Coaxing Anti-Inflammatory Granulocytes to Prevent Ischemic Kidney Injury: A Fine Balance
Authors: Alan D. Salama, Mark A. Little
Journal: Journal of the American Society of Nephrology (2020)

Neutrophils adhere to activated endothelium in peritubular capillaries and accumulate in the kidney interstitium <30 minutes after reperfusion following a period of ischemia. The traditional view is that these cells are proinflammatory and result in tubular injury. However, neutrophils are increasingly being characterized as a mixed population with pro- and anti-inflammatory effects. Unfortunately, the nomenclature and definition of these immunomodulatory cell types vary with the disease context, with diverse terms being used, such as “myeloid-derived suppressor cell” (MDSC), “tumor-associated neutrophil,” “low-density granulocyte,” and “interphase neutrophil.” In the cancer field, these cells are generally felt to suppress the antitumor T cell response and portend a poor prognosis, whereas in autoimmune disease, such as SLE, they may be proinflammatory.1 Given their effect on the immune response, their utility as a clinical tool in inflammatory disorders is a valid strand of investigation. However, dissecting out the context-specific effects of the various granulocyte subsets is an important first challenge. In the current issue of JASN, Yan et al.2 propose that G-CSF–induced MDSCs have a protective role in renal ischemia-reperfusion injury (IRI) in mice. This recapitulates data from previous rodent models of IRI in which G-CSF attenuated renal injury and promoted antiapoptotic pathways. However, the critical role of the G-CSF–induced suppressor cells was not appreciated in prior work. Flow cytometric and functional delineation of the MDSC populations, flawed as it is, is in line with current best practice in the manuscript by Yan et al.2 The authors used G-CSF to induce MDSCs, and they found that there was increased renal infiltration of these cells after IRI, that these facilitated renal recovery, and that, ultimately, they attenuated renal fibrosis. They also found that administering G-CSF before IRI ameliorated acute tissue injury, renal apoptosis, and inflammation, all associated with induction of MDSCs. Of note, all experiments were performed in male mice, and therefore, it is not known whether there is a gender effect. It is difficult to distinguish tissue-based MDSCs from mature neutrophils and M2 macrophages; the authors recognize this and have taken extensive measures to try to account for it. To investigate the functional activity of the G-CSF–induced MDSCs, they found that the cells expressed increased levels of IL-10, Arginase-1, and reactive oxygen species. MDSCs were cocultured with nonspecifically activated splenic T cells and showed effective suppression of T cell function. Adoptive transfer of the purified MDSCs reduced the severity of renal IRI. Although G-CSF therapy alone was a good preventive measure for IRI, combining G-CSF treatment with an anti–Gr-1 antibody aimed at depleting potentially damaging mature neutrophils maintained MDSCs and augmented the benefit on IRI-mediated renal dysfunction over and above G-CSF treatment alone. Unfortunately, most of the therapeutic experiments involved pretreatment with the agents, which would only have a therapeutic correlate in situations such as kidney transplantation or elective surgery in high-risk populations. G-CSF is used clinically in patients with congenital or acquired neutropenia and for mobilization of stem cells. It acts through its receptor on neutrophils and bone marrow precursor cells through the JAK/STAT, mitogen-activated protein kinase, and PI3/Akt pathways. G-CSF is also implicated in neurogenesis and has been tested for protective effects in cerebral ischemia. It has also shown beneficial effects in promoting coronary artery collateralization following ischemia and is undergoing trials in the postmyocardial infarction setting. Of note, therapeutic intervention with G-CSF is occasionally associated with adverse events, such as arteritis,3 Sweet syndrome,4 and exacerbation of ANCA-associated vasculitis and GN.5,6 The results of this study should be viewed in the light of a recent paper by Pegues et al.7 examining the increase of g-MDSCs in response to C-reactive protein administration in IRI mice. This paper showed that direct depletion of g-MDSCs (using the same anti–Gr-1 mAb) reduced the albuminuria caused by renal IRI, suggesting that they play a deleterious role, in direct contrast with the manuscript by Yan et al.2 Whereas Pegues et al.7 used anti–GR-1 antibody to deplete MDSCs, Yan et al.2 suggest that the anti–GR-1 antibody depletes the mature neutrophils, allowing the MDSCs to remain, even though both mature neutrophils and MDSC express Gr-1. Anti–Gr-1 antibody-mediated MDSC depletion, in the absence of inducible transgenic GR-1+ cell knockouts or bone marrow transplant techniques, is thus fraught with difficulty.8,9 The outcome will depend on the degree of relative depletion of mature murine neutrophils and MDSCs, which is difficult to determine accurately using flow cytometry. The authors should be commended by addressing this through additional use of peptide-Fc fusion protein, pep-G3, and gemcitabine, although concerns about specificity inevitably remain. The significance of this paper is that it suggests a positive therapeutic role for G-CSF–induced MDSCs in IRI in mice and a potential useful therapeutic for human IRI. However, translation of the combined G-CSF treatment with anti–Gr-1 may prove difficult because there is no known direct human homolog of Gr-1. Human CD177, the binding partner of surface-expressed proteinase-3 (of great interest in the ANCA vasculitis field), is structurally related to this molecule and may be a potential surrogate human target.10 Alternatively, other agents targeting mature neutrophil recruitment may also be testable substitutes, such as CXCR2 antagonists or C5a receptor antagonists, both currently in human clinical trials. The use of human G-CSF in a murine study also limits translational potential. There is only 73% amino acid sequence homology between human and mouse G-CSF. The authors used a markedly elevated dose to overcome this species difference, although this does raise some concerns about unmeasured off-target effects from these supranormal doses and how this effect could be translated to the clinical arena. In addition, it is known that renal ischemia/hypoxia itself promotes human renal tubular cell expression of G-CSF, which at physiologic concentrations, may already be mediating some renoprotective effects, and therefore, the degree of benefit attainable at appropriate pharmacologic concentrations is uncertain. Because G-CSF therapy is already clinically approved and undergoing human trials in other forms of ischemic injury, it could be readily tested in renal IRI in the context of transplantation or in high-risk subjects undergoing vascular surgery. However, to progress this, it would be essential to first develop mechanisms to define the various neutrophil subsets in humans. This would allow tailoring of therapy to ensure that the correct balance of pro- and anti-inflammatory neutrophils is entrained. The exact human correlate of the MDSCs studied by Yan et al.2 remains unclear. Crossdisciplinary agreement on characterization of low-density granulocytes and MDSCs is thus urgently needed. Disclosures None. Funding This work is funded by Charitable Infirmary Charitable Trust grant 210046 and Science Foundation Ireland grant 11/Y/B2093.