IL-9 production by regulatory T cells recruits mast cells that are essential for regulatory T cell-induced immune-suppression

Abstract

Both, mast cells (MC) and regulatory T cells (Treg) have gained attention as immunosuppressive cell populations. To investigate a possible interaction, we used the Th1- and Th17-dependent model of nephrotoxic serum nephritis (NTS), in which both MC and Treg have been shown to play a protective role.

Transfer of wild-type (wt) Treg into wt recipients almost completely prevents development of NTS and leads to a profound increase of MC in the renal draining lymph nodes (LN). By contrast, transfer of wt Treg into animals deficient in MC, which are characterized by an exaggerated susceptibility to NTS, no longer exhibited protective effects. Blocking the pleiotropic cytokine IL-9, known to be involved in MC recruitment and proliferation, by means of a monoclonal antibody in mice receiving Treg abrogated protection from NTS. Moreover, transfer of IL-9 deficient Treg also failed to protect from NTS. In the absence of Treg-derived IL-9, MC fail to accumulate in the LN, despite the fact that IL-9 deficiency does not alter the general suppressive activity of Treg.

In summary, we provide the first direct in vivo evidence that the nephroprotective, anti-inflammatory effects of Treg cells critically depend on IL-9-mediated attraction of MC into kidney-draining LN.

INTRODUCTION

Tipping the balance between effector and regulatory cell populations is of critical importance in the pathogenesis of various autoimmune disorders. According to a current paradigm, the pro-inflammatory axis of Th1 and Th17 cells is counterbalanced by the cell populations Th2 cells and regulatory T cells (Treg) (1).

CD4⁺CD25⁺FoxP3⁺ cells are thought to have a huge therapeutic potential as cellular immunosuppressants (2). In line with this idea, various groups including our own have demonstrated the therapeutic efficacy of Treg in murine models of inflammation (3-5). It is generally accepted that the pre-dominant target cell effect of Treg is a direct cell-to-cell contact dependent inhibition primarily mediated by membrane-bound TGF-ß (6). Moreover, soluble factors such as IL-10 have also been attributed to the Treg-induced immune-inhibitory effects (7, 8). However, various research groups have provided evidence that Treg also modify the function of non-lymphatic cell types, such as dendritic cells (DC) (9, 10), monocytes (11), endothelial cells (12) and mast cells (MC) (13). The latter are also known to play a critical role for immune regulation in allergy and autoimmunity (14). Very recently, MC have been demonstrated to exhibit immunomodulatory functions (15). They seem to exert either pro- or anti-inflammatory effects depending on the surrounding milieu (15).

For a more detailed analysis of the complex orchestration of these immunoregulatory networks, the murine model of acute nephrotoxic serum nephritis (NTS) has proven to be both informative and robust. The role of T cells, including Th1 and Th17 cells for NTS induction and maintenance is well documented (16-19). We recently provided evidence that CD4⁺CD25⁺FoxP3⁺ Treg have a therapeutic potential to control the onset and course of NTS (5). Moreover, Treg pre-dominantly migrate to LN but not to the end-organ, suggesting that lymphatic organs are the prime sites of their immunosuppressive action (5). This hypothesis is further supported by our latest observation showing that CCR7-deficient Treg lose their immunosuppressive potential due to their inability to enter the LN (20). Moreover, we and others clearly demonstrated that MC limit kidney-damaging immune responses (21, 22), as MC-deficient Kit^W/Kit^W-v mice display a profound exaggeration of NTS when compared to wild-type (wt) animals. Lu and colleagues support the concept of an important immune-regulatory function of MC by showing that they regulate allograft tolerance in a skin transplantation model (23). In this particular model, MC have been described to be protective by interacting with Treg (23). In contrast to the immune-inhibitory function of MC in acute inflammation models (21, 22), MC seem to play a central role in the development of inflammation-induced tissue fibrosis in chronic kidney diseases, since their kidney-infiltrating numbers tightly correlate with the grade of renal fibrosis (24-27).

In this report, we provide for the first time direct evidence that the Treg/MC interaction is also of critical importance for limiting endogenous inflammatory disease. As exemplified in a model of acute renal inflammation, Treg-induced immune-suppression critically depends on the recruitment of MC into kidney-draining LN. This process is mediated by Treg-derived IL-9 and is a prerequisite for the prevention of end-organ damage by effector immune cells.

