineate a TGF-–dependent Treg–macrophage inhibitory interaction that can explain cognate-independent protection by Treg.

	Introduction

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The CD4⁺CD25⁺ T cell is the most well-characterized CD4⁺ cell with regulatory properties and is thought to play a pivotal role in the maintenance of tolerance in rodents and human (1). Recently, models of organ-specific autoimmune disease have provided convincing evidence that the activity of regulatory T cells (Treg) not only protects against the development of autoimmune disease but also can suppress the response of T cells to exogenous antigens (2). Foxp3, a member of the forkhead protein family of transcription factors, is essential for Treg cell differentiation and possibly function and is a specific molecular marker for Treg (3).

The ability of Treg to suppress T cell–mediated pathology has been investigated extensively. For example, it was shown recently that CD4⁺CD25⁺ Treg can inhibit murine anti–glomerular basement membrane glomerulonephritis, a model in which cognate immune responses play a central role (4). However, the potential role of Treg in innate immune responses is less studied. Infiltration of macrophages, an important component of innate immunity, is one of the most striking and constant features of chronic renal injury, and the degree of mononuclear cell infiltrate is predictive of subsequent disease progression (5). Macrophages can contribute extensively to tissue damage and progressive renal failure via a number of mechanisms, including their production of proinflammatory cytokines such as TNF- and IL-12 and via their T cell–stimulatory capacity (6,7). To date, the possibility that the proinflammatory actions of macrophages in chronic renal disease can be modified by Treg has not been examined.

Current evidence indicates that Treg are able to use multiple mechanisms to suppress immune responses. In vitro studies have demonstrated the requirement for cell–cell contact in cytokine-independent pathways (8,9), whereas in vivo Treg have been shown to exert their effects by both cytokine-independent and cytokine-dependent pathways (10,11). Furthermore, the immunosuppressive cytokines IL-10 and TGF- have been reported to suppress both T cells and cells of the innate immune system (12,13). Although these studies raise the possibility that Treg also can inhibit innate immune activation, this possibility has been explored only in burn injury and inflammatory bowel disease (14,15); its relevance to renal injury has not been examined.

We showed previously (16) that depletion of CD4⁺ T cells in established Adriamycin nephropathy (AN) aggravated glomerular and interstitial injury, indicating that there may be a regulatory subset in the CD4⁺ population that protects against disease progression. In this study, we explored the effect of Treg by reconstituting these cells into SCID mice with established AN. The lymphocyte-independent protective role of Treg has been demonstrated by their reduction of renal injury in this noncognate model of chronic renal disease. Furthermore, the mechanism underlying this lymphocyte-independent protective effect of CD4⁺CD25⁺ Treg has been investigated in vitro. The data of our in vitro studies indicate for the first time that CD4⁺CD25⁺ Treg can downregulate the effector phenotype of macrophages and inhibit their production of cytokines in a TGF-–dependent manner, thereby hampering the innate effector function of these cells. Moreover, we present evidence that TGF-, which is accepted as a key mediator of renal fibrosis, can via Treg protect against renal injury.

	Materials and Methods

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Mice
Male SCID mice that were approximately 6 to 8 wk of age and weighed 22 to 24 g were purchased from Australian Research Council (Perth, Australia). All mice were housed in the Department of Animal Care at Westmead Hospital under standard conditions and allowed free access to standard food and water. Experiments were carried out in accordance with protocols approved by the Animal Ethics Committee of Sydney West Area Health Service. AN was induced in SCID mice by a single tail-vein injection of Adriamycin (5.2 mg/kg; David Bull Laboratories, Victoria, Australia). This model has been described in detail in BALB/c mice previously (17). AN in SCID mice is similar to that in BALB/c mice. In SCID AN, proteinuria was observed from days 3 to 5 and remained elevated throughout the 4 wk of observation. A focal increase in reabsorption droplets in tubular cells appeared at week 1, and tubular and glomerular injury appeared after week 2 and progressed to become severe by week 4. Macrophage infiltration was observed from days 7 to 9 within both interstitium and glomeruli. CD4⁺CD25⁺ T cells were reconstituted at a time when proteinuria and renal injury with macrophage infiltration was already apparent.

