FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2018594927> ?p ?o ?g. }

• W2018594927 endingPage "986" @default.
• W2018594927 startingPage "977" @default.
• W2018594927 abstract "Foxp3+ T-regulatory cells (Tregs) may suppress pathogenic inflammation; however, although transferred Tregs lessen glomerulonephritis in mice, the role of endogenous foxp3+ cells is not known. To study this, we characterized endogenous foxp3+ cells in accelerated anti-glomerular basement membrane (GBM) nephritis by using foxp3GFP reporter mice to track their responses in early and established disease. Further, diphtheria toxin was used to ablate foxp3+ Tregs in foxp3DTR mice after establishing an immune response. In this model, mice were immunized with sheep globulin in adjuvant, and sheep anti-mouse GBM globulin was injected after 4 days to initiate progressive histological and functional injury. Intrarenal leukocytic infiltrates were increased by day 3 but intrarenal foxp3+ Tregs, present in interstitial and periglomerular areas, were only increased at day 7. Ablation of foxp3+ Tregs after injection of anti-GBM globulin increased renal injury and systemic T-cell responses, including increased interferon-γ and interleukin-17A (IL-17A) production, but no change in antibody titers. Compared with foxp3+ Tregs isolated from naive mice, those from immunized mice produced more IL-10 and more effectively regulated CD4+foxp3− responder T cells. Thus, endogenous foxp3+ Tregs infiltrate the kidney in glomerulonephritis, and deleting foxp3+ cells after the induction of immune responses upregulated T-cell reactions and enhanced disease. Hence, endogenous foxp3+ cells have increased suppressive capacity after immune stimuli. Foxp3+ T-regulatory cells (Tregs) may suppress pathogenic inflammation; however, although transferred Tregs lessen glomerulonephritis in mice, the role of endogenous foxp3+ cells is not known. To study this, we characterized endogenous foxp3+ cells in accelerated anti-glomerular basement membrane (GBM) nephritis by using foxp3GFP reporter mice to track their responses in early and established disease. Further, diphtheria toxin was used to ablate foxp3+ Tregs in foxp3DTR mice after establishing an immune response. In this model, mice were immunized with sheep globulin in adjuvant, and sheep anti-mouse GBM globulin was injected after 4 days to initiate progressive histological and functional injury. Intrarenal leukocytic infiltrates were increased by day 3 but intrarenal foxp3+ Tregs, present in interstitial and periglomerular areas, were only increased at day 7. Ablation of foxp3+ Tregs after injection of anti-GBM globulin increased renal injury and systemic T-cell responses, including increased interferon-γ and interleukin-17A (IL-17A) production, but no change in antibody titers. Compared with foxp3+ Tregs isolated from naive mice, those from immunized mice produced more IL-10 and more effectively regulated CD4+foxp3− responder T cells. Thus, endogenous foxp3+ Tregs infiltrate the kidney in glomerulonephritis, and deleting foxp3+ cells after the induction of immune responses upregulated T-cell reactions and enhanced disease. Hence, endogenous foxp3+ cells have increased suppressive capacity after immune stimuli. T cells that express the transcription factor foxp3 are a regulatory subset of CD4+ helper T cells, necessary for the maintenance of peripheral tolerance1.Fontenot J.D. Gavin M.A. Rudensky A.Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.Nat Immunol. 2003; 4: 330-336Crossref PubMed Scopus (5710) Google Scholar, 2.Hori S. Nomura T. Sakaguchi S. Control of regulatory T cell development by the transcription factor foxp3.Science. 2003; 299: 1057-1061Crossref PubMed Scopus (36) Google Scholar and immune homeostasis.3.Kim J.M. Rasmussen J.P. Rudensky A.Y. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice.Nat Immunol. 2007; 8: 191-197Crossref PubMed Scopus (1224) Google Scholar, 4.Lahl K. Loddenkemper C. Drouin C. et al.Selective depletion of foxp3+ regulatory T cells induces a scurfy-like disease.J Exp Med. 2007; 204: 57-63Crossref PubMed Scopus (670) Google Scholar Foxp3+ T-regulatory cells (Tregs) are the most well-characterized subset of Tregs, but not all subsets of Tregs express foxp3.5.Maynard C.L. Harrington L.E. Janowski K.M. et al.Regulatory T cells expressing interleukin 10 develop from foxp3+ and foxp3- precursor cells in the absence of interleukin 10.Nat Immunol. 2007; 8: 931-941Crossref PubMed Scopus (440) Google Scholar Foxp3+CD4+ cells are reciprocally linked with the development of Th17 effector cells,6.Lee Y.K. Mukasa R. Hatton R.D. et al.Developmental plasticity of Th17 and Treg cells.Curr Opin Immunol. 2009; 21: 274-280Crossref PubMed Scopus (307) Google Scholar, 7.Zhou L. Chong M.M. Littman D.R. Plasticity of CD4+ T cell lineage differentiation.Immunity. 2009; 30: 646-655Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar and recent studies suggest that these foxp3+ Tregs cells can regulate both Th1-8.Koch M.A. Tucker-Heard G. Perdue N.R. et al.The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation.Nat Immunol. 2009; 10: 595-602Crossref PubMed Scopus (848) Google Scholar and Th17-9.Reynolds A.D. Stone D.K. Hutter J.A. et al.Regulatory T cells attenuate Th17 cell-mediated nigrostriatal dopaminergic neurodegeneration in a model of Parkinson's disease.J Immunol. 2010; 184: 2261-2271Crossref PubMed Scopus (245) Google Scholarmediated effector responses. In experimental murine glomerulonephritis, both Th1 and Th17 effector subsets, and their respective signature cytokines, interferon-γ (IFN-γ) and interleukin-17A (IL-17A), can mediate severe glomerular disease.10.Phoon R.K. Kitching A.R. Odobasic D. et al.T-bet deficiency attenuates renal injury in experimental crescentic glomerulonephritis.J Am Soc Nephrol. 