MATERIAL AND METHODS

Induction of accelerated nephrotoxic serum nephritis (NTS)

C57BL/6 mice (purchased from Charles River Laboratories, Sulzfeld, Germany), MC-deficient WBB6F₁-Kit^W/Kit^W-v (Kit^W/Kit^W-v) mice, and congenic wild-type WBB6F₁-Kit+/+ (Kit+/+) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). IL9-deficient mice have been back-crossed with C57BL/6 mice for 8 generations (28). Eight to 10 week old male animals were used in all studies. Accelerated NTS was induced as described previously (29). In brief, mice were pre-immunized subcutaneously with 100μl of 2 mg/ml rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) dissolved in incomplete Freund́s adjuvant (Sigma, St. Louis, MI, USA) and 0.01g/ml non-viable desiccated Mycobacterium tuberculosis H37a (Difco Laboratories, Detroit, MI, USA). After 3 days, heat-inactivated rabbit anti-mouse GBM antiserum was injected via the tail vein. All animal experiments were performed according to the austrian law (GZ 66.011/0.111-11/10b/2008).

Detection of urinary albumin and creatinine

Urinary albumin was determined by a double-sandwich ELISA (Abcam, Cambridge, MA, USA) as reported previously (29). Urinary creatinine was quantitated spectrophotometrically using a picric acid-based method (Sigma, St. Louis, MI, USA).

Histo- and immunomorphological evaluation of renal pathology and lymph nodes

Formalin-fixed renal and LN tissue was embedded in paraffin and cut in 4 μm sections. Renal sections were stained with periodic acid Schiff́s (PAS). In all cases a minimum of 50 equatorial glomerular cross sections were evaluated as previously described (30).

LN sections were stained with Wright-Giemsa. The three-layer immunoperoxidase staining of frozen tissue 4 μm sections was used for the detection of macrophages and T cell subpopulations in the kidney as well as in LN sections (29). Macrophages were stained with rat anti-mouse mAb (clone F4/80 or anti-CD68; both from Serotec, Oxford, UK). A semiquantitative scoring system for kidney infiltrating macrophages was performed as follows: 0 = 0 to 4 cells stained positive, 1+ = 5 to 10 cells, 2+ = 10 to 50 cells, 3+ = 50 to 200 cells and 4+ = over 200 cells stained positive per low-power field. For the detection of CD4⁺, CD8⁺ T cells and CD117⁺ cells we used rat anti-mouse mAbs (clone YTS191.1 and clone KT15, both from Serotec and clone ACK2 from eBiosciences, San Diego, CA, USA, respectively). T cell and MC quantitation was performed by counting the number of positive cells in 6 adjacent high-power fields (Hpf) of renal cortex and medulla or of the LN. Samples were blinded before evaluation. Fibrin deposition was evaluated on renal cryosections by using a FITC-conjugated fibrinogen mAb (Dako, Glostrup, Denmark).

Detection of circulating mouse anti-rabbit IgG

For detection of circulating mouse anti-rabbit IgG 96-well plates (Greiner, Kremsmuenster, Austria) were coated with 100μg/ml rabbit IgG (Jackson ImmunoResearch Laboratories Inc.) in carbonate/bicarbonate buffer (pH 9.5). After blocking with 1% BSA plates were incubated with serial-doubling dilutions of mouse serum. Bound mouse IgG was detected by HRP-conjugated goat-anti-mouse IgG (Dako, Glostrup, Denmark).

Reverse transcription (RT) real-time polymerase chain reaction (PCR)

Total RNA was isolated using TRIzol® (Sigma) according to a standard protocol. Thereafter, 2 μg of total RNA was reverse transcribed using Superscript III Transcription Kit (Invitrogen, Carlsbad, CA, USA) and random primers (Roche, Basel, Switzerland). Real-time PCR was performed on an ABI Prism 7700 (Applied Biosystems, Foster City, CA, USA). For linear amplification of β-actin (reference gene) and FoxP3 SYBR Green Master Mix (Invitrogen) and primers as described before (31) were used. For quantification of mouse MC tryptase 1, Cxcl-1, Cxcl-2, Cxcl-5, IFN-γ, IL-6, IL-17A, IL-10, Gata-3 and TGF-β1 TaqMan Mastermix (Applied Biosystems) and the gene expression assays Mm00600091_m1, Mm00433859_m1, Mm00436450_m1, Mm00436451_m1, Mm00801778_m1, Mm00446190_m1, Mm00439619_m1, Mm00439616_m1, Mm00484683_m1, Mm03024053_m1 (Applied Biosystems) were used.

