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Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation

† Kazumi Taguchi,* Atsushi Okada,* Hiroshi Kitamura, Takahiro Yasui,* Taku Naiki,* Shuzo Hamamoto,* Ryosuke Ando,* Kentaro Mizuno,* Noriyasu Kawai,* Keiichi Tozawa,* ‡ ‡ † Kenichi Asano, Masato Tanaka, Ichiro Miyoshi, and Kenjiro Kohri*

Departments of *Nephro-urology, and †Comparative and Experimental Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; and ‡Laboratory of Immune Regulation, School of Science, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan

ABSTRACT We recently reported evidence suggesting that migrating macrophages (Mws) eliminate renal crystals in hyperoxaluric mice. Mwscanbeinflammatory (M1) or anti-inflammatory (M2), and colony-stimulating factor-1 (CSF-1) mediates polarization to the M2Mw phenotype. M2Mws promote renal tissue repair and regeneration, but it is not clear whether these cells are involved in suppressing renal crystal formation. We investigated the role of M2Mws in renal crystal formation during hyperoxaluria using CSF-1–deficient mice, which lack M2Mws. Compared with wild-type mice, CSF-1–deficient mice had significantly higher amounts of renal calcium oxalate crystal deposition. Treatment with recombinant human CSF-1 increased the expression of M2-related genes and markedly decreased the number of renal crystals in both CSF-1– deficient and wild-type mice. Flow cytometry of sorted renal Mws showed that CSF-1 deficiency resulted in a smaller population of CD11b+F4/80+CD163+CD206hi cells, which represent M2-like Mws. Additionally, transfusion of M2Mws into CSF-1–deficient mice suppressed renal crystal deposition. In vitro phagocytosis assays with calcium oxalate monohydrate crystals showed a higher rate of crystal phagocytosis by M2- polarized Mws than M1-polarized Mws or renal tubular cells. Gene array profiling showed that CSF-1 de- ficiency resulted in disordered M2- and stone-related gene expressions. Collectively, our results provide compelling evidence for a suppressive role of CSF-1 signaling in renal crystal formation.

J Am Soc Nephrol 25: 1680–1697, 2014. doi: 10.1681/ASN.2013060675

We previously used microarray analysis to show a processing, although the mechanisms by which correlation between renal calcium oxalate (CaOx) they interact with crystals remain unclear. crystal formation and macrophages (Mws).1 We There are two subtypes of Mws, classically acti- also observed the migration of Mws to renal tubular vated (M1) and alternatively activated (M2) Mws, cells (RTCs) that include crystals followed by the which exert pro- and anti-inflammatory effects, respec- disappearance of crystals in the tubular lumen from tively.7,8 Resident tissue Mws differentiate after stimu- thecorticomedullaryjunctioninhyperoxaluric lation by various cytokines.9,10 Associations were mice.2 Mws and multinucleated giant cells also en- reported between kidney stones and atherosclerotic capsulated interstitial crystals in the kidneys of hy- peroxaluric rats and humans with acute and chronic oxalosis.3 In addition, normal rat kidney Received June 30, 2013. Accepted December 4, 2013. epithelial cells expressed monocyte chemotactic Published online ahead of print. Publication date available at protein-1 (MCP-1), the levels of which increased www.jasn.org. after exposure to CaOx monohydrate (COM) crys- Correspondence: Dr. Atsushi Okada, Department of Nephro- 4 5 tals and oxalate. Finally, several in vitro and in urology, Nagoya City University Graduate School of Medical vivo6 studies reported that urinary COM crystals Sciences, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467- couldbedegradedanddissolvedbyMws. These 8601, Japan. Email: [email protected] findingssuggestthatMwsinfluence crystal Copyright © 2014 by the American Society of Nephrology

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Figure 1. Morphologic and quantitative distribution of renal CaOx crystals. (A) Representative micrographs and three-dimensional imaging of renal sections harvested on day 6. In op/op mice, renal crystal deposits were detected inside the tubular lumen, predominantly at the papilla, using Pizzolato staining and polarized light optical microphotography. Pizzolato staining showed large crystals in the intratubular space of the renal papillary region in op/op mice. Crystallization throughout the kidney tissue is observed as white dots in the renal pa- renchyma with three-dimensional CT images. Original magnification, 340; 3400 in inset. Scanning EM showed that crystals in op/op mice were disordered and large compared with those crystals in the other groups of mice. Original magnification, 33000. Scale bar: 5 mm. CSF- 1–treated groups had the same intratubular crystal formation as the untreated groups, but the crystals were more disordered when assessed by scanning EM. (B) Qualitative analysis of crystals formed by the administration of glyoxylate using x-ray diffraction. X-ray diffraction analysis showed that the major component of renal crystals in op/op mice was COM, because multiple diffraction peaks characteristic of COM and no peaks characteristic of CaOx dihydrate or other components were detected. plaques in which various types of Mw migrate.11–14 In these Some reports suggested that colony-stimulating factor-1 studies, M2Mwsinfiltrated early atherosclerotic lesions, (CSF-1) signaling mediates tissue regeneration after injury whereas M1Mws were found predominantly in advanced le- and also, alters Mw polarization to the M2 phenotype.18–20 sions. Plaque formation correlated with the dominance of the In addition, several studies21–23 have used CSF-1–deficient M1 phenotype.15 Several reports suggested that M2Mws de- (op/op)mice,24 which contain a point mutation in CSF1 creased tissue inflammation and promoted tissue healing in and exhibit fewer circulating monocytes and tissue Mws GN16 and ischemia/reperfusion kidneys.17 than mice with CSF-1. Although these studies showed that

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Figure 2. Quantitative estimation of renal and urinary CaOx crystals. (A) The ratio of areas with renal crystal deposition. Crystallization in each kidney section was quantified by calculating the ratio (percent) of the area containing crystals to the entire kidney section using Image Pro Plus. (B) The number of urinary crystals per high-powered field; 24-hour urine samples on days 0 and 6 were collected in a metabolic cage, and CaOx crystals were counted at 3400 magnification. Day 6 (CSF-1) indicates that all mice in each genotype group were treated with 5.0 mgCSF-1.n=6 for all time points in each group. Data are presented as means6SEMs. *P,0.05 compared † between the +/+, +/op,andop/op groups at the same time points; P,0.05 compared with day 0 in each group; §P,0.05 compared with day 6 in each group.

loss of CSF-1 significantly reduces atherosclerotic severity, RESULTS they did not elucidate the role of Mw polarization in these outcomes. Morphologic and Quantitative Analysis of Renal and In this study, we examined and compared renal crystal Urinary CaOx Crystals formation and Mw polarization in wild-type and CSF-1– CaOx crystals were present predominantly in the renal tubular deficient mice after hyperoxaluric treatment. We showed lumenatthepapillainop/op mice and the renal tubular lumen at that CSF-1 deficiency results in increased renal crystal depo- the corticomedullary junction in both +/+ and +/op mice, con- sition and fewer M2-like Mws. We also found that the in vitro sistent with a previous study.25 Three-dimensional computed to- phagocytosis of COM crystals was higher in M2-polarized mography (CT) images of whole kidneys showed increased renal Mws from bone marrow-derived Mws (BMMs). Overall, this crystal deposits in op/op mice compared other groups (Figure 1A). study showed that CSF-1 suppresses renal crystal formation in Scanning electron microscopy (EM) revealed large disor- hyperoxaluric mice. dered crystals in op/op mice compared with radial ordered

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crystals in +/+ and +/op mice. Moreover, the crystals in op/op b,c a mice caused dilation of the renal tubular lumen. CSF-1–treated 0.22 0.37 0.00 4.92 0.14 1.54 0.33 9.15 1.70 6 6 6 6 6 6 6 6 mice had intratubular crystals comparable with those crystals 6 op/op

observed in the untreated groups, but the crystals were more 0.01 disordered by scanning EM. X-ray diffraction analysis showed 125.94

that the major component of renal crystals in op/op mice was groups with CSF-1 a a

COM (Figure 1B). /op 0.11 1.60 0.08 9.44 0.01 8.98 0.140 6.63 0.40 9.30 0.20 3.56 18.57 246.66 4.55 69.33 6 6 6 6 6 6 6 6 The number of renal crystal deposits on day 6 was 6 significantly higher in op/op mice than in +/+ or +/op mice. +/op 0.03 Day 6 (CSF-1)

CSF-1 treatment decreased the number of crystals in all 112.90 groups (Figure 2A). After treatment with glyoxylate, all groups exhibited CaOx crystalluria at day 6, which was mark- a a 0.19 1.98 0.01 0.07 9.23 5.67 0.10 6.60 0.28 7.58 0.27 2.93 9.15 221.04 edly higher in op/op mice. Importantly, CSF-1 treatment de- 7.59 61.32 6 6 6 6 6 6 6 6 6 creased CaOx crystalluria in op/op mice (Figure 2B). +/+ 108.92 Serum and Urinary Variables

Serum biochemistry and urinary variables are shown in a 0.24 2.00 0.01 0.04 6.94 0.17 9.02 0.12 6.40 0.40 8.06 0.51 2.54 11.88 209.93 Table 1. 1.77 66.03 cantly higher on day 6 in all groups (with or without CSF-1 treatment) 6 6 6 6 6 6 6 6 6 fi CSF-1–Deficient Mice Had Increased Expression of op/op Crystal Binding and Related Genes 132.28

Quantitative RT-PCR of whole-kidney tissue revealed a a marked increase in the expression of several genes from day 0.33 1.48 0.00 0.02 0.36 9.44 19.37 0.12 6.30 0.92 7.86 0.15 3.89 16.62 235.45 2.61 77.95 6 6 6 6 6 6 6 6 0 to 6 in all genotypes, including secreted phosphoprotein 1 6 groups and each group with CSF-1 treatment, except for +/+ and + +/op (Spp1; encoding osteopontin [OPN]), Cd44 (encoding CD44), Day 6

annexin A2 (Anxa2; encoding annexin II [ANX2)]), and che- 150.71 op/op mokine (CC motif) ligand 2 (Ccl2; encoding MCP-1). Their a expression was significantly higher in op/op mice than +/+ , and mice. Urinary oxalate levels were signi /op 0.28 1.71 0.00 0.01 7.32 0.09 9.23 0.11 6.22 0.46 8.22 0.31 3.00 19.13 238.93 and +/op mice (Figure 3A). On day 6, the expressions of 3.19 66.80 6 6 6 6 6 6 6 6 6 /op

Spp1, Cd44,andCcl2 increased in op/op mice and were 4-, +/+ 3-, and 5-fold higher than in the +/+ group, respectively. In 1.97 addition, the expressions of Spp1, Cd44, Anxa2,andCcl2 in 148.44 fi each group receiving CSF-1 were signi cantly lower than in b,c untreated mice. Nevertheless, the expressions of Spp1, Cd44, 0.00 0.01 6.93 0.12 9.35 0.14 6.45 0.58 9.15 0.09 0.20 2.88 16.90 245.26 5.62 71.48 6 6 6 6 6 6 6 6 6 and Ccl2 in op/op mice on day 6 (CSF-1) remained higher than mice than in +/+ and + in +/+ or +/op mice. op/op op/op

Strong immunohistochemical staining for OPN, CD44, cantly different among the +/+, + ANX2, and MCP-1 was observed on the luminal side of renal fi tubular epithelial cells, predominantly in the pericrystal region 0.00 0.01 2.03 52.33 0.32 10.25 0.12 6.75 1.42 9.13 0.41 0.70 0.58 3.22 17.49 248.98 (Figure 3B). The staining intensity of these proteins was ab- 3.86 77.99 6 6 6 6 6 6 6 6 6 cantly lower in Day 0 +/op rogated by CSF-1 treatment in all groups. fi

Glyoxylate Treatment Increased F4/80hi pan-Mw in All Mice SEMs. 6 0.00 0.01 0.10 9.47 0.12 6.70 0.72 9.53 0.29 1.80 0.23 2.94 21.20 281.03 3.45 63.98 The mRNA expression of EGF-like module-containing 4.70 68.30 6 6 6 6 6 6 6 6 6

– +/+ mucin-like hormone receptor like 1 (Emr1; encoding F4/80) mice. 0.01 9.10 9.10 w 1.46

and Cd68 (encoding CD68), which are pan-M markers, is /op shown in Figure 4A. Emr1 expression was lower in the +/op and op/op groups than in +/+ mice on day 0, but there was no significant difference between groups by day 6, irrespective of CSF-1 treatment. There was no significant difference in Cd68 expression between any groups of mice. Serum and urinary biochemistry mol/g per Cr) 55.57 mol/g per Cr) 58.80 mol/g per Cr) 2.35

fi m m mol/g per Cr) 275.67 Immunohistochemically, F4/80-positive cells in ltrated the m Genotypes m 0.05 compared with the +/+ mice. 0.05 compared with day 0 in each group. fi 0.05 compared with the +

renal interstitium in all six groups; no signi cant differences Treatment Days Cr (mg/dl) Ca (mg/dl) pH 6.7 P (mg/dl) Ca ( Volume (ml) P( Mg ( Ox ( , , , P P P Serum Table 1. Serum calcium, creatinine (Cr), and phosphorus levels were not signi a b c treatment on day 6. Urinary volumes onthan day 0 on were day signi 0. Data are given as means were identified between groups (Figure 4B). Urine

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Figure 3. Evaluation of crystal binding and related gene expressions. (A) The expression of each gene indicated was determined by quantitative RT-PCR using TaqMan assays. Control values represent the average of the data for the +/+ mice on day 0. The data are presented as means6SEMs. Day 6 (CSF-1) indicates that all mice in each genotype group were treated with 5.0 mgCSF-1.n=6 for each group. *P,0.05 † compared between the +/+, +/op,andop/op groups at the same time points; P,0.05 compared with day 0 in each group; §P,0.05 compared with day 6 in each group. (B) Immunohistochemical distribution of the expression of crystal binding and binding-related molecules in mouse kidneys harvested at day 6. Pizzolato staining indicated CaOx crystal deposits (arrows). Original magnification, 3400.

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Figure 4. Evaluation of pan-Mw–related gene expression. (A) The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the averages of the data for the +/+ mice on day 0. The data are presented as means6SEMs. Day 6 (CSF-1) indicates that all mice in each genotype group were administered 5.0 mgCSF-1.n=6 for each group. *P,0.05 compared † between the +/+, +/op,andop/op groups at the same time points; P,0.05 compared with day 0 in each group. (B) Immunohis- tochemical distribution of F4/80 expression in mouse kidneys harvested on day 6. Original magnification, 3400.