2008; 19: 477-485Crossref PubMed Scopus (57) Google Scholar, 11.Kitching A.R. Holdsworth S.R. Tipping P.G. IFN-gamma mediates crescent formation and cell-mediated immune injury in murine glomerulonephritis.J Am Soc Nephrol. 1999; 10: 752-759PubMed Google Scholar, 12.Kitching A.R. Tipping P.G. Holdsworth S.R. IL-12 directs severe renal injury, crescent formation and Th1 responses in murine glomerulonephritis.Eur J Immunol. 1999; 29: 1-10Crossref PubMed Scopus (73) Google Scholar, 13.Paust H.J. Turner J.E. Steinmetz O.M. et al.The IL-23/Th17 axis contributes to renal injury in experimental glomerulonephritis.J Am Soc Nephrol. 2009; 20: 969-979Crossref PubMed Scopus (180) Google Scholar In renal autoimmunity, regulation of autoreactive T-cell responses to the Goodpasture antigen by CD4+CD25+ T cells has been demonstrated in convalescent Goodpasture's disease patients.14.Salama A.D. Chaudhry A.N. Holthaus K.A. et al.Regulation by CD25+ lymphocytes of autoantigen-specific T-cell responses in Goodpasture's (anti-GBM) disease.Kidney Int. 2003; 64: 1685-1694Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar In murine models of crescentic glomerulonephritis, transfer of exogenous naive CD4+CD25+ T cells before induction of nephritis attenuates disease,15.Wolf D. Hochegger K. Wolf A.M. et al.CD4+CD25+ regulatory T cells inhibit experimental anti-glomerular basement membrane glomerulonephritis in mice.J Am Soc Nephrol. 2005; 16: 1360-1370Crossref PubMed Scopus (156) Google Scholar and affecting the migration of Tregs to secondary lymphoid organs16.Eller K. Weber T. Pruenster M. et al.CCR7 deficiency exacerbates injury in acute nephritis due to aberrant localization of regulatory T cells.J Am Soc Nephrol. 2010; 21: 42-52Crossref PubMed Scopus (38) Google Scholar or the kidney17.Turner J.E. Paust H.J. Steinmetz O.M. et al.CCR6 Recruits Regulatory T Cells and Th17 Cells to the Kidney in Glomerulonephritis.J Am Soc Nephrol. 2010; 21: 974-985Crossref PubMed Scopus (125) Google Scholar worsens injury. In murine models of toxic and ischemic renal injury, transfer of foxp3-transduced CD4+ cells or naive CD25+ cells, or anti-CD25 antibody administration, attenuates injury.18.Wang Y.M. Zhang G.Y. Wang Y. et al.Foxp3-transduced polyclonal regulatory T cells protect against chronic renal injury from adriamycin.J Am Soc Nephrol. 2006; 17: 697-706Crossref PubMed Scopus (70) Google Scholar, 19.Lee H. Nho D. Chung H.S. et al.CD4(+)CD25(+) regulatory T cells attenuate cisplatin-induced nephrotoxicity in mice.Kidney Int. 2010; 78: 1100-1109Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 20.Loser K. Hansen W. Apelt J. et al.In vitro-generated regulatory T cells induced by foxp3-retrovirus infection control murine contact allergy and systemic autoimmunity.Gene Ther. 2005; 12: 1294-1304Crossref PubMed Scopus (78) Google Scholar, 21.Gandolfo M.T. Jang H.R. Bagnasco S.M. et al.Foxp3+ regulatory T cells participate in repair of ischemic acute kidney injury.Kidney Int. 2009; 76: 717-729Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 22.Kinsey G.R. Sharma R. Huang L. et al.Regulatory T cells suppress innate immunity in kidney ischemia-reperfusion injury.J Am Soc Nephrol. 2009; 20: 1744-1753Crossref PubMed Scopus (269) Google Scholar, 23.Kinsey G.R. Huang L. Vergis A.L. et al.Regulatory T cells contribute to the protective effect of ischemic preconditioning in the kidney.Kidney Int. 2010; 77: 771-780Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 24.Monteiro R.M. Camara N.O. Rodrigues M.M. et al.A role for regulatory T cells in renal acute kidney injury.Transpl Immunol. 2009; 21: 50-55Crossref PubMed Scopus (29) Google Scholar Although studies suggest a role for foxp3+ Tregs in suppressing renal disease, most have focused on cell transfer; therefore, any role for endogenous foxp3+ cells in limiting glomerular disease directed by antigen-specific CD4+ cells has not been demonstrated directly. This question can be addressed using foxp3GFP reporter mice25.Fontenot J.D. Rasmussen J.P. Williams L.M. et al.Regulatory T cell lineage specification by the forkhead transcription factor foxp3.Immunity. 2005; 22: 329-341Abstract Full Text Full Text PDF PubMed Scopus (1830) Google Scholar and foxp3DTR mice.4.Lahl K. Loddenkemper C. Drouin C. et al.Selective depletion of foxp3+ regulatory T cells induces a scurfy-like disease.J Exp Med. 2007; 204: 57-63Crossref PubMed Scopus (670) Google Scholar Foxp3GFP mice25.Fontenot J.D. Rasmussen J.P. Williams L.M. et al.Regulatory T cell lineage specification by the forkhead transcription factor foxp3.Immunity. 2005; 22: 329-341Abstract Full Text Full Text PDF PubMed Scopus (1830) Google Scholar possess a green fluorescent protein (GFP)–foxp3 fusion protein-reporter knock-in allele that allows foxp3+ cells to be identified and isolated for functional experiments. Foxp3DTR mice4.Lahl K. Loddenkemper C. Drouin C. et al.Selective depletion of foxp3+ regulatory T cells induces a scurfy-like disease.J Exp Med. 2007; 204: 57-63Crossref PubMed Scopus (670) Google Scholar express the human diphtheria toxin (DT) receptor under the control of the foxp3 locus, allowing specific ablation of this regulatory T-cell subset on administration of DT. The aim of the current studies is to define the role of endogenous foxp3+ Tregs in anti-glomerular basement membrane (GBM) nephritis.26.Kitching A.R. Holdsworth S.R. Tipping P.G. Crescentic glomerulonephritis-a manifestation of a nephritogenic Th1 response?.Histol Histopathol. 