Cell Isolation and Purification
CD4⁺ T cells were isolated from blood, spleen, and lymph nodes of BALB/c mice. Single-cell suspensions were prepared and fractionated by Lymphoprep, and CD4⁺ T cells were isolated from leukocytes by positive selection with DYNAL separation system (Dynal Biotech, Lake Success, NY). CD4⁺CD25⁺ T cells and CD4⁺CD25^– T cells were isolated from blood, spleen, and lymph nodes of BALB/c mice using Miltenyi Biotech isolation kit (Bergisch Gladbach, Germany). Briefly, non-CD4⁺ T cells were depleted using a biotinylated antibody cocktail and anti-biotin microbeads. CD4⁺CD25⁺ T cells then were positively selected using phycoerythrin (PE)-labeled anti-CD25 mAb and anti-PE microbeads. The purity of CD4⁺CD25⁺ Treg and CD4⁺CD25^– T cells was >96 and 95%, respectively. Macrophages were isolated by positive selection using anti-CD11b mAb (Miltenyi Biotech), followed by incubation at 37°C in a CO₂ incubator for 2 h and collection of only the adherent cells (average purity >95%).

T Cell Reconstitution
SCID mice were reconstituted by intravenous injections of 3 x 10⁶ CD4⁺ or non-CD4⁺ T cells or of 1 x 10⁶ CD4⁺CD25⁺ or CD4⁺CD25^– T cells at day 8 after Adriamycin administration. Antibody treatment commenced the day after T cell reconstitution. Where indicated, mice were treated with 250 µg of anti–TGF- (1D11.16.8; Bioexpress, West Lebanon) every fourth day after reconstitution for the duration of the experiment or an isotype control by intraperitoneal injection. All mice were killed 4 wk after Adriamycin administration.

Assessment of Renal Injury
For assessment of renal function, fasting mice were placed in metabolic cages for 12 h to collect urine for determination of urinary protein and creatinine. Creatinine clearance (Ccr) was calculated as urinary creatinine excretion divided by plasma creatinine concentration. Venous blood for serum creatinine and albumin was collected when the mice were killed. All urine and blood specimens were analyzed by the Institute of Clinical Pathology and Medical Research, Westmead Hospital, using a BM/Hitachi 747 analyser (Tokyo, Japan).

Histology and Immunohistochemistry
Formalin-fixed coronal sections of renal tissue were embedded in paraffin, cut in 5-µm sections, and stained with periodic acid-Schiff for histologic analysis. The degree of renal injury was estimated by evaluating the percentage of renal injury per field and was graded on a scale of 0 to 3. A semiquantitative score from two blinded trained observers was used to evaluate the degree of renal injury, and a minimum of eight consecutive fields at a magnification of x400 were assessed and scored in each section. For the CD4⁺ reconstitution experiment, a computer-assisted image analysis system was used to quantify glomerular and tubulointerstitial structure in the renal cortex, as described previously (16).

Sections that were cut from frozen tissue (5 µm) were used for immunohistochemistry using standard techniques. Sections were incubated with antibodies (Becton Dickinson, Mountain View, CA) against macrophages (ED1) and CD4 T cells for 60 min at room temperature. After blocking with 3% H₂O₂ to eliminate endogenous peroxidases, sections were incubated in biotinylated rabbit anti-IgG (1:200) and then in an avidin-biotin-horseradish peroxidase complex. Reaction was visualized by the addition of freshly prepared 3,3-diaminobenzidine tetrahydrochloride (7.5 mg in 15 ml of PBS with 15 µl of 30% H₂O₂) and examined by light microscopy.

Reverse Transcription–PCR
Total RNA was extracted from renal cortical cells using TRIZOL Reagent (Invitrogen, Life Technologies, Scotland, UK) and reverse-transcribed using Superscript 11 reverse-transcriptase (Invitrogen Japan K.K., Tokyo, Japan). Foxp3 mRNA levels were quantified by real-time PCR using the ABI PRISM 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). The primers and TaqMan probe sequences for real-time PCR were as follows: Foxp3 primers 5'-TTG GCC AGC GCC ATC TT-3' and 5'-TGC CTC CTC CAG AGA GAA GTG-3'; Foxp3 probe 5'-6FAMCAG CTG CTG CTC CAGMGBNFQ-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers: 5'-TGC ACC ACC AAC TGC TTA GC –3' and 5'-GGA AGG CCA TGC CAG TGA-3'; and GAPDH probe 5'-VICCCT GGC CAA GGT CAT CCA TGA CAA CTTMGBNFQ-3'. A normalized value for Foxp3 mRNA expression in each sample was calculated as the relative quantity of Foxp3 divided by the relative quantity of GAPDH from five animals that were picked randomly from each group. All samples were run in triplicate.

