Regulatory T Cells Suppress Innate Immunity in KidneyIschemia-Reperfusion Injury

Gilbert R. Kinsey, Rahul Sharma, Liping Huang, Li Li, Amy L. Vergis, Hong Ye, Shyr-Te Ju,and Mark D. Okusa

Division of Nephrology and Center for Immunity, Inflammation and Regenerative Medicine, University of VirginiaHealth System, Charlottesville, Virginia

ABSTRACTBoth innate and adaptive mechanisms participate in the pathogenesis of kidney ischemia-reperfusioninjury (IRI), but the role of regulatory immune mechanisms is unknown. We hypothesized that theanti-inflammatory effects of CD4�CD25�FoxP3� regulatory T cells (Tregs) protect against renal IRI.Partial depletion of Tregs with an anti-CD25 mAb potentiated kidney damage induced by IRI. Reducingthe number of Tregs resulted in more neutrophils, macrophages, and innate cytokine transcription in thekidney after IRI but did not affect CD4� T cells or B cells. We performed adoptive transfer of lymph nodecells from wild-type mice or FoxP3-deficient Scurfy mice into T cell– and B cell–deficient RAG-1 knockoutmice to generate mice with and without FoxP3� Tregs, respectively. FoxP3� Treg–deficient miceaccumulated a greater number of inflammatory leukocytes after renal IRI than mice containing Tregs. Toconfirm that a lack of Tregs potentiated renal injury, we co-transferred isolated Tregs and Scurfy lymphnode cells; Treg repletion significantly attenuated IRI-induced renal injury and leukocyte accumulation.Furthermore, although adoptive transfer of wild-type Tregs into RAG-1 knockout mice was sufficient toprevent kidney IRI, transfer of IL-10–deficient Tregs was not. Taken together, these results demonstratethat Tregs modulate injury after kidney IRI through IL-10–mediated suppression of the innate immunesystem.

J Am Soc Nephrol 20: 1744–1753, 2009. doi: 10.1681/ASN.2008111160

Accumulation and activation of immune systemcells in the kidney during ischemia-reperfusion(IR) is a major mediator of kidney injury. Numer-ous studies have demonstrated the involvement ofthe immune system and inflammation in the patho-genesis of IR-induced acute kidney injury (AKI).1– 6

Experiments using animal models have revealedthat both the innate and adaptive immune systemsmediate IR injury (IRI) in the kidney. Accumula-tion of neutrophils and macrophages is a consistentearly finding in the kidney during reperfusion, anddepletion of either leukocyte subset protects micefrom IR-induced AKI.4,5 Conversely, mice lackingT cells and/or B cells sustain less kidney damageafter IR,1–3 demonstrating that the adaptive im-mune system also contributes to renal IRI.

Regulatory T cells (Tregs) are lymphocytes withimmunosuppressive properties. Tregs are com-

monly identified by their expression of CD4 andCD25 on the cell surface and upregulation of thetranscription factor FoxP3.7 The actions of Tregscan be mediated by the production of anti-inflam-matory cytokines such as IL-10 or TGF-�, by directcell– cell contact or CTLA-4 –mediated inhibition,or by production of extracellular adenosine.8 –11

Furthermore, Tregs have the ability to traffic to ar-

Received November 11, 2008. Accepted March 17, 2009.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Mark D. Okusa, Division of Nephrology,Box 800133, University of Virginia Health System, Charlottesville,VA 22908. Phone: 434-924-2187; Fax: 434-924-5848; E-mail:mdo7y@virginia.edu

Copyright � 2009 by the American Society of Nephrology

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eas of inflammation to mitigate immunereactions.12,13

In addition to Tregs’ widely known abil-ity to prevent effector T cell proliferationand function, Tregs can suppress the innateimmune response.14 –20 In vivo, CD4�-CD25�FoxP3� Tregs inhibit Adriamycin-induced macrophage accumulation andkidney injury and, in vitro, suppress themacrophage inflammatory phenotype.18

Neutrophil reactive oxygen species genera-tion and cytokine production are inhibitedin the presence of Tregs, and co-culturewith Tregs markedly increases the rate ofneutrophil cell death.19 CD4�CD25�-FoxP3� Tregs were recently identified innormal mouse kidneys by flow cytom-etry.21 In summary, Tregs are intrinsic anti-inflammatory leukocytes that reside in thekidney under normal conditions, but theirinvolvement in renal IRI has not been in-vestigated.