2000; 15: 993-1003PubMed Google Scholar The availability of foxp3DTRmice allows depletion of Tregs after initiating an immune response to the nephritogenic antigen (in this model, sheep globulin). Previously used strategies for depleting endogenous Tregs using anti-CD25 antibodies would, in this situation, deplete effector T cells as well.27.Couper K.N. Lanthier P.A. Perona-Wright G. et al.Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma gondii infection.J Immunol. 2009; 182: 3985-3994Crossref PubMed Scopus (55) Google Scholar The availability of foxp3DTR mice, used in the current studies, overcomes this problem by allowing specific deletion of Tregs on the basis of their lineage-specific expression of foxp3. Renal injury and infiltrates in this model of accelerated anti-GBM disease were characterized using foxp3GFP reporter mice.24.Monteiro R.M. Camara N.O. Rodrigues M.M. et al.A role for regulatory T cells in renal acute kidney injury.Transpl Immunol. 2009; 21: 50-55Crossref PubMed Scopus (29) Google Scholar Mice were immunized with normal sheep globulin 4 days before intravenous injection of sheep anti-GBM globulin on day 0, and humanely killed on day 3 or 7. Control mice were immunized with sheep globulin, and killed on day 7. Mice injected with anti-GBM globulin developed focal and segmental necrosis, glomerular crescent formation, tubulointerstitial injury (tubular dilation and atrophy, sloughing of tubular epithelial cells and cast formation), proteinuria and hematuria (described in Figure 1). Immunochemical staining revealed glomerular CD4+ T cells, macrophages, and neutrophils at day 3 but no further increase at day 7 (Figure 2a, c and e) and progressively increased numbers of tubulointerstitial CD4+ cells, macrophages, and neutrophils (Figure 2b, d and f).Figure 2Infiltration of cellular effectors into glomerular and tubulointerstitial compartments in early, day 3, and established, day 7, disease. Immunohistological staining on periodate lysine paraformaldehyde-fixed frozen kidney sections of renal (a, b) CD4+ T cells, (c, d) macrophages, and (e, f) neutrophils in control (ctrl), day 3, and day 7 mice. *P<0.05, **P<0.01, ***P<0.001 by analysis of variance, Tukey's post-test. gcs, glomerular cross-section; hpf, high-power field.View Large Image Figure ViewerDownload (PPT) Immunohistological staining for foxp3 on paraffin-embedded kidney sections showed that foxp3+ Tregs were rarely found in control or in day 3 mice but were readily detected in day 7 mice, predominantly in periglomerular and tubulointerstitial areas (Figure 3a–f and g). Foxp3+ cells were rarely found in glomeruli. Using flow cytometry, endogenous foxp3GFP numbers were increased at day 7 (compared with control and day 3 mice; Figure 3h). As a proportion of all intrarenal leukocytes (CD45+ cells), foxp3+ Tregs were increased at day 7 compared with control and day 3 mice: 1.9 versus 1.1 and 1.2%, respectively (Figure 3i). As a proportion of intrarenal CD4+ T cells, compared with control, foxp3+ Tregs were decreased at day 3, but increased by day 7: 11.0, 7.3, and 13.8%, respectively (Figure 3j). Representative flow cytometry plots of the intrarenal CD45+ cells, gated on CD4+ and foxp3GFP+ cells, are shown in Figure 3k. Dual immunofluorescence confocal microscopy of kidney sections stained with an antigen-presenting cell-conjugated anti-CD4+ antibody (using endogenous GFP expressed by foxp3GFP mice) confirmed the presence of intrarenal CD4+foxp3+ Tregs in kidneys of day 7 mice (Figure 4a–c).Figure 4Detection of intrarenal CD4+foxp3− cells and CD4+foxp3+ T-regulatory cells using dual immunofluorescence confocal microscopy in established disease. Kidney sections from mice with established anti-glomerular basement membrane nephritis (day 7) were stained with anti-CD4 antibodies, and foxp3+ cells were identified by green fluorescent protein expression. (a) CD4+ cells (red). (b) Foxp3+ cells (green). (c) Merge: double-positive cells (yellow arrows) are seen along with single-positive CD4+foxp3− cells (red). Micrographs were taken at × 600.View Large Image Figure ViewerDownload (PPT) Foxp3+ Tregs in the draining inguinal lymph nodes enumerated by flow cytometry and expressed as a proportion of CD45+ or CD4+ cells were not different between control, day 3, and day 7 groups (Table 1). There was a trend toward a decrease in the total number of foxp3+ Tregs and an increase in foxp3+ Tregs as a proportion of CD45+ cells. In this model of anti-GBM nephritis, the majority of foxp3+ Tregs in the kidney and inguinal draining lymph nodes were CD25+, whereas others were CD25− (Supplementary Figure S1 online). By day 7, a higher proportion of foxp3+ cells in the kidney were CD25− compared with control mice.Table 1Foxp3+ T-regulatory cell numbers and proportions of CD45+ and CD4+ cells in the draining lymph nodeCD4+foxp3+CD4+foxp3+CD4+foxp3+No. (× 103)CD45+ (%)CD4+ (%)Ctrl28.3±6.81.29±0.15.65±0.4Day 330.2±10.21.20±0.15.40±0.5Day 713.3±4.31.68±0.25.40±0.6Abbreviations: Ctrl, control; Tregs, T-regulatory cells.Total numbers of foxp3+ Tregs were enumerated by flow cytometry from the two inguinal draining lymph nodes and expressed as proportions of CD45+ as well as CD4+ cells in control-immunized mice at day 3 or at day 7. Open table in a new tab Download .jpg (.04 MB) Help with files Supplementary Figure 1 Abbreviations: Ctrl, control; Tregs, T-regulatory cells. Total numbers of foxp3+ Tregs were enumerated by flow cytometry from the two inguinal draining lymph nodes and expressed as proportions of CD45+ as well as CD4+ cells in control-immunized mice at day 3 or at day 7. Foxp3DTR mice were studied to determine the role of endogenous foxp3+ Tregs in the effector phase of disease. Mice were immunized with normal sheep globulin, then after 4 days injected with sheep anti-mouse