Amandeep Bajwa

IL-17 produced by neutrophils regulates IFN-γ–mediated neutrophil migration in mouse kidney ischemia-reperfusion injury

The IL-23/IL-17 and IL-12/IFN-gamma cytokine pathways have a role in chronic autoimmunity, which is considered mainly a dysfunction of adaptive immunity. The extent to which they contribute to innate immunity is, however, unknown. We used a mouse model of acute kidney ischemia-reperfusion injury (IRI) to test the hypothesis that early production of IL-23 and IL-12 following IRI activates downstream IL-17 and IFN-gamma signaling pathways and promotes kidney inflammation. Deficiency in IL-23, IL-17A, or IL-17 receptor (IL-17R) and mAb neutralization of CXCR2, the p19 subunit of IL-23, or IL-17A attenuated neutrophil infiltration in acute kidney IRI in mice. We further demonstrate that IL-17A produced by GR-1+ neutrophils was critical for kidney IRI in mice. Activation of the IL-12/IFN-gamma pathway and NKT cells by administering alpha-galactosylceramide-primed bone marrow-derived DCs increased IFN-gamma production following moderate IRI in WT mice but did not exacerbate injury or enhance IFN-gamma production in either Il17a-/- or Il17r-/- mice, which suggested that IL-17 signaling was proximal to IFN-gamma signaling. This was confirmed by the finding that IFN-gamma administration reversed the protection seen in Il17a-/- mice subjected to IRI, whereas IL-17A failed to reverse protection in Ifng-/- mice. These results demonstrate that the innate immune component of kidney IRI requires dual activation of the IL-12/IFN-gamma and IL-23/IL-17 signaling pathways and that neutrophil production of IL-17A is upstream of IL-12/IFN-gamma. These mechanisms might contribute to reperfusion injury in other organs.

Dendritic cells tolerized with adenosine A2AR agonist attenuate acute kidney injury

DC-mediated NKT cell activation is critical in initiating the immune response following kidney ischemia/reperfusion injury (IRI), which mimics human acute kidney injury (AKI). Adenosine is an important antiinflammatory molecule in tissue inflammation, and adenosine 2A receptor (A₂AR) agonists protect kidneys from IRI through their actions on leukocytes. In this study, we showed that mice with A₂AR-deficient DCs are more susceptible to kidney IRI and are not protected from injury by A₂AR agonists. In addition, administration of DCs treated ex vivo with an A₂AR agonist protected the kidneys of WT mice from IRI by suppressing NKT production of IFN-γ and by regulating DC costimulatory molecules that are important for NKT cell activation. A₂AR agonists had no effect on DC antigen presentation or on Tregs. We conclude that ex vivo A₂AR-induced tolerized DCs suppress NKT cell activation in vivo and provide a unique and potent cell-based strategy to attenuate organ IRI.

Activation of Sphingosine-1-Phosphate 1 Receptor in the Proximal Tubule Protects Against Ischemia-Reperfusion Injury

Agonists of the sphingosine-1-phosphate receptor (S1PR) attenuate kidney ischemia-reperfusion injury (IRI). Previous studies suggested that S1P1R-induced lymphopenia mediates this protective effect, but lymphocyte-independent mechanisms could also contribute. Here, we investigated the effects of S1PR agonists on kidney IRI in mice that lack T and B lymphocytes (Rag-1 knockout mice). Administration of the nonselective S1PR agonist FTY720 or the selective S1P1R agonist SEW2871 reduced injury in both Rag-1 knockout and wild-type mice. In vitro, SEW2871 significantly attenuated LPS- or hypoxia/reoxygenation-induced apoptosis in cultured mouse proximal tubule epithelial cells, supporting a direct protective effect of S1P1R agonists via mitogen-activated protein kinase and/or Akt pathways. S1P1Rs in the proximal tubule mediated IRI in vivo as well: Mice deficient in proximal tubule S1P1Rs experienced a greater decline in renal function after IRI than control mice and their kidneys were no longer protected by SEW2871 administration. In summary, S1PRs in the proximal tubule are necessary for stress-induced cell survival, and S1P1R agonists are renoprotective via direct effects on the tubule cells. Selective agonists of S1P1Rs may hold therapeutic potential for the prevention and treatment of acute kidney injury.