RESULTS

Antibody-Mediated Treg DepletionPotentiates Kidney IRITo investigate the role of Tregs in kidneyIRI in normal C57Bl/6 mice, we used ananti-mouse CD25 mAb (PC61).22–24 Ad-ministration of PC61 (300 �g intrave-nously) significantly decreased (approxi-mately 50%) spleen, kidney, and bloodTregs 5 d after injection compared withmice treated with control IgG (300 �g in-travenously; Figure 1, A through E). Thisdosing strategy was chosen on the basis ofthe kinetic analysis of single-dose PC61 onFoxP3� Tregs in C57Bl/6 mice.22 In addi-tion to decreasing the total number of CD4�FoxP3� cells,PC61 treatment significantly decreased the percentage ofCD4� cells expressing FoxP3 (Figure 1F). No significant effect ofPC61 administration was observed on kidney neutrophils, mac-rophages or dendritic cells, spleen B cells, CD4� T or CD8� Tcells, or natural killer (NK) or NK T cells measured at 5 d afterinjection (Figure 1G). Five days after PC61 or IgG injection, miceunderwent 24 min of bilateral renal ischemia or sham surgery,and kidneys were allowed to reperfuse for 24 h. Whereas there wasno difference in plasma creatinine (PCr) levels before surgery orafter sham surgery, mice with decreased Tregs (PC61) developedmore severe IRI compared with IgG-treated mice as measured byPCr levels (Figure 2A) and outer medulla acute tubular necrosis(ATN; Figure 2, B and C; ATN scores: IgG � IRI 1.2 � 0.3 andPC61 � IRI 2.5 � 0.3, P � 0.05).

PC61-Induced Reduction in Kidney Treg NumberEnhances the Innate Immune Response in the Kidneyafter IRITwenty four hours after kidney IR or sham surgery, kidney leu-kocyte accumulation was determined by flow cytometry. Therewas no difference in kidney neutrophil (CD45�7-AAD�-CD11b�GR-1high) or macrophage (CD45�7-AAD�F4/80int-CD11b�) number after sham operation in the IgG- and PC61-treated WT mice (Figure 3, A and B). The number of neutrophilsand macrophages in the kidney after IR in the PC61-treated mice(with significantly fewer Tregs) was significantly higher than theIgG-treated controls after IR (Figure 3, A and B). Previous studiesfrom our laboratory demonstrated that GR-1highIFN-�� leuko-cytes are activated neutrophils that accumulate in the kidney earlyduring reperfusion.6 Decreasing Treg numbers with PC61 caused

Figure 1. CD25 mAb (PC61) administration reduces Treg numbers in mouse spleen,kidney, and blood. (A) Five days after intravenous injection of 300 �g of PC61 or controlIgG in C57Bl/6 mice, spleen, kidney, and blood cells were analyzed by flow cytometry todetermine the number of Tregs (CD4�FoxP3�) per gram of tissue or 100 �l of blood asdescribed in the Concise Methods section. (B through E) Representative flow cytometryresults from mouse spleen or kidney 5 d after IgG (B and D) and PC61 (C and E) injection;total spleen or kidney cells were gated on the CD45� and Fixable Green Live/DeadStain–negative populations before the analysis shown. (F) The percentage of total CD4�

cells staining positive for intracellular FoxP3 in spleen and kidney was determined by flowcytometry. (G) The effect of PC61 administration versus IgG was determined by comparingin each group the percentage of CD45� cells that were kidney neutrophils (PMN, GR-1�/CD11b�), macrophages (F4/80low/CD11b�), or dendritic cells (F4/80high/CD11b�) orspleen B cells (B220�/CD19�), CD4� (CD3�/CD4�) or CD8� (CD3�/CD8�) T cells, or NK(CD3-/NK1.1�) or NK T (CD3�/NK1.1�) cells. Data are means � SEM (A, F, and G); n � 7for spleen and kidney and n � 3 for blood. *P � 0.05 versus IgG for same organ.

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a significant increase in the number of IFN-�–producing acti-vated neutrophils observed in the kidney after IR (Figure 3C). Incontrast, the number of kidney CD3�CD4� T cells andCD3�CD19� B cells was not different at 24 h after IR (Figure 3D).Immunohistochemical analysis of kidney sections using the anti-body 7/4 confirmed the flow cytometry results, demonstrating amarked increase in the number of kidney infiltrating neutrophilsand recently emigrated monocytes in the outer medulla at 24 hafter IR in PC61-treated mice (Figure 4). Real-time PCR analysisof total kidney RNA from IgG- or PC61-treated mice after shamor IR revealed that in addition to enhanced innate leukocyte ac-cumulation, greater transcription of the innate inflammatory cy-tokines IL-6 and TNF-� was observed after IR in mice with fewerTregs. Similarly TGF-� expression was increased in the kidneyafter IRI in the PC61-treated mice versus IgG-treated mice,whereas IL-10 expression was not different between the two treat-ment groups after IRI (Supplemental Figure 1).

