The calcium-binding protein complex S100A8/A9 has a crucial role in controlling macrophage-mediated renal repair following ischemia/reperfusion

Upon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by Toll-like receptor-4 and its activation is known to deleteriously contribute to renal I/R-induced injury. To further test this, wild-type and S100A9 knockout mice (deficient for S100A8/A9 complex) were subjected to renal I/R. The expression of S100A8/A9 was significantly increased 1 day after I/R and was co-localized with Ly6G (mouse neutrophil marker)-positive cells. These knockout mice displayed similar renal dysfunction and damage and neutrophil influx compared with wild-type mice at this early time point. Interestingly, S100A9 knockout mice displayed altered tissue repair 5 and 10 days post I/R, as reflected by increased renal damage, sustained inflammation, induction of fibrosis, and increased expression of collagens. This coincided with enhanced expression of alternatively activated macrophage (M2) markers, while the expression of classically activated macrophage (M1) markers was comparable. Similarly, S100A9 deficiency affected M2, but not M1 macrophage polarization in vitro. During the repair phase following acute kidney injury, S100A9 deficiency affects M2 macrophages in mice leading to renal fibrosis and damage. Thus, S100A8/A9 plays a crucial part in controlling macrophage-mediated renal repair following I/R. Upon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by Toll-like receptor-4 and its activation is known to deleteriously contribute to renal I/R-induced injury. To further test this, wild-type and S100A9 knockout mice (deficient for S100A8/A9 complex) were subjected to renal I/R. The expression of S100A8/A9 was significantly increased 1 day after I/R and was co-localized with Ly6G (mouse neutrophil marker)-positive cells. These knockout mice displayed similar renal dysfunction and damage and neutrophil influx compared with wild-type mice at this early time point. Interestingly, S100A9 knockout mice displayed altered tissue repair 5 and 10 days post I/R, as reflected by increased renal damage, sustained inflammation, induction of fibrosis, and increased expression of collagens. This coincided with enhanced expression of alternatively activated macrophage (M2) markers, while the expression of classically activated macrophage (M1) markers was comparable. Similarly, S100A9 deficiency affected M2, but not M1 macrophage polarization in vitro. During the repair phase following acute kidney injury, S100A9 deficiency affects M2 macrophages in mice leading to renal fibrosis and damage. Thus, S100A8/A9 plays a crucial part in controlling macrophage-mediated renal repair following I/R. Renal ischemia/reperfusion (I/R)-induced injury is a major clinical problem and is the most common cause of acute renal failure after renal transplantation, shock, sepsis, and renal artery stenosis.1Alkhunaizi A.M. Schrier R.W. Management of acute renal failure: new perspectives.Am J Kidney Dis. 1996; 28: 315-328Abstract Full Text PDF PubMed Scopus (80) Google Scholar Upon I/R-induced renal injury, several damage-associated molecular patterns are released like heat shock proteins, high mobility group box 1, hyaluronan, biglycan, and S100 calcium-binding proteins.2Wu H. Chen G. Wyburn K.R. et al.TLR4 activation mediates kidney ischemia/reperfusion injury.J Clin Invest. 2007; 117: 2847-2859Crossref PubMed Scopus (680) Google Scholar,3Zhang P.L. Lun M. Schworer C.M. et al.Heat shock protein expression is highly sensitive to ischemia-reperfusion injury in rat kidneys.Ann Clin Lab Sci. 2008; 38: 57-64PubMed Google Scholar These proteins can be recognized by several pattern recognition receptors including Toll-like receptor (TLR)2 and TLR4, and signaling through these receptors are known to play a deleterious role in renal I/R-induced injury.2Wu H. Chen G. Wyburn K.R. et al.TLR4 activation mediates kidney ischemia/reperfusion injury.J Clin Invest. 