Flow Cytometric Analysis
FACS analysis was performed using FITC-conjugated anti-mouse CD25 (IL-2 receptor chain), PE-conjugated anti-mouse CD3 (145 2C11), and PE-conjugated anti-mouse CD4 (L3T4; BD Bioscience, New South Wales, Australia). For detection and quantification of cell surface markers, cells were treated with mAb that were directed against these molecules. Cells were stained with adequate concentration of FITC- or PE-labeled antibodies and allowed to stand at room temperature for 30 min. The cells were washed and resuspended in PBS for analysis. All samples were analyzed on a FACScan analyzer (Becton Dickinson). CellQuest software was used for acquisition and analysis (Becton Dickinson).

In Vitro Experiments
Purified macrophages as well as CD4⁺CD25⁺ and CD4⁺CD25^– T cells were stimulated with LPS (2 µg/µl) and anti-CD3 antibody, respectively, for 2 h in flat-bottom plates (Nunc, Roskilde, Denmark). Cells were cultured in RPMI 1640 medium (Life Technologies BRL Life Technologies, Merelbeke, Belgium) supplemented with 1% streptomycin and 10% heat-inactivated FCS. All cells were washed three times after stimulation and co-cultured in varying responder:suppressor ratios for 16 h, 24 h, and 3 d. In another set of experiments, stimulated CD4⁺CD25⁺ T cells were co-cultured with stimulated macrophages in the presence of neutralizing anti–TGF- (2 to 10 µg/ml) and anti–IL-10 (2 µg/ml) antibodies. Expression of TNF-, IL-12, CCL3, CCL17, inducible nitric oxide synthetase (iNOS), IL-10, and TGF- was assessed using reverse transcription–PCR. Because no difference was observed in the suppression using the three different time points above, cells were co-cultured for 24 h for all of the experiments.

RNA was isolated using TRIZOL Reagent (Invitrogen, Life Technologies) according to the manufacturer’s instructions. The total amount of RNA was reverse transcribed using Superscript 11 reverse-transcriptase and oligo (dT) primer in a final volume of 20 µl. Semiquantitative PCR to assess cytokines, chemokines, and iNOS was carried out after reverse transcription of equal amounts of RNA (2 µg) from each sample. PCR amplification was tested on each primer pair at different cycles to ensure amplification in the linear range.

Statistical Analyses
Values are expressed as mean ± SD. Statistical analysis was performed using SPSS 11.0 (SPSS Inc., Chicago, IL). Statistical significance of differences among groups was determined by one-way ANOVA. The post test of ANOVA was the post hoc least significant difference test. P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
CD4⁺CD25⁺ Treg Suppress Macrophage Phenotype and Proinflammatory Cytokine Production
Because macrophages secrete and synthesize TNF- and IL-12, proinflammatory cytokines that are known to induce glomerular damage (18), we examined in vitro the effect of stimulated CD4⁺CD25⁺ T cells on macrophage TNF- and IL-12 expression. Macrophages that were cultured alone, without any stimulus, did not produce any cytokines but when stimulated with LPS showed increased expression of TNF- and IL-12. CD4⁺CD25⁺ T cells showed a suppressive effect on the expression of both of these cytokines (Figure 1, A and B) when co-cultured with stimulated macrophages irrespective of responder:suppressor ratio of cells (data not shown). CD4⁺CD25⁺ T cells, being more potent, exhibited complete suppression even at a ratio of 1:0.5.