Sphingosine-1-phosphate receptors: Biology and therapeutic potential in kidney disease

The major sphingolipid metabolite, sphingosine-1-phosphate (S1P), has important biological functions. S1P is the ligand for a family of five G-protein-coupled receptors with distinct signaling pathways that regulate angiogenesis, vascular maturation, immunity, chemotaxis, and other important biological pathways. Recently, clinical trials have targeted S1P receptors (S1PRs) for autoimmune diseases and transplantation and have generated considerable interest in developing additional, more selective compounds. This review summarizes current knowledge on the biology of S1P and S1PRs that forms the basis for future drug development and the treatment of kidney disease.

Ultrasound Prevents Renal Ischemia-Reperfusion Injury by Stimulating the Splenic Cholinergic Anti-Inflammatory Pathway

AKI affects both quality of life and health care costs and is an independent risk factor for mortality. At present, there are few effective treatment options for AKI. Here, we describe a nonpharmacologic, noninvasive, ultrasound-based method to prevent renal ischemia-reperfusion injury in mice, which is a model for human AKI. We exposed anesthetized mice to an ultrasound protocol 24 hours before renal ischemia. After 24 hours of reperfusion, ultrasound-treated mice exhibited preserved kidney morphology and function compared with sham-treated mice. Ultrasound exposure before renal ischemia reduced the accumulation of CD11b(+)Ly6G(high) neutrophils and CD11b(+)F4/80(high) myeloid cells in kidney tissue. Furthermore, splenectomy and adoptive transfer studies revealed that the spleen and CD4(+) T cells mediated the protective effects of ultrasound. Last, blockade or genetic deficiency of the α7 nicotinic acetylcholine receptor abrogated the protective effect of ultrasound, suggesting the involvement of the cholinergic anti-inflammatory pathway. Taken together, these results suggest that an ultrasound-based treatment could have therapeutic potential for the prevention of AKI, possibly by stimulating a splenic anti-inflammatory pathway.

Immune Mechanisms and Novel Pharmacological Therapies of Acute Kidney Injury

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and both innate and adaptive immunity contribute to the pathogenesis. Kidney resident cells promote inflammation after IRI by increasing endothelial cell adhesion molecule expression and vascular permeability. Kidney epithelial cells bind complement and express toll-like receptors and resident and infiltrating cells produce cytokines/chemokines. Early activation of kidney dendritic cells (DCs) initiates a cascade of events leading to accumulation of interferon-gamma-producing neutrophils, infiltrating macrophages, CD4(+) T cells, B cells and invariant natural killer T (NKT) cells. Recent studies from our laboratory now implicate the IL23/IL17 pathway in kidney IRI. Following the initial early phase of inflammation, the late phase involves infiltration of anti-inflammatory cells including regulatory T cells, alternatively activated macrophages and stem cells leading to attenuation of inflammation and initiation of repair. Based upon these immune mechanisms of injury, recent studies hold promise for novel drug therapies. These pharmacological agents have been shown to reduce inflammation or cytotoxicity in rodent models of AKI and some show early promise in clinical trials. This review summarizes recent advances to further our understanding of the immune mechanisms of AKI and potential pharmacological therapies.

Divergent roles of sphingosine kinases in kidney ischemia-reperfusion injury

Sphingosine-1-phosphate (S1P), produced by sphingosine kinase 1 (SphK1) or kinase 2 (SphK2), mediates biological effects through intracellular and/or extracellular mechanisms. Here we determined a role for these kinases in kidney injury of wild-type mice following ischemia-reperfusion. SphK1 but not SphK2 mRNA expression and activity increased in the kidney following injury relative to sham-operated animals. Although SphK1(-/-) mice had no alteration in renal function following injury, mice with a disrupted SphK2 gene (SphK2(tr/tr)) had histological damage and impaired function. The immune-modulating pro-drug, FTY720, an S1P agonist failed to provide protection in SphK2(tr/tr) mice. Injured kidneys of these mice showed increased neutrophil infiltration and neutrophil chemokine expression along with a 3- to 5-fold increase in expression of the G-protein-coupled receptor S1P(3) compared to heterozygous SphK2(+/tr) mice. Kidney function and reduced vascular permeability were preserved in S1P(3)(-/-) compared to S1P(3)(+/-) mice after ischemia-reperfusion injury, suggesting increased S1P(3) mRNA may play a role in the injury of SphK2(tr/tr) mice. Our study suggests that constitutive expression of SphK2 may contribute to reduced ischemia-reperfusion injury of the kidney, and its absence may enhance injury due to increased neutrophil infiltration and S1P(3) activation. We also confirm that SphK2 is necessary to mediate the protective effects of FTY720.