2007; 117: 2847-2859Crossref PubMed Scopus (680) Google Scholar, 4Leemans J.C. Stokman G. Claessen N. et al.Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney.J Clin Invest. 2005; 115: 2894-2903Crossref PubMed Scopus (470) Google Scholar, 5Shigeoka A.A. Holscher T.D. King A.J. et al.TLR2 is constitutively expressed within the kidney and participates in ischemic renal injury through both MyD88-dependent and -independent pathways.J Immunol. 2007; 178: 6252-6258Crossref PubMed Scopus (234) Google Scholar, 6Pulskens W.P. Teske G.J. Butter L.M. et al.Toll-like receptor-4 coordinates the innate immune response of the kidney to renal ischemia/reperfusion injury.PLoS ONE. 2008; 3: e3596Crossref PubMed Scopus (191) Google Scholar, 7Kruger B. Krick S. Dhillon N. et al.Donor Toll-like receptor 4 contributes to ischemia and reperfusion injury following human kidney transplantation.Proc Natl Acad Sci USA. 2009; 106: 3390-3395Crossref PubMed Scopus (287) Google Scholar S100A8 and S100A9 proteins were characterized as new endogenous TLR4 activators.8Vogl T. Tenbrock K. Ludwig S. et al.Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock.Nat Med. 2007; 13: 1042-1049Crossref PubMed Scopus (1016) Google Scholar S100A8 (also named myeloid-related protein 8) and S100A9 (myeloid-related protein 14) are members of the S100 calcium-binding proteins and exist mainly as a biologically functional S100A8/A9 heterodimer. S100A8 and S100A9 are constitutively expressed by granulocytes, monocytes, and early differentiation stages of macrophages and can be released upon necrosis or actively secreted by phagocytes during inflammation.9Foell D. Wittkowski H. Vogl T. et al.S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules.JLeukoc Biol. 2007; 81: 28-37Crossref PubMed Scopus (664) Google Scholar,10Ehrchen J.M. Sunderkotter C. Foell D. et al.The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer.J Leukoc Biol. 2009; 86: 557-566Crossref PubMed Scopus (579) Google Scholar S100A8/A9 proteins were linked to innate immunity because of the ability to activate TLR4.8Vogl T. Tenbrock K. Ludwig S. et al.Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock.Nat Med. 2007; 13: 1042-1049Crossref PubMed Scopus (1016) Google Scholar S100A8/A9 amplified endotoxin-mediated inflammatory response thought TLR4 and S100A9-deficient mice were resistant to endotoxin-induced lethal shock and abdominal sepsis.8Vogl T. Tenbrock K. Ludwig S. et al.Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock.Nat Med. 2007; 13: 1042-1049Crossref PubMed Scopus (1016) Google Scholar Next to sepsis, S100A8/A9 has been associated with several human diseases including renal transplantation.10Ehrchen J.M. Sunderkotter C. Foell D. et al.The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer.J Leukoc Biol. 2009; 86: 557-566Crossref PubMed Scopus (579) Google Scholar, 11Goebeler M. Roth J. Burwinkel F. et al.Expression and complex formation of S100-like proteins MRP8 and MRP14 by macrophages during renal allograft rejection.Transplantation. 1994; 58: 355-361Crossref PubMed Scopus (79) Google Scholar, 12Eikmans M. Roos-van Groningen M.C. Sijpkens Y.W. et al.Expression of surfactant protein-C, S100A8, S100A9, and B cell markers in renal allografts: investigation of the prognostic value.J Am Soc Nephrol. 2005; 16: 3771-3786Crossref PubMed Scopus (60) Google Scholar, 13Heller F. Frischmann S. Grunbaum M. et al.Urinary calprotectin and the distinction between prerenal and intrinsic acute kidney injury.Clin J Am Soc Nephrol. 2011; 6: 2347-2355Crossref PubMed Scopus (58) Google Scholar, 14Seibert F.S. Pagonas N. Arndt R. et al.Calprotectin and neutrophil gelatinase-associated lipocalin in the differentiation of pre-renal and intrinsic acute kidney injury.Acta Physiol (Oxf). 