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Alternatively activated macrophages as therapeutic agents for kidney disease: in vivo stability is a key factor

2014; Elsevier BV; Volume: 85; Issue: 4 Linguagem: Inglês

10.1038/ki.2013.405

1523-1755

Senthilkumar Alagesan, Matthew D. Griffin,

Phagocytosis and Immune Regulation

Infusing ex vivo–generated alternatively activated macrophages (AAM) has shown promise in experimental systems as a therapeutic strategy for inflammatory kidney disease. In the mouse Adriamycin nephropathy model, however, Cao et al. report that AAM derived from bone marrow precursors fail to ameliorate disease severity. Absence of the anticipated protective effect resulted from a loss of macrophage anti-inflammatory (M2) phenotype following trafficking to injured kidney—an effect that was mediated by localized colony-stimulating factor-1–dependent macrophage proliferation. Infusing ex vivo–generated alternatively activated macrophages (AAM) has shown promise in experimental systems as a therapeutic strategy for inflammatory kidney disease. In the mouse Adriamycin nephropathy model, however, Cao et al. report that AAM derived from bone marrow precursors fail to ameliorate disease severity. Absence of the anticipated protective effect resulted from a loss of macrophage anti-inflammatory (M2) phenotype following trafficking to injured kidney—an effect that was mediated by localized colony-stimulating factor-1–dependent macrophage proliferation. The healthy mammalian kidney contains an abundant population of interstitial mononuclear phagocytes with phenotypic and functional characteristics that overlap with the classical profiles of both macrophages and dendritic cells.1.Nelson P.J. Rees A.J. Griffin M.D. et al.The renal mononuclear phagocytic system.J Am Soc Nephrol. 2012; 23: 194-203Crossref PubMed Scopus (193) Google Scholar During acute and chronic kidney disease, these resident mononuclear phagocytic cells undergo a range of activation responses and become intermingled with macrophage- and dendritic cell–like cells derived from infiltrating blood monocytes.1.Nelson P.J. Rees A.J. Griffin M.D. et al.The renal mononuclear phagocytic system.J Am Soc Nephrol. 2012; 23: 194-203Crossref PubMed Scopus (193) Google Scholar Among the resident and infiltrating mononuclear cells within injured kidney are mature macrophages—large cells with specialized capacity for phagocytosis, intracellular microbial killing, receptor-mediated molecular pattern recognition, and orchestration of localized inflammatory response via secretion of cytokines and chemokines.2.Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3877) Google Scholar,3.Ricardo S.D. van Goor H. Eddy A.A. Macrophage diversity in renal injury and repair.J Clin Invest. 2008; 118: 3522-3530Crossref PubMed Scopus (562) Google Scholar The elucidation of distinct macrophage activation states, often referred to as classical, proinflammatory (M1) and alternative, anti-inflammatory (M2) polarization, has generated strong interest in better understanding macrophage roles in regulating tissue injury and repair (Figure 1).1.Nelson P.J. Rees A.J. Griffin M.D. et al.The renal mononuclear phagocytic system.J Am Soc Nephrol. 2012; 23: 194-203Crossref PubMed Scopus (193) Google Scholar, 2.Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3877) Google Scholar, 3.Ricardo S.D. van Goor H. Eddy A.A. Macrophage diversity in renal injury and repair.J Clin Invest. 2008; 118: 3522-3530Crossref PubMed Scopus (562) Google Scholar, 4.Lee S. Huen S. Nishio H. et al.Distinct macrophage phenotypes contribute to kidney injury and repair.J Am Soc Nephrol. 2011; 22: 317-326Crossref PubMed Scopus (619) Google Scholar, 5.Wang Y. Harris D.C. Macrophages in renal disease.J Am Soc Nephrol. 2011; 22: 21-27Crossref PubMed Scopus (182) Google Scholar Although undoubtedly an oversimplification of the biological reality, the M1/M2 dichotomy has also given rise to the translational goal of therapeutically manipulating macrophage activation states in a wide range of diseases.2.Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3877) Google Scholar,3.Ricardo S.D. van Goor H. Eddy A.A. Macrophage diversity in renal injury and repair.J Clin Invest. 2008; 118: 3522-3530Crossref PubMed Scopus (562) Google Scholar,5.Wang Y. Harris D.C. Macrophages in renal disease.J Am Soc Nephrol. 