Mice Completely Deficient in Tregs Are MoreSusceptible to IRIAs an additional model to investigate the role of Tregs in IR, weused RAG-1 knockout (KO) mice, which lack T and B cells.RAG-1 KO mice were reconstituted with 5 or 10 million lymphnode (LN) cells from either wild-type C57Bl/6 (WT) mice orScurfy (Sf) mice, which lack expression of FoxP3 and thusCD4�CD25�FoxP3� Tregs. Two weeks later, the efficiency of

adoptive transfer was confirmed using spleen cells from thevarious groups of mice by flow cytometry (Figure 5). Tregswere absent from spleens of RAG-1 KO mice and RAG-1 KOmice reconstituted with Sf LN cells (Figure 5, SupplementalFigure 2). Additional groups of RAG-1 KO mice received iso-lated CD4�CD25�FoxP3� Tregs from a WT mouse alongwith Sf cells. Reconstitution with WT LN cells or co-transfer ofisolated Tregs with Sf LN cells resulted in the presence of adistinct CD4�FoxP3� population in the spleen 2 wk afteradoptive transfer (Figure 5, Supplemental Figure 2). RAG-1KO mice that were not reconstituted with LN cells were pro-tected from mild IRI (Figure 6) as described previously.3

Adoptive transfer of 10 million WT LN cells 2 wk before IRresulted in renal injury, and reconstitution of RAG-1 KO micewith 10 million Sf LN cells significantly exacerbated the sever-ity of IRI compared with adoptive transfer of an equivalentnumber of WT LN cells (Figure 6; ATN scores: 10 million WTLN cells � IRI 2.0 � 0.4 and 10 million Sf LN cells � IRI 3.6 �0.2, P � 0.05). To confirm that the potentiation of renal injurywas due to the lack of Tregs in Sf 3 RAG-1 KO mice, weco-transferred isolated Tregs with 5 million Sf cells at a ratio of1:5 (Tregs:Sf LN cells). The purity of the isolated Tregs wasdetermined to be �85% CD4�CD25�, and of theCD4�CD25� population, 80 to 95% expressed FoxP3 as de-termined by flow cytometry (Supplemental Figure 3). Co-transfer experiments confirmed the protective role of Tregs inthis model; Treg repletion (1:5 ratio) significantly inhibited theIR-induced kidney dysfunction (Figure 7A) and tubular ne-crosis (Figure 7, B and C; ATN scores: 5 million Sf LN cells �IRI 1.3 � 0.4 and 5 million Sf LN cells � 1 million Tregs 0.2 �0.1, P � 0.05). Co-transfer of Tregs at a 1:10 ratio was insuffi-cient to prevent kidney IRI (data not shown).

Treg Deficiency Exacerbates Kidney Inflammatory CellAccumulation after IRTwenty four hours after kidney IR or sham surgery, we deter-mined kidney leukocyte accumulation by flow cytometry. Thenumber of neutrophils (CD45�7-AAD�GR-1highCD11b�)and macrophages (CD45�7-AAD�F4/80intCD11b�) in the kid-ney after IR in the RAG-1 KO mice reconstituted with 5 million SfLN cells was significantly higher than those reconstituted with 5million WT LN cells after IR (Figure 8). Immunohistochemicalanalysis of kidney sections using the antibody 7/4 confirmed theflow cytometry results (Supplemental Figure 4). In contrast toPC61-treated mice, the number of CD4� T cells in the kidneyafter IR was also higher in Treg-deficient mice. Co-transfer ofTregs with Sf LN cells completely blocked the increase in inflam-matory leukocyte accumulation caused by Sf LN cell transferalone (Figure 8).

Tregs Directly Suppress Innate Responses to IRI in anIL-10–Dependent MannerTo test directly the ability of Tregs to suppress the innate re-sponse to kidney IRI and the role of IL-10 produced by Tregs,we adoptively transferred WT or IL-10 KO Tregs alone to

Figure 2. Partial Treg depletion potentiates kidney IRI. Five daysafter intravenous injection of 300 �g of PC61 or control IgG,C57Bl/6 mice underwent sham surgery or bilateral renal ischemiafor 24 min. (A) After 24 h of reperfusion, renal function wasassessed by measurement of PCr levels. Data are means � SEM;n � 6 to 11 per group. n.s., P � 0.05. The extent of outer medullatubular necrosis was determined by hematoxylin and eosin (H&E)staining. (B, C) Images are representative of six to eight animalsper group. Magnifications: �200 in B and C; �600 in insets.