CSF-1 Deficiency Correlated with Insufficient chitinase 3–like 3 (Chi3l3; encoding Ym-1) were significantly Expression of M2- but Sufficient Expression of lower in op/op than +/+ mice (Figure 6A). The expressions of M1-Related Genes Cd163 in +/+ mice and Mrc1 and arginase 1 (Arg1)in+/+ and The expressions of integrin-aX(Itgax; encoding CD11c), in- +/op mice were increased markedly from day 0 to 6. In op/op ducible nitric oxide synthase 2 (Nos2; encoding iNOS), Tnf mice, the expressions of Cd163, Mrc1, Arg1,andChi3l3 on day (encoding TNFa), and Il6 (encoding IL-6) at day 0 were com- 6 were significantly lower than in +/+ mice. All genes were parable between the three genotypes. Expressions of Nos2 in increased by CSF-1 treatment. the +/+ and +/op groups and Itgax and Il6 in +/op and op/op Fluorescence double immunohistochemical staining re- mice increased significantly from day 0 to 6. The expression of vealed CD163 and CD206 double positive Mwsintheinter- Tnf in all genotypes increased from day 0 to 6. The initial stitial cells of the corticomedullary junction in +/+ and +/op expressions of Nos2, Tnf,andIl6 in all CSF-1–treated groups mice; however, neither protein was expressed in op/op mice were decreased significantly compared with the respective on day 0 or 6 (Figure 6B). The expressions of CD163 and groups on day 6. After CSF-1 treatment, the expressions of CD206 were detected in the interstitial space of the cortico- Itgax, Tnf,andIl6 were higher in the op/op group than the +/+ medullary junction in op/op mice after CSF-1 treatment. group (Figure 5A). iNOS-positive Mws were detected in the interstitial space of CSF-1 Deficiency Significantly Reduced the M2-Like the corticomedullary junction in all six groups (Figure 5B). Population The expression of iNOS was higher on day 6 than day 0 in all To quantify M1/M2-polarization Mws, we measured their sur- three genotypes, and it was decreased by CSF-1 treatment. face markers (M1, CD11c and Ly6C; M2, CD163 and CD206) iNOS expression was higher in op/op mice than +/+ and by flow cytometry. Approximately 70% of renal interstitial +/op mice after CSF-1 treatment (Figure 5C). Mws were CD11b+F4/80+ (Figure 7A). This population was On day 0, the expressions of Cd163 (encoding CD163), then separated into six populations based on differences in the mannose receptor C type 1 (Mrc1; encoding CD206), and expression of CD11c/Ly6C (Figure 7B, upper panel) and

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Figure 5. Evaluation of M1-like Mw-related genes. (A) The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the average of the data for the +/+ mice on day 0. The data are presented as means6SEMs. Day 6 (CSF-1) indicates that all mice in each genotype group were administered 5.0 mg CSF-1. n=6 for each group. *P,0.05 compared † between the +/+, +/op,andop/op groups at the same time points; P,0.05 compared with day 0 in each group; §P,0.05 compared

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CD163/CD206 (Figure 7B, lower panel). There were no sig- number of M1-like Mws, whereas M2-like Mws increased in nificant differences in M1-like Mws expressing CD11b+F4/80+ op/op mice after M2Mw transfusion (Figure 9D). CD11cintLy6Chi between +/+ and op/op mice, except at day 0, and CSF-1 treatment did not change the characteristics of CSF-1 Deficiency Resulted in Severely Disordered those populations (Figure 7, C and D). There were markedly Stone- and M2-Related Related Gene Expressions in fewer CD11b+F4/80+CD163+CD206hi cells, representing M2- Renal Mws like Mws (Figure 7, C and E), in op/op than +/+ mice on days Finally, we characterized the gene expression profiles of renal 0 and 6. These M2-like Mws were increased after CSF-1 treat- Mws isolated from +/+ and op/op mice using DNA microarrays. ment in the op/op group. As shown in Figure 10A, renal Mws from +/+ and op/op mice were distinguished by the hybridization signals of 925 probes M2Mws Phagocytized COM Crystals In Vitro corresponding to M1- and M2-abundant genes (Figure 10A, BMM-derived M1Mws (Figure 8A) and M2Mws (Figure 8B) Supplemental Table 2). Moreover, Mwsinop/op mice treated were detected by flow cytometry with staining for F4/80+Ly6C+ with glyoxylate for 6 days exhibited reduced expression of and F4/80+CD206+, respectively. BMM-derived M2Mwswere stone- and M2-related genes compared with Mwsin+/+ differentiated using IL-4 and CSF-1, which showed that the M2a mice. In contrast, there was negligible difference in the expres- subtype was CD163hiCD206hiArginasehiPparghiIL-10low (Figure sions of M1-related genes, except for Il6 and Tnf (Figure 10B). 8D). M2Mws had a higher phagocytic rate of COM crystals than RTCs or M1Mws at each time point. The phagocytic ability of M2Mws increased gradually with COM exposure. DISCUSSION The expressions of Spp1, Cd44, Arg1, and Chi3l3 were sig- nificantly higher in M2MwsthanRTCsandM1Mws(Figure Previous studies of the mechanism of kidney stone formation 8D). Spp1 and Cd44 levels decreased in M2Mws but increased using hyperoxaluric animal models showed intratubular in M1Mws over time. The expressions of Nos2, Il6,andIl10 crystals at the corticomedullary region.26,27 Two major path- were significantly lower in M2Mws compared with M1Mws ways lead to kidney stone formation.28 One pathway involves and decreased after 24 hours of COM exposure. In M1Mws, overgrowth on interstitial apatite plaques (Randall’s plaques), the expression levels of Nos2 at 6 and 24 hours, Tnf from 1 to which was observed in idiopathic CaOx stone formers.28–30 24 hours, and Il6 at 6 hours were higher than at 0 hours. The other pathway involves intratubular crystal plugs in the Neutralization of crystal binding molecules showed that sup- duct of Bellini, which was observed with all other stone for- pressing CD44 increased COM phagocytosis by M1 and mers.28,31 Several studies revealed that the primary causative M2Mw significantly, whereas suppressing MCP-1 increased factors for crystal formation were increased supersaturation of phagocytic M2Mw. Neutralizing OPN did not affect COM the tubular fluid and attachment of crystals to undifferentiated phagocytosis by these Mws (Figure 8E). tubular epithelial cells by crystal binding molecules, such as OPN, CD44, and ANX2.32–35 In this study, CSF-1–deficient M2Mw Transfusion Suppressed Renal Crystal mice retained large crystals in the tubular lumen at the papilla, Deposition and the Expression of Crystal Binding similar to human nephrolithiasis with hyperoxaluric status.28 Molecules Serum biochemistry showed no statistical differences Adoptive transfusion of BMM-derived M2Mwsdecreasedrenal between the three genotypes. On day 0, the urinary volumes crystal deposition in +/op and op/op mice on day 6, whereas were significantly lower in op/op mice than in the other two there were no differences in the number of urinary crystals be- groups. However, there were no differences in urinary volumes tween genotypes (Figure 9B). The renal crystals in each genotype on days 0 and 6, urinary biochemistries with both concen- were intratubular CaOx crystals from the corticomedullary trations (Supplemental Table 1), and 24-hour urine excre- junction to the papilla (Figure 9A). The expressions of Spp1, tion between the +/+ and op/op mice. Therefore, it remains Cd44,andCcl2 in whole-kidney tissue of op/op mice after unclear how differences in urinary volume affect crystal for- M2Mw transfusion were lower than in untreated mice (Figure mation. There were no other differences in Ca, P, Mg, and 9C). Flow cytometric analyses revealed no differences in the oxalate excretion on day 6, irrespective of CSF-1 treatment;

with day 6 in each group. (B) Fluorescence immunohistochemical distribution of iNOS expression in mouse kidneys harvested on days 0 and 6. In the interstitial space of the corticomedullary junction, iNOS-positive Mws (white arrows) were detected in all six groups. Blue, nucleus; green, iNOS. Original magnification, 3400; 3800 in inset. (C) Western blotting of mouse kidneys for iNOS protein expression. The molecular mass of iNOS was confirmed by bands at 135 kD. Control values are the average of the data for the +/+ mice on day 0. The data are presented as means6SEMs. n=3 for each group. *P,0.05 compared between the +/+, +/op,andop/op groups at the same time † points; P,0.05 compared with day 0 in each group; §P,0.05 compared with day 6 in each group.

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Figure 6. Evaluation of M2-like Mw-related genes. (A) The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the average of the data for the +/+ mice on day 0. The data represent mean6SEM values. Day 6 (CSF-1) indicates that every mouse in each genotype group was administered 5.0 mg CSF-1. n=6 for each group. *P,0.05 compared † between the +/+, +/op,andop/op groups at the same time points; P,0.05 compared with day 0 in each group; §P,0.05 compared

1688 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1680–1697, 2014 www.jasn.org BASIC RESEARCH therefore, CSF-1 treatment may not affect these minerals or of these genes in the kidney. According to flow cytometry oxalate metabolism. analysis, there were no differences in M1-like Mw expression Oxalate exposure promotes the adherence of crystals to between the +/+ and op/op groups, suggesting that CSF-1 de- renal epithelial cells and differentially modulates the genes ficiency did not contribute to a shift to M1-type renal Mws. required for molecular functions, biologic pathways, and However, the increased expression of these genes from day 0 to cellular components, particularly by suppressing genes.36,37 6 and the decreased expression of Nos2, Tnf,andII6 after CSF- Therefore, we investigated changes in the expression of genes 1 treatment suggest that hyperoxaluria promotes the expres- encoding molecules related to crystal binding. OPN is an in- sion of inflammatory M1-related genes, which was suppressed flammatory chemokine and major component of the stone by CSF-1 treatment. Our data provide no evidence of a corre- matrix,38,39 and it plays a key role in CaOx stone forma- lation between M1-like Mws and the promotion of renal crys- tion.40,41 The CD44 antigen acts as a receptor for hyaluronic tal formation. acid and OPN, and it is related to the phagocytic function of In contrast, there were significantly fewer renal M2-like Mws42 and OPN-induced Mw accumulation.43 It also plays a Mws (CD11b+F4/80+CD163+CD206hi)intheop/op group role in Mw migration during kidney stone formation.2 MCP-1 than the +/+ group. In +/+ mice, the M2-like Mw population promotes the migration and aggregation of monocytes and increased after glyoxylate treatment, which was enhanced by Mwstoinflammatory regions of injured tissues.3 Exposure CSF-1. These results suggest that M2-like Mws were recruited to COM crystals increased MCP-1 expression in RTCs.4 Spp1, through a CSF-1–dependent pathway in the hyperoxaluric Cd44, Anxa2,andCcl2 expression and crystal deposition were kidney. Our finding that the expression of M2-related genes, higher in op/op mice than in other genotypes. Immunohisto- such as Arg1 and Chi3l3, was lower in op/op than +/+ mice but chemical staining revealed OPN, CD44, ANX2, and MCP-1 increased by CSF-1 treatment strengthens this hypothesis in expression in tubular epithelial cells in both pericrystal re- terms of renal crystal formation. Because a significant decrease gions and other lumens. In addition, the expression of these in M2Mw levels in op/op mice resulted in an increase in both genes was decreased significantly at both the mRNA and pro- renal crystal deposition and urinary crystal excretion, M2Mws tein levels after CSF-1 treatment. Thus, CSF-1 deficiency is could reduce renal crystal deposition by reducing crystal for- associated with the promotion of crystal formation by in- mation or crystal retention and deposition in the kidney. creased expression of crystal binding and related molecules. To identify the specificroleofM2Mws, we assessed the in There were no differences between the three genotypes in vitro crystal phagocytic ability of BMM-derived M1/M2Mws the resident pan-Mws F4/80 and CD68 during hyperoxaluric and the direct effect of M2Mw transfusion on crystal forma- statement. Therefore, renal resident pan-Mws were induced tion in vivo.M2Mwshadasignificantly greater ability to simultaneously in the kidney during crystal formation, and phagocytize COM crystals than RTCs and M1Mws, and this induction was independent of CSF-1 function. These re- transfusion of M2Mws suppressed renal crystal deposition. sults suggest that CSF-1 deficiency induced inflammation in- These results suggest that M2Mwsdirectlysuppressrenal dependent of the undifferentiated immune cell function. crystal formation by phagocytizing crystals. In addition, Membrane surface markers, such as CD11c, identify Spp1 and Cd44 expressions were higher in M2Mws at 0 hours M1Mws. They also produce chemokines and cytokines, such in the in vitro study, and microarray analysis revealed in- as iNOS, TNFa, IL-6, and MCP-1. In contrast, M2Mws express creased Spp1 and Cd44 expressions in Mws from +/+ com- the scavenger receptor CD163, mannose receptor CD206, and pared with op/op mice. Neutralization of CD44 and MCP-1 Ym-1 and produce chemokines and cytokines, including increased the COM phagocytic ability of M2Mws. Because arginase-1, TGF-b, and IL-10.44–47 M2Mws promote cell pro- both MCP-1 and CD44 are related to tissue inflammation liferation, reduce apoptosis, stimulate angiogenesis,44 facili- and inflammatory-related M w migration,51,52 inhibiting tate muscle injury repair,48 and regenerate the growth re- these molecules could reduce renal crystal formation by sponse of sensory axons after spinal cord injury.49 They also increasing M2 phagocytic activity. Interestingly, renal Mws reduce glomerular cell injury and proteinuria through the an- sorted from op/op mice expressed lower levels of stone- and giotensin type 1 receptor,50 and they affect the resolution of M2-related genes based on microarray analysis. Some genes ischemia-reperfusion injury.17,19,20 listed in Figure 10A (such as Aqp1, Aurka,andSlc34a1)in- Because the expression of M1-related genes and iNOS in the hibit the development of renal stones,53,54 suggesting that renal interstitial space on day 0 was comparable between the CSF-1 deficiency resulted in severely impaired defense three genotypes, CSF-1 deficiency did not affect the expression against crystal formation in Mws.

with day 6 in each group. (B) Fluorescence immunohistochemical distribution of CD163 and CD206 expression in mouse kidneys harvested on days 0 and 6. CD163 and CD206 double positive Mws are shown in the interstitial cells of the corticomedullary junction in the +/+ and +/op groups; however, neither protein was expressed in the op/op group on day 0 or 6. The expression of CD163 and CD206 was in- creased by CSF-1 treatment. Blue, nucleus; green, CD206; red, CD163. Original magnification, 3400; 3800ininset.