GBM globulin. Foxp3+ Tregs were ablated in foxp3DTR mice by intraperitoneal 1 μg DT injections after anti-GBM globulin on the day of injection, with further injections after 24 and 72 h, according to previously published protocols.4.Lahl K. Loddenkemper C. Drouin C. et al.Selective depletion of foxp3+ regulatory T cells induces a scurfy-like disease.J Exp Med. 2007; 204: 57-63Crossref PubMed Scopus (670) Google Scholar, 28.Schildknecht A. Brauer S. Brenner C. et al.FoxP3+ regulatory T cells essentially contribute to peripheral CD8+ T-cell tolerance induced by steady-state dendritic cells.Proc Natl Acad Sci USA. 2010; 107: 199-203Crossref PubMed Scopus (71) Google Scholar This depletion strategy ensured that immune responses to sheep globulin had been established, with depletion occurring after the induction of anti-GBM nephritis. Control C57BL/6 wild-type (WT) mice with glomerulonephritis received equal doses of DT. WT mice developed moderate histological disease at day 10, characterized by glomerular focal and segmental necrosis, crescent formation, and tubulointerstitial injury. All disease parameters were exacerbated in foxp3DTR mice after depleting foxp3+ Tregs (Figure 5a–h). Compared with WT mice, endogenous foxp3+ depletion in foxp3DTR mice resulted in an increase in CD4+ T cells, macrophages, and neutrophils both in glomeruli and the tubulointerstitium (Figure 6a–f). The proportions and activation status of intrarenal CD4+ T cells were analyzed by flow cytometry. As a proportion of CD45+ cells, foxp3DTR mice had increased CD4+ cells (Figure 7a and b), but proportions of intrarenal CD4+ cells expressing the effector memory phenotype (CD4+CD44+) were unchanged (Figure 7c and d).Figure 7Renal CD4+ cell proportion is increased by flow cytometry. Kidneys of WT and foxp3DTR mice were digested, and single cell suspensions were analyzed for CD45+, CD4+, and CD44+ expression by flow cytometry. (a) Analysis of renal CD4+ cells as a proportion of CD45+ cells. (b) Illustrative FACS histograms showing a marked increased in the proportion of CD4+ cells in the kidney (numbers are the percentages of CD4+ cells). (c) Analysis of the proportion of CD4+ cells that are of effector memory, CD4+CD44+, phenotype. (d) Illustrative FACS histograms of proportions of renal CD4+ cells expressing CD44. *P<0.05 by Student's t-test. WT, wild type.View Large Image Figure ViewerDownload (PPT) Supernatant from sheep globulin-stimulated splenocyte cultures was analyzed by cytometric bead array for IFN-γ, IL-17A, tumor necrosis factor, and IL-6. The prototypic Th1 cytokine IFN-γ or the Th17 cytokine IL-17A was not detected in splenocyte supernatants from WT mice (limit of assay: 20 pg/ml), but both were found in supernatants from all foxp3DTR mice (Figure 8a and b). Tumor necrosis factor and IL-6 were also increased in foxp3DTR mice (Figure 8c and d). Titers of mouse anti-sheep immunoglobulin G (IgG) antibodies were unchanged in foxp3DTR mice (Figure 8e). CD4+ effector T cells were assessed in lymph nodes by removing the draining inguinal lymph nodes at day 10, counting cell numbers, then analyzing the proportions of CD4+CD44+ effector memory T cells. Compared with WT mice, foxp3+ cell-depleted mice had more total leukocytes in the draining nodes (Figure 9a). The proportion of CD4+ T cells within the lymph node cell population was not different between groups, but foxp3+ cell-depleted mice had a higher proportion of CD4+ cells expressing CD44 (Figure 9b–d).Figure 9CD4+ T-cell differentiation into CD4+CD44+ effector memory cells is upregulated in the draining inguinal lymph node following ablation of foxp3+ T-regulatory cells. Single cell suspensions from the draining inguinal lymph nodes were enumerated, stained for CD4+ and CD44+ expression, and analyzed by flow cytometry. (a) Total numbers of leukocytes from the two inguinal nodes. (b) Percentage of leukocytes expressing CD4+. (c) Percentage of CD4+CD44+ cells as a proportion of CD4+ cells. (d) Illustrative FACS plot showing a marked increase in the percentages of CD44-expressing CD4+ cells in foxp3DTR mice. **P<0.01 and ***P<0.001 by Student's t-test. WT, wild type.View Large Image Figure ViewerDownload (PPT) To determine whether endogenous foxp3+ Tregs have an increased suppressive capacity following immune stimulation, the potency of ‘activated’ foxp3+ Tregs (from immunized mice) in suppressing effector T-cell responses was compared with that of naive foxp3+ Tregs. Responder CD4+foxp3− T cells from sheep globulin-immunized mice were cultured with either CD4+foxp3+ Tregs from naive mice or with CD4+foxp3+ Tregs from mice immunized with sheep globulin. Different foxp3+ Treg/responder T-cell ratios were used, with mitomycin C-treated, CD4-depleted naive splenocytes, and sheep IgG included in cultures. Compared with foxp3+ Tregs from naive mice, foxp3+ Tregs from immunized mice had a greater capacity to inhibit proliferation of CD4+foxp3− responder T cells (Figure 10a). IFN-γ secretion from CD4+foxp3− responder T cells was suppressed below the assay's detection limit (20 pg/ml) when cocultured with foxp3+ Tregs from immunized mice, but was detectable at a ratio of 1:16 and 1:8 when mixed with foxp3+ Tregs from naive mice (Figure 10b). Tregs from immunized mice were also more potent inhibitors of IL-17A (Figure 10c). IL-2 and tumor necrosis factor secretion levels were also markedly reduced by foxp3+ Tregs and more effectively with Tregs from immunized mice. Tregs (2 × 104 cells) from immunized mice secreted more IL-10 when cultured without responder T cells compared with foxp3+ Tregs from naive mice (Figure 10f). At a ratio of 1:2 (foxp3+ Treg (1 × 104 cells) to responder T cells (2 × 104 cells)), IL-10 was