View larger version (48K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. (A) Reverse transcription–PCR analysis of purified macrophages and CD4+CD25+ T cells alone and in coculture (1:1) without inserts, showing that the effects were contact-dependent. After 24 h, purified RNA was reverse transcribed, and primers for the indicated genes were used to amplify the cDNA. IL-10 and TGF- were expressed only by CD4+CD25+ T cells; other genes were expressed only by macrophages. Representative of three independent experiments, each performed in triplicate. (B) Relative intensity of gene expression by macrophages alone or in coculture (1:1) with CD4+CD25+ T cells, with respect to housekeeping gene (18S). Data are mean ± SD from three independent experiments, each performed in triplicate. *P < 0.001 versus unstimulated macrophages, CD4+CD25+ T cells + stimulated (s) macrophages, and CD4+CD25+ T cells + s macrophages + anti–IL-10; P < 0.001 versus all other groups. (C) Inhibitory effect of CD4+CD25– T cells on the expression of TNF-, IL-12, CCL3, and inducible nitric oxide synthase (iNOS) depended on the responder:suppressor ratio of macrophages and CD4+CD25– T cells. CD4+CD25+ T cells were fully suppressive at each ratio (data not shown), whereas no effect was seen when macrophages were cultured in excess of CD4+CD25– T cells. Data are mean ± SD from three independent experiments, each performed in triplicate. *P < 0.05, **P < 0.001 versus the other two ratios. (D) Dose-response effect of anti–TGF-, showing an inhibition of the suppressive effect of CD4+CD25+ T cells on macrophage expression of TNF- and IL-12 (from 2 µg/ml) and of CCL3 and iNOS (from 5 µg/ml). Data are mean ± SD from three independent experiments, each performed in triplicate. P < 0.001 versus 0 anti–TGF- for all comparisons (except CCL3 and iNOS at 2 µg/ml).

The phenotype of LPS-induced macrophages was investigated on the basis of expression of chemokines CCL3 and CCL17 and iNOS. LPS-induced macrophages showed increased expression of CCL3 and iNOS, which was absent in the unstimulated macrophages, but there was no expression of CCL17 or IL-10 in either of the macrophage populations (Figure 1, A and B), suggesting that LPS-stimulated macrophages are type I or classically activated macrophages (19,20). IL-10 and TGF- expression was observed in the CD4⁺CD25⁺ T cells alone and when co-cultured with macrophages. Macrophages alone and CD4⁺CD25^– T cells, however, did not produce any TGF- in vitro under the conditions described in our study. In addition, CD4⁺CD25⁺ T cells inhibited the expression of CCL3 and iNOS by stimulated macrophages when co-cultured together (Figure 1A). CD4⁺CD25^– T cells also showed an inhibitory effect, which depended on the responder:suppressor ratio (Figure 1C).

Neutralizing anti–TGF- and anti–IL-10 antibodies were used to define the role of these cytokines in the suppressive effect shown by CD4⁺CD25⁺ Treg. When macrophages were co-cultured with CD4⁺CD25⁺ T cells in the presence of neutralizing antibodies, anti–TGF- reversed the suppressive effect of CD4⁺CD25⁺ Treg in a dose-dependent manner (Figure 1, A, lane 6, and D). Anti–IL-10 antibodies, however, did not show any effect on the suppressive effect of CD4⁺CD25⁺ Treg (Figure 1A, lane 7).

SCID Mice Are More Sensitive to AN
All SCID mice that received an injection of Adriamycin developed severe AN, characterized by proteinuria, hypoalbuminemia, hypercreatininemia, extensive interstitial inflammatory infiltrates, and progressive renal injury. The histologic changes were dominated by tubulointerstitial injury, including tubule cell atrophy with brush border loss and interstitial expansion with monocytic infiltration, and glomerular sclerosis. The mean body weight of mice with AN fell >15% at the end of week 1, which was greater than shown previously in immunocompetent BALB/c mice (17) despite use of a much lower dose (5.2 mg/kg body wt in SCID and 9.8 mg/kg body wt in BALB/c mice).

Adoptive Transfer of CD4⁺ T Cells Protects against T Cell–Independent Injury
Mice that were reconstituted with CD4⁺ T cells had significantly reduced glomerular sclerosis and mesangial matrix expansion than mice that were treated with Adriamycin alone and mice that were reconstituted with non-CD4⁺ leukocytes (Table 1). Tubular atrophy and interstitial volume were significantly decreased in mice that were reconstituted with CD4⁺ T cells but increased in mice that were reconstituted with non-CD4⁺ cells (Figure 2). Urine protein levels were significantly lower in mice that were reconstituted with CD4⁺ T cells as compared with other groups (Table 2), consistent with the histologic changes.

View larger version (124K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Representative renal cortical sections stained by periodic acid-Schiff (PAS) from normal SCID mice (A), SCID mice with Adriamycin nephropathy (AN; 4 wk after Adriamycin; B), SCID mice with AN (4 wk after Adriamycin and 3 wk after reconstitution with CD4+ T cells; C), and SCID mice with AN (4 wk after Adriamycin and 3 wk after reconstitution with non-CD4+ cells; D). The results of morphometric analysis of the histologic changes are summarized in Table 1.