2011; 22: 21-27Crossref PubMed Scopus (182) Google Scholar Rodent models have proved to be of particular value for studying macrophage phenotypes in the context of acute and chronic kidney disease. For example, mononuclear phagocyte depletion strategies, including liposomal clodronate administration, macrophage-specific gene deletions, and transgenic expression of the diphtheria toxin receptor in cells expressing the myeloid markers CD11b and CD11c, have been widely applied to rodent renal disease models with a view to elucidating macrophage influences on acute kidney injury, repair, and fibrosis.3.Ricardo S.D. van Goor H. Eddy A.A. Macrophage diversity in renal injury and repair.J Clin Invest. 2008; 118: 3522-3530Crossref PubMed Scopus (562) Google Scholar,6.Griffin M.D. Mononuclear phagocyte depletion strategies in models of acute kidney disease: what are they trying to tell us?.Kidney Int. 2012; 82: 835-837Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar Although interpretation of disease outcomes following such in vivo depletions is complex,6.Griffin M.D. Mononuclear phagocyte depletion strategies in models of acute kidney disease: what are they trying to tell us?.Kidney Int. 2012; 82: 835-837Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar a large body of literature has now accumulated in support of a model whereby a time-dependent, coordinated transition from predominantly classically activated (M1) to alternatively activated (M2) macrophages within the renal interstitium serves to maximize functional and structural recovery following injury.1.Nelson P.J. Rees A.J. Griffin M.D. et al.The renal mononuclear phagocytic system.J Am Soc Nephrol. 2012; 23: 194-203Crossref PubMed Scopus (193) Google Scholar, 2.Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3877) Google Scholar, 3.Ricardo S.D. van Goor H. Eddy A.A. Macrophage diversity in renal injury and repair.J Clin Invest. 2008; 118: 3522-3530Crossref PubMed Scopus (562) Google Scholar, 4.Lee S. Huen S. Nishio H. et al.Distinct macrophage phenotypes contribute to kidney injury and repair.J Am Soc Nephrol. 2011; 22: 317-326Crossref PubMed Scopus (619) Google Scholar, 5.Wang Y. Harris D.C. Macrophages in renal disease.J Am Soc Nephrol. 2011; 22: 21-27Crossref PubMed Scopus (182) Google Scholar Dysregulation of this process may be detrimental, with resulting chronic inflammation and/or excessive fibrosis.3.Ricardo S.D. van Goor H. Eddy A.A. Macrophage diversity in renal injury and repair.J Clin Invest. 2008; 118: 3522-3530Crossref PubMed Scopus (562) Google Scholar The concept and initial functional definitions of alternatively activated macrophages (AAMs) stemmed primarily from in vitro characterization of macrophage-like cells differentiated under modified conditions from tissue macrophages, monocytes, or bone marrow–derived myeloid precursors.2.Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3877) Google Scholar,3.Ricardo S.D. van Goor H. Eddy A.A. Macrophage diversity in renal injury and repair.J Clin Invest. 2008; 118: 3522-3530Crossref PubMed Scopus (562) Google Scholar Such AAMs are typically generated during several days' culture in the presence of modulating agents such as interleukin (IL)-4, IL-13, IL-10, transforming growth factor-β, glucocorticoid, and others.1.Nelson P.J. Rees A.J. Griffin M.D. et al.The renal mononuclear phagocytic system.J Am Soc Nephrol. 2012; 23: 194-203Crossref PubMed Scopus (193) Google Scholar, 2.Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3877) Google Scholar, 3.Ricardo S.D. van Goor H. Eddy A.A. Macrophage diversity in renal injury and repair.J Clin Invest. 2008; 118: 3522-3530Crossref PubMed Scopus (562) Google Scholar, 4.Lee S. Huen S. Nishio H. et al.Distinct macrophage phenotypes contribute to kidney injury and repair.J Am Soc Nephrol. 2011; 22: 317-326Crossref PubMed Scopus (619) Google Scholar, 5.Wang Y. Harris D.C. Macrophages in renal disease.J Am Soc Nephrol. 2011; 22: 21-27Crossref PubMed Scopus (182) Google Scholar Protocols in which cultured AAMs are derived from monocytes or bone marrow–derived myeloid precursors also require the addition of a differentiation factor, colony-stimulating factor-1 (CSF-1; also known as macrophage colony-stimulating factor (M-CSF)). Defining characteristics of in vitro–generated rodent AAMs, some of which have been confirmed in the in vivo setting, include expression of specific surface proteins, transcription factors, enzymes, and anti-inflammatory mediators (Figure 1).1.Nelson P.J. Rees A.J. Griffin M.D. et al.The renal mononuclear phagocytic system.J Am Soc Nephrol. 2012; 23: 194-203Crossref PubMed Scopus (193) Google Scholar, 2.Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3877) Google Scholar, 3.Ricardo S.D. van Goor H. Eddy A.A. Macrophage diversity in renal injury and repair.J Clin Invest. 