Isolation and transfer of CD4⁺CD25⁺ regulatory T cells

CD4⁺CD25⁺ Treg were isolated from minced spleens and lymph nodes obtained from C57BL/6 or from IL-9 deficient mice using magnetic bead separation (CD4⁺CD25⁺ regulatory T-cell kit, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The purity of both populations was controlled by flow cytometric analysis of CD4 together with intracellular FoxP3 and reached routinely >90%. Mice received intravenously 2-5×10⁶ CD4⁺CD25⁺ Treg on the day before antiserum injection.

Flow cytometry of CD4⁺FoxP3⁺ cells

Cell suspensions from LN, spleens and kidneys were stained for FoxP3⁺ Treg using co-staining of CD4 (BD Biosciences, San Diego, CA, USA) and FoxP3⁺ (eBiosciences) strictly adhering to the manufacturer’s instructions. Data collection and analysis was done on a FACSCalibur (BD Biosciences).

Treg suppression assays

Treg and the respective CD4⁺CD25⁻ control T cell populations were isolated from i) IL-9 deficient and from wt animals or from ii) Kit^W/Kit^W-v and the respective wt control animals. In both systems we used responder cells from wt animals and tested the respective IL-9 or MC-deficient animal-derived Treg population versus wt Treg. The responder to suppressor ratio was 5:1 and 1:1. The cells were stimulated by plate-bound anti-CD3 mAb (clone 17A2, BD Biosciences, San Diego, CA, USA, coating concentration 5 μg/ml). As control the respective T cell populations (i.e. Treg and CD4⁺CD25⁻ T cells) were cultured alone. Proliferation was measured between day 5 and 7 by ³H-thymidine incorporation in a β-scintillator.

Statistics

When comparing two groups, the non-parametric Mann-Whitney-U Test was used and p<0.05 was considered as significant. When comparing three groups we performed the Kruskal-Wallis-Test. When significances were detected, the different groups were compared by the Mann-Whitney-U Test. The level of significance was corrected to the number of groups and p<0.025 was considered to be significant. All statistical analyses were done using SPSS 13.0.1 for Windows (SPSS, Inc., Chicago, IL, USA).

RESULTS

MC increase in lymph nodes of Treg-treated mice after induction of NTS

C57BL/6 mice received either Treg or CD4⁺CD25⁻ control T cells isolated from healthy C57BL/6 mice and were subjected to NTS. Fourteen days after induction of NTS, LN of mice were evaluated for the infiltration of MC. MC tryptase 1 was significantly elevated as compared to NTS animals receiving CD4⁺CD25⁻ control T cells (Figure 1A). Accordingly, Giemsa-stained LN sections of Treg-injected NTS mice showed an almost 3-fold increase of MC (Figure 1B) with most of the MC being located in the LN sinusoids close to the T cell area (Figure 1C). In contrast to Treg-induced MC recruitment, Treg occupancy of LN was not affected by MC, as we did not detect any significant difference in LN Treg content between nephritic MC-deficient Kit^W/Kit^W-v when compared to the respective Kit+/+ controls suffering from NTS, as determined by quantification of FoxP3 mRNA (Figure 2A) and by FACS co-staining of CD4⁺FoxP3⁺ (Figure 2B).

Figure 1. Treg transfer increases MC in kidney-draining LN.

Kidney-draining LN of animals receiving either Treg (white bar) or control T cells (black bar; n=13 per group) were analyzed for MC infiltration fourteen days after disease induction. (A) Real-time PCR for the expression of MC-tryptase is shown. Data are presented as x-fold increase as compared to LN of healthy controls. (B) Giemsa staining of LN. The number of MC per mm² is given. The data are expressed as mean ± SEM. *p<0.05. (C) A representative example of a Giemsa-stained LN section from a Treg-injected mouse 14 days after induction of NTS is shown. MC are marked by arrows. Magnification x400.