View larger version (44K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Urinary protein (A) and creatinine clearance (Ccr; B) at 4 wk in SCID mice with AN, untreated except for Adriamycin (n = 12); reconstituted with CD4+CD25+ T cells (n = 12) or CD4+CD25– T cells (n = 12); reconstituted with CD4+CD25+ T cells (n = 7) or CD4+CD25– T cells (n = 7) and treated with 250 µg of anti–TGF- (1D11.16.8; Bioexpress) intraperitoneally every fourth day after reconstitution for the duration of the experiment; or treated with anti–TGF- alone (n = 7). Reconstituted mice that were treated with an isotype control antibody (n = 5; data not shown) were no different from reconstituted mice that received no antibody. Urinary protein was significantly lower and Ccr significantly higher in AN mice with CD4+CD25+ T cells than in each of the other groups. *P < 0.05 versus AN alone and AN/anti–TGF-; **P < 0.001 versus all other groups. Data are mean ± SD.

View larger version (38K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Semiquantification of glomerular sclerosis (A) tubular atrophy (B), and interstitial injury (C) at 4 wk in SCID mice with AN, untreated except for Adriamycin (n = 12); reconstituted with CD4+CD25+ T cells (n = 12) or CD4+CD25– T cells (n = 12); reconstituted with CD4+CD25+ T cells (n = 7) or CD4+CD25– T cells (n = 7) and treated with 250 µg of anti–TGF- intraperitoneally every fourth day after reconstitution for the duration of the experiment; or treated with anti–TGF- alone (n = 7). Reconstituted mice that were treated with an isotype control antibody (n = 5; data not shown) were no different from reconstituted mice that received no antibody. SCID mice that had AN and were reconstituted with CD4+CD25+ T cells showed significantly less glomerular sclerosis, tubular atrophy, and interstitial injury than AN mice without reconstitution. *P < 0.001 versus all other groups.

View larger version (109K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Representative renal cortical sections stained by PAS. (A) Normal SCID mice showed morphologically normal glomeruli and tubules. (B) SCID mice with AN (4 wk after Adriamycin) showed glomerulosclerosis, tubular atrophy, interstitial expansion, and hyaline casts. (C) SCID mice that had AN and were reconstituted with CD4+CD25+ T cells from day 7 had less renal injury at 4 wk than mice with AN alone. SCID mice that had AN and were reconstituted with CD4+CD25– T cells (D), reconstituted with CD4+CD25+ T cells and treated with 250 µg of anti–TGF- (ID11) intraperitoneally every fourth day after reconstitution for the duration of the experiment (E), or reconstituted with CD4+CD25– T cells and treated with anti–TGF- (F) each had injury that was no different from SCID mice with AN alone.

View larger version (74K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Representative immunostaining for renal macrophages (A) and quantification of interstitial macrophages (B) in normal SCID mice (n = 5; A), SCID mice with AN (4 wk after Adriamycin; n = 12; B), SCID mice that had AN and were reconstituted with CD4+CD25+ T cells (n = 12; C), SCID mice that had AN and were reconstituted with CD4+CD25– T cells (n = 12; D), and SCID mice that had AN and were reconstituted with CD4+CD25+ T cells (n = 7; E) or CD4+CD25– T cells (n = 7) and treated with 250 µg of anti–TGF- (ID11; F). Interstitial macrophage infiltration was significantly reduced in SCID mice with AN after reconstitution with CD4+CD25+ T cells. *P < 0.001 versus all other groups. Data are mean ± SD.

View larger version (46K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Representative immunostaining (A) and quantification (B) for cortical CD4+ T cells in normal SCID mice (n = 5; A), SCID mice with AN (4 wk after Adriamycin; n = 12; B), SCID mice that had AN and were reconstituted with CD4+CD25+ T cells (n = 12; C), SCID mice that had AN and were reconstituted with CD4+CD25– T cells (n = 12; D), and SCID mice that had AN and were reconstituted with CD4+CD25+ T cells (n = 7; E) or CD4+CD25– T cells (n = 7) and treated with 250 µg of anti–TGF- (ID11; F). *P < 0.001 versus AN/CD4+CD25+, AN/CD4+CD25–, AN/CD4+CD25+/anti–TGF- and AN/CD4+CD25–/anti–TGF-; P < 0.001 versus all other groups. Data are mean ± SD.