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Figure 7. Flow cytometric analysis of renal Mws. (A) Gating strategy for CD11b+F4/80+ of renal mononuclear cells. The population of F4/80hiCD11b+ cells was determined from the representative graph. Isotype controls of both F4/80 and CD11b proteins are shown as red dot plots (upper panel). Back gates for live cells and CD45 are also shown. Most F4/80hiCD11b+ cells were CD45-positive and 7AAD-negative (lower left panel). The dotted line shows the isotype control of CD45 protein (lower right panel). (B) The CD11cintLy6Chi (upper panel) and CD163+CD206hi (lower panel) subpopulations of CD11b+F4/80+ cells are shown. Isotype controls for CD11c and Ly6C (upper panel) and CD163 and CD206 (lower panel) are shown on each graph as red dotted lines. (C) Quantification of CD11b+F4/ 80+CD11cintLy6Chi (upper left panel) and CD11b+F4/80+CD163+CD206hi Mws (lower left panel). There were no significant differences between +/+ and op/op mice except on day 0, and treatment with CSF-1 did not change the characteristics of those populations; op/ op mice had a significantly lower number of CD11b+F4/80+CD163+CD206hi Mws than +/+ mice on days 0 and 6. The number of CD11b+F4/80+CD163+CD206hi Mws was increased after CSF-1 treatment in op/op mice. The median fluorescent intensities (MFIs) of Ly6C, CD11c, CD206, and CD163 in CD11b+F4/80+ cells are shown in right panel; op/op had lower intensities of CD206 and CD163 staining than +/+ mice, but CSF-1 treatment increased CD206 intensity. The data represent mean6SEM values. Day 6 (CSF-1) indicates that all mice in each genotype group were treated with 5.0 mg CSF-1. n=4 for each group. *P,0.05 compared with the +/+ group; † P,0.05 compared with day 0 in each group; §P,0.05 compared with day 6 in each group. (D) M1-like Mw expression. Representative

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In conclusion, this study provides compelling evidence that recombinant human CSF-1, with concomitant glyoxylate adminis- CSF-1–deficient mice had markedly increased renal crystal de- trationdailyfor6daysasdescribedpreviously.19,56 Recombinant position and that the reintroduction of CD11b+F4/80+CD163+ human CSF-1 was purchased from Miltenyi Biotec, Inc. (Auburn, CD206hi M2-like Mws by CSF-1 treatment reduced renal crystal CA). deposition and CaOx crystal formation. We also showed that M2Mw transfusion directly suppressed crystal formation and Induction of M1Mws and M2Mws from Bone Marrow the marked crystal phagocytic ability of M2Mws. This study is Cell Cultures the first report showing that CSF-1 signaling suppresses renal BMMs grown in lymphocytes and 10% L-conditioned medium were crystal formation by inducing M2Mws. generated as described previously.57 On day 7, adherent cells were harvested and seeded at a density of 1.03105 cells/cm2. For priming experiments, BMMs were stimulated for 20 hours with 100 ng/ml CONCISE METHODS LPS (Sigma-Aldrich) and 20 ng/ml GM-CSF (R&D Systems) for M1Mws or 50 ng/ml IL-4 (R&D Systems) and 10 ng/ml M-CSF Animal Procedures and Genotyping (Miltenyi Biotec, Inc.) for M2Mws.58,59 All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Transfusion of In Vitro–Derived M2Mws Animals, conformed to the Regulations for Animal Experimentation After the culture and differentiation of M2Mws from BMMs, the at Nagoya City University, and were reviewed by the Institutional Lab- M2Mws were counted, and 1.03106 viable cells were suspended in oratory Animal Care and Use Committee of Nagoya City University. 0.2 ml serum-free medium. These cells were then transfused by a A breeding pair of heterozygous B6C3Fe-a/a-Csf1op mice (+/op) single tail vein injection 1 day before glyoxylate administration as was purchased from Charles River Japan Inc. (Yokohama, Japan). described previously.60,61 Heterozygous (+/op) mice were mated to yield mice with a homoge- neous genetic background. The mice were fed standard chow (con- Evaluation of COM Crystal Internalization by RTCs, taining 1.01/100 g calcium, 0.78/100 g phosphorus, and 0.21/100 g M1Mws, and M2Mws magnesium; Oriental Yeast Co., Tokyo, Japan) and had free access to Fluorescence-labeled COM crystals were prepared as described in water. CSF-1–deficient mice were fed a powdered diet, because they detail previously.62 The COM crystals were made from a mixture of 5 lack incisors. The genotypes of the progeny from the heterozygous mM CaCl2z2H2O and 0.5 mM Na2C2O4 in buffer containing 90 mM crosses were determined using TaqMan genotyping assays. Genomic TriszHCl and 10 mM NaCl. Briefly, the COM crystals were crystallized DNA samples were extracted from tail tissue using custom designed as described earlier but in the presence of 0.58 mg/ml rabbit anti- TaqMan probe sets (Applied Biosystems, Foster City, CA). mouse IgG conjugated with Alexa Fluor 488 (Life Technologies, We divided the 8-week-old male mice into three groups according Carlsbad, CA). to their genotypes: wild type (+/+), heterozygous (+/op), and homo- Weused murine renal tubular epithelial cells (M-1; American Type zygous (op/op). Based on our previous report,25 the mice were given Culture Collection, Manassas, VA) and M1Mws and M2Mwsthathad daily intra-abdominal injections of 80 mg/kg glyoxylate; then, the been induced from BMMs as described above. Cells of each type kidneys were extracted, and blood and 24-hour urine samples were (1.03105 cells/cm2) were grown in six-well culture dishes (Nagle collected on days 0 and 6 (n=6 per group) to examine crystal formation. Nunc, Naperville, IL). The cells were maintained in a humidified

Blood biochemistry and urinary calcium, phosphorus, creatinine, and incubator at 37°C with 5% CO2 for 24 hours and then treated with magnesium analyses were conducted by Mitsubishi Chemical Medi- 25 mg/cm2 fluorescence-labeled COM crystals. Each group of cul- cine (Tokyo, Japan). Urinary pH and volumes were measured manu- tured cells was harvested after 0, 1, 6, and 24 hours of COM treatment ally. Urinary oxalate concentrations were analyzed using FOM-110A and used for flow cytometry and RT-PCR. (Hokuto Denko Co., Tokyo, Japan).55 The urine was centrifuged at For the neutralization of crystal binding molecules, 2 mg/ml OPN 15003g for 15 minutes, and 100-ml sediment samples were observed antibody (R&D Systems),63 2 mg/ml MCP-1 antibody (R&D Sys- at 3400 magnification using light optical microphotography (AX80; tems),64 and 10 mg/ml CD44 antibody (Calbiochem)65 were added Olympus, Tokyo, Japan) to detect and quantify the number of CaOx to dishes of M1Mws and M2Mws 6 hours before COM treatment. crystals. Enrichment of Renal Mononuclear Cells by Magnetic Supplementation with Recombinant Human CSF-1 Sorting To investigate the role of CSF-1 in kidney crystal formation, mice of We prepared single cell suspensions from mouse kidneys by digestion each genotype were injected subcutaneously with 5 mgpurified with 500 mg/ml collagenase II and 10 ng/ml DNase I (Sigma-Aldrich).

FACS histograms of either CD11c or Ly6C in CD11b+F4/80+ cells are shown. Dotted lines show isotype controls of CD11c and Ly6C proteins. (E) M2-like Mw expression. Representative FACS histograms of either CD206 or CD163 in CD11b+F4/80+ cells are shown. Dotted lines show isotype controls of CD206 and CD163 proteins.

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Figure 8. Analysis of the ability of M1- and M2Mws to phagocytize COM crystals in vitro. (A) Schematic representation using flow cytometry. Detection of M1Mws (left panel) and FITC-labeled COMs phagocytized by M1Mws (right panel). (B) Detection of M2Mws (left panel) and FITC-labeled COMs phagocytized by M2Mws (right panel). (C) Quantitative analysis of phagocytic Mws. The ratios of COM phagocytic Mws were evaluated by flow cytometry according to the strategy described above. M2Mws had a higher rate of

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CD11b+ and/or CD11c+ cells in the kidney single cell suspensions the op/op genotype was frozen, crushed, and placed in a sample 2 2 were then enriched using mouse CD11b or CD11c microbeads and holder, and qualitative and quantitative analyses were performed auto-MACS (Miltenyi Biotec, Inc.) using the manufacturer’sprotocol. with SmartLab (Rigaku, Tokyo, Japan).

Flow Cytometry Immunohistochemical Staining Single cell suspensions of CD11b and/or CD11c cells from mouse OPN, CD44, ANX2, MCP-1, and F4/80 were immunohistochemically kidneys and BMM-derived M1/M2Mws were stained with V450-labeled stained on 4-mm-thick renal cross-sections as described previously.2 rat anti-mouse Ly-6C (BD Horizon), V510-labeled anti-mouse CD45 Anti-mouse OPN rabbit polyclonal antibody (IBL Co. Ltd., Gunma, (BioLegend, San Diego, CA), FITC-labeled rat anti-mouse CD11b (BD Japan), anti-mouse CD44 rat monoclonal antibody (BioLegend), Pharmingen), PE-labeled hamster anti-mouse CD11c (BD Pharmin- anti-mouse ANX2 rabbit polyclonal antibody (Abcam, Inc., Cambridge, gen), CD163 (M-96; Santa Cruz Biotechnology), goat anti-rabbit IgG, MA), anti-mouse MCP-1 rat monoclonal antibody (Monosan, Uden, F(ab9)2-PE-Cy7 (Santa Cruz Biotechnology), APC-labeled anti- The Netherlands), and anti-mouse F4/80 rat monoclonal antibody mouse CD206 (MMR; BioLegend), and APC/Cy7-labeled anti-mouse (Abcam) were used as primary antibodies. The reacted antibodies F4/80 antibodies (BioLegend). Renal mononuclear (1.0–8.03105) were detected using a Histofine Simple Stain Kit for rat or rabbit and cultured M1/M2Mw (1.0–5.03105) cells were harvested using a IgG (Nichirei Biosciences, Inc., Tokyo, Japan), according to the man- FACS CantoII (Becton Dickinson, San Jose, CA), and data were ana- ufacturer’s instructions. lyzed using FlowJo software 7.6.5 (Tree Star, Palo Alto, CA). The Mw subtypes were detected by immunohistochemical stain- ing for iNOS, CD163, and CD206 using the color modification Detection of Kidney CaOx Crystals method. Slides were incubated for 1 hour at 37°C with anti-mouse Crystal formation in the extracted mouse kidneys was examined using iNOS rabbit polyclonal antibody (Thermo Fisher Scientific, Chesh- Pizzolato staining as described previously.66 To confirm that the pos- ire, UK), anti-mouse CD163 rabbit polyclonal antibody (Santa Cruz itively stained materials were crystals, nonstained sections were Biotechnology), or anti-mouse CD206 rat monoclonal antibody observed using polarized light optical microphotography (AX80; (AbD Serotec, Oxford, UK). Anti-rat IgG (Alexa Fluor 594; Life Tech- Olympus, Tokyo, Japan). We also observed crystal deposits in renal nologies) and anti-rabbit IgG (Alexa Fluor 488; Life Technologies) parenchyma with three-dimensional imaging using an in vivo x-ray secondary antibodies were added and incubated for 30 minutes at mCT system (R_mCT2; Rigaku, Tokyo, Japan) to determine spatial 37°C. Nuclei were counterstained with 49,6-diamidino-2-phenylin- structure. Crystal formation, calculated as the percent area of CaOx dole (Dojindo Laboratories, Kumamoto, Japan) solution for 10 crystal deposition per kidney section, was assessed quantitatively us- minutes at 37°C. After washing several times in reverse osmosis water, ing Image Pro Plus (Media Cybernetics, Inc., Bethesda, MD). the slides were mounted with water-soluble mounting medium Scanning EM was performed to observe the superstructure of (Cosmo Bio Co. Ltd., Tokyo, Japan) and examined by confocal laser generated crystals. The paraffin-embedded sections were dewaxed and scanning microscopy (Pascal, Osaka, Japan). washedwithphosphoricacidbuffer.Then,sectionswererefixed with 2.5% glutaraldehyde first and then, 2% osmium tetroxide. Dehydration was Quantitative RT-PCR performed using a 50%–100% ethanol series. The samples were embed- For quantitative RT-PCR, total RNAwas extracted from kidney tissues ded in epoxy resin, coated with platinum, and then photographed with a using an RNeasy Midi Kit (Qiagen, Hilden, Germany). All RNA scanning electron microscope (S-4800; Hitachi, Tokyo, Japan). samples were reverse-transcribed into cDNA using a high-capacity cDNA Reverse Transcription Kit (Applied Biosystems). To assess the X-Ray Diffraction of Crystals expression of genes encoding crystal binding and related molecules, The components of inorganic calcification were determined with the annotation information for Spp1 (Mm00436767_m1), Cd44 x-ray diffraction. One half of the section of the extracted kidney of (Mm01277163_m1), Anxa2 (Mm01150673_m1), and Ccl2

phagocytosis for COM crystals than RTCs and M1Mw. These relative cell numbers of phagocytic Mws were increased time-dependently; 0, 1, 6, and 24 hours indicate the time elapsed after COM treatment in each cell type. The data represent mean6SEM values. n=4 for each † group. *P,0.05 compared between the RTC, M1Mw, and M2Mw groups; P,0.05 compared with 0 hours in each group. (D) The ex- pression of each gene was determined by quantitative RT-PCR using TaqMan assays. Black bar, RTCs; slashed bar, M1Mws; dotted bar, M2Mws. Control values are the average of the data for the M1Mw group at 1 hour; 0, 1, 6, and 24 hours indicate the time elapsed after COM treatment in the RTC, M1Mw, and M2Mw groups. The data represent mean6SEM values. n=4 for each group. *P,0.05 compared be- † tween the RTC, M1Mw, and M2Mw groups; P,0.05 compared with 0 hours in each group. (E) Quantitative analysis of phagocytic M1Mws and M2Mws after neutralization with crystal binding molecules. The ratios of COM phagocytic Mws were evaluated by flow cytometry according to the strategy described above. Cells were incubated with COM for 6 hours in all groups. In both M1 and M2Mw groups, CD44 suppression increased the relative number of phagocytic cells. MCP-1 neutralization also increased the relative number of COM phagocytic M2Mws. There were no differences in the relative number of phagocytic M1- or M2Mws after OPN neutralization. OPN-Ab, MCP-1-Ab, and CD44-Ab indicate groups that were treated with each respective neutralization antibody. The data represent mean6SEM values. n=4 for each group. *P,0.05 compared between four groups.