2013; 207: 700-708Crossref PubMed Scopus (48) Google Scholar In a microarray study to identify genes of which expression during acute rejection is associated with progression to chronic allograft nephropathy, Eikmans et al.12Eikmans M. Roos-van Groningen M.C. Sijpkens Y.W. et al.Expression of surfactant protein-C, S100A8, S100A9, and B cell markers in renal allografts: investigation of the prognostic value.J Am Soc Nephrol. 2005; 16: 3771-3786Crossref PubMed Scopus (60) Google Scholar showed that S100A8 and S100A9 expression could distinguish between patients with graft loss later on due to chronic allograft nephropathy and those who retained stable graft function. Relatively higher S100A8 and S100A9 mRNA and protein levels in infiltrating cells were associated with a favorable prognosis supporting the interest for S100A8/A9 in renal disease. Recently, Fujiu et al.15Fujiu K. Manabe I. Nagai R. Renal collecting duct epithelial cells regulate inflammation in tubulointerstitial damage in mice.J Clin Invest. 2011; 121: 3425-3441Crossref PubMed Scopus (188) Google Scholar showed that S100A8/A9 is induced in a model for progressive renal injury and contributes in monocyte recruitment and development. The role for S100A8/A9 in acute kidney injury is unknown. To determine the contribution of S100A8/A9 in acute renal damage, we subjected wild type (WT) and S100A9 knockout (KO) mice to renal I/R. We showed for the first time that S100A9 deficiency affects M2 macrophage polarization both in vitro and in vivo. In vivo, S100A9 deficiency leads to an uncontrolled repair phase resulting in renal fibrosis through an unrestrained M2 macrophage polarization. Compared with sham WT mice, renal protein level of the biologically relevant S100A8/A9 heterodimer8Vogl T. Tenbrock K. Ludwig S. et al.Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock.Nat Med. 2007; 13: 1042-1049Crossref PubMed Scopus (1016) Google Scholar,16Vogl T. Roth J. Sorg C. et al.Calcium-induced noncovalently linked tetramers of MRP8 and MRP14 detected by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry.J Am Soc Mass Spectrom. 1999; 10: 1124-1130Crossref PubMed Scopus (122) Google Scholar,17Vogl T. Ludwig S. Goebeler M. et al.MRP8 and MRP14 control microtubule reorganization during transendothelial migration of phagocytes.Blood. 2004; 104: 4260-4268Crossref PubMed Scopus (253) Google Scholar was elevated in renal tissue, especially 1 day post I/R (Figure 1a). In plasma from WT mice, S100A8/A9 levels remained similar at all time points (sham: 859±132, I/R day 1: 980±127, I/R day 5: 814±114, I/R day 10: 619±131, data are ng/ml with mean±s.e.m., N=7–8 per group). We further determined S100A8 and S100A9 expression in detail. Renal S100A8 and/or S100A9 staining was observed especially 1 day after I/R in corticomedular region, which is most susceptible to I/R (Figure 1 and Supplementary Figure S1 online). S100A8 and S100A9 co-localized with Ly6G-positive cells, most presumably granulocytes (Figure 1d). Only very few S100A8 cells were observed in S100A9 KO mice, 1 day after I/R (Figure 1b). As expected8Vogl T. Tenbrock K. Ludwig S. et al.Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock.Nat Med. 2007; 13: 1042-1049Crossref PubMed Scopus (1016) Google Scholar,18Manitz M.P. Horst B. Seeliger S. et al.Loss of S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro.Mol Cell Biol. 2003; 23: 1034-1043Crossref PubMed Scopus (251) Google Scholar, renal S100A8/A9 heterodimer and S100A9 staining was undetectable in sham and I/R-treated S100A9 KO mice (Figure 1c and data not shown). Download .jpg (.04 MB) Help with files Supplementary Figure 1 In order to unravel the role of S100A8/A9 in renal I/R, WT and S100A9 KO mice were subjected to bilateral renal I/R. Naïve WT and S100A9 KO mice displayed similar renal function (plasma creatinine and urea; Figure 2), renal collagen deposition (Figure 3), urinary albumin levels, and