View larger version (29K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Foxp3 mRNA levels normalized against glyceraldehyde-3-phosphate dehydrogenase. (A) Levels of Foxp3 mRNA in freshly isolated cells. (B) SCID mice that had AN and were reconstituted with CD4+CD25+ T cells (n = 5) showed a significant increase in renal cortical Foxp3 mRNA in comparison with mice with AN alone (n = 5) as well as mice that had AN and were reconstituted with CD4+CD25– T cells (n = 5). The results shown are mean ± SD of two independent measurements, each with triplicate wells. *P < 0.001 versus other groups.

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Quantitative real-time PCR analysis for the detection of Foxp3 mRNA expression in renal cortex of SCID mice that had AN and were reconstituted or not with CD4+CD25+ or CD4+CD25– T cells and treated or not with anti–TGF-. The degree of protection, against glomerulosclerosis (A), tubular atrophy (B), and interstitial injury (C) correlated with the levels of cortical Foxp3 mRNA. P < 0.001 for each correlation. Each symbol represents the average value from three independent measurements in an individual mouse.

CD4⁺CD25⁺ Treg–Mediated Protection against T Cell–Independent Injury Is TGF-–Dependent

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
Natural CD4⁺CD25⁺ Treg have been shown to regulate cognate immunity in autoimmune diseases and allotransplantation, whereas there has been little exploration of their potential role in innate immune mechanisms of chronic inflammatory diseases. In chronic renal disease, as in other chronic inflammatory diseases, monocytes/macrophages and their mediators make an important contribution to the inflammatory process. We present here evidence for a novel suppressive activity of CD4⁺CD25⁺ Treg by showing that these cells can downregulate macrophage function and phenotype. Data from our in vitro study indicate that CD4⁺CD25⁺ Treg and, to a lesser extent, CD4⁺CD25^– T cells are capable of suppressing macrophages, leading to reduced proinflammatory cytokine production. Because macrophages and their mediators are known to be major players in lymphocyte-independent renal injury, these findings provide support for the potential use of CD4⁺CD25⁺ Treg to treat both cognate and innate components of injury. Recently, Taams et al. (21) showed a similar effect, demonstrating that human CD4⁺CD25⁺ Treg can exert direct suppressive effects on macrophage function in vitro.

Our study in SCID mice emphasizes that innate immune mechanisms are central to the interstitial inflammation and injury in this toxin-induced model of chronic renal disease. In addition, we showed for the first time that such T cell–independent renal injury could be inhibited by transfer of CD4⁺CD25⁺ T cells through cytokine-dependent mechanisms. These results are consistent with those of Maloy et al. (22), who demonstrated the ability of Treg to inhibit innate immunity in a model of inflammatory bowel disease. Our in vitro studies have demonstrated a mechanism whereby Treg could inhibit pathology in a lymphocyte-independent model by downmodulation of macrophage activity. Previously, we showed a pathogenic role for macrophages in this model (23). Others have shown a direct effect of Treg on antigen-presenting cells (24,25). Recently, an effect of Treg on NK cells was demonstrated (26); however, we showed recently that NK cells are not important in the murine AN (27), so it is unlikely that such an interaction explains the protective effect of CD4⁺CD25⁺ Treg in our study. We are investigating whether an interaction of CD4⁺CD25⁺ Treg with dendritic cells or tubular epithelial cells could be important to their effect.

Our previous studies showed that in vivo CD4⁺ cell depletion aggravated histologic and functional damage in AN, indicating that a dominant subset of CD4⁺ T cells was protective in this model (16). This prompted us to examine the effect of reconstitution of CD4⁺ T cells in SCID mice with established AN. CD4⁺ T cell reconstitution resulted in a significant reduction in glomerular sclerosis, tubular atrophy, and interstitial injury, which was accompanied by congruent functional protection. Because CD4⁺CD25⁺ T cells have emerged as one of the major populations of Treg that exhibit potent suppressor activity in vivo and in vitro (28), SCID mice with AN were reconstituted with CD4⁺CD25⁺ T cells and showed significant protection against both functional and structural injury. That the number of CD4⁺ T cells in renal cortex of mice that were reconstituted with CD4⁺CD25⁺ or CD4⁺CD25^– T cells was no different suggests that the effect of CD4⁺CD25⁺ reconstitution was related to a specific effect of CD4⁺CD25⁺ Treg and not just the number of cells that reached the kidney.