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Figure 9. Evaluation of the effect of M2Mw transfusion on crystal formation. In each graph, the data from day 6 experiments of each genotype (described previously) were used as the control to compare the differences between groups treated with M2Mw and those groups not treated. Day 6 (M2Mw) indicates that every mouse in each genotype group was transfused with 1.03106 cells of M2Mws. Data represent means6SEMs. (A) Representative micrographs of renal sections harvested on day 6 from M2Mw-transfused mice. Each

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which was used as the internal control. The cor- rected expression of each gene was normalized to the average value on day 0 (+/+ group) for the in vivo study and at 0 hours (M1Mw group) for the in vitro study.

Western Blotting Whole-protein extracts prepared by sonication were separated on 12.5% SDS polyacrylamide gels and transferred to Immobilon-H polyviny- lidene difluoride membranes (EMD Millipore). After blocking with Tris-buffered saline (pH 7.5) –Tween 20 containing 5% skimmed milk, the membranes were incubated with anti-mouse iNOS antibody (Thermo Fisher Scientific) fol- lowed by horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). Chemiluminescent signals were visualized using enhanced chemiluminescence Western Figure 10. Microarray analysis of renal Mws sorted from +/+ and op/op mice and M1- blotting detection reagents and scanned using a and M2Mws derived from mouse bone marrow cells. M1Mws (M1) and M2Mws(M2) were prepared from wild-type mouse bone marrow. Renal Mwsweresortedfrom LAS4000 analyzer (GE Healthcare). glyoxylate-treated op/op mice (op/op) or wild-type littermates (+/+) after 6 days of treatment. (A) Cluster analysis of renal Mw samples obtained from +/+ and op/op mice using the expression pattern of M1- and M2-abundant genes. For the clustering Microarray Analyses fi analysis, we used 925 probes that gave significantly different (P,0.01 and more than cDNAs ampli ed using the Ovation Pico Sys- 4-fold) signals between the +/+ and op/op mice. (B) Heat map representation of the tem (Nugen, San Carlos, CA) were subjected to expression of renal stone- (left panel), M1- (center panel), and M2-related (right panel) transcriptome analysis using Agilent SurePrint genes. G3 microarrays. Microarray data were ana- lyzed using the GeneSpring GX 12.1 program (Agilent Technologies, Santa Clara, CA). (Mm00441242_m1) were used to generate a TaqMan Gene Expres- Changesingeneexpressionthatweremorethan3-foldbetween sion Assay product and a TaqMan MGB probe (6-carboxyfluorescein groups were deemed to be significantly different (P,0.01). All dye-labeled) for each complete mRNA sequence. For the detection of microarray data were deposited inGeneExpressionOmnibus(ac- Mw-related genes, we used probe sets for Emr1 (Mm00802529_m1), cession number GSE51466). Cd68 (Mm03047340_m1), Itgax (Mm00498698_m1), Nos2 (Mm00440502_m1), Tnf (Mm00443258_m1), Il6 (Mm00446190_m1), Statistical Analyses Cd163 (Mm00474091_m1), Mrc1 (Mm00485148_m1), Arg1 All data are expressed as means6SEMs. Statistical analyses were (Mm00475988_m1), Chi3l3 (Mm04213363_u1), and Il10 performed by two-way repeated measures ANOVA (comparisons (Mm00439614_m1). Quantitative PCR was performed using a TaqMan among three or more groups) or the Mann–Whitney U test (com- FAST Universal PCR Master Mix (4352042; Applied Biosystems) with a parisons between two groups) using SAS, version 9.1 (SAS Institute, 7500 FAST Real-Time PCR System (Applied Biosystems).23 The ex- Inc., Cary, NC). Values of P,0.05 were considered to be statisti- pression of each gene was normalized to the expression of b-actin, cally significant.

genotype had the same intratubular CaOx crystals from the corticomedullary junction to papilla. n=5 for each group. Original magnifi- cation, 340; 3400 in inset. (B) Quantitative estimation of renal and urinary CaOx crystals. (Left panel) The ratio of the area containing renal crystal deposition. Crystallization in each kidney section was quantified by calculating the ratio (percent) of the area containing crystals to the entire kidney section using Image Pro Plus. (Right panel) The number of urinary crystals per high-powered field; 24-hour urine samples were collected in a metabolic cage, and CaOx crystals were counted at 3400 magnification. n=5 for each group. (C) Evaluation of crystal binding and related gene expressions. The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the average of the data for the +/+ mice on day 0. The data are presented as means6SEMs. n=5 for each group. (D) Flow cytometric analysis of renal Mws. The numbers of CD11b+F4/80+CD11cintLy6Chi (left panel) and CD11b+F4/80+CD163+CD206hi (right panel) Mwswerecalculatedbyflow cytometry. The gating strategy was the same as the strategy used in Figure 7 (described above). n=4 for each group. *P,0.05 compared with the +/+ group.

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ACKNOWLEDGMENTS 14. Reiner AP, Kahn A, Eisner BH, Pletcher MJ, Sadetsky N, Williams OD, Polak JF, Jacobs DR Jr., Stoller ML: Kidney stones and subclinical atherosclerosis in young adults: The CARDIA study. JUrol185: 920–925, 2011 This work was supported in part by Grants-in-Aid for Scientific 15. Khallou-Laschet J, Varthaman A, Fornasa G, Compain C, Gaston AT, Research 23249074, 23592374, 23592375, 23791770, 23791774, Clement M, Dussiot M, Levillain O, Graff-Dubois S, Nicoletti A, Caligiuri 22791481, and 21791517 from the Ministry of Education, Culture, G: Macrophage plasticity in experimental atherosclerosis. PLoS One 5: Sports, Science, and Technology and the Ninth Research Grant of e8852, 2010 Asahi Kasei Pharma Urological Academy. 16. Fujita E, Shimizu A, Masuda Y, Kuwahara N, Arai T, Nagasaka S, Aki K, Mii A, Natori Y, Iino Y, Katayama Y, Fukuda Y: Statin attenuates ex- perimental anti-glomerular basement membrane glomerulonephritis together with the augmentation of alternatively activated macro- DISCLOSURES phages. Am J Pathol 177: 1143–1154, 2010 None. 17. Lee S, Huen S, Nishio H, Nishio S, Lee HK, Choi BS, Ruhrberg C, Cantley LG: Distinct macrophage phenotypes contribute to kidney injury and repair. J Am Soc Nephrol 22: 317–326, 2011 18. Zhang MZ, Yao B, Yang S, Jiang L, Wang S, Fan X, Yin H, Wong K, REFERENCES Miyazawa T, Chen J, Chang I, Singh A, Harris RC: CSF-1 signaling mediates recovery from acute kidney injury. J Clin Invest 122: 4519– 1. Okada A, Yasui T, Hamamoto S, Hirose M, Kubota Y, Itoh Y, Tozawa K, 4532, 2012 Hayashi Y, Kohri K: Genome-wide analysis of genes related to kidney 19. Alikhan MA, Jones CV, Williams TM, Beckhouse AG, Fletcher AL, Kett stone formation and elimination in the calcium oxalate nephrolithiasis MM, Sakkal S, Samuel CS, Ramsay RG, Deane JA, Wells CA, Little MH, model mouse: Detection of stone-preventive factors and involvement Hume DA, Ricardo SD: Colony-stimulating factor-1 promotes kidney of macrophage activity. JBoneMinerRes24: 908–924, 2009 growth and repair via alteration of macrophage responses. Am J Pathol 2. Okada A, Yasui T, Fujii Y, Niimi K, Hamamoto S, Hirose M, Kojima Y, Itoh 179: 1243–1256, 2011 Y, Tozawa K, Hayashi Y, Kohri K: Renal macrophage migration and 20. Menke J, Iwata Y, Rabacal WA, Basu R, Yeung YG, Humphreys BD, crystal phagocytosis via inflammatory-related gene expression during Wada T, Schwarting A, Stanley ER, Kelley VR: CSF-1 signals directly to kidney stone formation and elimination in mice: Detection by associa- renal tubular epithelial cells to mediate repair in mice. JClinInvest119: tion analysis of stone-related gene expression and microstructural ob- 2330–2342, 2009 servation. JBoneMinerRes25: 2701–2711, 2010 21. Qiao JH, Tripathi J, Mishra NK, Cai Y, Tripathi S, Wang XP, Imes S, 3. de Water R, Noordermeer C, van der Kwast TH, Nizze H, Boevé ER, Kok Fishbein MC, Clinton SK, Libby P, Lusis AJ, Rajavashisth TB: Role of DJ, Schröder FH: Calcium oxalate nephrolithiasis: Effect of renal crystal macrophage colony-stimulating factor in atherosclerosis: Studies of deposition on the cellular composition of the renal interstitium. Am osteopetrotic mice. Am J Pathol 150: 1687–1699, 1997 JKidneyDis33: 761–771, 1999 22. Wiktor-Jedrzejczak W, Bartocci A, Ferrante AW Jr., Ahmed-Ansari A, 4. Umekawa T, Chegini N, Khan SR: Oxalate ions and calcium oxalate Sell KW, Pollard JW, Stanley ER: Total absence of colony-stimulating crystals stimulate MCP-1 expression by renal epithelial cells. Kidney Int factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse. 61: 105–112, 2002 Proc Natl Acad Sci U S A 87: 4828–4832, 1990 5. Grover PK, Thurgood LA, Fleming DE, van Bronswijk W, Wang T, Ryall 23. Cecchini MG, Dominguez MG, Mocci S, Wetterwald A, Felix R, Fleisch RL: Intracrystalline urinary proteins facilitate degradation and dissolu- H, Chisholm O, Hofstetter W, Pollard JW, Stanley ER: Role of colony tion of calcium oxalate crystals in cultured renal cells. Am J Physiol stimulating factor-1 in the establishment and regulation of tissue Renal Physiol 294: F355–F361, 2008 macrophages during postnatal development of the mouse. De- 6. Vervaet BA, Verhulst A, Dauwe SE, De Broe ME, D’Haese PC: An active velopment 120: 1357–1372, 1994 renal crystal clearance mechanism in rat and man. Kidney Int 75: 41–51, 24. Yoshida H, Hayashi S, Kunisada T, Ogawa M, Nishikawa S, Okamura H, 2009 Sudo T, Shultz LD, Nishikawa S: The murine mutation osteopetrosis is in 7. Gordon S: Alternative activation of macrophages. Nat Rev Immunol 3: the coding region of the macrophage colony stimulating factor gene. 23–35, 2003 Nature 345: 442–444, 1990 8. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M: The 25. Okada A, Nomura S, Higashibata Y, Hirose M, Gao B, Yoshimura M, Itoh chemokine system in diverse forms of macrophage activation and po- Y, Yasui T, Tozawa K, Kohri K: Successful formation of calcium oxalate larization. Trends Immunol 25: 677–686, 2004 crystal deposition in mouse kidney by intraabdominal glyoxylate in- 9. Verreck FA, de Boer T, Langenberg DM, Hoeve MA, Kramer M, Vaisberg jection. Urol Res 35: 89–99, 2007 E, Kastelein R, Kolk A, de Waal-Malefyt R, Ottenhoff TH: Human IL-23- 26. Khan SR, Glenton PA, Byer KJ: Modeling of hyperoxaluric calcium ox- producing type 1 macrophages promote but IL-10-producing type 2 alate nephrolithiasis: Experimental induction of hyperoxaluria by macrophages subvert immunity to (myco)bacteria. Proc Natl Acad Sci hydroxy-L-proline. Kidney Int 70: 914–923, 2006 USA101: 4560–4565, 2004 27. Khan SR, Canales BK: Genetic basis of renal cellular dysfunction and the 10. Verreck FA, de Boer T, Langenberg DM, van der Zanden L, Ottenhoff formation of kidney stones. Urol Res 37: 169–180, 2009 TH: Phenotypic and functional profiling of human proinflammatory 28. Coe FL, Evan AP, Worcester EM, Lingeman JE: Three pathways for type-1 and anti-inflammatory type-2 macrophages in response to mi- human kidney stone formation. Urol Res 38: 147–160, 2010 crobial antigens and IFN-gamma- and CD40L-mediated costimulation. 29. Evan A, Lingeman J, Coe FL, Worcester E: Randall’splaque:Patho- J Leukoc Biol 79: 285–293, 2006 genesis and role in calcium oxalate nephrolithiasis. Kidney Int 69: 11. van der Wal AC, Das PK, Tigges AJ, Becker AE: Macrophage differ- 1313–1318, 2006 entiation in atherosclerosis. An in situ immunohistochemical analysis in 30. Miller NL, Gillen DL, Williams JC Jr., Evan AP, Bledsoe SB, Coe FL, humans. Am J Pathol 141: 161–168, 1992 Worcester EM, Matlaga BR, Munch LC, Lingeman JE: A formal test of 12. Woollard KJ, Geissmann F: Monocytes in atherosclerosis: Subsets and the hypothesis that idiopathic calcium oxalate stones grow on Randall’s functions. Nat Rev Cardiol 7: 77–86, 2010 plaque. BJU Int 103: 966–971, 2009 13. Yasui T, Itoh Y, Bing G, Okada A, Tozawa K, Kohri K: Aortic calcification 31. Khan SR: Experimental calcium oxalate nephrolithiasis and the forma- in urolithiasis patients. Scand J Urol Nephrol 41: 419–421, 2007 tion of human urinary stones. Scanning Microsc 9: 89–100, 1995

1696 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1680–1697, 2014 www.jasn.org BASIC RESEARCH

