original article
http://www.kidney-international.org & 2011 International Society of Nephrology
Regulatory T cells control the Th1 immune response in murine crescentic glomerulonephritis Hans-Joachim Paust1,6, Annett Ostmann2,6, Annette Erhardt2, Jan-Eric Turner1, Joachim Velden3, Hans-Willi Mittru¨cker4, Tim Sparwasser5, Ulf Panzer1 and Gisa Tiegs2 1
III Medizinische Klinik, Universita¨tsklinikum Hamburg-Eppendorf, Hamburg, Germany; 2Institut fu¨r Experimentelle Immunologie und Hepatologie, Universita¨tsklinikum Hamburg-Eppendorf, Hamburg, Germany; 3Institut fu¨r Pathologie, Universita¨tsklinikum Hamburg-Eppendorf, Hamburg, Germany; 4Institut fu¨r Immunologie, Universita¨tsklinikum Hamburg-Eppendorf, Hamburg, Germany and 5Institut fu¨r Infektionsimmunologie, Medizinische Hochschule Hannover, Hannover, Germany
Crescentic glomerulonephritis is mediated by inappropriate humoral and cellular immune responses toward self-antigens that may result from defects in central and peripheral tolerance. Evidence now suggests that regulatory T cells (Tregs) may be of pathophysiological importance in proliferative and crescentic forms of glomerulonephritis. To analyze the role of endogenous Tregs in a T celldependent glomerulonephritis model of nephrotoxic nephritis, we used ‘depletion of regulatory T cell’ (DEREG) mice that express the diphtheria toxin receptor under control of the FoxP3 (forkhead box P3) gene promoter. Toxin injection into these mice efficiently depleted renal and splenic FoxP3 þ Treg cells as determined by fluorescentactivated cell sorting (FACS) and immunohistochemical analyses. Treg depletion exacerbated systemic and renal interferon-c (IFNc) expression and increased recruitment of IFNc-producing Th1 cells into the kidney without an effect on the Th17 immune response. The enhanced Th1 response, following Treg cell depletion, was associated with an aggravated course of glomerulonephritis as measured by glomerular crescent formation. Thus, our results establish the functional importance of endogenous Tregs in the control of a significantly enhanced systemic and renal Th1 immune response in experimental glomerulonephritis. Kidney International (2011) 80, 154–164; doi:10.1038/ki.2011.108; published online 27 April 2011 KEYWORDS: cytokines; glomerulonephritis; immunology and pathology; immunosuppression; inflammation
Correspondence: Gisa Tiegs, Universita¨tsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. E-mail:
[email protected] 6
These authors contributed equally to this work.
Received 21 July 2010; revised 9 February 2011; accepted 15 February 2011; published online 27 April 2011 154
Crescentic glomerulonephritis is mediated by inappropriate humoral and cellular immune responses toward not wellcharacterized (self) antigens that might result from defects in central and peripheral tolerance. CD4 þ CD25 þ FoxP3 þ regulatory T cells (Tregs) are generally thought to be of critical importance for the maintenance of peripheral tolerance.1 Until now, little is known about the suppressive mechanisms mediated by CD4 þ CD25 þ FoxP3 þ Tregs and their behavior during proliferative and crescentic glomerulonephritis.2,3 With respect to human disease, one report indicates that CD25 þ T cells emerge following acute episodes of Goodpasture’s disease suppressing the response to the Goodpasture autoantigen.4 Furthermore, the potential of Tregs to suppress the pathogenic immune responses in kidney disease has recently been proven in a model of crescentic glomerulonephritis in mice. The adoptive transfer of CD4 þ CD25 þ Tregs showed that Tregs downregulate the nephritogenic immune response directly at the systemic site of antigen-specific T-cell priming, namely the renal lymph node.5 In consecutive studies, Eller et al.6 demonstrated that the chemokine receptor CCR7 (chemokine (C-C motif) receptor 7) regulates the trafficking of Tregs to the lymph node in experimental glomerulonephritis, which is essential for their anti-inflammatory properties. Our group recently provided evidence that Treg cells migrate in a CCR6–CCL20dependent manner into the inflamed kidney in crescentic glomerulonephritis and might locally suppress immunemediated renal inflammation.7 This study was designed to examine the potential role of endogenous Tregs in crescentic glomerulonephritis. We therefore induced the T cell-dependent model of nephrotoxic nephritis (NTN) in ‘depletion of regulatory T cell’ (DEREG) mice that express the diphtheria toxin (DTx) receptor and green fluorescent protein (GFP) under control of the FoxP3 (forkhead box P3) promoter, allowing selective and efficient depletion of FoxP3 þ Treg cells by DTx injection.8 Hence, we intended to address whether Treg depletion influences the clinical course of experimental glomerulonephritis, and if so, to study their potential mechanisms of action. Kidney International (2011) 80, 154–164
