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http://www.kidney-international.org & 2013 International Society of Nephrology

see basic research on page 62

Heat-shock protein-70 and regulatory T cell–mediated protection from ischemic injury Stephen O’Neill1 and Jeremy Hughes1 Heat preconditioning induces heat-shock protein-70 upregulation and protects from renal ischemia/reperfusion injury. Kim et al. report that heat-shock protein-70 and regulatory T cells play a key role in protective heat preconditioning. This work reinforces the utility of inducing heat-shock protein-70 expression by pharmacological agents as a novel therapy for the prevention and treatment of ischemic kidney injury. Kidney International (2013) 85, 5–7. doi:10.1038/ki.2013.304

Heat-shock proteins (HSPs) are ubiquitously expressed and highly conserved molecular chaperones with diverse functions including the stabilization of misfolded proteins and elimination of denatured proteins as well as regulation of cell-signaling pathways and cell survival. Multiple HSPs are described according to their molecular mass, with each HSP exhibiting specific properties. HSPs are constitutively expressed but can be upregulated by cell stresses including heat (hence the name ‘heat-shock protein’), oxidative stress, infection, and so on. HSPs play an important role in immunity and inflammation; recent work indicates that they modulate the function of regulatory T cells (Tregs).1,2 HSP70 is a highly studied member of the HSP family, as it has a wide tissue distribution, interacts with a multitude of other molecules, and has significant anti-inflammatory properties, such that it represents an attractive target that can be modulated for therapeutic benefit.3 Indeed, the induction of increased 1

MRC Centre for Inflammation Research, Queen’s Medical Research Institute, Edinburgh, UK Correspondence: Jeremy Hughes, MRC Centre for Inflammation Research, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh, EH164TJ, UK. E-mail: [email protected] Kidney International (2014) 85

HSP70 expression either before or after renal ischemia/reperfusion injury (IRI) protects kidney function.4 REGULATORY T CELLS AND RENAL ISCHEMIA/REPERFUSION INJURY

Renal IRI is a highly complex form of tissue injury involving many biological processes, including acute inflammation, cell death, microvascular dysfunction, and immune activation together with multiple myeloid and lymphoid cell types that may play a role in both the initiation and the resolution of tissue injury.5,6 Although the complex pathophysiology of renal IRI represents a challenge, it also offers an opportunity to manipulate these cellular responses in order to limit injury and facilitate subsequent repair. CD25 þ Foxp3 þ Tregs are a pivotal subtype of lymphocytes that display potent immunosuppressive function and can regulate both immunological function and inflammatory injury. Treg immunodepletion using antiCD25 antibody worsens renal IRI,7 and increasing Treg recruitment to the kidney ameliorates renal IRI.8 In addition, the fact that Tregs both inhibit the innate immune injury of renal IRI9 and are involved in renal repair following renal IRI10 highlights their potential

utility for therapy in patients with acute kidney injury. Various preconditioning strategies, including transient ischemia or heat preconditioning, have been used to induce renoprotection from subsequent renal IRI, and it is highly pertinent that previous studies have demonstrated a role for both Tregs and HSP70 in mediating the protective effect of preconditioning.11,12 TREGS, HEAT-SHOCK PROTEIN-70, AND RENAL ISCHEMIA/REPERFUSION INJURY

Kim et al.13 (this issue) examined the role of HSP70 in the protection from renal IRI afforded by heat preconditioning comprising the heating of mice to 41 1C for 15 min 24 h before the induction of bilateral renal IRI. They present data supporting the concept that HSP70 expressed by CD11c þ dendritic cells plays an important role in Tregmediated protection from renal IRI. Heat preconditioning of mice provided significant protection from the adverse effects of renal IRI, as previously observed by this group,12 with heat-preconditioned mice exhibiting a reduced serum creatinine and less tubular injury. Treg immunodepletion induced by the administration of an anti-CD25 antibody prior to the induction of renal IRI significantly abrogated the protective effect of heat preconditioning. The restoration of the protective effect of heat preconditioning by adoptive transfer of Tregs to anti-CD25-treated mice strongly suggested an important mechanistic role for Tregs, and experiments were therefore undertaken to explore the specific effect of heat preconditioning on Tregs. Heat preconditioning increased the number of splenic CD4 þ CD25 þ Tregs and reduced the proliferative response of cultured splenic mononuclear cells to a mitogenic stimulus. Although HSP70 expression was noted in spleen, kidney, liver, and lung under basal conditions, HSP70 expression was increased by heat preconditioning, with CD11c þ HSP70 þ dendritic cells evident 5