without occurrence of tubular damage or glomerular scenescence (data not shown). One day after I/R, WT and S100A9 KO mice displayed similar renal dysfunction (Figure 2a and b). In contrast to WT mice, several parameters of renal damage in S100A9 KO mice did not return to basal level. Serum creatinine and urea levels remained increased pointing toward an impaired renal function after I/R. In addition, tubular damage remained severe in S100A9 KO mice as displayed by a higher renal necrosis injury score, KIM1 and NGAL expression and increasing amount of apoptotic tubular epithelial cells (Figure 2c–g). Strikingly, in S100A9 KO mice, tubules remained damaged and cast formation was still present 10 days after I/R, whereas in WT mice, kidney tissue was repaired with functional, brush-border-positive tubuli (Figure 2d). Caspase-3- and Ki67-positve cells were mainly tubular epithelial cells and not characterized as macrophages or fibroblasts (data not shown). In S100A9 KO mice, ongoing tissue damage and apoptosis of tubular epithelial cells was in line with ongoing proliferating tubular epithelial cells, most likely because of the necessity for tissue repair in these mice (Figure 2g and h).Figure 3Renal fibrosis in wild-type (WT) and S100A9 knockout (KO) mice during renal ischemia/reperfusion (I/R) injury. Markers of fibrosis transforming growth factor-beta (TGF-β) (a), hepatocyte growth factor (HGF) (b), collagen type 1 (c, e, and f), and collagen type 3 (d, g, and h) in kidney tissue from WT mice (black bars) and S100A9 KO mice (white bars). TGF-β and HGF expression was corrected for total protein level in tissue. Collagen type 1 and type 3 mRNA levels are expressed relative to housekeeping gene TATA box-binding protein (TBP). Collagen type 1 (e) and type 3 (g) staining on renal tissue slides were digitally analyzed and presented as % positive staining per high-power field (HPF). Representative photos of collagen type 1 (f) and type 3 (h) staining on renal tissue slides from WT and S100A9 KO, 10 days after I/R (original magnification × 200). Graph data are mean±s.e.m. *P<0.05, **P<0.005, ***P<0.001 versus WT.View Large Image Figure ViewerDownload (PPT) The impaired renal tissue repair in S100A9 KO mice upon I/R could lead to renal fibrosis. Therefore, we determined major markers for fibrosis. Transforming growth factor (TGF)-β and hepatocyte growth factor (HGF) are important opposite key players in fibrosis during the progression of renal disease with TGF-β being profibrotic and HGF anti-fibrotic.19Stahl P.J. Felsen D. Transforming growth factor-beta, basement membrane, and epithelial-mesenchymal transdifferentiation: implications for fibrosis in kidney disease.Am J Pathol. 2001; 159: 1187-1192Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar,20Liu Y. Hepatocyte growth factor in kidney fibrosis: therapeutic potential and mechanisms of action.Am J Physiol. 2004; 287: F7-16Crossref PubMed Scopus (217) Google Scholar Total TGF-β levels were elevated in S100A9 KO mice 5 and 10 days after I/R, whereas HGF levels were lower in S100A9 KO mice 5 days after I/R (Figure 3a and b). TGF-β is a known inducer of myofibroblast accumulation, which is characterized by induced expression of α-smooth muscle actin (α-SMA) and production of collagens.21Zeisberg M. Neilson E.G. Mechanisms of tubulointerstitial fibrosis.J Am Soc Nephrol. 