2008; 118: 3522-3530Crossref PubMed Scopus (562) Google Scholar, 4.Lee S. Huen S. Nishio H. et al.Distinct macrophage phenotypes contribute to kidney injury and repair.J Am Soc Nephrol. 2011; 22: 317-326Crossref PubMed Scopus (619) Google Scholar Given this background, it is not surprising that in vitro–generated AAMs have drawn attention as a potential cellular therapy for inflammatory diseases of the kidney and other organs.5.Wang Y. Harris D.C. Macrophages in renal disease.J Am Soc Nephrol. 2011; 22: 21-27Crossref PubMed Scopus (182) Google Scholar For instance, Lee et al. have shown that infusion of IL-4-treated macrophages promotes renal tubular epithelial cell proliferation (a feature of tissue repair) in mice following ischemia–reperfusion injury.4.Lee S. Huen S. Nishio H. et al.Distinct macrophage phenotypes contribute to kidney injury and repair.J Am Soc Nephrol. 2011; 22: 317-326Crossref PubMed Scopus (619) Google Scholar Conversely, in this and other studies, adoptive transfer of macrophages classically activated with interferon-γ resulted in worsening of inflammatory renal disease.4.Lee S. Huen S. Nishio H. et al.Distinct macrophage phenotypes contribute to kidney injury and repair.J Am Soc Nephrol. 2011; 22: 317-326Crossref PubMed Scopus (619) Google Scholar,7.Wang Y. Cao Q. Zheng G. et al.By homing to the kidney, activated macrophages potently exacerbate renal injury.Am J Pathol. 2008; 172: 1491-1499Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Over the past 6 years, the group of David Harris has contributed a series of important rodent-based studies that have documented the effects on acute and chronic kidney disease models of infusing in vitro–generated macrophages of various types and have investigated the mechanisms underlying these effects.5.Wang Y. Harris D.C. Macrophages in renal disease.J Am Soc Nephrol. 2011; 22: 21-27Crossref PubMed Scopus (182) Google Scholar,7.Wang Y. Cao Q. Zheng G. et al.By homing to the kidney, activated macrophages potently exacerbate renal injury.Am J Pathol. 2008; 172: 1491-1499Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar,8.Wang Y. Wang Y.P. Zheng G. et al.Ex vivo programmed macrophages ameliorate experimental chronic inflammatory renal disease.Kidney Int. 2007; 72: 290-299Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar In the mouse adriamycin nephropathy model of acute proteinuric renal disease, this group has previously demonstrated that a single intravenous injection of 106 AAMs, generated by culture of splenic CD11b+ cells for 48h in the presence of IL-4 and IL-13 and administered 5 days after adriamycin, was associated with amelioration of renal injury.8.Wang Y. Wang Y.P. Zheng G. et al.Ex vivo programmed macrophages ameliorate experimental chronic inflammatory renal disease.Kidney Int. 2007; 72: 290-299Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar In this study, spleen-derived AAMs were shown to home to injured kidneys and to maintain a stable, non-proliferative M2-like phenotype for as long as 4 weeks.8.Wang Y. Wang Y.P. Zheng G. et al.Ex vivo programmed macrophages ameliorate experimental chronic inflammatory renal disease.Kidney Int. 2007; 72: 290-299Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar Now, Cao et al.9.Cao Q. Wang Y. Zheng D. et al.Failed renoprotection by alternatively activated bone marrow macrophages is due to a proliferation-dependent phenotype switch in vivo.Kidney Int. 2014; 85 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar (this issue) report surprising findings from a follow-up study in this same model that have potentially important implications for the clinical translation of AAMs as a cellular therapy. As a starting point, the authors demonstrate that mouse bone marrow cells can be polarized to an M2-like phenotype very similar to that of spleen-derived AAMs, via CSF-1-driven differentiation culture in the presence of IL-4 and IL-13. The most obvious rationale for performing this experiment rests with the fact that, unlike the spleen, the bone marrow represents an accessible source of macrophage precursors for AAM therapy in human subjects. However, when the bone marrow M2–like macrophages (BM-M2 macrophages) were administered to mice with adriamycin nephropathy, it was found that the amelioration of proteinuria and renal functional impairment associated with transfer of spleen-derived cells (SP-M2 macrophages) was absent. In fact, the outcome for BM-M2 macrophage–treated animals was no different from