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RAG-1 KO mice before IRI. No difference in CD25 or FoxP3expression levels was observed between WT and IL-10 KOTregs (data not shown). In contrast to 24 min of ischemia,which did not result in a significant increase in PCr in RAG-1KO mice (Figure 6), 28 min of ischemia caused an increase inkidney neutrophil accumulation, PCr levels, and ATN (Figures9 and 10). Adoptive transfer of 1 � 106 WT CD4�CD25�

Tregs significantly reduced kidney injury, whereas the samenumber of CD4�CD25� T cells or CD4�CD25� Tregs fromIL-10 KO mice had no protective effect on IRI in RAG-1 KOmice (Figure 9; ATN scores: No cells � IRI 2.7 � 0.4, 1 millionWT Tregs � IRI 0.6 � 0.3,a,b 1 million IL-10 KO Tregs � IRI

3.1 � 0.2, aP � 0.02 versus no cells � IRI, bP � 0.001versus IL-10 KO Tregs). WT Tregs but not IL-10 KOTregs blocked the accumulation of GR-1highCD11b�

neutrophils in the kidney at 24 h after IRI (Figure10).

DISCUSSION

We have established a protective role for Tregs inkidney IRI using three different animal models tomodulate Tregs. Use of PC61, which resulted in ap-proximately 50% reduction in Tregs in the spleen,kidney, and blood, potentiated kidney IRI, as mea-sured by PCr levels and ATN. Similar results wereobtained using mice that completely lack Tregs (SfLN cells 3 RAG-1 KO mice), and renal injury inthese mice was significantly inhibited by addingTregs along with Treg-deficient Sf cells. Adoptivetransfer of WT Tregs alone blocked kidney IRI inlymphocyte-deficient RAG-1 KO mice. In contrast,IL-10 KO Tregs were ineffective at blocking IRI in

RAG-1 KO mice, illustrating the important role for IL-10 fromTregs in protection from IRI. In each model of Treg deficiency,the innate immune response to kidney IRI was enhanced, aneffect that was reversed by addition of WT Tregs. Collectively,these results demonstrate that Tregs function to protect thekidney from inflammation and dysfunction associated withIRI. In addition, our observations suggest that a major mech-anism of Treg-mediated kidney protection is by inhibition ofthe innate immune response to kidney injury.

One method to investigate functions of Tregs is to depletethem partially using an anti-CD25 antibody (PC61), becauseTregs express high levels of CD25 under normal conditions. In

Figure 3. Partial Treg depletion enhances kid-ney leukocyte accumulation after IR. Five daysafter intravenous injection of 300 �g of PC61 orcontrol IgG, C57Bl/6 mice underwent sham sur-gery or bilateral renal ischemia for 24 min. (A–C)After 24 h of reperfusion, renal leukocyte num-bers were assessed by flow cytometry as de-scribed in the Concise Methods section. Allgroups were gated previously on CD45� and7-AAD� cell populations before the analysisshown. (D) CD4� T cells were identified asCD3�/CD4�, and B cells were identified andCD3�CD19�. Data are means � SEM; n � 6 to11 per group. n.s., P � 0.05.

Figure 4. Partial Treg depletion promotes kidney innate inflammatory cellaccumulation after IR. (A and B) Five days after intravenous injection ofcontrol IgG (A) or PC61 (B), C57Bl/6 mice underwent renal ischemia for 24min. After 24 h of reperfusion, renal neutrophil and recently emigratedmonocyte accumulation was assessed by staining frozen sections with the7/4 antibody (bright green). DAPI was used to identify nuclei (blue). Imagesare representative of at least three separate mice per group.

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our hands, PC61 was effective at decreasing the total numberand percentage of CD4�FoxP3� Tregs in the kidney, spleen,and blood. One potential caveat of this method is that otherleukocytes can also express CD25, including B cells, activatedeffector T cells, and NK cells. No significant effect on any ofthese non-Treg subsets was observed after PC61 administra-tion. These results demonstrate that the use of PC61 specifi-cally reduces Treg numbers in control mice without unin-tended effects on other leukocyte populations.

The second method used to study the role of Tregs in IR wasthe establishment of a mouse completely deficient inCD4�CD25�FoxP3� cells. FoxP3 is a transcription factor thatis vital for the regulatory functions of Tregs.7 Sf mice lackFoxP3 and develop severe autoimmune disease, resulting inpremature death by 3 to 4 wk of age. Before death, Sf LN cellswere harvested and used to reconstitute T cell– and B cell–deficient, RAG-1 KO mice. This strategy results in a mousewith an intact innate immune system, T cells and B cells, butdevoid of FoxP3� Tregs.25 These Treg-deficient mice werefound to be significantly more sensitive to kidney IRI, addingfurther support to the hypothesis that Tregs are protective inthis setting. To confirm the importance of Tregs, we co-admin-istered highly pure isolated Tregs with Sf LN cells, which weresufficient to inhibit IRI significantly compared with Treg-de-ficient mice.