original article
H-J Paust et al.: Tregs and Th1 cells in experimental glomerulonephritis
RESULTS Functional involvement of endogenous Tregs in NTN
To investigate the role of endogenous Tregs, we determined the frequency of CD4 þ CD25 þ FoxP3 þ Tregs upon induction of NTN in a time course experiment. Interestingly, the number of Tregs was increased from day 5 onward, as shown by immunohistological FoxP3 staining (Figure 1a and b). To evaluate the immunosuppressive capacity of Tregs from nephritic mice compared with their counterparts from healthy control mice, we isolated splenic CD4 þ CD25 þ Tregs from both nephritic and healthy control mice and performed T-cell co-culture experiments. Enzyme-linked immunosorbent assay (ELISA) analysis of supernatants from co-cultures of Tregs and CD4 þ CD25 responder T cells indicated that Tregs from nephritic mice maintained their in vitro suppressive capacity and might be even more potent than Tregs from healthy controls to inhibit the production of interleukin (IL)-2 and interferon-g (IFNg) by responder T cells (Figure 1c, IL-2 (pg/ml): single culture of responder T cells: 334.3±22.0; responder T cells þ Tregs (control): 79.3±10.5; responder T cells þ Tregs (NTN): 43.7±13.5; ***Po0.0001; IFNg (pg/ml): single culture of responder T cells: 8490±772, responder T cells þ Tregs (control): 6084±2949, responder T cells þ Tregs (NTN): 4137±845.2; *Po0.05). Depletion of Tregs in DEREG mice by injection of DTx
To examine the involvement of endogenous Tregs in the model of crescentic glomerulonephritis, we used C57BL/6
DEREG mice in which specific depletion of FoxP3 þ Tregs by injection of DTx and tracking of green fluorescent Tregs by fluorescent-activated cell sorting (FACS) analysis can be performed. To assess the efficiency of Treg depletion after repetitive injection of 1 mg DTx per mouse (day 1 and day 3 upon NTN induction), FoxP3 expression was quantified in spleen and kidney at 7 days after NTN induction. Indeed, frequency of CD4 þ FoxP3 þ Tregs was significantly reduced in both spleen (Figure 2a; 7.8 vs o1%) and kidney tissue (Figure 2b; 20.1 vs 2.6%) of DTx-treated mice in contrast to control nephritic mice as shown by intracellular FoxP3 staining with subsequent FACS analysis. GFP expression was also reduced in accordance to FoxP3 expression (data not shown). Successful renal Treg depletion was confirmed by histological staining of FoxP3 (Figure 2c). The frequency of Tregs was significantly reduced by B80% at 4 days after the second DTx challenge (Figure 2d: FoxP3 þ Tregs (cells per low power field): 24.7±2.7 vs 5.3±1.5; **Po0.01). Aggravated NTN in Treg-depleted nephritic mice
Next, we induced NTN in C57BL/6 wild-type (wt) and DEREG mice with and without Treg depletion. Examination of periodic acid-Schiff (PAS)-stained kidney sections in the T cell-mediated autologous phase at day 7 after injection of the nephritogenic sheep serum revealed glomerular and tubulointerstitial damage. First, PAS-positive material was deposited intraglomerularly. Moreover, formation of cellular crescents and hypercellularity account for glomerular NTN
30 25 20 15 10 5 0
Renal Tregs
C on tro 24 l 3 h D 5 ays Da 7 D ys 10 ays Da ys
FoxP3
FoxP3+ cells/lpf
Control
Responder T cells IFNγ : ELISA
Tr eg s Tr Tr eg eg s s w t w t( N TN )
*
–
+
–
+
+
15,000 12,500 10,000 7500 5000 2500 0
+
*** ***
(pg/ml)
IL-2: ELISA
Tr e Tr gs Tr eg eg s s w w t( t N TN )
(pg/ml)
Responder T cells 600 500 400 300 200 100 0
Figure 1 | Endogenous regulatory T cells (Tregs) in the murine model of nephrotoxic nephritis (NTN). (a) Representative immunohistochemistry for Tregs (forkhead box P3 (FoxP3)) in kidney sections from nephritic mice and nonnephritic controls (original magnification 400). (b) Quantification of tubulointerstitial FoxP3 þ T-cell infiltration per low power field (lpf; original magnification 100) during the time course of NTN (nX4). (c) 1 105 Splenic CD4 þ CD25 responder T cells were cultured without or with 1 105 splenic CD4 þ CD25 þ Tregs from nonnephritic wild-type (wt) controls or nephritic wt mice and stimulated with plate-bound anti-CD3 monoclonal antibody (mAb) for 72 h. Secretion of the cytokines interleukin-2 (IL-2) and interferon-g (IFNg) was assessed in supernatants by enzymelinked immunosorbent assay (ELISA; *Po0.05, ***Po0.0001). Kidney International (2011) 80, 154–164
155
original article
H-J Paust et al.: Tregs and Th1 cells in experimental glomerulonephritis
Spleen
5
7.8
5
5