that of mice receiving no treatment or non-polarized (M0) macrophages. Although these phenomena are of interest, the strength of the study lies in the subsequent experiments carried out to elucidate the mechanistic basis for the failure of BM-M2 macrophages to improve the outcome of adriamycin nephropathy. An initial clue came from the higher expression by BM-M2 macrophages of the CSF-1 receptor CD115 and the mouse monocyte–associated marker Gr-1, which is normally absent on mature tissue macrophages. However, the more revealing data derive from the application of techniques to track the injected macrophages in vivo and to retrieve them for analysis of proliferation and phenotype in comparison with the therapeutically active SP-M2 macrophages. By repurifying infused macrophages from diseased kidneys at different time points, Cao et al. convincingly showed that BM-M2 macrophages lost expression of several AAM-defining transcripts between 2 and 7 days after administration while increasing the expression of proinflammatory transcripts such as IL-6, tumor necrosis factor-α, and inducible nitric oxide synthase.9.Cao Q. Wang Y. Zheng D. et al.Failed renoprotection by alternatively activated bone marrow macrophages is due to a proliferation-dependent phenotype switch in vivo.Kidney Int. 2014; 85 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Next, by prelabeling injected cells with a fluorescent dye that becomes diluted following cell division, the authors make the key observation that BM-M2 macrophages, but not SP-M2 macrophages, underwent multiple rounds of proliferation within diseased kidneys. Reverting to in vitro studies, Cao et al. were able to demonstrate that loss of the M2 phenotype of bone marrow–derived AAMs was directly linked to their proliferation after removal from polarizing culture conditions and could be partially prevented by the blocking of CSF-1-mediated signaling. Finally, in an elegant application of two-color immunofluorescence microscopy, it is shown that clusters of (presumably proliferative) BM-M2 macrophages were strongly colocalized with CSF-1-expressing tubular cells of affected kidneys—a phenomenon that was absent in kidneys containing SP-M2 macrophages. This observation, combined with the study's other experimental results, provides strong evidence that infused M2-polarized macrophages that retain precursor-like features, including high expression of CD115, may be induced to proliferate and to revert to a neutral or even proinflammatory phenotype within the inflamed kidney through the local production of CSF-1 (Figure 1). The results of this study provide a striking example of macrophage phenotypic plasticity in vivo whereby individual cells or cell populations exhibit varying functional characteristics at different time points under the influence of 'programming' factors within the immediate microenvironment.1.Nelson P.J. Rees A.J. Griffin M.D. et al.The renal mononuclear phagocytic system.J Am Soc Nephrol. 2012; 23: 194-203Crossref PubMed Scopus (193) Google Scholar, 2.Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3877) Google Scholar, 3.Ricardo S.D. van Goor H. Eddy A.A. Macrophage diversity in renal injury and repair.J Clin Invest. 2008; 118: 3522-3530Crossref PubMed Scopus (562) Google Scholar, 4.Lee S. Huen S. Nishio H. et al.Distinct macrophage phenotypes contribute to kidney injury and repair.J Am Soc Nephrol. 2011; 22: 317-326Crossref PubMed Scopus (619) Google Scholar, 5.Wang Y. Harris D.C. Macrophages in renal disease.J Am Soc Nephrol. 2011; 22: 21-27Crossref PubMed Scopus (182) Google Scholar Considering the results obtained in parallel for SP-M2 macrophages in this study, it could also be concluded that polarization of fully differentiated macrophages, as opposed to growth factor–induced myeloid precursors, results in an inherently more stable phenotype that is relatively resistant to reprogramming even with prolonged exposure to an inflammatory environment. This particular insight may merit further consideration by researchers investigating the phenotypic characteristics of naturally occurring macrophages in localized inflammatory diseases, as such cells may derive either from terminally differentiated tissue-resident mononuclear phagocytes or from precursor monocytes.1.Nelson P.J. Rees A.J. Griffin M.D. et al.The renal mononuclear phagocytic system.J Am Soc Nephrol. 