BASIC RESEARCH www.jasn.org GM-CSF Promotes Macrophage Alternative Activation after Renal Ischemia/Reperfusion Injury † ‡ Sarah C. Huen,* Larry Huynh, Arnaud Marlier,* Yashang Lee,* Gilbert W. Moeckel, and Lloyd G. Cantley* *Section of Nephrology, Department of Medicine, †Yale Cancer Center, and ‡Department of Pathology, Yale University School of Medicine, New Haven, Connecticut ABSTRACT After kidney ischemia/reperfusion (I/R) injury, monocytes home to the kidney and differentiate into activated macrophages. Whereas proinflammatory macrophages contribute to the initial kidney damage, an alternatively activated phenotype can promote normal renal repair. The microenvironment of the kidney during the repair phase mediates the transition of macrophage activation from a proinflammatory to a reparative phenotype. In this study, we show that macrophages isolated from murine kidneys during the tubular repair phase after I/R exhibit an alternative activation gene profile that differs from the canonical alternative activation induced by IL-4–stimulated STAT6 signaling. This unique activation profile can be reproduced in vitro by stimulation of bone marrow-derived macrophages with conditioned media from serum-starved mouse proximal tubule cells. Secreted tubular factors were found to activate macro- phage STAT3 and STAT5 but not STAT6, leading to induction of the unique alternative activation pattern. Using STAT3-deficient bone marrow-derived macrophages and pharmacologic inhibition of STAT5, we found that tubular cell-mediated macrophage alternative activation is regulated by STAT5 activation. Both in vitro and after renal I/R, tubular cells expressed GM-CSF, a known STAT5 activator, and this pathway was required for in vitro alternative activation of macrophages by tubular cells. Furthermore, administration of a neutralizing antibody against GM-CSF after renal I/R attenuated kidney macrophage alternative activation and suppressed tubular proliferation. Taken together, these data show that tubular cells can instruct macrophage activation by secreting GM-CSF, leading to a unique macrophage reparative phe- notype that supports tubular proliferation after sterile ischemic injury. J Am Soc Nephrol 26: 1334–1345, 2015. doi: 10.1681/ASN.2014060612 Macrophages are key participants in regulating the in- subsequently been detected in multiple types of in- flammatory response to infections and sterile injuries. fectious and sterile tissue injuries. M2 macrophages Macrophages are activated in response to pathogens have been typically subcategorized into M2a wound- and endogenous injury stimuli. The inflammation healing macrophages induced by IL-4/IL-13 activation that ensues acts to eliminate pathogens/toxins, restore of the IL-4Ra–STAT6 signaling pathway and regula- tissue homeostasis, andpromote tissue repair.Broadly tory M2b/c macrophages, which are activated by im- defined, macrophage activation has been divided into mune complexes and IL-10 or TGF-b, respectively.1 two major subtypes: classically activated proinflam- matory M1 macrophages and alternatively activated anti-inflammatory M2 macrophages. Classically ac- Received June 25, 2014. Accepted October 9, 2014. tivated M1 macrophages are induced by IFN-g and Published online ahead of print. Publication date available at activation of Toll-like receptors and promote antimi- www.jasn.org. crobial responses downstream of STAT1 and NF-kB Correspondence: Dr. Sarah C. Huen, Yale University School of signaling, respectively. Alternatively activated macro- Medicine, 333 Cedar Street, PO Box 208029, New Haven, CT phages were first defined as IL-4/IL-13–induced mac- 06510. Email: [email protected]. rophages seen in parasitic infections but have Copyright © 2015 by the American Society of Nephrology 1334 ISSN : 1046-6673/2606-1334 JAmSocNephrol26: 1334–1345, 2015 www.jasn.org BASIC RESEARCH These categories have largely been defined in vitro with individ- RESULTS ual stimuli. In vivo, macrophage differentiation and activation of effector functions during the inflammatory response are dic- Tubular-Mediated Alternative Activation Is IL-4–STAT6 tated by a dynamic and complex microenvironment. Investiga- Independent tion of in vivo macrophages in a wide variety of disease models We have found that proinflammatory macrophages cocultured associated with inflammation, both infectious and sterile, has with MPT cells or primary tubular epithelial cells show increased shown a diverse spectrum of macrophage phenotypes that are expression of the classic alternative activation genes arginase-1 temporally and functionally dynamic.2 Identification of the in (Arg1)andCd206 (mannose receptor), which is independent of vivo signaling pathways that govern macrophage activation and IL-4Ra–STAT6 signaling10 (Supplemental