32. Asselman M, Verhulst A, De Broe ME, Verkoelen CF: Calcium oxalate 51. Lan HY: Role of macrophage migration inhibition factor in kidney dis- crystal adherence to hyaluronan-, osteopontin-, and CD44-expressing ease. Nephron, Exp Nephrol 109: e79–e83, 2008 injured/regenerating tubular epithelial cells in rat kidneys. JAmSoc 52. Kang HS, Liao G, DeGraff LM, Gerrish K, Bortner CD, Garantziotis S, Nephrol 14: 3155–3166, 2003 Jetten AM: CD44 plays a critical role in regulating diet-induced adipose 33. Kumar V, Farell G, Deganello S, Lieske JC: Annexin II is present on renal inflammation, hepatic steatosis, and insulin resistance. PLoS One 8: epithelial cells and binds calcium oxalate monohydrate crystals. JAm e58417, 2013 Soc Nephrol 14: 289–297, 2003 53. Yasui T, Okada A, Urabe Y, Usami M, Mizuno K, Kubota Y, Tozawa K, 34. Verhulst A, Asselman M, De Naeyer S, Vervaet BA, Mengel M, Gwinner Sasaki S, Higashi Y, Sato Y, Kubo M, Nakamura Y, Matsuda K, Kohri K: A W, D’Haese PC, Verkoelen CF, De Broe ME: Preconditioning of the replication study for three nephrolithiasis loci at 5q35.3, 7p14.3 and distal tubular epithelium of the human kidney precedes nephrocalcinosis. 13q14.1 in the Japanese population. J Hum Genet 58: 588–593, 2013 Kidney Int 68: 1643–1647, 2005 54. Fujii Y, Okada A, Yasui T, Niimi K, Hamamoto S, Hirose M, Kubota Y, 35. Vervaet BA, Verhulst A, De Broe ME, D’Haese PC: The tubular epi- Tozawa K, Hayashi Y, Kohri K: Effect of adiponectin on kidney crystal thelium in the initiation and course of intratubular nephrocalcinosis. formation in metabolic syndrome model mice via inhibition of in- Urol Res 38: 249–256, 2010 flammation and apoptosis. PLoS One 8: e61343, 2013 36. Koul S, Khandrika L, Meacham RB, Koul HK: Genome wide analysis of 55. Honda K, Yoshimura M, Rao TN, Fujishima A: Electrogenerated chem- differentially expressed genes in HK-2 cells, a line of human kidney iluminescence of the ruthenium Tris(2,29)bipyridyl/amines system on a epithelial cells in response to oxalate. PLoS One 7: e43886, 2012 boron-doped diamond electrode. JPhysChemB107: 1653–1663, 2003 37. Koul HK, Koul S, Fu S, Santosham V, Seikhon A, Menon M: Oxalate: 56. Umeda S, Takahashi K, Shultz LD, Naito M, Takagi K: Effects of mac- From crystal formation to crystal retention. J Am Soc Nephrol 10[Suppl rophage colony-stimulating factor on macrophages and their related 14]: S417–S421, 1999 cell populations in the osteopetrosis mouse defective in production of 38. Kohri K, Suzuki Y, Yoshida K, Yamamoto K, Amasaki N, Yamate T, functional macrophage colony-stimulating factor protein. Am J Pathol Umekawa T, Iguchi M, Sinohara H, Kurita T: Molecular cloning and 149: 559–574, 1996 sequencing of cDNA encoding urinary stone protein, which is identical 57. Lari R, Fleetwood AJ, Kitchener PD, Cook AD, Pavasovic D, Hertzog PJ, to osteopontin. Biochem Biophys Res Commun 184: 859–864, 1992 Hamilton JA: Macrophage lineage phenotypes and osteoclastogenesis 39. Umekawa T, Yamate T, Amasaki N, Kohri K, Kurita T: Osteopontin —complexity in the control by GM-CSF and TGF-beta. Bone 40: 323– mRNA in the kidney on an experimental rat model of renal stone for- 336, 2007 mation without renal failure. Urol Int 55: 6–10, 1995 58. Fleetwood AJ, Lawrence T, Hamilton JA, Cook AD: Granulocyte- 40. Okada A, Nomura S, Saeki Y, Higashibata Y, Hamamoto S, Hirose M, macrophage colony-stimulating factor (CSF) and macrophage CSF- Itoh Y, Yasui T, Tozawa K, Kohri K: Morphological conversion of calcium dependent macrophage phenotypes display differences in cytokine oxalate crystals into stones is regulated by osteopontin in mouse kid- profiles and transcription factor activities: Implications for CSF block- ney. J Bone Miner Res 23: 1629–1637, 2008 adeinin flammation. JImmunol178: 5245–5252, 2007 41. Hamamoto S, Nomura S, Yasui T, Okada A, Hirose M, Shimizu H, Itoh Y, 59. Hamers AA, Vos M, Rassam F, MarinkovicG,KurakulaK,vanGorpPJ, Tozawa K, Kohri K: Effects of impaired functional domains of osteo- de Winther MP, Gijbels MJ, de Waard V, de Vries CJ: Bone marrow- pontin on renal crystal formation: Analyses of OPN transgenic and OPN specificdeficiency of nuclear receptor Nur77 enhances atherosclerosis. knockout mice. JBoneMinerRes25: 2712–2723, 2010 Circ Res 110: 428–438, 2012 42. Vachon E, Martin R, Plumb J, Kwok V, Vandivier RW, Glogauer M, Kapus 60. Zheng D, Wang Y, Cao Q, Lee VW, Zheng G, Sun Y, Tan TK, Wang Y, A, Wang X, Chow CW, Grinstein S, Downey GP: CD44 is a phagocytic Alexander SI, Harris DC: Transfused macrophages ameliorate pancre- receptor. Blood 107: 4149–4158, 2006 atic and renal injury in murine diabetes mellitus. Nephron, Exp Nephrol 43. Marcondes MC, Poling M, Watry DD, Hall D, Fox HS: In vivo osteo- 118: e87–e99, 2011 pontin-induced macrophage accumulation is dependent on CD44 61. Oh DY, Morinaga H, Talukdar S, Bae EJ, Olefsky JM: Increased mac- expression. Cell Immunol 254: 56–62, 2008 rophage migration into adipose tissue in obese mice. Diabetes 61: 44. Nelson PJ, Rees AJ, Griffin MD, Hughes J, Kurts C, Duffield J: The renal 346–354, 2012 mononuclear phagocytic system. J Am Soc Nephrol 23: 194–203, 2012 62. Chaiyarit S, Thongboonkerd V: Changes in mitochondrial proteome of 45. Ricardo SD, van Goor H, Eddy AA: Macrophage diversity in renal injury renal tubular cells induced by calcium oxalate monohydrate crystal and repair. J Clin Invest 118: 3522–3530, 2008 adhesion and internalization are related to mitochondrial dysfunction. J 46. Shaul ME, Bennett G, Strissel KJ, Greenberg AS, Obin MS: Dynamic, Proteome Res 11: 3269–3280, 2012 M2-like remodeling phenotypes of CD11c+ adipose tissue macro- 63. Li H, Liu Y, Zhang Q, Jing Y, Chen S, Song Z, Yan J, Li Y, Wu X, Zhang X, phages during high-fat diet—induced obesity in mice. Diabetes 59: Zhang Y, Case J, Yu M, Ingram DA, Yang FC: Ras dependent paracrine 1171–1181, 2010 secretion of osteopontin by Nf1+/- osteoblasts promote osteoclast 47. Liao X, Sharma N, Kapadia F, Zhou G, Lu Y, Hong H, Paruchuri K, activation in a neurofibromatosis type I murine model. Pediatr Res 65: Mahabeleshwar GH, Dalmas E, Venteclef N, Flask CA, Kim J, Doreian BW, 613–618, 2009 Lu KQ, Kaestner KH, Hamik A, Clément K, Jain MK: Krüppel-like factor 4 64. Kawada M, Seno H, Kanda K, Nakanishi Y, Akitake R, Komekado H, regulates macrophage polarization. J Clin Invest 121: 2736–2749, 2011 Kawada K, Sakai Y, Mizoguchi E, Chiba T: Chitinase 3-like 1 promotes 48. Ruffell D, Mourkioti F, Gambardella A, Kirstetter P, Lopez RG, Rosenthal macrophage recruitment and angiogenesis in colorectal cancer. On- N, Nerlov C: A CREB-C/EBPbeta cascade induces M2 macrophage- cogene 31: 3111–3123, 2012 specific gene expression and promotes muscle injury repair. Proc Natl 65. Raheja LF, Genetos DC, Yellowley CE: Hypoxic osteocytes recruit hu- Acad Sci U S A 106: 17475–17480, 2009 man MSCs through an OPN/CD44-mediated pathway. Biochem Bio- 49. Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, phys Res Commun 366: 1061–1066, 2008 Popovich PG: Identification of two distinct macrophage subsets with 66. Pizzolato P: Histochemical recognition of calcium oxalate. JHistochem divergent effects causing either neurotoxicity or regeneration in the Cytochem 12: 333–336, 1964 injured mouse spinal cord. JNeurosci29: 13435–13444, 2009 50. Ma LJ, Corsa BA, Zhou J, Yang H, Li H, Tang YW, Babaev VR, Major AS, Linton MF, Fazio S, Hunley TE, Kon V, Fogo AB: Angiotensin type 1 receptor modulates macrophage polarization and renal injury in obe- This article contains supplemental material online at http://jasn.asnjournals. sity. Am J Physiol Renal Physiol 300: F1203–F1213, 2011 org/lookup/suppl/doi:10.1681/ASN.2013060675/-/DCSupplemental.

J Am Soc Nephrol 25: 1680–1697, 2014 CSF-1 Suppresses Renal Crystals 1697 SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure 1 Dose-dependent effects of CSF-1 treatment in op/op mice.

Seven groups were administered glyoxylate by daily intra-abdominal injection for

6 days. To investigate the role of CSF-1 in kidney crystal formation, mice of each genotype were injected subcutaneously with purified recombinant human CSF-1

(rhCSF-1), with concomitant glyoxylate administration, daily for 6 days.

Data are presented as mean ± SE. *p < 0.05 compared with untreated day 6 op/op mice. op + CSF-1(0.1), (1.0), (5.0), and (10) indicate op/op mice administered 0.1, 1.0, 5.0, and 10 μg of CSF-1 per mouse, respectively.

A. The ratio of area with renal crystal deposition. The ratio of renal crystal depositions in op+CSF-1 (1.0), (5.0), and (10) groups were significantly lower than in the op/op group.

B. The concentrations of serum CSF-1. CSF-1 levels were calculated using multiple quantitative immunoassays with an analysis device (MAGPIX; Merck

Millipore, Billerica, MA). The serum CSF-1 concentrations of op/op mice were significantly lower than those of the +/+ group, but those in the op+CSF-1 (5.0) and (10) groups were increased significantly compared with op/op. Based on these results, we used a CSF-1 concentration of 5.0 μg per mouse for subsequent experiments.

Supplementary Table 1.

Body weight, water intake, and urinary concentrations of +/+ and op/op mice

Day 0 Day 6

+/+ op/op +/+ op/op

BW (g) 23.8 ± 0.6 20.8 ± 1.1 22.4 ± 1.0 20.8 ± 0.9

WI (mL) 3.0 ± 0.3 3.2 ± 0.4 5.4 ± 0.8§ 4.6 ± 0.4§

UV (mL) 1.14 ± 0.10 0.62 ± 0.10* 1.84 ± 0.76 1.48 ± 0.29§

U-Ca (mg/dL) 3.0 ± 0.3 3.7 ± 0.6 3.1 ± 0.7 4.1 ± 1.2

U-Ox (mg/L) 173.8 ± 8.0 192.0 ± 7.9 288.0 ± 28.4§ 291.3 ± 44.5§

On day 0, the urinary volume (UV) of the op/op mice was lower than that of the

+/+ group; otherwise there were no significant differences in body weight (BW), water intake (WI), urinary calcium (U-Ca), and urinary oxalate (U-Ox) between groups. The WI of both +/+ and op/op mice increased on day 6 compared with day 0, whereas the UV of op/op mice was higher on day 6 than on day 0. The concentrations of U-Ox on day 6 were higher than on day 0 in both groups.

Data are presented as mean ± SE. *p < 0.05 compared with +/+ mice, §p < 0.05 compared with day 0 in each group.

BW, body weight; WI, water intake; UV, urinary volume; U-Ca, urinary calcium;

U-Ox, urinary oxalate.

Supplementary Table 2.

A list of genes showing a different expression pattern between M1 and M2 macrophages. M1 and M2 macrophages were prepared from the bone marrow cells of wild-type mice, then subjected to microarray analysis using Agilent

SurePrint G3 Mouse GE microarrays. The analysis was performed using triplicates of each macrophage subpopulation. Probes showing significant (p <

0.01, and > 4-fold) differences between M1 and M2 macrophages are listed. The probe ID of the Agilent array, official gene symbol, and average values of the fold-difference are shown.

Supplementary Table2. A list of genes showing a different expression pattern between M1 and M2 macrophages