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in the spleen by immunohistochemistry and flow cytometry. Also, in vitro treatment of cultured splenocytes with the HSP70 inducer geranylgeranylacetone increased HSP70 expression and the proportion of CD4 þ cells expressing CD25. Increased numbers of CD4 þ CD25 þ Tregs were detected by flow cytometry in enzyme-dissociated kidneys following renal IRI compared with sham kidneys, indicating localization of Tregs to the injured kidney. Heat preconditioning significantly expanded the intrarenal CD4 þ CD25 þ Treg population following renal IRI in comparison with control injured kidneys, with increased expression of Foxp3 mRNA also evident. In order to probe the role of HSP70 in heat preconditioning, Kim and colleagues13 administered the HSP70 inhibitor quercetin to mice 3 hours before heat preconditioning. Quercetin decreased HSP70 expression in splenocytes, reduced the expansion of splenic Tregs, and blocked the protective effect of heat preconditioning on renal IRI. Furthermore, the adoptive transfer of Tregs to quercetintreated mice restored the protective effects of heat preconditioning. These data suggest that the protective effect of heat preconditioning might be mediated through an interaction between HSP70 and Tregs. However, because of the lack of specificity of HSP70 inhibition by quercetin and the possible upregulation of other anti-inflammatory HSPs by heat preconditioning, Kim et al. undertook further experiments using T cell-deficient nude mice and the adoptive transfer of cells derived from HSP70 knockout mice. Nude mice exhibited reduced injury following renal IRI, as previously demonstrated by other groups. The adoptive transfer of T cells purified from control mice reconstituted injury and significantly increased serum creatinine in nude mice following renal IRI. In contrast, the transfer of T cells from heat-preconditioned wild-type mice to nude mice did not increase injury. Importantly, nude mice given T cells from heat-preconditioned HSP70 knockout mice before renal IRI exhib6

ited increased creatinine and partial reconstitution of injury compared with nude mice given T cells from heatpreconditioned wild-type mice, as the latter mice retained the protected phenotype of nude mice. The totality of these data suggests that heat preconditioning induces renoprotection that is Treg dependent and associated with increased numbers of both splenic and infiltrating renal Tregs. It is suggested that splenic Treg expansion may result from CD11c þ HSP70 þ dendritic cells. Studies involving the administration of HSP70 inhibitors and inducers as well as adoptive transfer of T cells from heatpreconditioned HSP70 knockout mice suggest that HSP70 is important for the generation of injury-limiting Tregs. DISCUSSION

The study by Kim et al.13 reinforces the capacity of both Tregs and HSP70 induction to modulate renal IRI, and, since HSP70 is amenable to pharmacological upregulation, the findings suggest translational potential for preventing ischemic acute kidney injury (Figure 1). Indeed, this area of research may bear fruit in other conditions, such as arthritis and diabetes; recent evidence from human trials suggests that exogenous administration of HSP peptides may exert anti-inflammatory effects via actions on T cells and Tregs.1 The study also raises a number of questions, and, as ever, there are several caveats to the work. For example, the molecular mechanism underlying the protective effect of Tregs induced by heat preconditioning is unclear, though previous work suggests that interleukin-10 is an important Treg effector molecule in renal IRI.9 It should also be noted that anti-inflammatory Tregs that protect from renal IRI may also be induced by treatment with an adenosine 2A receptor (A2AR) agonist,14 and it would be of interest to determine whether concurrent HSP70 induction and treatment with an A2AR agonist augments protection. Although the exact nature of the renoprotective Tregs generated has not been definitively determined, it is likely that