still detectable in immunized mice Tregs, but only marginally detectable in naive Treg cocultures. The role of endogenous Tregs in nephritis has not yet been defined. The current studies characterize the evolution of a T-regulatory cell response by endogenous foxp3+ Tregs during early inflammatory injury, at day 3, and when disease is established, at day 7, and demonstrates that depleting endogenous foxp3+ Tregs after the induction of immune responses enhances T-cell effector responses and leads to more severe glomerulonephritis. Furthermore, following immunization, foxp3+ Tregs have an increased ability to regulate helper T effector-mediated responses. In this model of anti-GBM nephritis, mice were immunized with sheep globulin to induce an immune response against this antigen, and renal disease was triggered using sheep anti-mouse GBM globulin. In a similar model of nephritis, nephritic kidneys exhibited increased foxp3+ Tregs at day 7.17.Turner J.E. Paust H.J. Steinmetz O.M. et al.CCR6 Recruits Regulatory T Cells and Th17 Cells to the Kidney in Glomerulonephritis.J Am Soc Nephrol. 2010; 21: 974-985Crossref PubMed Scopus (125) Google Scholar However, whether these endogenous foxp3+ Tregs were recruited into the kidney before or after effector cell infiltrates was not known. Tracking of foxp3+ Tregs at days 3 and 7 within the kidney by immunohistology and flow cytometry showed that intrarenal foxp3+ Tregs are increased in the kidney only at day 7, but not at day 3. As a proportion of CD4+ cells, foxp3+ Tregs are decreased at day 3, consistent with the influx of CD4+foxp3− effector helper T cells into the kidney. Confocal microscopy confirmed that foxp3+CD4+ cells were readily found in the kidney at day 7. Therefore, tracking of endogenous renal foxp3+ Tregs in the early and established phase of disease suggests that intrarenal foxp3+ Tregs are unlikely to be important at the onset of nephritis but could be relevant after nephritic insult by cellular effectors has occurred. In the draining lymph nodes, total numbers of foxp3+ Tregs and foxp3+ Treg percentages as proportions of CD45+ or CD4+ were not significantly different between control, day 3, and day 7 mice, but there was a trend to decreased numbers of foxp3+ Tregs at day 7, which may reflect emigration of Tregs from the node. Before the development of foxp3DTR mice, studies in other models of disease have used anti-CD25 antibodies to deplete Tregs.29.McHugh R.S. Whitters M.J. Piccirillo C.A. et al.CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor.Immunity. 2002; 16: 311-323Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar This method does not deplete CD25− foxp3+ cells,2.Hori S. Nomura T. Sakaguchi S. Control of regulatory T cell development by the transcription factor foxp3.Science. 2003; 299: 1057-1061Crossref PubMed Scopus (36) Google Scholar, 25.Fontenot J.D. Rasmussen J.P. Williams L.M. et al.Regulatory T cell lineage specification by the forkhead transcription factor foxp3.Immunity. 2005; 22: 329-341Abstract Full Text Full Text PDF PubMed Scopus (1830) Google Scholar and, in the context of an active immune response, may deplete activated T cells.27.Couper K.N. Lanthier P.A. Perona-Wright G. et al.Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma gondii infection.J Immunol. 2009; 182: 3985-3994Crossref PubMed Scopus (55) Google Scholar It is likely that depleting foxp3+ cells before a nephritogenic immune response was established would enhance the subsequent adaptive immune response and therefore injury. However, as using foxp3DTR mice means that Tregs can be depleted specifically, we sought to address the role of endogenous foxp3+ cells in the context of an active immune response. In the current studies, foxp3+ Treg ablation after inducing nephritogenic immune responses led to more severe glomerulonephritis. Although there was also an increase in renal CD4+ T cells as a proportion of CD45+ cells, the proportion of CD4+ T cells that were CD4+CD44+ effector memory phenotype was similar, showing that endogenous foxp3+ Tregs limited recruitment to the kidney of both CD44+ and CD44− CD4+ cells. Systemic immunity to the nephritogenic antigen, measured by splenocyte cytokine production, showed that ablation of endogenous foxp3+ Tregs resulted in increased Th1 and Th17 cytokines (IFN-γ and IL-17A), as well as in increased proinflammatory cytokines tumor necrosis factor and IL-6. The increase in both IFN-γ and IL-17A suggests that, in this model, foxp3+ Tregs regulate both Th1 and Th17 effector subsets. In the draining lymph node, foxp3DTR mice had increased total number of cells and a marked increase in the proportions of CD4+ T cells that were CD4+CD44+ effector memory T cells, showing that foxp3+ Tregs regulate T-cell activation in secondary lymphoid organs in this model.16.Eller K. Weber T. Pruenster M. et al.CCR7 deficiency exacerbates injury in acute nephritis due to aberrant localization of regulatory T cells.J Am Soc Nephrol. 2010; 21: 42-52Crossref PubMed Scopus (38) Google Scholar The timing of the depletion of foxp3+ cells, after the induction of immune responses, allowed the development of intact humoral immunity, as serum anti-sheep IgG levels were unchanged. The near absence of Tregs in glomeruli of mice with glomerulonephritis in this model, together with the markedly increased systemic T-cell response, implies, at least in the glomerular lesion, that Tregs exert their effects predominantly within secondary lymphoid organs. However, the presence of intrarenal Tregs within the tubulointerstitium raises the possibility of an additional local role for Tregs in regulating the tubulointerstitial component of this disease. The availability of foxp3GFP reporter allows for Tregs to be isolated from an activated immune system and used in functional assays without compromising viability. Reliably isolating regulatory T cells for functional assays was previously possible only in naive mice, as selecting Tregs on the basis of CD25 expression meant that in active immune responses effector CD4+ cells expressing CD25 would be included as ‘regulatory’ cells.27.Couper K.N. Lanthier P.A. Perona-Wright G. et al.Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma gondii infection.J Immunol. 2009; 182: 3985-3994Crossref PubMed Scopus (55) Google Scholar In our ex vivo coculture experiments, foxp3+ Tregs from immunized mice were better at suppressing T-cell effector functions (proliferation and secretion of proinflammatory cytokines) than were Tregs from naive mice. This increased suppressive effect of foxp3+ Tregs from immunized mice suggests that endogenous Tregs upregulate their suppressive function in response to immune stimuli. Tregs from immunized mice secreted more IL-10, consistent with studies showing that this anti-inflammatory cytokine regulates disease in this model.30.Kitching A.R. Tipping P.G. Timoshanko J.R. et al.Endogenous interleukin-10 regulates Th1 responses that induce crescentic glomerulonephritis.Kidney Int. 2000; 57: 518-525Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar The effects of foxp3+ Tregs from naive mice when cocultured at higher ratios of Tregs to T-responder cells are consistent with those of previous experiments demonstrating that transfer of exogenous CD4+CD25+ Tregs before inducing an immune response results in suppression of nephritis.15.Wolf D. Hochegger K. Wolf A.M. et al.CD4+CD25+ regulatory T cells inhibit experimental anti-glomerular basement membrane glomerulonephritis in mice.J Am Soc Nephrol. 2005; 16: 1360-1370Crossref PubMed Scopus (156) Google Scholar In conclusion, the current studies characterize the infiltration of endogenous foxp3+ Tregs into the kidney, demonstrate their necessity in attenuating glomerulonephritis, and show that foxp3+ Tregs upregulate their regulatory capacity in nephritogenic inflammation. Foxp3GFP female mice (n=4 per group) were housed at Monash Animal Services (Melbourne, Australia). Age- and sex-matched foxp3DTR mice (n=6), and control C56BL/6J mice (n=5) were housed at the Institute for Molecular Medicine and Experimental Immunology, Bonn, Germany. Accelerated anti-GBM glomerulonephritis was induced by immunizing mice subcutaneously with 0.5 mg (100 μl) of sheep globulin in Freund's complete adjuvant (100 μl) at day −4, followed by intravenous injection of sheep anti-mouse GBM globulin (0.15 or 0.20 mg/g) at day 0. Mice were humanely killed at day 3, 7, or 10. Foxp3+ Tregs were specifically ablated by injection of DT (Merck, Darmstadt, Germany) at days 0, 1, and 3. Control C57BL/6 mice with glomerulonephritis received equal doses of DT at the same time points. This method of foxp3+ cell depletion achieved an efficiency of ~90% when measured by flow cytometry at day 4 in the kidney, inguinal draining lymph nodes, and spleen (Supplementary Figure S2 online). Animal studies were conducted under specific pathogen-free conditions and approved by the Monash University Animal Ethics Committee or the German Institutional and Government Review Boards. Results are expressed as means±s.e.m. Statistical analyses were conducted using analysis of variance when comparing three groups, followed by Tukey's post-test or Student’s unpaired t-test when comparing two groups, *P<0.05, **P<0.01, ***P<0.001 (GraphPad Prism; GraphPad Software, San Diego, CA). Download .jpg (.03 MB) Help with files Supplementary Figure 2 Glomerular necrosis and crescents and tubulointerstitial injury were assessed on 3 μm-thick, periodic acid-Schiff-stained, Bouin's-fixed, paraffin-embedded sections, ≥50 glomeruli per mouse. Glomerular necrosis was defined as accumulation of periodic acid-Schiff-positive material in ≥50% of the glomerulus, and glomerular crescents as two or more layers of cells in Bowman's space. Tubulointerstitial injury was assessed in 15 randomly selected cortical areas from each animal at × 250 magnification using a 10 mm2 graticule, and defined as tubular dilation, tubular atrophy, sloughing of tubular epithelial cells, and cast formation. Injury was graded according to a scale of 0–4: grade 0, 0%, grade 1, 0–25%; grade 2, 25–50%, grade 3; 50–75%; and grade 4, 75–100% of the tubulointerstitium injured. Urine was collected either by bladder puncture at the time of death or by metabolic cage 24 h before death. Proteinuria and hematuria was measured by Combur-Test strips (Roche Diagnostics, Castle Hill, Australia) or proteinuria by modified Bradford's method as previously described.31.Huang X.R. Tipping P.G. Apostolopoulos J. et al.Mechanisms of T cell-induced glomerular injury in anti-glomerular basement membrane (GBM) glomerulonephritis in rats.Clin Exp Immunol. 1997; 109: 134-142Crossref PubMed Scopus (91) Google Scholar CD4+ T cells, macrophages, and neutrophils were shown by immunoperoxidase staining of 6 μm-thick, periodate lysine paraformaldehyde-fixed, frozen kidney sections as previously described.32.Kitching A.R. Turner A.L. Wilson G.R. et al.IL-12p40 and IL-18 in crescentic glomerulonephritis: IL-12p40 is the key Th1-defining cytokine chain, whereas IL-18 promotes local inflammation and leukocyte recruitment.J Am Soc Nephrol. 