The model of adoptive transfer of CD4�CD25� Tregsalone into RAG-1 KO mice, which contain only an innateimmune system, has been used by others to demonstrate thepotential for Tregs to inhibit the innate immune responsedirectly.16,20 For example, Murphy et al.16 showed thatadoptive transfer of CD4�CD25� Tregs could suppress theinnate response to burn injury, but an equivalent number ofCD4�CD25� leukocytes could not. Kim et al.20 reportedthat both CD4�CD25� Tregs and CD4�CD25� non-Tregscould prevent lethal overactivation of the innate immuneresponse to infection. Similar to these two studies, weobserved that CD4�CD25� Tregs could protect RAG-1KO mice from kidney IRI. In contrast to Kim et al.,20

CD4�CD25� cells did not offer protection from IRI. Thisdifference may be explained by the methods used to activatethe innate immune system in the different studies. In addi-tion, the number of T cells (Tregs and non-Tregs) adop-tively transferred was dramatically higher than in our study.

CD25 is the �-subunit of the IL-2 receptor, whose activationdrives proliferation to sustain Treg numbers in vivo26–28 and isessential for maximal suppressive activity.29,30 In our model, tar-geting CD25 with PC61 was sufficient to exacerbate kidney IRI. Inaddition, in the adoptive transfer experiments, the Tregs, whichwere selected on the basis of CD25 expression, were competent toreduce kidney IRI in these models and prevent Sf LN cell–medi-ated multiorgan damage in a previous study.25 These findings sug-gest that Treg CD25 expression is an important phenotypicmarker for protection against kidney IRI.

The role of the innate immune response in the pathogen-esis of IRI is well established.4 – 6 Depletion of neutrophils ormacrophages is sufficient to protect the kidney from IRI.4,5

In mice with decreased Tregs or mice completely deficientin Tregs, we observed significant increases in accumulationof neutrophils and macrophages in the kidney after IR. Inaddition, more neutrophils exhibiting an activated pheno-type (IFN-� producing) were detected in kidneys from micewith fewer Tregs after IR. We did not detect any differencein the numbers of adaptive immune cells in the kidney afterIR in PC61-treated mice compared with controls. Examples

Figure 5. Effectiveness of LN cell transfer to RAG-1 KO recipi-ents. (A through C) Representative flow cytometry results ofsplenocytes 2 wk after adoptive transfer of no cells (A), 10 millionWT LN cells (B), or 10 million Sf LN cells (C). Total spleen cellswere gated on the CD45� and Fixable Green Live/Dead Stain–negative populations before the analysis shown. (D, E) Quantifi-cation of the percentage of CD45� spleen cells that wereCD4�FoxP3� or CD4�FoxP3� 2 wk after adoptive transfer. Dataare means � SEM; n � 3 to 5 per group. n.s., P � 0.05 (D and E).

Figure 6. Complete Treg deficiency potentiates kidney IRI. Twoweeks after adoptive transfer of no cells, 10 million WT LN cells,or 10 million SF LN cells, RAG-1 KO mice underwent either shamsurgery or 24 min of bilateral renal ischemia. Twenty-four hoursafter surgery, renal function was determined by measurement ofPCr levels. Data are means � SEM; n � 3 to 5 per group.

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of direct actions of Tregs on the innate immune responsehave been previously reported.14,16,18 –20 To test whetherTregs could directly suppress the innate immune responseto kidney IRI, we performed adoptive transfer of CD4�-CD25� Tregs or CD4�CD25� T cells into lymphocyte-defi-cient RAG-1 KO mice before IRI. We observed that the transferof Tregs (CD4�CD25�) could prevent kidney neutrophil ac-cumulation and IRI, but non-Tregs (CD4�CD25�) could not.These findings demonstrate that, in IR, Tregs can directly in-hibit innate immune responses.