interleukin-10-producing T regulatory type 1 cells are involved, though this should be formally examined. Increased HSP70 expression by splenic CD11c þ dendritic cells was noted, and it is suggested that these are key to Treg induction. However, heat preconditioning also induced renal HSP70 expression, and it is possible that HSP70 upregulation in renal immune cells may also play a role in renal protection. Also, HSP70 present within T cells may physically interact with Foxp3 and directly support survival and proliferation of Tregs and their immunosuppressive function15 such that CD11c þ dendritic cells may not be required. In this regard, it would be interesting to investigate the degree of dependence on CD11c þ dendritic cells by ablating CD11c þ dendritic cells before pharmacological HSP70 induction and adoptive T cell transfer. Preconditioning strategies are administered before the ischemic insult and are thus of use in patients at risk of renal ischemia or acute kidney injury, such as patients about to undergo cardiac surgery. Preconditioning is thus of no use in patients who present with established or evolving acute kidney injury. However, it has been shown that the induction of HSP70 upregulation by geranylgeranylacetone administered after renal IRI is still protective,4 and it is unknown whether this results from the induction of antiinflammatory Tregs or more direct beneficial effects on renal parenchymal cells, including tubular cells. The specificity of both pharmacological inhibitors and inducers of HSPs is an issue with some drugs; for instance, geldanamycin and its analogs induce both HSP70 and HSP2716—though this may not be problematic if all the induced HSPs exert a protective action. Renal IRI occurring in the context of renal transplantation merits particular consideration, as transplantation presents possibilities to promote antiinflammatory recipient Tregs as well as induce cytoprotective HSPs in the donor kidney before harvesting the organ. However, it is noteworthy that the commonly used immunosuppressive drug mycophenolate mofetil Kidney International (2014) 85

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Other proteins

HSP90 ATP

PP

HSF1

REFERENCES

Other proteins P

+

+ Inducer HSF1 P

HSP90

ATP

ATP

Unfolded proteins due to heat preconditioning

PP

1.

ATP

Pharmacological inducer of HSP70 (pre- or post-renal IRI)

2. Other proteins

P PP

+

ATP

HSP90 +

HSF1

HSP90

ATP

ATP

HSF1

HSF1 P

P HSF1 PP

HSF1 P HSF1

P

P

HSP70

HSP27

? Induction of other HSPs ? Off-target effects Induction of HSP70 expression

HSP70 upregulation in tubular cells RENAL IRI

3.

4.

P

P HSF1 P

HSF1

+

ATP

HSF1 release + activation

HSF1 release + activation

PP

Other proteins

P PP

HSP70 upregulation in DCs

? HSP70-mediated DC-independent induction of Tregs

5.

6.

7.

8.

9. ? Mediator (? TGF-β, ? IL-10)

Increased Tregs (spleen & kidney)

Induction of Tregs by A2AR agonist

Decreased apoptosis Decreased effector T cells ? Altered or decreased activation of monocytes/macrophages

10.

11.

Reduced serum creatinine and acute tubular injury

Figure 1 | Heat preconditioning upregulates heat-shock protein-70, leading to regulatory T-cell expansion and recruitment and suppression of inflammation in renal ischemia/reperfusion injury. Schematic diagram illustrating the potential beneficial effects on renal ischemia/reperfusion injury (IRI) of heat-shock protein-70 (HSP70) upregulation induced by either heat preconditioning or pharmacological agents. Induction of HSP70 expression in splenic CD11c þ cells induces expansion of regulatory T cells (Tregs) that ameliorate renal injury, as reported by Kim et al.13 Tregs may also be induced by adenosine 2A receptor (A2AR) agonists. Also, HSP70 induction in renal parenchymal cells such as tubular epithelial cells may exert a protective action by limiting cell death. DCs, dendritic cells; HSF1, heat-shock transcription factor 1; HSP90, heat-shock protein-90; IL-10, interleukin-10; TGF-b, transforming growth factor-b.

12.

13.

14.

worsens experimental IRI of native kidneys via effects on lymphocytes and renal Treg recruitment,17 though it is unclear whether this is of clinical relevance. Also, the upregulation of the renal heat-shock response is associated with increased tubular major histocompatibility complex class II expression and immunogenicity.18 This association may result from the increased action of heat-shock transcription factor 1 (HSF1) that underlies both endogenous and pharmacologically induced HSP70 upregulation (Figure 1).