2005; 16: 2023-2033Crossref PubMed Scopus (70) Google Scholar Foxp3 was detected on formalin-fixed paraffin-embedded 3 μm sections. The primary monoclonal antibodies used were GK1.5 (anti-mouse CD4; American Type Culture Collection, Manassas, VA), FA/11 (macrophages, anti-mouse CD68; from Dr Gordon L. Koch, Cambridge, England), RB6–8C5 (neutrophils, anti-Gr-1), and anti-mouse foxp3 (FJK-16s; eBiosciences, San Diego, CA), with rabbit anti-rat biotin as a secondary antibody (BD Biosciences, North Ryde, Australia). To enumerate positive-stained cells, ≥20 consecutively viewed glomeruli and ≥15 high-power cortical interstitial fields (excluding perivascular regions) were assessed per animal as described previously.32.Kitching A.R. Turner A.L. Wilson G.R. et al.IL-12p40 and IL-18 in crescentic glomerulonephritis: IL-12p40 is the key Th1-defining cytokine chain, whereas IL-18 promotes local inflammation and leukocyte recruitment.J Am Soc Nephrol. 2005; 16: 2023-2033Crossref PubMed Scopus (70) Google Scholar Confocal images were captured using a NikonC1 inverted confocal laser and Nikon Ti-E scanning microscope from 6 μm SNAP frozen sections, blocked with 10% rat serum in 5% bovine serum albumin/phosphate buffered saline before incubating with antigen-presenting cell-conjugated CD4 antibodies (eBioscience). Kidneys were digested with 1 mg/ml collagenase D (Roche Diagnostics) and 0.1 mg/ml DNAse I (Roche Diagnostics) in RPMI 1640/10% fetal calf serum (Invitrogen, Mount Waverley, Australia) and 10 mmol/l HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Spleens and lymph nodes were minced and filtered, erythrocytes lysed with ammonium chloride, and viability assessed by trypan blue staining. Single cell suspensions were stained with antibodies against CD4, CD25, CD44, and CD45 (all BD Biosciences). Foxp3+ cells were assessed by endogenous GFP expression. Propidium iodide or Hoechst-positive cells were excluded from analyses. Total cells were enumerated with BD Calibrite Beads (BD Biosciences). Cell data were acquired on FACSCanto flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR). Cytokines were measured from supernatants of cultured splenocytes using single cell suspensions of splenocytes from each mouse. Splenocytes were cultured for 72 h (24-well tissue culture plates, 1 ml aliquots, 4 × 106cells/ml, RPMI-1640, 10% fetal calf serum, 1% penicillin/streptomycin, 2 mmol/l L-glutamine, 50 μmol/l 2-mercaptoethanol), with 10 μg/ml protein G-purified sheep IgG, as previously described32.Kitching A.R. Turner A.L. Wilson G.R. et al.IL-12p40 and IL-18 in crescentic glomerulonephritis: IL-12p40 is the key Th1-defining cytokine chain, whereas IL-18 promotes local inflammation and leukocyte recruitment.J Am Soc Nephrol. 2005; 16: 2023-2033Crossref PubMed Scopus (70) Google Scholar, 33.Tipping P.G. Kitching A.R. Huang X.R. et al.Immune modulation with interleukin-4 and interleukin-10 prevents crescent formation and glomerular injury in experimental glomerulonephritis.Eur J Immunol. 1997; 27: 530-537Crossref PubMed Scopus (115) Google Scholar using a BD Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine kit according to the manufacturer's instructions (BD Biosciences). Spleens from each individual mouse in both groups were assessed for proportions of CD4+ cells and CD4+foxp3+ cells. The proportions of CD4+ cells and CD4+foxp3+ Tregs in the spleen were similar between WT and foxp3DTR mice at day 10 (CD4+ WT 16.2±3.0, foxp3DTR 14.9±1.7, of CD45+ splenocytes; CD4+foxp3+ WT 3.5±0.8, foxp3DTR 3.5±0.3, of CD4+ cells), consistent with previous published data showing the return of CD4+foxp3+ Tregs to normal levels after 5–10 days after DT injections.4.Lahl K. Loddenkemper C. Drouin C. et al.Selective depletion of foxp3+ regulatory T cells induces a scurfy-like disease.J Exp Med. 2007; 204: 57-63Crossref PubMed Scopus (670) Google Scholar In separate experiments, cytokines were measured from cocultures of foxp3+ Treg cells (from immunized or naive foxp3GFP mice) with foxp3− responder cells at the ratios indicated. Circulating mouse anti-sheep IgG levels were assessed from serum as described previously33.Tipping P.G. Kitching A.R. Huang X.R. et al.Immune modulation with interleukin-4 and interleukin-10 prevents crescent formation and glomerular injury in experimental glomerulonephritis.Eur J Immunol. 1997; 27: 530-537Crossref PubMed Scopus (115) Google Scholar using horseradish peroxidase-conjugated sheep anti-mouse IgG (1:800 dilution, Amersham Biosciences, Rydalmere, Australia). CD4+ cells were isolated by magnetic cell sorting according to the manufacturer's instructions (MACS CD4+ T cell Isolation Kit; Miltenyi Biotec, North Ryde, Australia), followed by sorting based on GFP expression using cell sorter, Beckman Coulter MoFlo XDP (Beckman Coulter, Gladesville, Australia). CD4+foxp3− responder T cells (2 × 104) were cocultured with increasing amounts of CD4+foxp3+ Tregs (1.25 × 103−2 × 104) and with erythrocyte-lysed, MACS CD4-depleted, mitomycin C-treated (50 μg/ml, 30 min, 37 °C, then washed repeatedly) splenocytes (8 × 104) and 100 μg/ml sheep IgG in 96-well round-bottom plates for 72 h. [3H]-thymidine (0.5 μCi) was added per well for the last 16 h of culture and proliferation measured as counts per million, using a liquid scintillation β-counter (Cambridge Scientific, Cambridge, MA). This study was supported by NHMRC Australia Program and Project Grants, Go8 Australia-Germany DAAD Co-operation Scheme, and DFG grants LU1387/2, Ku1063/7, and KFO228. We thank Professor Alexander Rudensky, University of Washington, Seattle, WA, USA for foxp3GFP mice and Professor Tim Sparwasser, Institute of Infection Immunology, Hannover, Germany for foxp3DTR mice. Figure 1. Foxp3+ Tregs in the kidney and in the inguinal draining lymph node are either CD25− or CD25+. Figure 2. Depletion efficiency of foxp3+ Tregs in foxp3DTR mice at day 4. Supplementary material is linked to the online version of the paper http://www.nature.com/ki" @default.