Foxp3 seems to be highly specific for Treg (29,30). In our study, renal expression of Foxp3 was significantly upregulated in the mice that were reconstituted with CD4⁺CD25⁺ T cells, and the level of renal Foxp3 expression correlated inversely and strongly with all aspects of tissue injury. It should be noted that the mice that were reconstituted with CD4⁺CD25^– T cells also expressed Foxp3, but the degree of expression was significantly less than that of CD4⁺CD25⁺ T cells. This is consistent with Fontenot et al. (30), who showed that although a significant percentage of Foxp3⁺ natural Treg are CD4⁺CD25^–, these constitute a minority of the overall CD4⁺CD25^– population. This means that CD4⁺CD25^– T cells have some regulatory activity, yet on a per-cell basis, they seem to be less potent than CD4⁺CD25⁺ T cells at exhibiting suppression. However, at the dosage used in our in vivo study, CD4⁺CD25^– T cells were largely ineffective because Treg in this population are diluted out by non-Treg. Zelenay et al. (31) showed that CD4⁺CD25^– T cells that express Foxp3 can regain CD25 expression and act as regulatory cells. However, at least under the conditions described in our study, there was no evidence of a regulatory effect after CD4⁺CD25^– reconstitution, except for a trivial improvement in Ccr.

Blockade of TGF- using neutralizing antibodies prevented the protective effect of CD4⁺CD25⁺ T cells in SCID mice with AN and also reduced significantly the renal expression of Foxp3 that was associated with CD4⁺CD25⁺ T cell reconstitution. This suggests that TGF- is important for the survival of Treg or for Foxp3 expression. This is in keeping with data of Marie et al. (32) that showed higher levels of Foxp3 in CD4⁺CD25⁺ Treg from mice that overexpressed active TGF-1, whereas Foxp3 expression was reduced in cells with impaired TGF- signaling. However, this contrasts with data of Fahlen et al. (33) that showed that CD4⁺CD25⁺ Treg could develop normally and retain suppressive function against colitis in the absence of TGF-1. The regulatory and protective role of TGF- in this model is remarkable in that it is at odds with previous data showing that TGF- is profibrotic in renal injury (34). A more direct demonstration of TGF- as the major protective element comes from our in vitro data showing that anti–TGF- mAb reversed the suppressive effect of CD4⁺CD25⁺ Treg on macrophages in a dose-dependent manner. In the same experiments, anti–TGF- administration also abolished production of TGF- message in Treg, suggesting that anti–TGF- mAb may be influencing the signaling pathway for TGF- gene expression. Anti–IL-10 antibodies, however, did not show any direct effect on the suppressive role of CD4⁺CD25⁺ Treg yet inhibited TGF- message production by Treg in vitro. The reason for the lack of effect of anti–IL-10 on Treg function is not clear and might be related to the suppressive capacity of IL-10. It has been reported that IL-10 acts on T cells to induce suppression only by altering the co-stimulatory CD28 signaling pathway. However, TGF- has broad effects on cell function by inhibiting both TCR/CD3 and the CD28 co-stimulatory pathway (35). Moreover, TGF-, unlike IL-10, can induce the suppressive function of Treg not only in its secreted form but also in its membrane-bound form, which has a critical role in cell contact–dependent effects of Treg (36). Reversal of suppression with anti–TGF- could be due to inhibition of surface-bound TGF- on Treg.

	Conclusion

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
We present in vitro evidence for the first time that the regulatory function of CD4⁺CD25⁺ Treg could involve TGF-–dependent downregulation of TNF-, IL-12, CCL3, and iNOS expression by macrophages. This capacity to affect innate immune responses is highlighted by further in vivo studies, which showed the ability of CD4⁺CD25⁺ T cells to protect against established macrophage-dependent, lymphocyte-independent injury. Finally, we have demonstrated that endogenous TGF- is required for full in vivo suppressive capacity of CD4⁺CD25⁺ T cells.

	Acknowledgments

 
This study was supported by a grant from the National Health and Medical Research Council of Australia (project grant 211147).

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

	References

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 

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