Kidney International (2014) 85

There is undoubtedly scope for much future work in this area to determine key cellular and molecular mediators together with an exploration of pharmacological agents that may be used to harness and manipulate the endogenous protection of Tregs and HSPs in an experimental setting prior to translation to human disease. DISCLOSURE

Jeremy Hughes has acted as a consultant for GlaxoSmithKline and received previous grant support from Genzyme.

15.

16.

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18.

Keijzer C, Wieten L, van Herwijnen M et al. Heat shock proteins are therapeutic targets in autoimmune diseases and other chronic inflammatory conditions. Exp Opin Ther Targets 2012; 16: 849–857. Brenu EW, Staines DR, Tajouri L et al. Heat shock proteins and regulatory T cells. Autoimmune Dis [online] 2013; 813256. Borges TJ, Wieten L, van Herwijnen MJ et al. The anti-inflammatory mechanisms of Hsp70. Front Immunol [online] 2012; 3: 95. Wang Z, Gall JM, Bonegio RG et al. Induction of heat shock protein 70 inhibits ischemic renal injury. Kidney Int 2011; 79: 861–870. Eltzschig HK, Eckle T. Ischemia and reperfusion—from mechanism to translation. Nat Med 2011; 17: 1391–1401. Bonventre JV, Yang L. Cellular pathophysiology of ischemic acute kidney injury. J Clin Invest 2011; 121: 4210–4221. Monteiro RM, Camara NO, Rodrigues MM et al. A role for regulatory T cells in renal acute kidney injury. Transpl Immunol 2009; 21: 50–55. Lai LW, Yong KC, Lien YH. Pharmacologic recruitment of regulatory T cells as a therapy for ischemic acute kidney injury. Kidney Int 2012; 81: 983–992. Kinsey GR, Sharma R, Huang L et al. Regulatory T cells suppress innate immunity in kidney ischemia-reperfusion injury. J Am Soc Nephrol 2009; 20: 1744–1753. Gandolfo MT, Jang HR, Bagnasco SM et al. Foxp3 þ regulatory T cells participate in repair of ischemic acute kidney injury. Kidney Int 2009; 76: 717–729. Kinsey GR, Huang L, Vergis AL et al. Regulatory T cells contribute to the protective effect of ischemic preconditioning in the kidney. Kidney Int 2010; 77: 771–780. Jo SK, Ko GJ, Boo CS et al. Heat preconditioning attenuates renal injury in ischemic ARF in rats: role of heat-shock protein 70 on NF-kB-mediated inflammation and on tubular cell injury. J Am Soc Nephrol 2006; 17: 3082–3092. Kim M-G, Cho EJ, Lee JW et al. The heat-shock protein-70–induced renoprotective effect is partially mediated by CD4 þ CD25 þ Foxp3 þ regulatory T cells in ischemia/reperfusioninduced acute kidney injury. Kidney Int 2013; 85: 62–71. Kinsey GR, Huang L, Jaworska K et al. Autocrine adenosine signaling promotes regulatory T cell-mediated renal protection. J Am Soc Nephrol 2012; 23: 1528–1537. de Zoeten EF, Wang L, Sai H et al. Inhibition of HDAC9 increases T regulatory cell function and prevents colitis in mice. Gastroenterology 2010; 138: 583–594. Harrison EM, Sharpe E, Bellamy CO et al. Heat shock protein 90-binding agents protect renal cells from oxidative stress and reduce kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 2008; 295: F397–F405. Gandolfo MT, Jang HR, Bagnasco SM et al. Mycophenolate mofetil modifies kidney tubular injury and Foxp3 þ regulatory T cell trafficking during recovery from experimental ischemia-reperfusion. Transpl Immunol 2010; 23: 45–52. Bidmon B, Kratochwill K, Rusai K et al. Increased immunogenicity is an integral part of the heat shock response following renal ischemia. Cell Stress Chaperones 2012; 17: 385–397.

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Heat-shock protein-70 and regulatory T cell-mediated protection from ischemic injury.

Heat preconditioning induces heat-shock protein-70 upregulation and protects from renal ischemia/reperfusion injury. Kim et al. report that heat-shock...
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