2010; 21: 1819-1834Crossref PubMed Scopus (667) Google Scholar In S100A9 KO mice, more renal α-SMA staining was observed compared with WT mice, 5 days after I/R (WT versus S100A9 KO mice: 1.5±0.5 vs. 3.7±0.7% positive area/HPF, P<0.05). In line with collagen type I and type III mRNA data, S100A9 KO mice displayed more collagens in renal tissue compared with WT mice 5 and 10 days following I/R (Figure 3e–h and data not shown). These data showed that S100A9 KO mice displayed and altered repair phase following I/R leading to the development of fibrosis. The enhanced fibrosis was in line with ongoing inflammation in S100A9 KO mice as displayed by enhanced production of pro-inflammatory mediators (Figure 4). The elevated levels of pro-inflammatory mediators could be related to the increased renal expression of damage-associated molecular patterns biglycan and HSP60 in S100A9 KO mice which are known to induce a TLR-mediated inflammatory response (Supplementary Figure S2 online). Download .jpg (.04 MB) Help with files Supplementary Figure 2 To further determine which cell type could be involved in the observed phenotype in S100A9 KO mice, we determined granulocyte, dendritic cell (DC) and macrophage content. Granulocyte content and activation (as determined by myeoloperoxidase levels and superoxide dismutase (SOD) activity in renal homogenate) increased similarly in WT and S100A9 KO mice 1 day after I/R (Supplementary Figure S3 online) and granulocyte content decreased thereafter in similar manner to sham level implicating no direct difference in renal granulocyte migration and function between the two strains. Next, we quantified renal CD11c cells, presumably DC. CD11c mRNA levels increased in WT and S100A9 KO mice 5 days after I/R, which remained high in S100A9 KO mice 10 days after I/R (Supplementary Figure S4 online). Increased mRNA levels in S100A9 KO mice were confirmed on protein level by immunohistochemistry. In addition, we determined activation state of renal DC in kidney single cells. DC activation state was similar between WT and S100A9 KO mice 10 days after I/R, as reflected by similar MHC-II expression on CD45+CD11c+F4/80 cells (MFI WT versus S100A9 KO: 1168±32 vs. 1205±60). We also observed increased level of F4/80-positive cells, presumably macrophages, in kidneys from S100A9 KO mice during late repair phase compared with WT mice (Figure 5). Most F4/80-positive cells were Ki67 negative implicating that F4/80-positive cells were not proliferating but infiltrating cells (data not shown). Macrophages can roughly be categorized into so-called ‘classically activated’ (M1) or ‘alternatively activated’ (M2) macrophages, depending on their stimuli and/or environment.22Ricardo 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, 23Gordon S. Alternative activation of macrophages.Nat Rev Immunol. 2003; 3: 23-35Crossref PubMed Scopus (4721) Google Scholar, 24Gordon S. Martinez F.O. Alternative activation of macrophages: mechanism and functions.Immunity. 2010; 32: 593-604Abstract Full Text Full Text PDF PubMed Scopus (2799) Google Scholar To determine if S100A9 KO mice displayed a different profile in macrophage phenotypes in their kidneys, several markers involved in M1 polarization or M2 polarization were analyzed (M1: nitric oxide synthase-2 and interferon-regulating factor-5: IRF5,25Krausgruber T. Blazek K. Smallie T. et al.IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses.Nat Immunol. 2011; 12: 231-238Crossref PubMed Scopus (859) Google Scholar M2: arginase-1: Arg1, macrophage galactose-type C-type lectin-1 and IRF426Satoh T. Takeuchi O. Vandenbon A. et al.The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection.Nat Immunol. 2010; 11: 936-944Crossref PubMed Scopus (848) Google Scholar). Emr1 mRNA levels (Figure 6a), which encode for macrophage F4/80 gene expression, reflected the influx of renal F4/80+ macrophages in kidneys of WT and S100A9 KO mice with similar pattern (Figure 5). The level of Emr1 mRNA and number of macrophages in the kidney of WT mice peaked at day 5 and declined thereafter, whereas these parameters remained higher in the S100A9 KO mice. M1 markers nitric oxide synthase-2 and IRF5 increased similarly in WT and S100A9 KO mice during the repair phase (Figure 6b and c). Interestingly, M2-associated macrophage markers macrophage galactose-type C-type lectin-1, IRF4, and especially Arg1 were elevated in S100A9 KO mice 5 days after I/R (Figure 