Figure 2. Treg in MC-deficient Kit^W/Kit^W-v and Kit+/+ mice.

Fourteen days after NTS was induced in Kit^W/Kit^W-v (white bar) and Kit+/+ mice (black bar; n=10 per group), LN were evaluated for Treg infiltration by (A) performing real-time PCR for the detection of FoxP3 and by (B) flow cytometric analysis for CD4⁺CD25⁺FoxP3⁺ cells. The real-time data are expressed as x-fold increase of FoxP3/beta-actin compared to mRNA isolated from healthy control LN (which was set as 1). (C and D) Evaluation of the immune-suppressive potential of CD4⁺CD25⁺ Treg from healthy (C) Kit+/+ mice and (D) MC-deficient Kit^W/Kit^W-v. Treg from Kit+/+ or MC-deficient Kit^W/Kit^W-v animals were co-incubated with CD4⁺CD25⁻ Kit+/+ cells in a 1:1 and 1:5 ratio. Proliferation was measured by ³H-thymidine incorporation after 7 days and the percentage of proliferation compared to the respective control CD4⁺CD25⁻ T cell population is shown. Moreover, both strains were subjected to Treg quantification, as shown by representative FACS-stainings shown in the inserts in C and D.

Adoptively transferred Treg do not protect MC-deficient animals from NTS

We next tested whether adoptive Treg transfer can compensate for MC-deficiency in Kit^W/Kit^W-v mice to further define, whether Treg-mediated immunosuppression is preserved in the absence of MC. Due to the well known role of Kit signalling for thymic T cell development (32), we first excluded a numerical or functional deficit of Treg isolated from Kit^W/Kit^W-v mice by performing standard Treg quantification by FACS (see representative FACS staining inserted into Figure 2C and D) as well as proliferation and suppression assays with isolated Treg populations from the respective genetic backgrounds (Figure 2C and D). We next transferred Treg to NTS-subjected Kit^W/Kit^W-v and Kit+/+ animals. Notably, in contrast to the well-known protective effects of Treg in wt animals with NTS, even high numbers of Treg (5×10⁶) were not able to confer protection from NTS in MC-deficient Kit^W/Kit^W-v mice. Accordingly, albuminuria, which is a marker for the severity of kidney damage was markedly reduced by Treg in wt nephritic animals but remained unchanged in MC-deficient Kit^W/Kit^W-v mice with NTS when compared to control T cell-transferred animals, respectively (Figure 3A). This observation was supported by histological data showing that hypercellularity and focal deposition of PAS positive material was reduced by Treg in wt animals, but was more pronounced in MC-deficient Kit^W/Kit^W-v mice. In the latter, adoptive Treg transfer did not affect morphological changes when compared to control T cell-injected animals as shown by histology (Figure 3B) or semiquantitative evaluation of PAS deposition (Figure 3C). Further analysis of kidney sections revealed that the increased disease susceptibility of MC-deficient Kit^W/Kit^W-v mice to NTS is also reflected by a significant increase of CD4⁺ and CD8⁺ T cells (Figure 3D) as well as F4/80⁺ and CD68⁺ cells in the kidney (Figure 3E-F). Of note, no F4/80⁺ cells were detected in healthy MC-deficient Kit^W/Kit^W-v and Kit+/+ mice (supplemental web figure 1). In line with the albuminuria and the histological data, Treg transfer in wt animals decreased inflammatory infiltrates (Figure 3D-F) whereas adoptive Treg transfer did not alter T cell or macrophage infiltration into kidneys of MC-deficient Kit^W/Kit^W-v mice (Figure 3D-F). To exclude an impaired production of anti-rabbit IgG of the respective animals, we quantified serum anti-rabbit IgG titers. Of note, we could not detect any significant alteration in the titres between the different groups (Figure 3G), suggesting that the initial priming of B cells during immunization remains unaffected by either MC deficiency or by Treg transfer.

Figure 3. MC-deficient animals with NTS are resistant to the anti-inflammatory Treg effects.