Probe ID Official Gene Symbol M1/M2(fold) A_51_P257951 Retnla -2291.429843 A_55_P2079009 Slco2b1 -193.0182164 A_55_P2030155 Slco2b1 -89.64720135 A_55_P1988108 Mrc1 -85.60838472 A_55_P2119772 Scn3a -85.56570377 A_52_P425839 Retnlg -68.87519533 A_52_P547662 P2ry1 -51.6391769 A_66_P106808 Scn3a -45.2257054 A_51_P455338 Ear11 -37.52441304 A_52_P71686 Atp6v0d2 -35.66962145 A_55_P2002291 Msx3 -29.46520156 A_52_P590396 Sort1 -29.43434503 A_52_P126403 Tmem26 -27.87923946 A_52_P16419 Gpd1 -26.85207645 A_55_P2085974 Igf1 -26.55213197 A_52_P161630 St6gal1 -21.71107416 A_51_P167876 Tfrc -21.47003444 A_55_P2097998 1500017E21Rik -20.19696038 A_55_P2026139 BC055004 -18.2303912 A_65_P06446 Tfrc -18.19677273 A_55_P2406802 4930511J24Rik -17.88859903 A_51_P303424 Itgax -17.84860851 A_51_P303160 Arg1 -17.61197309 A_55_P2128853 Ear12 -17.34376369 A_55_P2082796 Gm14461 -17.07099033 A_51_P433083 4930510E17Rik -16.73492291 A_55_P2036394 Slc39a12 -16.14410995 A_66_P124179 Atp6v0d2 -16.13305554 A_51_P366672 Slc36a2 -15.80344541 A_55_P2031636 Igf1 -15.62547238 A_65_P13459 Cd300lb -15.49681791 A_52_P228236 Tfrc -15.33087038 A_51_P229498 Lrfn2 -14.21049791 A_55_P2220683 4833419A21Rik -14.18143481 A_55_P1952618 Ear2 -14.02326226 A_51_P517145 Sort1 -13.23131182 A_51_P106799 Pparg -12.65224669 A_55_P2252891 D11Ertd717e -12.4725591 A_51_P398723 Flt1 -12.4709497 A_51_P324287 Kif23 -12.2346642 A_55_P2105783 Mmp12 -12.00312455 A_55_P2097478 Gda -11.82350524 A_55_P2114863 Mgll -11.79527378 A_55_P2045129 5830410O09Rik -11.7622414 A_55_P2344244 4930556H04Rik -11.66942039 A_55_P2067041 Agxt2 -11.39357269 A_55_P1984596 Stxbp5 -11.33321282 A_51_P520936 Bcar3 -11.26745357 A_52_P412585 Ear1 -11.01401711 A_55_P2120469 Armc2 -10.98069451 A_55_P2025523 Arhgap6 -10.93242041 A_51_P279100 Ptgs1 -10.90917396 A_55_P2036176 Mcf2l -10.4517598 A_55_P2057430 Lipn -10.31938787 A_66_P121459 Cenpa -10.20463333 A_55_P2163774 Crip1 -10.04088341 A_52_P257812 Lpl -9.972980297 A_55_P2084739 Gcnt1 -9.832105526 A_55_P1988623 Add3 -9.744211179 A_52_P21550 Gcnt1 -9.727608049 A_51_P103397 Vwf -9.655974057 A_52_P375323 Slc9a9 -9.594972024 A_52_P116006 Gda -9.406386223 A_52_P585124 Cxcr4 -9.347969714 A_55_P2114187 Gm6522 -9.333124884 A_66_P133397 Chi3l4 -9.27371699 A_66_P138915 Fyn -9.217805523 A_55_P1974028 Stab2 -9.166438104 A_51_P513716 Ankrd55 -9.112114654 A_51_P469285 Nrp1 -9.078790432 A_66_P115682 Tectb -9.057170397 A_55_P2256601 AU022434 -8.81816456 A_55_P2109445 Irf4 -8.74831883 A_55_P2036180 Mcf2l -8.699922022 A_55_P2063316 Mgll -8.659932621 A_51_P459661 Lipa -8.626074643 A_51_P409452 Cldn11 -8.584080467 A_52_P139716 Olfr112 -8.51571419 A_52_P553890 Itgb3 -8.473368312 A_55_P2014882 Dhrs9 -8.42979969 A_51_P302139 Mastl -8.278280882 A_52_P308413 1810011H11Rik -8.274175739 A_52_P628067 Cdca3 -8.240277943 A_55_P2157008 Ear10 -8.232400101 A_51_P322640 Ccl24 -8.104442798 A_55_P2062418 Atp6v0a1 -8.053147736 A_55_P2025528 Arhgap6 -8.001363287 A_55_P1981135 Olfr114 -7.97770126 A_51_P347240 Lrr1 -7.974071824 A_51_P122582 Daglb -7.82054023 A_51_P167292 Chi3l3 -7.793409911 A_52_P577662 Ednrb -7.769729728 A_51_P259296 Lpl -7.732994034 A_52_P404942 ORF63 -7.722895232 A_51_P195825 Tsnaxip1 -7.720016869 A_66_P117729 Awat1 -7.694009441 A_55_P2036392 Slc39a12 -7.659897569 A_51_P460048 Cnrip1 -7.568499995 A_55_P1952240 Slitrk2 -7.543305733 A_55_P2082679 Aph1b -7.519703615 A_55_P2086575 Arhgap19 -7.448640325 A_55_P2113051 Fosb -7.314751737 A_55_P2084723 LOC635918 -7.205361931 A_55_P2288285 2310007J06Rik -7.125249831 A_51_P114634 Amz1 -7.10351966 A_55_P2116385 Gpr183 -7.095143189 A_52_P591473 Armc2 -7.088917794 A_51_P391367 Tcfap4 -7.086270561 A_55_P2043862 Stmn1 -6.978005332 A_52_P171019 Man1a -6.927564797 A_55_P2409088 BB163080 -6.904117128 A_55_P2085979 Igf1 -6.899735703 A_52_P366047 Rpp40 -6.886686484 A_51_P220317 Cdca7l -6.868540869 A_55_P2140118 Qpct -6.824434483 A_55_P2005320 Tm7sf4 -6.815976399 A_52_P571371 Tmem144 -6.796466121 A_55_P2065726 Snx29 -6.791396673 A_55_P1970895 Olfr889 -6.779481786 A_52_P299832 Plxnc1 -6.759539852 A_52_P552589 Map4k1 -6.755473999 A_55_P2112560 Ern1 -6.747341584 A_55_P2068560 Lyzl4 -6.706685544 A_55_P2046648 LOC641221 -6.689131467 A_55_P2033362 Egr2 -6.640668401 A_55_P1961736 Rcan2 -6.61322506 A_55_P2065294 Scn3a -6.602621753 A_52_P222696 Poteg -6.59955432 A_55_P2011436 Gm11223 -6.587492892 A_55_P2065288 Scn3a -6.533916418 A_55_P2178084 Dnmt3a -6.518953389 A_55_P1994927 Rab3il1 -6.447121624 A_55_P2088028 Mtss1 -6.443791885 A_55_P1960735 Gdf15 -6.438122377 A_55_P2221901 Gm10797 -6.400520802 A_55_P2086664 Tspan33 -6.388357943 A_51_P251352 Slc25a13 -6.324277358 A_51_P397925 Smap2 -6.323373776 A_55_P2068663 Stmn1 -6.317014133 A_52_P381430 Tbc1d4 -6.314804367 A_52_P33831 Lrrc27 -6.299386584 A_52_P251366 Neil3 -6.294772769 A_55_P2119907 Dnahc11 -6.136536291 A_55_P2065671 Ccnb1 -6.104058971 A_51_P349495 Mboat1 -6.078918598 A_55_P2345853 3830612M24 -6.078824931 A_51_P302520 Myom1 -6.011078664 A_66_P133404 2810417H13Rik -5.948624035 A_51_P445841 Deptor -5.945832411 A_51_P415905 Pola1 -5.871368882 A_51_P351896 Fam198b -5.836336679 A_55_P2005946 Lyzl4 -5.831030666 A_55_P1979491 Cd28 -5.827714777 A_51_P501018 Nek2 -5.821897945 A_55_P2003541 Nrcam -5.798575653 A_55_P2037662 Atp1a3 -5.773417354 A_52_P442169 Bsn -5.771540722 A_52_P15388 Ltf -5.770900793 A_52_P11174 Hip1 -5.756245327 A_55_P2155859 Olfr243 -5.749093523 A_55_P2176280 Prkar1b -5.745078477 A_52_P385606 Ckb -5.74483971 A_52_P507498 Plxnc1 -5.730491885 A_51_P236412 Cdkl2 -5.726022344 A_55_P2101757 Dscc1 -5.72401241 A_55_P2024953 Tbc1d4 -5.695964284 A_55_P2109857 Rgs2 -5.659829135 A_66_P136186 Wee1 -5.649084183 A_52_P708792 Gm5547 -5.630467442 A_55_P2060193 Mcf2l -5.627508263 A_55_P1976224 Ckb -5.528309551 A_55_P2122020 Klf4 -5.51108787 A_52_P142191 Aph1b -5.504476639 A_55_P1963184 LOC100504348 -5.495648109 A_55_P2336558 Chn2 -5.495575237 A_55_P2025033 Cep55 -5.476542631 A_55_P2056496 Tk1 -5.454398848 A_51_P247249 Alox5 -5.451472615 A_51_P368009 E2f2 -5.443142704 A_66_P104980 Tceal5 -5.440263763 A_55_P2092661 Nup210 -5.436535994 A_66_P134542 Anln -5.415171916 A_55_P2013268 Gm13391 -5.390185861 A_51_P485862 Eef1a2 -5.384735571 A_51_P272283 Cmbl -5.357973924 A_52_P231075 Fcrls -5.338307608 A_55_P2001891 Prps1 -5.313940374 A_55_P1977008 Gfi1 -5.308712454 A_55_P2033557 AB041803 -5.226018023 A_52_P354373 1190002F15Rik -5.222816078 A_52_P325527 Edil3 -5.204042341 A_55_P2089710 Ednrb -5.169462204 A_55_P2030030 Adssl1 -5.137601103 A_51_P481920 Ccna2 -5.096663837 A_55_P2170681 Cdca8 -5.057357623 A_51_P172054 Gas6 -5.055812638 A_51_P125205 Aqp1 -5.052998158 A_55_P1983450 S100a4 -5.038056844 A_51_P389988 Slc40a1 -5.033425461 A_51_P166023 Hip1 -5.012601587 A_55_P2030583 Lrriq1 -5.008032761 A_55_P2177578 Atp6v0a1 -4.998318032 A_55_P2008417 Mnd1 -4.972271866 A_55_P2098539 Itgb3bp -4.970865611 A_52_P239320 Syngr1 -4.967491848 A_55_P2045055 Clec10a -4.965589499 A_51_P410949 Polr3g -4.941749563 A_55_P1972772 Gsg1 -4.894188948 A_55_P2129514 Trerf1 -4.89405953 A_55_P1983768 Birc5 -4.881025538 A_55_P2016652 Cdk1 -4.877592558 A_55_P1983448 S100a4 -4.832943068 A_65_P14951 Cblb -4.828199034 A_51_P191649 Ndc80 -4.824403248 A_55_P2164534 Dtl -4.803811696 A_55_P1983769 Birc5 -4.78884586 A_55_P1952256 Ccnb1 -4.770373323 A_55_P1953723 Rab13 -4.760924004 A_51_P155152 Ank -4.757171247 A_51_P481398 Kif11 -4.726222665 A_51_P246653 Clec7a -4.720529843 A_51_P240453 Nusap1 -4.708246648 A_52_P523503 Tmem154 -4.691094293 A_55_P1983773 Birc5 -4.684343267 A_52_P168567 Cebpa -4.674365674 A_51_P132978 Idh1 -4.64597007 A_55_P1996941 Ube2c -4.63652849 A_51_P320852 Cd9 -4.625390914 A_51_P155142 Cdca8 -4.6158387 A_51_P281380 Tspan5 -4.613240012 A_66_P105422 Lonrf3 -4.590843812 A_52_P193925 Sulf2 -4.575744826 A_51_P461319 Gatm -4.553098909 A_51_P303749 Depdc1b -4.524111857 A_66_P120074 Uhrf1 -4.502704404 A_52_P134195 Ceacam10 -4.480646159 A_55_P1976127 Ect2 -4.479036708 A_51_P204402 Shcbp1 -4.451632508 A_51_P408071 Kntc1 -4.435473424 A_55_P1973783 Tex9 -4.39818738 A_55_P2149931 Arap2 -4.393634016 A_55_P2008651 Qdpr -4.376746421 A_51_P472829 Aif1l -4.37395052 A_52_P540219 Timp2 -4.368717748 A_55_P2076048 Nuf2 -4.346081925 A_55_P2074736 Prkar1b -4.334362149 A_66_P102528 Tspan5 -4.329959107 A_55_P1991475 Sesn1 -4.323844171 A_51_P220150 Angptl7 -4.318999352 A_55_P2176240 Pdxk -4.313165576 A_55_P2130388 Mical1 -4.300263577 A_55_P1968178 Smagp -4.287784562 A_55_P2180301 Clcn5 -4.287540176 A_51_P101006 Ak8 -4.267666766 A_51_P309589 2700099C18Rik -4.26562835 A_55_P1998601 Slc17a9 -4.254681507 A_55_P2152427 Zwilch -4.248997015 A_55_P2118684 Idh2 -4.238806472 A_51_P473888 Il6st -4.225158902 A_51_P123405 Bub1 -4.218512861 A_55_P2109717 Kif20b -4.218341808 A_55_P1987043 Tpp1 -4.215660684 A_55_P2156304 Kcnj16 -4.209759454 A_55_P2069659 9930013L23Rik -4.200165862 A_55_P2007878 Rnf144b -4.19074942 A_52_P227391 Kif15 -4.18288316 A_52_P104824 Diap3 -4.179882536 A_52_P588881 Iqgap3 -4.177494416 A_55_P2063626 Matk -4.170169603 A_51_P148105 Rad51 -4.169961345 A_52_P136138 Fdft1 -4.164321835 A_55_P2158011 D2Ertd750e -4.15787331 A_55_P2031631 Igf1 -4.149596046 A_52_P231170 Megf9 -4.142621181 A_55_P2165120 Cblb -4.138949368 A_51_P415220 Zmat3 -4.135413065 A_51_P513530 Spag5 -4.134959503 A_55_P2150003 Gnat3 -4.131155026 A_51_P241843 Tmem202 -4.111539531 A_51_P375146 Cd36 -4.099095778 A_55_P2058348 Tmem206 -4.096541414 A_55_P2292046 D230018H15Rik -4.088002277 A_55_P2085984 Igf1 -4.085554508 A_66_P138584 Mnd1 -4.084029819 A_55_P2062255 4831426I19Rik -4.073067434 A_55_P2294295 Tanc2 -4.072608402 A_55_P2080774 Qdpr -4.053935177 A_51_P133137 Kif20a -4.053879764 A_52_P529570 Nsl1 -4.053225588 A_51_P248387 Pcyox1l -4.047112632 A_51_P140368 2310061C15Rik -4.036750582 A_51_P422625 