6d–f). The difference in Arg1 mRNA was confirmed at protein level (Figure 6g). Arg1 protein expression co-localized with F4/80 staining (Figure 6h). These data implicate that M2, but not M1 polarization, is affected in S100A9 KO mice. Download .jpg (.03 MB) Help with files Supplementary Figure 3 Download .jpg (.03 MB) Help with files Supplementary Figure 4Figure 6Classical and alternative macrophage markers in ischemia/reperfusion (I/R)-damaged kidney from wild-type (WT) and S100A9 knockout (KO) mice. mRNA levels of general macrophage marker Emr1 (a), classical macrophage marker nitric oxide synthase-2 (NOS-2) (b), interferon-regulating factor-5 (IRF5) (c), and alternative macrophage marker arginase-1 (Arg1) (d), macrophage galactose-type C-type lectin-1 (MGL1) (e), and interferon-regulating factor-4 (IRF4) (f) in kidneys from WT mice (black bars) and S100A9 KO mice (white bars) after I/R or in sham (Sh) mice. mRNA levels are expressed relative to housekeeping gene TATA box-binding protein (TBP). (g) Representative photos for Arg1 single staining on kidney tissue slides from WT and S100A9 KO mice, 5 days after I/R (original magnification × 200). (h) Representative photos for co-localization of Arg1 (red) and F4–80 (blue) on kidney tissue slides from WT and S100A9 KO mice, 5 days after I/R (original magnification × 200). Graph data are mean±s.e.m. *P<0.05, **P<0.005 versus WT.View Large Image Figure ViewerDownload (PPT) We determined S100A8 and S100A9 mRNA kinetics during macrophage development in vitro. Expression of S100A8 and S100A9 mRNA reduced during differentiation of bone marrow cells into macrophages (Figure 7a and b). However, M1 polarization increased S100A8 mRNA level whereas M2 polarization further decreased S100A8 and S100A9 mRNA levels (Figure 7c and d). To determine effect of extracellular S100A8 and/or S100A9 on macrophage development, WT bone marrow–derived macrophages (BMDMs) were incubated with recombinant S100A8, S100A9, or S100A8/A9 complex. S100A8, S100A9, and S100A8/A9 induced a dose-dependent tumor necrosis factor-α response and nitric oxide synthase-2 mRNA expression (Figure 8a and data not shown). In contrast, S100A8 and/or S100A9 did not affect Arg1 mRNA expression (Figure 8b and c). These data showed that in vitro, endogenous S100A8 and S100A9 does not induce M2-type macrophage markers, but is involved in macrophage M1 activation.Figure 8Macrophage response to recombinant S100A8 and/or S100A9. Wild-type bone marrow–derived macrophages (WT BMDMs) were cultured in vitro and incubated with medium, 1 or 5μg/ml S100A8 (A8), S100A9 (A9), S100A8/A9 (A8/A9), lipopolysaccharide+interferon (IFN)-γ (M1 polarization), or interleukin (IL)-4+IL-13 (M2 polarization) for 48h. Tumor necrosis factor (TNF)-α was measured in supernatant (a); arginase-1 (Arg1) (b) and interferon-regulating factor 4 (IRF4) (c) were measured on mRNA level. Data are mean±s.e.m. *P<0.05, **P<0.01 versus control (N=3 individuals mice per group).View Large Image Figure ViewerDownload (PPT) We further investigated the contribution of S100A9 deficiency in macrophage polarization in vitro. Nitric oxide synthase-2 mRNA levels increased similarly in WT and S100A9 KO macrophages during M1 polarization (Figure 9a and c). During M2 polarization, a striking induction of Arg1 mRNA level was observed in S100A9 KO macrophages compared with WT macrophages (Figure 9b and d) which matched our in vivo Arg1 findings. Similar results were observed in a second independent experiment (data not shown). In vitro, IRF4 but not macrophage galactose-type C-type lectin-1 mRNA levels were higher in M2-polarized peritoneal macrophages from S100A9 KO mice compared with WT mice (Supplementary Figure S5 online) indicating that the characteristics of M2 polarization of S100A9 KO mice in vitro and in vivo did not perfectly match. These data showed that in vitro, S100A9 deficiency intrinsically