In Treg-deficient mice, we observed significantly greaterCD4� T cell accumulation after IR. This finding could bethe result of the more highly activated phenotype of CD4� Tcells from Sf mice.25,31 Compared with T cells from a WTmouse, these Sf T cells express more activation markers andmay be more highly responsive to IRI in the kidney. Anotherpossibility is that a greater degree of Treg depletion than weachieved using PC61 is required to promote CD4� T cellaccumulation in the kidney after IR. In either case, the co-transfer of isolated Tregs with Sf LN cells prevented thekidney accumulation of CD4� T cells, induced by Sf LN celltransfer alone, confirming that the enhanced lymphocyteaccumulation was due to the lack of Tregs. It should be

noted that our observations of infiltrating leukocytes afterIR all were made at 24 h of reperfusion, and CD4� T cells arereported to enter the kidney very early (within 30 min) dur-ing reperfusion and then exit shortly thereafter.6,32–34 Al-though the first two models used in this study leave open thepossibility that Tregs act early to suppress initial CD4� Tcell activation (e.g., invariant NK T cells),6 results of theadoptive transfer of WT Tregs alone into RAG-1 KO micebefore IRI demonstrate that Tregs can directly inhibit in-nate immune responses to IRI.

We observed that kidney Treg numbers (per gram) aresignificantly decreased 24 h after IRI compared with sham-operated mice (1564 � 152 versus 10,075 � 3450; n � 4 and3, respectively; P � 0.05). This result is in agreement recentfindings of Liesz et al.,35 who reported that after ischemicstroke, FoxP3� Tregs were undetectable in the ischemicbrain until 3 d of reperfusion. Our observations may be dueto the increased kidney levels of IL-6,36 which is known tosuppress FoxP3 expression alone or in the presence of TGF-�,37,38 which is also elevated after IRI.39 These results suggestthat the protection afforded by Tregs is due to the Tregs inthe kidney during ischemia and early reperfusion (beforethe 24-h time point) and/or the global pool of Tregs (out-side the kidney, up to 24 h of reperfusion).

Although TGF-� and IL-10 are products of Tregs, thesecytokines are also produced by damaged renal tubular cells39

and by other T cells and macrophages,40 respectively. The in-volvement of all of these non-Tregs in kidney IRI is well doc-umented3,4,39 and may explain our inability to detect decreasesin IL-10 and TGF-� in Treg-deficient mouse kidneys after IRI.

A major mechanism of Treg-mediated suppression of innateinflammatory responses is IL-10 production.19,36 IL-10 inhibitsleukocyte activation through suppression of Jak-Stat and NF-�Bsignaling pathways,40 and in mice, administration of exogenousIL-10 before IRI is sufficient to protect mice from kidney injury.41

For these reasons, we used Tregs isolated from the IL-10 KOmouse to determine its role in Treg-mediated protection fromkidney IRI. The use of IL-10 KO Tregs completely abrogated theprotection afforded to RAG-1 KO mice by adoptive transfer of thesame number of WT Tregs, confirming that IL-10 from Tregs isindispensable for preventing kidney IRI. These results are similarto a recent report in an ischemic stroke model: WT Tregs pre-vented injury, whereas IL-10 KO Tregs had no effect.36

In conclusion, our findings demonstrate a potent protectiverole for CD4�CD25�FoxP3� Tregs in kidney IRI. Measurementsof infiltrating leukocytes in the postischemic kidney suggest thatthe protection is mediated, at least in part, by IL-10–dependentblockade of innate immune cell activation and accumulation.

CONCISE METHODS

Mice and Adoptive TransferMale WT mice that were 6 to 8 wk of age and weighed 20 to 25 g were

obtained from Charles River Laboratories (Wilmington, MA). Male

Figure 7. Treg repletion inhibits kidney IRI in Treg-deficient mice.Two weeks after adoptive transfer of no cells, 5 million WT or Sf LNcells, or 5 million Sf LN cells � 1 million isolated Tregs, RAG-1 KOmice underwent either sham surgery or 24 min of bilateral renalischemia. Twenty-four hours after surgery, renal function was deter-mined by measurement of PCr levels (A). Data are means � SEM.*Significantly different from respective sham operated mice, P �0.05 (A). (B, C) Twenty-four hours after surgery, the extent of outermedulla tubular necrosis was determined by H&E staining. Imagesare representative of 5 to 6 animals per group. Magnifications: �200in B and C; �600 in insets.

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B6.129S7-Rag1tm1Mom/J (RAG-1 KO) and B6.129P2-Il10tm1Cgn/J

(IL-10 KO) mice that were 6 to 8 wk of age and weighed 20 to 25 g were

obtained from the Jackson Laboratories (Bar Harbor, ME). Heterozy-

gous female B6.Cg-Foxp3sf/x/J mice (Jackson Laboratories) were bred

with male C57Bl/6 mice to produce Sf mice. C57Bl/6 and Sf LN cells

were isolated as described previously.42 For adoptive transfer, 5 or

10 � 106 WT or Sf LN cells were administered by intravenous injec-

tion to RAG-1 KO mice 2 wk before IR. Tregs were isolated from

spleen and LNs of WT or IL-10 KO mice using the Dynal (Carlsbad,

CA) CD4� negative selection kit and Miltenyi Biotec (Auburn, CA)

CD25� positive selection kit according to the manufacturer’s proto-

cols. In some experiments, isolated Tregs were mixed and co-trans-

ferred with Sf LN cells at a ratio of 1:5 or 1:10 (Tregs:total Sf LN cells).