Arl11 -4.036439589 A_55_P2115851 9930013L23Rik -4.012378038 A_51_P490509 Bub1b -4.003832166 A_55_P1969341 Brip1 -4.001781674 A_55_P1995092 H2-Q10 4.014280975 A_55_P1971010 Gzme 4.016123794 A_55_P2133248 Gm8995 4.02784152 A_51_P383032 Clec4d 4.043728117 A_55_P1968568 Rapgef5 4.0495221 A_55_P2123987 Arid3b 4.049579755 A_52_P30550 Acp2 4.050530863 A_55_P2032659 Tox 4.050620707 A_55_P1972040 Nox4 4.051317632 A_51_P149714 Ms4a6d 4.051870316 A_55_P1969002 Tbx3 4.065993996 A_55_P2170309 Ppfibp2 4.081006674 A_55_P2086933 Gm4368 4.084122106 A_55_P2033255 Prdx5 4.08872774 A_55_P1955309 Zfp800 4.097444562 A_51_P384318 C1ra 4.09820059 A_66_P109986 Cd33 4.098274845 A_52_P367774 Zfp821 4.098550176 A_52_P496924 Mgat4a 4.100495204 A_55_P1999339 Inpp5b 4.102719951 A_55_P1958250 Rnf114 4.107886156 A_55_P1985850 Timp1 4.113290599 A_51_P195958 Phlda1 4.113464891 A_52_P364140 Itga5 4.116160392 A_55_P2090880 Stk40 4.134985012 A_55_P2111790 Gem 4.141418908 A_55_P2009121 Agrn 4.143397405 A_51_P374726 Ptx3 4.144475976 A_51_P413059 Mcpt8 4.1451675 A_52_P226348 F730043M19Rik 4.153008141 A_55_P1953353 Triobp 4.161805376 A_55_P2236291 Ppap2a 4.185141496 A_51_P137419 Cst7 4.18785009 A_52_P552550 Agtrap 4.189884371 A_55_P1957213 3930401B19Rik 4.18996444 A_55_P2149763 Tapbp 4.193720462 A_51_P302566 Maob 4.195036607 A_52_P532227 S1pr1 4.195415013 A_55_P2277171 Gm10134 4.197819691 A_55_P1963364 Fam55c 4.199156041 A_55_P1977929 Itgam 4.223128659 A_55_P2037235 Sp110 4.227050652 A_55_P2072115 AW011738 4.232599314 A_55_P1989738 4930426L09Rik 4.232651771 A_51_P464703 Ccl8 4.233301522 A_52_P658122 Ets2 4.242290571 A_55_P2092904 Ddb2 4.243470359 A_51_P513568 Stx11 4.259611929 A_51_P515605 Col3a1 4.281821755 A_66_P114784 Pla2g7 4.284382837 A_52_P90363 Ifi27l2a 4.289863342 A_55_P1977926 Itgam 4.29605094 A_51_P234539 Nfxl1 4.297537713 A_52_P488779 Pilrb2 4.306760807 A_52_P183181 Adar 4.308156324 A_55_P2010038 Tnfsf9 4.308615326 A_51_P295192 Nfkbia 4.324942643 A_55_P2044953 Lst1 4.328033602 A_66_P138790 Glis3 4.331971646 A_55_P2165286 Gmfg 4.334635506 A_52_P308875 LOC100504219 4.339068915 A_51_P234113 Nod1 4.342496993 A_55_P1952269 Xylt2 4.342987806 A_66_P119376 Kctd12 4.344760644 A_51_P345367 Psmb8 4.352250212 A_52_P645410 Apobec3 4.364509114 A_55_P2031125 Ube2l6 4.374666384 A_51_P350048 Gstt2 4.374667294 A_55_P2038882 Niacr1 4.375552933 A_52_P160955 Sox11 4.38040086 A_55_P2119257 Serpine1 4.407955013 A_51_P472621 Zfp719 4.42609096 A_51_P369803 Psmb9 4.426792038 A_51_P149349 Nfxl1 4.427575462 A_52_P466090 Samhd1 4.439654474 A_55_P1993263 Chic1 4.447114037 A_55_P2108763 Sp100 4.45031637 A_52_P479269 Sdc1 4.461547068 A_55_P1969078 Adar 4.461779136 A_55_P2087265 Ifitm1 4.464226418 A_55_P1959938 Pde7b 4.467386574 A_51_P387608 Hif1a 4.473858105 A_55_P2087796 Fam176b 4.474298165 A_55_P1955637 Trp53i11 4.475491102 A_55_P1960989 Nlgn2 4.493374786 A_51_P184484 Mmp13 4.49599428 A_52_P274496 Tspan18 4.501033181 A_55_P2152566 Sp110 4.511187342 A_51_P121031 MARCH1 4.514057826 A_55_P2192819 BB157357 4.514231484 A_55_P2115442 Clec2d 4.528169617 A_55_P2014229 Sema4a 4.532547377 A_51_P503822 Slitrk6 4.536458937 A_55_P1958991 A530032D15Rik 4.537388213 A_52_P597461 Skil 4.565805807 A_55_P2003883 Gm9909 4.581222712 A_55_P2052924 Sepw1 4.581791155 A_51_P320022 Atp10a 4.601584432 A_55_P1971009 Gzme 4.60736406 A_55_P2018417 Osbpl3 4.608637198 A_55_P2367878 Ncrna00085 4.60936112 A_51_P458584 Fpr-rs3 4.613783156 A_55_P2123502 Jam2 4.629142904 A_55_P1959425 Slc16a3 4.630413073 A_51_P363187 Cxcl1 4.631588562 A_52_P634090 Jag1 4.640832411 A_66_P109733 1700123I01Rik 4.643785711 A_51_P207988 Ptger4 4.677138012 A_52_P520408 Serp1 4.67929051 A_51_P417854 Rab20 4.691540762 A_55_P2065601 Sytl5 4.69437307 A_55_P2053838 Tnfaip3 4.695795562 A_55_P2305623 Cmklr1 4.700857865 A_55_P2064659 Trim12a 4.708252359 A_51_P302738 Upp1 4.721744901 A_52_P665675 Abca1 4.75232977 A_55_P2023637 Prg4 4.758607952 A_55_P2094524 Gm14005 4.773569288 A_55_P1975560 Ifi204 4.781442744 A_52_P363216 Gcnt2 4.784773392 A_55_P2135526 Gzmc 4.788395774 A_55_P2179413 Lgals3bp 4.791357626 A_55_P2185589 Rbms3 4.79184818 A_55_P2067505 Slc16a3 4.799167777 A_51_P326191 Serpina3g 4.821689669 A_55_P2045741 Fyb 4.822736341 A_55_P2004511 Cd300lf 4.82351276 A_55_P2020050 Gm8120 4.833734389 A_52_P604515 Zfp319 4.839688858 A_55_P2114938 Irf9 4.843230817 A_51_P222657 Tspo 4.843510268 A_55_P2048378 Gm5595 4.878540821 A_55_P2179599 Gbp8 4.887209162 A_55_P2024841 Gstt1 4.90728571 A_55_P2095311 Ly6g 4.917761179 A_55_P2014675 Olfr800 4.927266485 A_52_P434055 Birc3 4.935760528 A_55_P2006869 Fyb 4.937815734 A_51_P319460 Osmr 4.943119486 A_55_P2031436 Ly6e 4.944333645 A_51_P417720 Itga11 4.948802108 A_55_P2168736 Relb 4.957596518 A_55_P2088131 Gm5595 4.965737158 A_52_P235102 Gm5595 4.965850056 A_55_P1983459 Cflar 4.982517941 A_51_P288876 Tmem45a 5.00224514 A_51_P412835 Daxx 5.005476451 A_55_P2033420 2010106G01Rik 5.006206425 A_51_P419017 Pde10a 5.0104907 A_55_P1970329 Gm10696 5.020343225 A_51_P501453 Ms4a8a 5.036705577 A_51_P464394 Klb 5.053978756 A_55_P2082096 Wdr66 5.078264362 A_51_P230507 Ell2 5.092693527 A_55_P1998892 Smox 5.100417128 A_51_P359636 Lgals3bp 5.106508476 A_52_P329185 Pde7b 5.12259722 A_55_P2059765 Foxf1a 5.129512176 A_51_P196844 Osbpl3 5.130248468 A_51_P469968 H2-M3 5.156810925 A_55_P2038525 C3 5.164765333 A_55_P2035504 Nt5c3 5.182230908 A_51_P279606 Socs1 5.18412297 A_52_P317627 Gm11435 5.209208563 A_51_P381558 Rasa4 5.230610707 A_52_P305279 Spata13 5.27366438 A_55_P2118675 Tnip1 5.287453608 A_55_P1981999 BC117090 5.299694139 A_55_P2091473 Bnip3 5.307460393 A_55_P2118674 Tnip1 5.311904649 A_55_P1960237 Slc2a6 5.320153973 A_66_P122110 Mitd1 5.323719465 A_55_P2160094 Plscr2 5.33232432 A_51_P489996 Dst 5.347574142 A_52_P61697 9930111J21Rik2 5.348355637 A_55_P1998416 Ifi47 5.357108812 A_52_P559919 Eif2ak2 5.365489144 A_51_P483454 Kif26b 5.371215629 A_52_P269158 Pid1 5.377240208 A_52_P267391 Trim12a 5.407474437 A_51_P182572 Phactr1 5.426367285 A_55_P2053032 Xkr8 5.44757053 A_55_P2043367 Apol9a 5.454047255 A_55_P1989225 Tgtp2 5.458662725 A_51_P153423 Fndc1 5.463050654 A_55_P2091472 Bnip3 5.474907158 A_55_P2146254 Ifitm1 5.481583599 A_66_P128997 Pml 5.498721277 A_55_P2034705 Nmi 5.503945864 A_55_P2142048 LOC100504062 5.518397575 A_51_P323180 Gbp9 5.52137795 A_52_P409833 Plat 5.521815944 A_52_P281702 Igfbp5 5.551835416 A_66_P138137 Pnp 5.55911952 A_51_P430929 Fam20a 5.569426976 A_55_P2151601 Samd9l 5.582204316 A_55_P2055612 Zfp800 5.598669432 A_52_P481957 Grem1 5.608721313 A_55_P1978576 Dtx2 5.672772864 A_51_P374900 P2ry13 5.708781717 A_52_P359965 Cpd 5.714356365 A_51_P318577 Sult6b1 5.727937695 A_55_P2006693 Pstpip2 5.740519769 A_55_P1955778 Adamts4 5.772313832 A_55_P2061739 Tmsb4x 5.773921006 A_55_P1984556 Ccl12 5.805107969 A_55_P2147081 Tnfaip6 5.827287135 A_55_P1971889 F3 5.841705221 A_51_P131888 Capns2 5.850367286 A_55_P2087118 Ly6i 5.855762737 A_55_P2049867 Ccrl2 5.864192408 A_55_P2011620 Gpr141 5.869622823 A_55_P2091461 Casp4 5.897966794 A_51_P385812 Il12b 5.898774261 A_55_P2168223 Aicda 5.920366957 A_55_P2104975 Serpina3f 5.922525068 A_66_P119034 Pla2g7 5.924541056 A_55_P1984168 Casp4 5.94616089 A_55_P2137121 Sp140 5.958728051 A_66_P121787 Samd9l 5.968756181 A_55_P2071176 Il1f9 5.984239228 A_51_P307559 6430704M03Rik 5.984808977 A_55_P2077068 Malt1 5.987351628 A_55_P2097828 Gmfg 5.992226262 A_51_P128876 Ifitm3 6.047026913 A_51_P367866 Egr1 6.052247977 A_55_P2182452 Tnfsf15 6.082785792 A_55_P2156855 Car13 6.084233282 A_66_P134453 I830077J02Rik 6.090320284 A_51_P503261 Lrrc8c 6.112254606 A_55_P2238406 6330407A03Rik 6.180792705 A_55_P1960238 Slc2a6 6.198262693 A_55_P2070079 Lgals9 6.204667628 A_55_P2090660 Mrpl39 6.212991153 A_55_P1961395 Pdpn 6.225280246 A_55_P1970710 Mllt6 6.228751429 A_55_P2116165 Pfkfb3 6.230624617 A_55_P2064652 Trim12c 6.236724798 A_55_P2101055 Alpk1 6.327338854 A_55_P2097259 Zufsp 6.340923054 A_51_P520966 Icosl 6.370800674 A_55_P2182736 Limd2 6.404432774 A_52_P582374 Epsti1 6.427179835 A_52_P362917 Pfkfb3 6.431465966 A_55_P2066578 Ifi204 6.460858147 A_51_P248666 Cd274 6.491419538 A_55_P2036693 Cd40 6.495722269 A_55_P1983468 Cflar 6.530218659 A_52_P552194 Il13ra1 6.547986285 A_51_P210395 Glrx 6.559429758 A_52_P642239 Pik3ap1 6.562006851 A_51_P389751 Relb 6.565972011 A_55_P2106175 Gch1 6.5733534 A_55_P2031836 Gpr85 6.600836209 A_51_P120830 Mmp10 6.633478237 A_51_P167527 Lum 6.655207278 A_55_P2085915 Tnfrsf14 6.737286264 A_55_P2077618 Csgalnact1 6.740492467 A_52_P78023 Limd2 6.784537167 A_52_P338066 Ubd 6.789680088 A_55_P2017769 B020031M17Rik 6.790325345 A_51_P154842 Oas1f 6.800134586 A_55_P1979463 Prdx5 6.80983055 A_51_P340699 Rasl11a 6.833997879 A_66_P108965 Smpdl3b 6.842373701 A_51_P497985 C2 6.861785843 A_66_P119283 LOC100417829 6.872215131 A_55_P1994194 Heph 6.872617179 A_52_P298002 Gch1 6.905903621 A_66_P129710 Pion 6.911100219 A_55_P2167040 Pvrl4 6.912261991 A_55_P1969032 Rgs9 6.917370738 A_52_P609868 Timd4 6.930461862 A_51_P469008 Ehd1 6.946804033 A_51_P496720 Dnmt3l 6.986282591 A_55_P2048607 Hp 6.98639702 A_51_P400543 Aif1 7.009042635 A_55_P1994654 1810029B16Rik 7.01392255 A_51_P110301 C3 7.019469976 A_55_P2174541 Dtx3l 7.030566541 A_55_P1955906 Stat1 7.043226815 A_55_P2052062 Cd200 7.106461443 A_52_P226929 Cflar 7.10727223 A_55_P1988202 Ifi203 7.116681643 A_55_P2001233 Pydc3 7.119185534 A_51_P228768 Slfn3 7.142145327 A_52_P66520 Clec12a 7.156145893 A_52_P155554 Cdc42ep2 7.158173241 A_55_P2097448 Rbpms 7.164658149 A_52_P304128 Mmp14 7.177142772 A_66_P119968 Clec4a1 7.179634493 A_51_P235123 Nfkbie 7.190013253 A_52_P577384 Il18bp 7.198488755 A_51_P351015 Lta 7.25509799 A_66_P132493 Siglece 7.294424197 A_51_P179258 Kif26b 7.315284938 A_51_P169693 Bst2 7.327440764 A_55_P2206605 5830444B04Rik 