2012; 23: 194-203Crossref PubMed Scopus (193) Google Scholar First and foremost, however, the study of Cao et al.9.Cao Q. Wang Y. Zheng D. et al.Failed renoprotection by alternatively activated bone marrow macrophages is due to a proliferation-dependent phenotype switch in vivo.Kidney Int. 2014; 85 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar gives pause for thought as regards the clinical feasibility of ex vivo–generated AAMs as a cellular therapy for inflammatory diseases of the kidney or other tissues. One factor worth considering is the scale-up that would be required to directly translate such experimental protocols from mouse to human. Given the typical weight of an adult laboratory mouse (about 20–25g), an inoculum of 106 cells per animal represents the equivalent of approximately 3 × 109 to 3.5 × 109 cells for a 70-kg person—roughly one-fifth of the total circulating monocyte number of a healthy adult. Thus, unless the potency and/or homing characteristics of ex vivo–generated human AAMs can be manipulated to allow for lower dosing ranges, cost and practicality represent important obstacles. A second key variable is the selection of an appropriate precursor cell from which to derive human AAMs. As the use of cells from spleen and other tissues containing significant numbers of mature macrophages is impractical for human cell therapy, the most likely starting materials would be peripheral blood monocytes, bone marrow monocytes/myeloid precursors, or, conceivably, induced pluripotent stem cells. With the potential need for high cell numbers, the latter two cell types, unlike blood monocytes, could bring the advantage of in vitro proliferative capacity. However, as we now learn from Cao et al.,9.Cao Q. Wang Y. Zheng D. et al.Failed renoprotection by alternatively activated bone marrow macrophages is due to a proliferation-dependent phenotype switch in vivo.Kidney Int. 2014; 85 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar this capacity may come with a considerable disadvantage—the potential loss of anti-inflammatory functions following in vivo transfer. For clinical translation, therefore, it will now be important to carefully examine the functional stability of human M2-like macrophages generated from different precursor cells and to test strategies (such as blockade of CSF-1-mediated signaling) for preventing 'depolarization' in vivo. It will also be of particular interest to learn whether AAMs derived from blood monocytes (which require the presence of CSF-1 for in vitro differentiation but are not highly proliferative) resemble more closely spleen- or bone marrow–derived M2-like macrophages. Furthermore, it is worth noting that, although the basic phenomenon of classical and alternative activation has been documented for human macrophages, there are significant differences between humans and rodents as regards the defining markers and effector molecules of AAMs.10.Murray P.J. Wynn T.A. Protective and pathogenic functions of macrophage subsets.Nat Rev Immunol. 2011; 11: 723-737Crossref PubMed Scopus (3323) Google Scholar As things currently stand, the in vivo characteristics of human AAMs are considerably less well understood than those of rodents. Despite these caveats, promising early clinical experiences have been reported in the settings of kidney transplantation11.Hutchinson J.A. Riquelme P. Sawitzki B. et al.Cutting Edge: Immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients.J Immunol. 2011; 187: 2072-2078Crossref PubMed Scopus (188) Google Scholar and critical limb ischemia12.Powell R.J. Marston W.A. Berceli S.A. et al.Cellular therapy with Ixmyelocel-T to treat critical limb ischemia: the randomized, double-blind, placebo-controlled RESTORE-CLI trial.Mol Ther. 2012; 20: 1280-1286Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar for cellular products composed of or containing AAMs (albeit not those generated with the use of IL-4 and IL-13). Furthermore, clinical trial activity for other immunomodulatory cells such as mesenchymal stromal (stem) cells and regulatory T cells continues to grow worldwide. The current study by Cao et al.9.Cao Q. Wang Y. Zheng D. et al.Failed renoprotection by alternatively activated bone marrow macrophages is due to a proliferation-dependent phenotype switch in vivo.Kidney Int. 2014; 85 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar reminds us of the unique complexities of cellular therapies compared with pharmacological agents and of the value of innovative animal model experiments for better understanding the disease- and tissue-specific fates of infused cells. M.D.G. is funded by grants from Science Foundation Ireland (SFI SRC 09/SRC/B1794) and the Health Research Board of Ireland (HRA/HSR/2010/63).