In other experiments, 1 � 106 WT or IL-10 KO CD4�CD25� Tregs or

WT CD4�CD25� leukocytes were administered by tail-vein injection

to RAG-1 KO mice 18 h before IRI.

Surgical ProtocolFor the renal IR protocol, bilateral flank incisions were made under

anesthesia. Both renal pedicles were exposed and cross-clamped for

24 or 28 min, clamps were removed, and kidneys were allowed to

reperfuse for 24 h as described previously by our laboratory.6 All an-

imal experiments were approved by the University of Virginia Insti-

tutional Animal Care and Use Committee.

Assessment of Renal FunctionPCr was measured after renal IR using a colorimetric assay (Sigma, St.

Louis, MO),6 and outer medullary tubular necrosis was assessed by

hematoxylin and eosin staining as described previously.43 Briefly,

stained kidney sections were scored in a blinded manner, and each

Figure 8. Treg repletion inhibits IR-induced leukocyte accumu-lation in Treg-deficient mice. Two weeks after adoptive transfer of5 million WT or Sf LN cells or 5 million Sf LN cells � 1 millionisolated Tregs (Sf � Treg), RAG-1 KO mice underwent 24 min ofbilateral renal ischemia. Twenty-four hours after surgery, the ac-cumulation of leukocytes in the kidney was determined by flowcytometry as described in the Concise Methods section. Data aremeans � SEM. #P � 0.05 versus WT; *P � 0.05 versus Sf.

Figure 9. IL-10� Tregs inhibit kidney IRI in lymphocyte-deficient RAG-1 KO mice. RAG-1 KO mice were administered PBS or 1 � 106

WT CD4�CD25� Tregs, 1 � 106 IL-10 KO CD4�CD25� Tregs, or 1 � 106 WT CD4�CD25� cells 18 h before 28 min of bilateral renalischemia or sham surgery. (A) After 24 h of reperfusion, renal function was estimated by measurement of PCr, and ATN was assessedby H&E staining. (B–E) Data are means � SEM; values with different superscripts are significantly different from each other. P � 0.05;n � 4 to 9 per group (A). Magnifications: �200 in B through E; �600 in insets.

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sample was assigned a number on the basis of the percentage of outer

medulla tubules displaying evidence of tubular necrosis (presence of

pink casts inside tubules). The scoring system used was as follows: 0,

no damage; 1, �10%; 2, 10 to 25%; 3, 25 to 75%; and 4, �75%.

Flow CytometryFive-color flow cytometry was used to determine the number and

phenotype of leukocytes in the kidney, spleen, and blood. Counting

beads (Caltag, Carlsbad, CA) were used as described previously6 to

determine the total number of CD45� cells per gram of kidney or

spleen tissue or 100 �l of blood. This leukocyte number was used to

convert the percentages of leukocytes displaying certain characteris-

tics into a leukocyte count used for comparisons between treatment

groups. All samples were treated with anti-mouse CD16/CD32

(2.4G2) to block the nonspecific FcR binding, 7-AAD or LIVE/DEAD

Fixable Green Dead Cell Stain (Invitrogen, Carlsbad, CA) to exclude

dead cells and anti-CD45 to gate on the live leukocyte populations.

CD4�CD25�FoxP3� Tregs were stained, fixed, and permeabilized

using the eBioscience (San Diego, CA) FoxP3 buffer set according to

the manufacturer’s protocol. Intracellular IFN-� staining was per-

formed using the BD Biosciences (San Jose, CA) Fix/Perm buffer set

according to the manufacturer’s protocol and as described previously

by our laboratory.6 Flow cytometry data acquisition and analysis were

performed on BD FACSCalibur using Flowjo software (Tree Star,

Ashland, OR). Anti-mouse fluorophore-labeled antibodies (clones)

used were as follows: CD45-PE (30-F11), CD45-FITC (30-F11),

CD45-APC-AlexaFluor750 (30-F11), CD25-PE (PC61), CD4 PE-Cy5

(GK1.5), CD4 AlexaFluor647 (GK1.5), FoxP3-APC (FJK-16s), GR-1-

FITC (RB6-8C5), CD11b-PE (M1/70), F4/80-APC (BM8), IFN-�–

AlexaFluor647 (XMG1.2), CD3-APC-AlexaFluor750 (17A2),

NK1.1-PE (PK136), B220-APC-AlexaFluor750 (RA3-6B2), and

CD8�-PE (53-6.7) from eBiosciences; CD19-PE (1D3) from BD

Pharmingen (San Jose, CA); and CD25-PE (7D4) from Southern Bio-

tech (Birmingham, AL).