Figure 1). To deter- regulate macrophage effector functions may, therefore, provide mine if tubular cell-induced alternative activation is distinct therapeutic targets to promote inflammation resolution and tis- from IL-4–induced alternative activation, naïve BMMs were sue repair. stimulated with IL-4 or CM from serum-starved MPT cells. In sterile ischemic kidney injury models, macrophages seem The gene expression profiles of IL-4– compared with MPT to play roles in all phases of the injury process, including the CM-treated macrophages show both overlapping (Arg1 and initial injury, subsequent repair, and late fibrosis.3,4 Within 24 Cd206) (Figure 1A) and divergent gene expression. Msr1 is in- hours after ischemia/reperfusion (I/R) injury, monocytes are duced by MPT CM but not IL-4 (Figure 1B), whereas Dectin-1, recruited to the kidney, where they differentiate into macro- Fizz1, Ym1,andIgf-1 are specifictoIL-4–induced macrophage phages. Proinflammatory macrophages predominate during alternative activation (Figure 1C). To determine the relevance of the early injury phase, during which tubular apoptosis is these distinct alternative activation profiles for in vivo macro- prominent. During the tubular repair phase, when tubular phage activation, macrophages were flow-sorted from control cells are proliferating and repopulating the denuded basement kidneys and ischemically injured kidneys on day 5 after reper- membrane, kidney macrophages begin to express markers of fusion (the time at which we detected increased Arg1 and Cd206 alternative activation. Macrophage depletion studies suggest expression10) and analyzed for alternative activation gene ex- that the functional phenotypes of the macrophages correlate pression. Consistent with the tubular cell activation profile, with each phase.5–11 Our previous in vivo studies using fluo- Msr1 expression is upregulated in macrophages at day 5 after rescently tagged ex vivo IFN-g–primed macrophages sugges- I/R injury compared with macrophages from uninjured kidneys, ted that signals within the injured kidney were sufficient to whereas the expression of downstream IL-4 gene targets (Dectin-1, induce this proinflammatory to anti-inflammatory pheno- Fizz1,andYm112) is not induced (Figure 1D). Of note, Igf-1 ex- typic switch in infiltrating macrophages.10 Both in vivo studies pression is upregulated on day 5 after I/R, suggesting that either using IL-4Ra null mice and in vitro coculture studies suggest there is selective IL-4 signaling to induce Igf-1 but not other IL-4 that the primary signal for alternative macrophage activation targets or a third pathway (nontubular and non–IL-4) may be in the injured kidney uses an IL-4Ra–independent pathway.10 responsible for Igf-1 expression. In this study, we show that the pattern of in vivo alternative macrophage activation after I/R injury is distinct from that Tubular Factors That Induce Macrophage Alternative seen after in vitro IL-4 stimulation but consistent with the Activation Are Basolaterally Secreted pattern of activation seen after macrophage exposure to tubular Proximal tubular cells are polarized epithelia, with the apical cell conditioned media (CM). To determine the mechanism by surface facing the tubule lumen exposed to the glomerular which tubular cells can promote macrophage alternative acti- filtrate and the basolateral surface attached to the basement vation, we defined the signaling pathways activated in macro- membrane facing the interstitium. After I/R injury, macro- phages by secreted tubular factors. In vitro studies with primary phages traffic to the interstitium of the kidney and line areas of bone marrow-derived macrophages (BMMs) and CM from tubular damage adjacent to the tubular basement membrane.10 serum-starved mouse proximal tubule (MPT) cells showed To model in vivo polarity and determine whether the polarity that tubular cell-secreted factors activate JAK–STAT (STAT3 of secreted tubular factors would be physiologically relevant in and STAT5) pathways in macrophages. Using BMMs from activating macrophages in the interstitium, MPT cells were fl fl LysM-Cre;Stat3 / mice as well as pharmacologic inhibition grown to confluency on 0.4-mm Transwell inserts, and CM of STAT5, we found that tubule cell-mediated macrophage al- were collected from either the apical- or basolateral-facing ternative activation is regulated by STAT5 activation. GM- chambers (Figure 2A). The basolateral MPT CM induced sig- CSF, a well known STAT5 activator, is upregulated in renal nificantly higher expression of Arg1 and Cd206 than the apical proximal tubule cells after I/R injury. We found that GM- CM (Figure 2, B and C), consistent with the model that tubu- CSF is secreted by MPT cells and required to induce alterna- lar cell-secreted factors can signal