Kit^W/Kit^W-v (grey and light grey bar) and Kit+/+ mice (black and white bar) received either 5×10⁶ Treg (light grey and white bar) or control T cells (grey and black bar) and were subjected to NTS (n=8 per group). (A) 7 and 14 days after induction of NTS albumin and creatinine in the urine were evaluated. The urinary albumin/creatinine ratio is given in mg/mg.

(B) Representative PAS staining from tissue sections of kidneys from the indicated groups 14 days after induction of NTS are shown. Magnification x400. Kit^W/Kit^W-v receiving either Treg or control T cells display crescent formation as highlighted by black arrows. Only few crescent formations were seen in Kit+/+ control mice, but they were found to have, as Kit^W/Kit^W-v mice, PAS-positive deposits in their glomeruli (black asterix) as well as infiltrating inflammatory cells. When Kit+/+ mice were treated with Treg no glomerular pathologies were detected. (C) Semiquantitative analysis of PAS deposits in kidney sections according to the PAS-score described in the Material and Methods section is shown. (D) T cell infiltration pattern in the indicated groups are shown by semiquantitative quantification of CD4 and CD8 stained tissue sections. The number of positive cells in 6 HPF is given. (E-F) Kidney infiltration patterns of (E) F4/80⁺ or (F) CD68⁺ cells, which were stained and counted according to the F4/80 and CD68-scoring procedure described in the Material and Methods section. (G) Rabbit anti-mouse titers were evaluated in the serum of respective mice 14 days after induction of NTS. All data are presented as mean ± SEM. * … provides the significance between Kit+/+ mice receiving either Treg or control cells (p<0.025). # … provides the significance between Kit^W/Kit^W-v and Kit+/+ mice receiving control cells (p<0.025). Three independent experiments were performed.

To further evaluate the potential mechanism of MC-mediated immunosuppression, we evaluated the T cell response in the LN 7 days after induction of NTS by performing real-time PCR for the mRNA expression of Th1, Th2 and Th17 markers. The Th1 cytokines IL-6 and IFN-γ and the Th17-cytokine IL-17A were significantly increased in LN of Kit^W/Kit^W-v mice as compared to Kit+/+ mice. Interleukin-10 was also found to be significantly increased in lymph nodes of Kit^W/Kit^W-v mice. In contrast, other Th2 marker Gata-3 and TGF-β1 were not affected by MC-deficiency (Figure 4).

Figure 4. Increased inflammation in MC-deficient animals suffering from NTS.

NTS was induced in Kit^W/Kit^W-v and Kit+/+ mice (n=6 per group). Seven days after induction of NTS the mRNA expression of IFN-γ, IL-6, IL-17A, IL-10, Gata-3 and TGF-β was evaluated in the LN. The real-time PCR data are expressed as x-fold increase of the respective parameter/beta-actin compared to the mean expression of Kit+/+ LN (which was set as 1). All data are presented as mean ± SEM. *p<0.05.

IL-9 links Treg and MC in NTS

We next focused on a potential candidate linking Treg and MC. Thus, we evaluated the functional role of IL-9 for Treg/MC interaction, as IL-9 functions as a key proliferation/differentiation factor and chemoattractant for MC (33), is produced by Treg (23) and has been implicated to be a key cytokine regulating the interaction between Treg and MC (23). To address this particular question, NTS mice receiving Treg were either i.p. injected with a blocking anti-IL-9 mAb or the respective isotype control antibody every second day. Systemic blockade of IL-9 reversed the protective effects of adoptively transferred Treg in NTS as shown by a significantly increased albuminuria when compared to the Treg/isotype control group (Figure 5A). Again, this was accompanied by the respective histological changes, i.e. increased PAS-positive deposits in the glomeruli (Figure 5B) and decreased renal inflammatory cell infiltrates (Table 1). Most importantly, the increased infiltration of MC (quantified by semiquantitative staining of LN for CD117 and by detection of MC tryptase 1 mRNA levels) in the LN of Treg-treated mice was reduced in the anti-IL-9 mAb-treated animals to the levels of mice receiving control cells (Figure 5C-D).

Figure 5. Systemic IL-9 blockade prevents nephroprotection by Treg.