Immunohistochemical AnalysisKidneys were fixed overnight in Periodate-Lysine Paraformalde-

hyde (1%), incubated for 48 h in 30% sucrose at 4°C, then embed-

ded and frozen in OCT compound (Ted Pella, Redding, CA). Fro-

zen kidney sections (10 �m) from each mouse were blocked with

goat serum and 2.4G2, then labeled with a FITC-conjugated anti-

body (clone 7/4; Cedarlane, Burlington, Ontario, Canada), which

recognizes neutrophils and recently emigrated monocytes.44,45

Nuclei were visualized using DAPI. Specimens were mounted with

ProLong Gold Antifade reagent (Molecular Probes, Eugene, OR)

and examined using a Zeiss Axiovert 200 microscope with Apo-

Tome (Zeiss, Thornwood, NY).

Figure 10. IL-10� Tregs inhibit IR-induced neutrophil accumulation in lymphocyte-deficient RAG-1 KO mice. RAG-1 KO mice wereadministered PBS or 1 � 106 WT CD4�CD25� Tregs, 1 � 106 IL-10 KO CD4�CD25� Tregs, or 1 � 106 WT CD4�CD25� cells 18 hbefore 28 min of bilateral renal ischemia or sham surgery. (A) After 24 h of reperfusion, renal neutrophil accumulation was measuredby flow cytometry as described in the Concise Methods section. (B through E) Total kidney cells were gated on the CD45� and 7-AAD�

populations before the analysis shown. Data are means � SEM; values with different superscripts are significantly different from eachother. P � 0.05; n � 4 to 9 per group (A).

BASIC RESEARCHwww.jasn.org

J Am Soc Nephrol 20: 1744–1753, 2009 Regulatory T Cells in Kidney IRI 1751

Real-Time Reverse Transcriptase–PCRKidney sections were immediately transferred into RNA Later

(Ambion, Austin, TX). RNA and cDNA were prepared from these

samples as described previously.43 Subsequently, real-time Reverse

transcriptase–PCR was performed using a MyIQ Single Color

Real-Time PCR Detection System (BioRad, Hercules, CA) as

described previously.46 Primers used were as follows: Glyceraldehyde-

3-phosphate dehydrogenase, 5�-ACGGCAAATTCAACGGCACA-

GTCA-3� (forward) and 5�-TGGGGGCATCGGCAGAAGG-3�

(reverse); IL-6, 5�-TGGCTAAGGACCAAGACCATCCAA-3� (for-

ward) and 5�-AACGCACTAGGTTTGCCGAGTAGA-3� (reverse);

TNF-�, 5�-CCTCCCTCTCATCAGTTCTATGG-3� (forward) and

5�-CGTGGGCTACAGGCTTGTC-3� (reverse); TGF-�, 5�-TAAA-

GAGGTCACCCGCGTGCTAAT-3� (forward) and 5�-ACTGCT-

TCCCGAATGTCTGACGTA-3� (reverse); and IL-10, 5�-TGCAC-

TACCAAAGCCACAAAGCAG-3� (forward) and 5�-TGCAGTT-

ATTGTCTTCCCGGCTGT-3� (reverse).

AntibodiesThe TIB-222 hybridoma was used by the Lymphocyte Culture Core

Facility at the University of Virginia to produce the anti-mouse CD25

mAb (clone PC61). Control IgG ImmunoPure Rat IgG was purchased

from Pierce (Rockford, IL).

Statistical AnalysisComparisons of two treatment groups were made using an unpaired t

test to determine statistical significance. ANOVA was performed for

comparisons of three or more groups, and the Holm-Sidak procedure

was used for pair-wise comparisons using the SigmaStat statistical

software (San Jose, CA). P � 0.05 was considered statistically signifi-

cant.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants

DK56223, DK58413, DK62324, T32 DK072922, PO1 HL073361, DE-

017579, and AR-051203.

We are grateful to Dr. Kenneth Tung and Hui Qiao for the use of

the TIB-222 hybridoma and assistance with purification of the PC61

mAb. We also thank Dr. Peter Lobo for helpful discussions and sug-

gestions about this project.

DISCLOSURESNone.

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