Harnessing Endogenous T-regulatory Cells in Acute Kidney Injury

Abstract

Acute kidney injury (AKI) affects 20–50% of hospitalized and intensive care patients, with a sizable proportion progressing to end-stage renal disease or death (https://www.kidney.org/atoz/content/AcuteKidneyInjury). Since inflammation contributes to the pathogenesis of AKI, studies on T-regulatory cells (Tregs), which suppress inflammation are important. Here we highlight advances that utilize Tregs for intervention in AKI that were presented at the 25^th International Conference on Advances in Critical Care Nephrology and UAB/UCSD O’Brien Center AKI & CRRT 2020.

Introduction

T-regulatory cells (Tregs) are a subset of helper T-cells that are identified by the expression of interleukin (IL)-2Rα (CD25) and the Forkhead BoxP3 (Foxp3) transcription factor (reviewed in [1]). Tregs are one of the major mechanisms for immune tolerance and preventing abnormal activation of immune system. Mutations in IL-2/IL-2R or Foxp3 induce Treg-deficiency and fatal autoimmune inflammation. Many cell-surface and intracellular molecules have been identified that are either expressed on Tregs and regulate their survival and function[1]. AKI occurs due to diverse conditions including ischemia, sepsis, nephrotoxicity, transplantation etc. and is exacerbated by inflammation[1].

Tregs in AKI

A role of Tregs in AKI was first identified when depletion of Tregs with an anti-CD25 antibody, administered before ischemia-reperfusion injury, increased the susceptibility of mice to inflammation and injury[2]. Tregs were found to persist in the kidneys upto 10 days post IRI[3]. Depletion of Treg-depletion one day after IRI, also worsened mortality and delayed recovery[3]. Increasing Tregs numbers by infusion of Tregs isolated from donor wild-type (WT) mice either before or after IRI, increased the resistance of the recipient mice to AKI[2] or improved recovery, respectively[3]. Lower expression of TNFRII or reduced suppressive activity of circulating Tregs pre-transplantation correlated with delayed graft function and AKI[4]. Since AKI is frequent in cardiac surgery patients, we evaluated the levels of circulating Tregs in these patients before surgery. Indeed, the numbers of circulating Tregs prior to cardiac surgery could be a predictor of AKI as they negatively correlated with the incidence of AKI (Rahul Sharma, Gilbert Kinsey, Murat Dogan and Mitchell Rosner, manuscript under preparation).

ATP released during cellular damage acts as a danger signal and is usually converted by a pair of ectonucleotidases CD39 and CD73 to the anti-inflammatory adenosine. In adoptive transfer studies, Tregs from CD39 overexpressing mice were more protective in IRI than those from WT mice[5]. On the other hand donor Tregs from mice deficient in CD73 or adenosine receptor (A2AR) failed to offer any protection[6]. Ex vivo treatment of Tregs with ATL1222, a synthetic agonist of A2AR increased the expression of the checkpoint inhibitor PD-1 on Tregs and enhanced their ability of Tregs to protect in IRI[6]. Injection of blocking antibodies to either ligands of PD-1 (PD-L1/L2) negated the ability of Tregs to protect from IRI[7]. Further, Tregs from PD-1-deficient mice are also unable to protect mice from IRI in adoptive transfer studies and may regulate the function of Tregs (Gilbert Kinsey, Murat Dogan and Rahul Sharma, manuscript under preparation). Indeed, treatment with the mTOR-inhibitor rapamycin enhanced the protective activity of Tregs in IRI[8]. Thus, Treg-mediated protection from AKI involves conversion of ATP to adenosine and its autocrine utilization, which promotes PD-1 on Tregs to stimulate mTOR signaling (Figure 1). Enhancing Treg number or function may be a therapeutic target for intervention in AKI.

Figure 1.

T-regulatory cells (Tregs) prevent the progression of acute kidney injury (AKI) by restricting inflammation and also promote repair and regeneration. Proliferation, activation or stabilization of endogenous Tregs by IL-2C (IL-2/anti-IL-2 Ab complex) or a novel hybrid cytokine IL233 (bearing IL-2 and IL-33 in a single molecule) is strongly protective. Conversion of ATP to Adenosine by CD39 and CD73 and its (or analog ATL1222) utilization through adenosine receptor (A_2AR) can promote Tregs through PD-1, which may enhance Treg metabolism through mTOR. Other mediators that promote endogenous Tregs in the settings of AKI include, but are not limited to, sphingosine receptor (S1P1) agonist FTY720, Rapamycin, P2X7 antagonist periodate-oxidized ATP (oATP) or bee-venom Phospholipase A2 (bvPLA2) that works through dendritic cells.

Therapeutic Approaches Targeting Endogenous Tregs

Although, autologous-Treg therapy is being explored in autoimmune diseases (NCT01210664, NCT02428309), it may not be suitable for AKI, because ex vivo expansion of autologous Tregs to required numbers takes several weeks and there is still doubts of their specificity and stability. Thus, approaches that could enhance endogenous Tregs are highly desirable. Some strategies that indirectly increased the levels of Tregs and found to be protective in AKI include Ischemic preconditioning [9] or pharmacologic treatment with the sphingosine-1-phosphate analog FTY720[10], Phospholipase-A2[11] or the purinergic P2X₇-antagonist periodate-oxidized ATP[12]. Among some of approaches that can directly act on Tregs, treatment with an IL-2/anti-IL2 antibody complex robustly increased Tregs in the spleen and kidneys, as well as attenuated ischemic injury[13]. We recently found that IL-2 upregulates IL-33 receptor (ST2) expression on Tregs, thus enabling IL-33 administration to directly increase Treg and partially protected from IRI by significantly reducing plasma creatinine and tubular injury levels halfway to normal[14]. Based on the observation that IL-2 and IL-33 that signal through independent pathways synergize to increase Tregs to protect mice from AKI, we generated a recombinant cytokine (IL233), bearing IL-2 and IL-33 in a single molecule, which outperformed a simple co-injection of IL-2 and IL-33 for increasing Tregs and protecting from IRI[14]. Importantly, administration of IL233 after IRI completely ameliorated renal injury and mortality that was accompanied by an increase in the ST2⁺ Tregs in kidneys[14]. In another study, treatment with IL233 as late as 2 weeks after doxorubicin-induced AKI restored renal structure and function, as well as prevented fibrosis and mortality[15]. Importantly, treatment with IL233 after the injury induced a regenerative program in the kidneys as observed by enrichment of tubular progenitors in a Treg-dependent manner[15].

Conclusion

Despite the major role of inflammation in the pathogenesis of AKI, current treatments do not include anti-inflammatory regimen. Tregs offer a mechanism to regulate inflammation and the evidences for their contribution to tissue repair is growing rapidly. The availability of several potential translational approaches discussed here warrants the utilization of endogenous Tregs for the prevention and treatment of AKI.