NTS animals received either Treg (white bar; n=5) or control T cells (black bar; n=5) in combination with an isotype antibody control. A third group was injected with Treg in combination with an anti-IL-9 blocking mAb, which was applied every second day (grey bar; n=5). (A) The albumin/creatinine ratio in the urine was evaluated 7 and 14 days after induction of NTS. (B) PAS stained sections were counted according to the PAS-score as described in the Material and Methods section. (C) Sections were stained with Giemsa and the number of MC per mm² was evaluated. (D) The kidney-draining LN was evaluated for MC infiltration by real-time PCR for MC tryptase 1 mRNA. The data are expressed as x-fold increase of MC tryptase 1/beta-actin compared to mRNA isolated from healthy control LN (which was set as 1). All data are presented as mean ± SEM. *p<0.025. Two independent experiments were performed.

Table 1. The role of systemic IL-9 blockade on renal inflammatory cell infiltrates after Treg transfer.

The infiltration of CD4⁺, F4/80⁺ and CD68⁺ cells was evaluated in the kidneys of the respective animals (n=5 per group) 14 days after induction of NTS. The data are presented as mean ± SEM.

Groups	control	Treg	Treg + anti-IL-9
CD4+ cells (number per 6Hpf)	54.8 ± 9.6	14.2 ± 3.5*	21.8 ± 3.4
F4/80+ cells (score)	1.05 ± 0.27	0.45 ± 0.08*#	0.75 ± 0.07
CD68+ cells (score)	1.75 ± 0.40	0.55 ± 0.11*	0.88 ± 0.21

^*p<0.025 between the control T cell and the Treg group.

^#p<0.025 when the Treg and Treg+anti-IL9 group are compared.

To rule out the possibility that IL-9 mAb antagonizes IL-9 secreted from various cell types [e.g. Th9 (34), Th17 cells (35)] in this complex inflammation model, we next sought to define the functional significance of Treg-derived IL-9 in this particular model by using Treg isolated from IL-9 knock-out mice for adoptive transfer. Treg from IL-9-deficient animals failed to reduce albuminuria, histological changes (Figure 6A-C) or the renal cellular infiltrates (Table 2), as well as the amount of the mouse anti-rabbit IgG titers remained unaltered (Table 2). Moreover, the increase of MC in the LN of NTS mice receiving wt Treg was blunted in NTS animals that received IL-9-deficient Treg (Figure 6D). Of note, no significant difference in the deposition of fibrin in the glomeruli of the three different groups was detected (Figure 6E). Additionally, we excluded that IL-9 deficient Treg per se are characterized by a defective immunosuppressive potential. Treg derived from IL-9 deficient animals exert a comparable target cell inhibition when cultured with wt responder T cells (Figure 6F). Finally, we evaluated chemokines known to be involved in the recruitment of MC. Interestingly, Cxcl-1 was found to be significantly decreased in the LN of mice receiving IL-9 deficient Treg as compared to mice receiving wt Treg or control cells (Figure 7A). In contrast, Cxcl-2 and -5 were found to be up-regulated in LN 7 days after NTS induction, but remained unaffected by any of the treatment modalities (Figure 7A). Comparable results were found in mice treated with the IL-9 blocking mAb (Figure 7B).

Figure 6. Treg-derived IL-9 is critical for MC-dependent nephroprotection in NTS.

NTS mice received either wt-Treg (white bar), control T cells (black bar) or Treg isolated from IL-9 deficient animals (grey bar). (A) The albumin/creatinine ratio in the urine was evaluated 7 (n=7-9) and 14 days (n=5-7) after induction of NTS. (B) Representative PAS stained kidney sections from the indicated groups 14 days after induction of NTS are shown. Magnification x400. Mice receiving either control cells or IL-9 deficient Treg displayed crescent formations (black arrows) and large PAS-positive deposits (black asterix) in their glomeruli, whereas Treg treated mice were found to have only marginal PAS-positive deposits and no crescent formations. (C) Quantification of PAS deposition by the PAS-score is given for the respective groups. (D) The kidney-draining LN was evaluated for MC infiltration by real-time PCR for MC tryptase 1 mRNA. The data are expressed as x-fold increase of MC tryptase 1/beta-actin compared to healthy control LN (which was set as 1). All data are presented as mean ± SEM. *p<0.05. (E) Renal cryosections of the three groups were stained for fibrin deposition. No significant differences were observed. Representative pictures are shown. Magnification x400. (F) Evaluation of the immune-suppressive potential of CD4⁺CD25⁺ Treg from IL-9 deficient animals. Treg from wt or IL-9 deficient animals were co-incubated with CD4⁺CD25⁻ wt cells in a 1:1 ratio. Proliferation was measured by ³H-thymidine incorporation after 7 days and the percentage of proliferation compared to the respective control CD4⁺CD25⁻ T cell population is shown. The data are presented as mean ± SEM; *p<0.025; n.s. not significant. Two independent experiments were performed.