Dendritic Cell Sphingosine 1-Phosphate Receptor-3 Regulates Th1–Th2 Polarity in Kidney Ischemia–Reperfusion Injury

Dendritic cells (DCs) are central to innate and adaptive immunity of early kidney ischemia–reperfusion injury (IRI), and strategies to alter DC function may provide new therapeutic opportunities. Sphingosine 1-phosphate (S1P) modulates immunity through binding to its receptors (S1P1–5), and protection from kidney IRI occurs in S1P3-deficient mice. Through a series of experiments we determined that this protective effect was owing in part to differences between S1P3-sufficient and -deficient DCs. Mice lacking S1P3 on bone marrow cells were protected from IRI, and S1P3-deficient DCs displayed an immature phenotype. Wild-type (WT) but not S1P3-deficient DCs injected into mice depleted of DCs prior to kidney IR reconstituted injury. Adoptive transfer (i.e., i.v. injection) of glycolipid (Ag)-loaded WT but not S1P3-deficient DCs into WT mice exacerbated IRI, suggesting that WT but not S1P3-deficient DCs activated NKT cells. Whereas WT DC transfers activated the Th1/IFN-γ pathway, S1P3-deficient DCs activated the Th2/IL-4 pathway, and an IL-4–blocking Ab reversed protection from IRI, supporting the concept that IL-4 mediates the protective effect of S1P3-deficient DCs. Administration of S1P3-deficient DCs 7 d prior to or 3 h after IRI protected mice from IRI and suggests their potential use in cell-based therapy. We conclude that absence of DC S1P3 prevents DC maturation and promotes a Th2/IL-4 response. These findings highlight the importance of DC S1P3 in modulating NKT cell function and IRI and support development of selective S1P3 antagonists for tolerizing DCs for cell-based therapy or for systemic administration for the prevention and treatment of IRI and autoimmune diseases.

Slit2 Prevents Neutrophil Recruitment and Renal Ischemia-Reperfusion Injury

Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeria monocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response.

Natural IgM Anti-Leukocyte Autoantibodies Attenuate Excess Inflammation Mediated by Innate and Adaptive Immune Mechanisms Involving Th-17

Little is known about the function of natural IgM autoantibodies, especially that of IgM anti-leukocyte autoantibodies (IgM-ALA). Natural IgM-ALA are present at birth and characteristically increase during inflammatory and infective conditions. Our prior clinical observations and those of other investigators showing fewer rejections in renal and cardiac allografts transplanted into recipients with high levels of IgM-ALA led us to investigate whether IgM-ALA regulate the inflammatory response. In this article, we show that IgM, in physiologic doses, inhibit proinflammatory cells from proliferating and producing IFN-γ and IL-17 in response to alloantigens (MLR), anti-CD3, and the glycolipid α-galactosyl ceramide. We showed in an IgM knockout murine model, with intact B cells and regulatory T cells, that there was more severe inflammation and loss of function in the absence of IgM after renal ischemia reperfusion injury and cardiac allograft rejection. Replenishing IgM in IgM knockout mice or increasing the levels of IgM-ALA in wild-type B6 mice significantly attenuated the inflammation in both of these inflammatory models that involve IFN-γ and IL-17. The protective effect on renal ischemia reperfusion injury was not observed using IgM preadsorbed with leukocytes to remove IgM-ALA. We provide data to show that the anti-inflammatory effect of IgM is mediated, in part, by inhibiting TLR-4–induced NF-κB translocation into the nucleus and inhibiting differentiation of activated T cells into Th-1 and Th-17 cells. These observations highlight the importance of IgM-ALA in regulating excess inflammation mediated by both innate and adaptive immune mechanisms and where the inflammatory response involves Th-17 cells that are not effectively regulated by regulatory T cells.