• W2018594927 created "2016-06-24" @default.
• W2018594927 creator A5017688992 @default.
• W2018594927 creator A5021992813 @default.
• W2018594927 creator A5022756309 @default.
• W2018594927 creator A5040346731 @default.
• W2018594927 creator A5043305850 @default.
• W2018594927 creator A5044898121 @default.
• W2018594927 creator A5049739608 @default.
• W2018594927 creator A5051556702 @default.
• W2018594927 creator A5052802894 @default.
• W2018594927 creator A5058750534 @default.
• W2018594927 creator A5071291097 @default.
• W2018594927 date "2011-05-01" @default.
• W2018594927 modified "2023-10-17" @default.
• W2018594927 title "Endogenous foxp3+ T-regulatory cells suppress anti-glomerular basement membrane nephritis" @default.
• W2018594927 cites W1546232808 @default.
• W2018594927 cites W1982373973 @default.
• W2018594927 cites W1987735044 @default.
• W2018594927 cites W1997702872 @default.
• W2018594927 cites W1998810352 @default.
• W2018594927 cites W1999227421 @default.
• W2018594927 cites W2002726823 @default.
• W2018594927 cites W2004829396 @default.
• W2018594927 cites W2007894666 @default.
• W2018594927 cites W2008954693 @default.
• W2018594927 cites W2016827757 @default.
• W2018594927 cites W2036812884 @default.
• W2018594927 cites W2060822348 @default.
• W2018594927 cites W2067885308 @default.
• W2018594927 cites W2068892733 @default.
• W2018594927 cites W2074006404 @default.
• W2018594927 cites W2075372904 @default.
• W2018594927 cites W2077629604 @default.
• W2018594927 cites W2086439124 @default.
• W2018594927 cites W2097811455 @default.
• W2018594927 cites W2099032903 @default.
• W2018594927 cites W2105551571 @default.
• W2018594927 cites W2117229973 @default.
• W2018594927 cites W2117872639 @default.
• W2018594927 cites W2117934757 @default.
• W2018594927 cites W2130486201 @default.
• W2018594927 cites W2140920572 @default.
• W2018594927 cites W2147652122 @default.
• W2018594927 cites W2149915530 @default.
• W2018594927 cites W2150692938 @default.
• W2018594927 cites W2155652150 @default.
• W2018594927 cites W2163693304 @default.
• W2018594927 doi "https://doi.org/10.1038/ki.2010.541" @default.
• W2018594927 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21248715" @default.
• W2018594927 hasPublicationYear "2011" @default.
• W2018594927 type Work @default.
• W2018594927 sameAs 2018594927 @default.
• W2018594927 citedByCount "50" @default.
• W2018594927 countsByYear W20185949272012 @default.
• W2018594927 countsByYear W20185949272013 @default.
• W2018594927 countsByYear W20185949272014 @default.
• W2018594927 countsByYear W20185949272015 @default.
• W2018594927 countsByYear W20185949272016 @default.
• W2018594927 countsByYear W20185949272017 @default.
• W2018594927 countsByYear W20185949272018 @default.
• W2018594927 countsByYear W20185949272019 @default.
• W2018594927 countsByYear W20185949272020 @default.
• W2018594927 countsByYear W20185949272021 @default.
• W2018594927 countsByYear W20185949272022 @default.
• W2018594927 countsByYear W20185949272023 @default.
• W2018594927 crossrefType "journal-article" @default.
• W2018594927 hasAuthorship W2018594927A5017688992 @default.
• W2018594927 hasAuthorship W2018594927A5021992813 @default.
• W2018594927 hasAuthorship W2018594927A5022756309 @default.
• W2018594927 hasAuthorship W2018594927A5040346731 @default.
• W2018594927 hasAuthorship W2018594927A5043305850 @default.
• W2018594927 hasAuthorship W2018594927A5044898121 @default.
• W2018594927 hasAuthorship W2018594927A5049739608 @default.
• W2018594927 hasAuthorship W2018594927A5051556702 @default.
• W2018594927 hasAuthorship W2018594927A5052802894 @default.
• W2018594927 hasAuthorship W2018594927A5058750534 @default.
• W2018594927 hasAuthorship W2018594927A5071291097 @default.
• W2018594927 hasBestOaLocation W20185949271 @default.
• W2018594927 hasConcept C126322002 @default.
• W2018594927 hasConcept C142724271 @default.
• W2018594927 hasConcept C16613235 @default.
• W2018594927 hasConcept C203014093 @default.
• W2018594927 hasConcept C2776115139 @default.
• W2018594927 hasConcept C2776193487 @default.
• W2018594927 hasConcept C2777058396 @default.
• W2018594927 hasConcept C2779727006 @default.
• W2018594927 hasConcept C2780091579 @default.
• W2018594927 hasConcept C2780368995 @default.
• W2018594927 hasConcept C71924100 @default.
• W2018594927 hasConcept C86803240 @default.
• W2018594927 hasConcept C8891405 @default.
• W2018594927 hasConcept C95444343 @default.
• W2018594927 hasConceptScore W2018594927C126322002 @default.
• W2018594927 hasConceptScore W2018594927C142724271 @default.
• W2018594927 hasConceptScore W2018594927C16613235 @default.
• W2018594927 hasConceptScore W2018594927C203014093 @default.
• W2018594927 hasConceptScore W2018594927C2776115139 @default.

• next