Table 2. The role of Treg-derived IL-9 on mouse anti-rabbit IgG antibody titres and renal inflammatory cell infiltrates after Treg transfer.

Rabbit anti-mouse IgG were evaluated in the serum of mice of the three different groups (n=5 per group) 14 days after induction of NTS. The infiltration of CD4⁺, F4/80⁺ and CD68⁺ cells is given. The data are presented as mean ± SEM.

Groups	control	wt Treg	IL-9 ko Treg
Mouse anti-rabbit IgG (OD450)	0.293 ± 0.040	0.354 ± 0.122	0.236 ± 0.060
CD4+ cells (number per 6Hpf)	30.0 ± 16.2	12.4 ± 4.5#	33.0 ± 5.5
F4/80+ cells (score)	3.38 ± 0.54	1.90 ± 0.26*#	3.40 ± 0.17
CD68+ cells (score)	3.25 ± 0.28	1.80 ± 0.33*	3.00 ± 0.37

^*p<0.025 between the control T cell- and the Treg-injected group.

^#p<0.025 when comparing the wt Treg and IL-9 deficient Treg group.

Figure 7. Regulation of the chemokines Cxcl-1, -2, -5 in the kidney draining LN.

(A) NTS mice received either wt Treg (white bar), control T cells (black bar) or Treg isolated from IL-9 deficient animals (grey bar). (B) NTS animals received either Treg (white bar; n=5) or control T cells (black bar; n=5) in combination with an isotype antibody control. A third group received Treg in combination with an anti-IL-9 blocking mAb, which was applied every second day (grey bar; n=5). (A and B) In both experiments, the kidney-draining LN was evaluated for the chemokines Cxcl-1, -2, -5 by real-time PCR. The data are expressed as x-fold increase of the respective chemokine/beta-actin ration compared to healthy control LN (which was set as 1). All data represent the mean value ± SEM. *p<0.025.

DISCUSSION

Using the well described murine complement-, Th1 and Th17 T cell-mediated NTS model (29), we here provide first evidence that the nephroprotective effects of adoptively transferred Treg depend on MC recruitment to the kidney draining LN, which is linked by the cytokine IL-9.

In line with various disease models [reviewed in (36)], Treg have been shown to be essential in the regulation of NTS (5). The importance of this cell population in renal disease such as human Goodpasture’s disease or kidney transplantation has also been convincingly demonstrated (37, 38). MC have long been thought to only exert effector cell functions since they contain an armada of pro-inflammatory cytokines and chemokines stored in their granules. But more recently, it has been discussed that they can also function as an immuno-suppressive cell population (15). In line, MC-deficient mice subjected to NTS were found to develop significantly increased disease indices as compared to respective wt control animals (21, 22). In the recent past, the interaction between Treg and MC gained attention in various disease models (13, 39-41). Supporting a possible interaction, we observed that NTS mice receiving Treg displayed significantly increased MC infiltrates in the kidney-draining LN. Intriguingly, Treg transfer cannot overcome the lack of MC in our hands. Gri and co-workers first provided evidence that Treg might interact with MC in allergic disorders by limiting the degranulation of MC in vitro. Favouring our hypothesis, in vivo Treg depletion resulted in an increased anaphylactic reaction (13). Other reports found MC to have the potential to regulate Treg function in vitro (39-41). They found MC either to suppress (39, 40) or enhance (41) Treg function by various mechanisms. The difference in the in vitro experiments might be explained by variations in the surrounding conditions, which seem to be of crucial importance for the function of MC. In line, MC-deficient mice were found to differentially develop various inflammatory disease models. Whereas they were found to be protected from inflammatory arthritis (42) or experimental allergic encephalomyelitis (43), they developed increased disease indices when subjected to NTS (21, 22), contact dermatitis (44) or skin transplantation (23). Here we provide clear in vivo evidence that Treg-mediated immunosuppression in the NTS model depends on the presence of MC.