predisposes macrophages to sustain a M2 phenotype. We then determined which T helper type 2-related response in vivo was involved in M2 polarization during the repair phase. Renal interleukin (IL)-2, IL-4, and IL-5 protein level and IL-13 and Gata3 (transcription factor T helper type 2 cells) mRNA levels remained similar at all time points in all groups (data not shown). It remains to be determined which mediator in vivo contributes to M2 polarization following I/R in S100A9 KO mice. Download .jpg (.03 MB) Help with files Supplementary Figure 5 The recent discovery of S100A8/A9 as a TLR4 activator leads to new interest in its contribution during inflammatory.8Vogl T. Tenbrock K. Ludwig S. et al.Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock.Nat Med. 2007; 13: 1042-1049Crossref PubMed Scopus (1016) Google Scholar In renal I/R, TLR-induced inflammation plays a deleterious role. Unexpectedly, S100A8/A9 did not contribute to renal I/R-induced acute injury. By contrast, at later stages, S100A9 KO mice displayed a phenotype with enhanced renal dysfunction, damage, fibrosis, and inflammation that is associated with an enhanced polarization of macrophages into a M2 subtype. We showed that S100A8/A9 plays a crucial part in controlling macrophage-mediated renal repair following I/R. Following renal I/R, granulocytes infiltrate the tissue which are known to play a detrimental role in I/R-induced injury. Granulocytes abundantly express S100A8 and S100A99Foell D. Wittkowski H. Vogl T. et al.S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules.JLeukoc Biol. 2007; 81: 28-37Crossref PubMed Scopus (664) Google Scholar,10Ehrchen J.M. Sunderkotter C. Foell D. et al.The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer.J Leukoc Biol. 2009; 86: 557-566Crossref PubMed Scopus (579) Google Scholar and apoptotic granulocytes are able to release S100A9,27Voganatsi A. Panyutich A. Miyasaki K.T. et al.Mechanism of extracellular release of human neutrophil calprotectin complex.J Leukoc Biol. 2001; 70: 130-134PubMed Google Scholar a known DAMP and activator of TLR4.8Vogl T. Tenbrock K. Ludwig S. et al.Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock.Nat Med. 2007; 13: 1042-1049Crossref PubMed Scopus (1016) Google Scholar TLR4 plays part in renal I/R-induced injury2Wu H. Chen G. Wyburn K.R. et al.TLR4 activation mediates kidney ischemia/reperfusion injury.J Clin Invest. 2007; 117: 2847-2859Crossref PubMed Scopus (680) Google Scholar,28Pulskens W.P. Rampanelli E. Teske G.J. et al.TLR4 promotes fibrosis but attenuates tubular damage in progressive renal injury.J Am Soc Nephrol. 2010; 21: 1299-1308Crossref PubMed Scopus (127) Google Scholar and we hypothesized that S100A8/A9–TLR4 signaling could play part in the mechanism behind renal I/R-induced injury. Unexpectedly, WT and S100A9 KO mice displayed similar granulocyte influx and activation, renal dysfunction and damage, 1 day after I/R. Compared with WT mice, S100A9 KO mice displayed a mild reduction in renal keratinocyte-derived chemokine levels implicating a limited role for TLR4-S100A8/A9 signaling at this time point. In a study by Ziegler et al.,29Ziegler G. Prinz V. Albrecht M.W. et al.Mrp-8 and -14 mediate CNS injury in focal cerebral ischemia.Biochim Biophys Acta. 2009; 1792: 1198-1204Crossref PubMed Scopus (60) Google Scholar S100A9 KO mice displayed less I/R-induced cerebral damage, most likely by absence of S100A8/A9-induced inflammation when signaling through TLR4.30Lehnardt S. Massillon L. Follett P. et al.Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway.Proc Natl Acad Sci USA. 2003; 100: 8514-8519Crossref PubMed Scopus (843) Google Scholar However, unlike in cerebral I/R,29Ziegler G. Prinz V. Albrecht M.W. et al.Mrp-8 and -14 mediate CNS injury in focal cerebral ischemia.Biochim Biophys Acta. 2009; 1792: 1198-1204Crossref PubMed Scopus (60) Google Scholar we did not obser