IL-9 has been implicated as a crucial factor in the regulation of MC recruitment and their effector functions (28, 33). IL-9 is secreted from various cell types, namely Th9 (34), Th17 cells (35) and Treg (23). Recent reports have suggested that IL-9 is an important factor driving Th17 T cell differentiation and supporting Treg function (35, 45). However, the exact role of IL-9 produced by Treg in an in vivo model remains elusive so far. We here show that Treg-derived IL-9 is the central mediator linking the nephro-protective Treg effects in NTS to the induction of protective MC responses. This is clearly demonstrated by the observation that Treg lacking IL-9 failed to suppress NTS. In contrast to Treg from IL-9 receptor deficient animals (45), Treg derived from IL-9 deficient animals exert a comparable target cell inhibition when cultured with wt responder T cells. The most likely reason for this observation is that IL-9 produced by the wt responder T cell population supports Treg function, whereas in vivo the Treg-derived IL-9 is the critical factor regulating MC accumulation in the inflammatory LN. Whether Treg-derived IL-9 promotes the recruitment and/or the proliferation of MC remains to be determined. Previous studies provided evidence that inflammation results in a remarkable MC hyperplasia, which is the result of both a limited expansion of mature MC and a more dramatic expansion of MC progenitors accompanied by their maturation within the inflamed tissue, where they are recruited to [reviewed in (46)]. CXCR-2 expressed on MC has been implicated in this recruitment process in an inflammatory model of the intestine (47). In our model only one CXCR-2 ligand, namely KC/Cxcl-1 was regulated by either IL-9 blocking antibody or transfer of IL-9 deficient Treg. In contrast, other CXCR2 ligands such as LIX/Cxcl-2 and MIP-2/Cxcl-5 remained unaltered in the absence of either systemic or Treg-derived IL-9. Whether the regulation of KC/Cxcl-1 is reflecting decreased MC infiltration or whether it is the key regulator in the recruitment of MC by Treg-derived IL-9 needs to be explored in future studies.

The mechanism how MC exert their immune-suppressive effects on kidney damaging effector T cells remains elusive so far. Histamine secreted by MC might be an interesting candidate, as exposure of DC to histamine induces a Th2-polarization (48, 49) and histamine injection itself inhibits NTS (50). Furthermore, it has been implicated that IL-10 or TGF-beta both produced by MC might contribute to the immunosuppressive state (51, 52). We found MC to influence the T cell response in the LN, as MC-deficiency leads to increased mRNA expression of Th1 and Th17 markers, which is reminiscent to the effects seen in Treg-depleted animals (own un-published data) and which suggests that, at least in the model of NTS, both MC and Treg are endogenous immuno-suppressive cells limiting inflammatory processes leading to renal tissue damage (5, 20).

In summary, despite some limitations of the NTS model (e.g. that parts of the antigen presentation depends on the initial immunization process and that the antigen might not be renal specific, which might at least in part be modified by Treg transfer), we are confident that Treg indeed recruit MC to the local LN, thus tipping the balance towards a more immuno-suppressive milieu inhibiting the various inflammatory processes involved in renal inflammation and subsequent organ dysfunction. The excess of immune complex deposition in the kidney is not altered in the Treg-injected animals. This strongly supports the idea that the initial priming process resulting in anti-rabbit IgG production is not altered by Treg, but that the Treg cells are indeed primarily involved in regulating the systemic T cell immune-response, which leads to amelioration of renal inflammation. The importance of Tregs localized to the regional LN is supported by our recent data showing that CCR7-mediated occupancy of the kidney-draining LN is required for their protective cues (20).

Thus, our data provide the first evidence that the immunosuppressive effects of adoptively transferred Treg depend on IL-9-mediated recruitment of MC to the kidney draining LN in NTS. This model is in perfect agreement with our previous report showing that CCR7-mediated LN occupancy of Treg is a prerequisite for their immune-suppressive potential and further adds a piece of information to the functional understanding of the in vivo anti-inflammatory effects of Treg.