7.328082269 A_55_P2173210 LOC100504287 7.350316893 A_52_P586944 Bmpr1b 7.392282545 A_55_P2082733 Cybb 7.392923578 A_66_P128795 Gm1045 7.408441103 A_52_P423247 Pde4b 7.411429843 A_66_P106060 Dtx3l 7.443399139 A_55_P2096917 Mreg 7.45480492 A_52_P441954 Ovol1 7.456051888 A_52_P650540 Nlrp3 7.465182344 A_51_P495581 Tlr1 7.488836021 A_55_P2004507 Ifi203 7.492273162 A_55_P1980287 Birc3 7.493218393 A_66_P109708 Il1f6 7.498832507 A_65_P01319 Pde4b 7.532277751 A_55_P1983162 Pstpip2 7.537738992 A_51_P466378 Itgal 7.54049535 A_55_P1963920 A430084P05Rik 7.555061818 A_55_P2045258 Ehd1 7.571557768 A_55_P2142863 Parp9 7.588053234 A_52_P368057 Slc7a11 7.598657755 A_51_P222280 Ikbke 7.636653334 A_55_P2273439 Nlrp3 7.683451858 A_55_P2051885 Bco2 7.716590968 A_55_P2073552 A930003A15Rik 7.78432368 A_55_P2066230 Hck 7.799060593 A_51_P100327 Tap1 7.812885656 A_55_P2006774 Gbgt1 7.813115817 A_55_P2166148 H2-M9 7.832176083 A_51_P239750 Inhba 7.884748838 A_55_P2064962 Pdpn 7.889933825 A_55_P2023542 Ccr7 7.896695716 A_55_P1989663 Slco3a1 7.932927148 A_55_P2052834 Lst1 7.959431001 A_55_P1963017 Stfa1 7.99512345 A_51_P286488 Ier3 8.084778789 A_52_P409578 Rassf4 8.096282925 A_66_P105689 Trim34a 8.105505712 A_55_P1991811 Gm5431 8.112782439 A_55_P2347906 B230303A05Rik 8.136851154 A_51_P401907 Gm5483 8.163977464 A_51_P381618 Pla1a 8.167355924 A_55_P2026233 Uba7 8.170680845 A_52_P15461 Il15 8.214217795 A_55_P2113256 Parp11 8.223514653 A_66_P122086 9030619P08Rik 8.243373708 A_55_P2184601 Itgal 8.244338037 A_66_P118906 Adora2b 8.247960491 A_51_P241995 Col5a3 8.266387707 A_55_P2026688 Rnf213 8.280826106 A_52_P507578 Slamf6 8.304439971 A_51_P333274 Gzmb 8.332991451 A_55_P1998979 Oas1g 8.340352734 A_66_P129976 Apba1 8.345119449 A_66_P116252 Dusp16 8.358397671 A_51_P427171 9030625A04Rik 8.364489543 A_55_P1993503 Adora2b 8.380225701 A_51_P477736 4932415M13Rik 8.417977187 A_55_P2073099 Trex1 8.440095949 A_55_P2135200 Slfn10-ps 8.463085255 A_55_P1998943 Oas1a 8.467653648 A_66_P125389 F830016B08Rik 8.522339237 A_55_P2010152 Sell 8.522503873 A_55_P2012989 Slamf7 8.523613641 A_55_P2143282 Nkd2 8.536369351 A_55_P1997696 Plbd1 8.584936124 A_52_P799815 Tmem171 8.585400974 A_55_P2470474 9530082P21Rik 8.58888289 A_66_P128525 Slfn5 8.599553529 A_55_P2091191 Slc28a2 8.612269704 A_52_P272778 Klrk1 8.649411643 A_52_P487686 BC100530 8.6823227 A_55_P2158227 Angpt1 8.689836422 A_55_P2106926 Kcna3 8.712719287 A_55_P1973229 LOC640746 8.742385525 A_52_P501875 4930578N16Rik 8.818216514 A_51_P520306 Alox12 8.853211578 A_55_P2130970 Parp10 8.889162742 A_55_P2001334 Gpr31c 8.906513735 A_55_P1998942 Oas1a 8.910124188 A_55_P2112500 Slco3a1 9.005146208 A_51_P423578 Slfn2 9.013862232 A_55_P1982628 1810029B16Rik 9.048275143 A_55_P2073452 Glp2r 9.052764974 A_51_P105380 2010005H15Rik 9.080239812 A_52_P627816 Tgm1 9.155172695 A_51_P334104 Dcn 9.212568427 A_55_P2264483 A730054J21Rik 9.232254536 A_55_P2059154 Dtx3l 9.254057095 A_51_P416137 Slc31a2 9.302977259 A_55_P1966731 Ifi203 9.402969967 A_55_P2059606 Stat2 9.413389559 A_55_P2010271 Samsn1 9.442911177 A_51_P389885 Spic 9.509177104 A_55_P2178578 Tmprss4 9.556421584 A_55_P1973415 Csprs 9.559245152 A_55_P1992889 Serping1 9.565848018 A_55_P2016034 Nlrc5 9.592716495 A_52_P332788 C130026I21Rik 9.687172621 A_55_P1962344 Trim21 9.74136066 A_55_P2282421 4933432I03Rik 9.770551516 A_55_P2108165 Gm6907 9.832969037 A_55_P2064771 Ly6c1 9.927796201 A_55_P2117656 Slfn8 9.963901986 A_55_P2182467 Slamf6 10.0071875 A_51_P463452 Acsl1 10.03304972 A_52_P223809 Dhx58 10.03706598 A_55_P2359797 Nod2 10.03990895 A_51_P504815 Stfa3 10.15992396 A_55_P2076757 Znfx1 10.22630262 A_51_P214747 Parp12 10.33820923 A_51_P187262 Mmp25 10.50658198 A_52_P472324 Slpi 10.51829821 A_55_P2011797 Sox30 10.63632972 A_55_P1971951 Cd86 10.6949677 A_51_P398766 Gbp1 10.75100259 A_55_P2147083 Tnfaip6 10.86470728 A_51_P114616 Batf 10.90066466 A_51_P327996 Ccl22 10.93946288 A_55_P2019601 Csprs 11.06648843 A_55_P1965000 Ddx58 11.12354659 A_51_P514712 Parp14 11.26411138 A_55_P2054491 Slc31a2 11.44724173 A_66_P135106 Slco3a1 11.48333431 A_55_P2100103 Htr7 11.55867162 A_51_P443403 Smoc1 11.59965909 A_52_P632601 Zfp811 11.63761243 A_55_P2110086 Rasgrp1 11.64157089 A_55_P2133195 Gm4951 11.71556235 A_52_P266132 Fgl2 11.74006507 A_51_P376238 Serping1 11.81734388 A_55_P2042988 Aoah 11.82449561 A_55_P1998843 Ifi203 11.88388818 A_55_P1978987 Ifih1 11.88772465 A_51_P496432 Acsl1 11.97700264 A_52_P99888 Cxcl16 11.98860649 A_52_P613241 Icam1 12.10003068 A_52_P385536 Ddx58 12.13179122 A_51_P265495 Ly6a 12.21471782 A_51_P193146 Ms4a6c 12.23782034 A_51_P488739 Niacr1 12.25937504 A_51_P517695 Ly6f 12.4726784 A_55_P2082961 Fnbp1l 12.47860252 A_55_P1978521 Gbp10 12.5024766 A_55_P2129354 Pilra 12.59837714 A_55_P2181222 Pydc3 12.60539429 A_52_P216613 Gpr18 12.60609644 A_55_P1990032 Cxcl5 12.69332618 A_51_P315391 Txlnb 12.78938778 A_55_P2064004 Rapgef2 12.82242298 A_55_P2088401 H2-T9 12.9531077 A_55_P2005783 Ifih1 12.95866235 A_55_P1959953 BC006779 13.14244835 A_51_P508259 Ppm1n 13.23899314 A_52_P56488 Zfp558 13.26828959 A_52_P466147 Rarres2 13.3058097 A_51_P159503 Rnf213 13.34679555 A_66_P105046 Il18 13.44252827 A_55_P2086279 Gm7609 13.46594246 A_51_P209183 Cxcl14 13.46632721 A_55_P2082180 Tnfrsf9 13.5755797 A_55_P2137049 AA467197 13.66321794 A_51_P202633 Ebi3 13.78483697 A_51_P324473 9130014G24Rik 13.82577359 A_51_P275454 Trim30a 13.93947866 A_51_P478722 Tgtp1 14.06194839 A_55_P2098971 Gm14085 14.10039233 A_51_P286357 Emr4 14.22598655 A_65_P17218 Mndal 14.32082211 A_55_P2004867 Serpinb2 14.39070374 A_55_P1981479 Irgm1 14.4458491 A_55_P2062246 Tgtp2 14.45939015 A_51_P262171 Irgm1 14.54076533 A_51_P172573 Sod2 14.60170264 A_51_P464918 Mefv 14.67782809 A_55_P2035018 Clec4n 14.72650015 A_55_P2129360 Ntng2 14.75995277 A_66_P116173 Il23r 14.84503408 A_55_P2048759 4930583H14Rik 14.92833085 A_55_P1982896 Lpcat2 14.98069758 A_66_P105585 Slfn8 15.00959454 A_55_P2107775 Apol9a 15.03418649 A_52_P199633 Trim30d 15.0850461 A_55_P2149083 Fpr-rs6 15.08730166 A_51_P254656 Hdc 15.19564491 A_55_P2001336 Gpr31c 15.2207815 A_51_P166728 Pou2f2 15.29289569 A_55_P2087628 LOC640793 15.46191385 A_55_P2042813 Gbp11 15.71241789 A_55_P2016099 Gpr31c 15.87484763 A_51_P405912 Lmcd1 15.95357339 A_52_P64514 Herc6 16.0188303 A_55_P2101585 Cd55 16.02101115 A_55_P2019719 Oas2 16.03173321 A_55_P2055587 Enpp4 16.31914378 A_55_P2192961 Trim30b 16.65971979 A_66_P133148 Aoah 16.86020882 A_55_P2011084 LOC100046632 16.95279683 A_51_P382152 Procr 17.07601962 A_52_P68893 Ifng 17.28027174 A_52_P483799 Pydc4 17.322174 A_55_P1970763 Pilrb2 17.83898344 A_51_P108020 Zmynd15 17.86379258 A_51_P233334 Stc1 17.88641934 A_51_P428372 Ppbp 17.88721531 A_66_P128761 Pydc3 17.89300261 A_51_P343833 Traf1 18.30754466 A_55_P1991812 Gm5431 18.34464232 A_51_P327657 H28 18.52788887 A_51_P213359 Has2 18.90553538 A_66_P112006 H2-T10 19.09255666 A_55_P1983668 Gm6377 19.36273844 A_55_P2183147 Tdgf1 19.62194842 A_51_P430766 Il10 19.6834659 A_55_P1966833 Xaf1 20.64536715 A_52_P386627 Irak3 20.91125944 A_55_P2018806 Trim30e-ps1 21.07910051 A_51_P452629 Tlr2 21.39305924 A_55_P2004562 Klra2 22.02537428 A_55_P2141943 Ube2l6 22.08104278 A_55_P2039359 Tnfsf11 22.27620239 A_52_P679860 Herc6 22.99864883 A_51_P255699 Mmp3 23.10908448 A_55_P2111302 Cp 23.23418606 A_51_P246066 Slamf9 23.95957645 A_55_P2129348 Pilra 24.26356107 A_52_P380369 D14Ertd668e 24.564034 A_55_P2026632 Pilrb1 24.69005252 A_52_P211956 Ms4a4d 25.57998033 A_51_P304170 Rtp4 26.01355382 A_55_P1974178 Pyhin1 26.08629229 A_51_P144180 Ifnb1 26.93523225 A_51_P265374 Vnn3 27.4483648 A_55_P2090617 Pilra 27.51324308 A_55_P1978465 H2-Q8 27.54844153 A_51_P509573 Ccl4 27.86152846 A_55_P2137611 Irgm2 28.00909181 A_51_P387123 Oasl2 28.09290867 A_51_P487690 Ifi44 28.42850643 A_55_P1969650 Rasgrp1 28.52035986 A_55_P1974189 Ptges 28.62890176 A_51_P257550 Marcksl1 28.652591 A_55_P2169247 Gm15056 28.71655937 A_55_P2038262 Gm4902 28.82752353 A_55_P2008740 Fcgr1 28.97969488 A_51_P254855 Ptgs2 29.12271795 A_51_P172853 Cd14 29.22729484 A_51_P472867 Oas3 29.76695719 A_55_P2073024 Gm4841 30.51235227 A_51_P420547 Clic5 30.64117996 A_66_P110633 Apol9b 30.71669307 A_51_P514085 Mx2 31.06717381 A_51_P385099 Tnf 31.08437572 A_55_P1988975 Ms4a4b 31.69342346 A_51_P492047 Fam3b 31.82471744 A_55_P2097219 Gbp1 32.61711962 A_51_P100852 Fam26f 32.63475945 A_51_P499838 Bst1 32.65468531 A_55_P2115401 Bst1 32.89965157 A_66_P101942 Gm9706 32.8998486 A_55_P2088033 Gm6904 33.41809755 A_51_P474459 Socs3 33.534061 A_51_P203955 Gbp2 35.22554201 A_51_P463846 Gbp7 36.29035941 A_55_P2015687 D14Ertd668e 37.15650086 A_51_P288138 Fpr2 37.27049048 A_51_P112355 Igtp 37.67714648 A_52_P203691 Arl5c 37.83124368 A_55_P1962918 Mnda 38.31130144 A_55_P1997756 Il6 38.40597579 A_55_P1977038 Cxcl9 38.43054684 A_55_P1994862 Ddx60 39.62792386 A_55_P2098398 Trim30c 40.59427855 A_51_P262515 Phf11 40.86506347 A_55_P2173892 Isg20 41.05001659 A_66_P139683 Zbp1 42.1716832 A_55_P2067518 Slc13a3 44.06144793 A_51_P140710 Ccl3 45.2371677 A_52_P203440 U90926 46.63009294 A_55_P1962400 Il1rn 47.43536851 A_66_P101835 Cd38 47.85684851 A_55_P2018017 Tnfsf10 52.53007357 A_55_P2114953 Usp18 56.78451662 A_55_P2044932 Gpr84 59.54424615 A_55_P2103698 Isg15 61.77374668 A_55_P2181963 Gm8369 62.60454095 A_66_P112573 Isg20 68.6538347 A_55_P2052385 Gbp6 75.51901277 A_55_P2052380 Gbp6 75.63008315 A_55_P2036627 Pydc3 77.33666355 A_55_P2135064 H2-M2 80.13855549 A_51_P432229 Gstt4 80.17787076 A_55_P2036357 Pyhin1 82.69900921 A_55_P2118441 Mx1 85.61088887 A_52_P670026 Rsad2 86.6898451 A_51_P421876 Irf7 87.16429007 A_55_P2103837 Gbp4 88.9894312 A_66_P128537 Isg15 100.7568769 A_55_P2035509 Pyhin1 101.7873383 A_55_P2472435 Gbp3 101.9376083 A_55_P2082319 BC094916 104.4920085 A_51_P371750 Marco 121.7635318 A_52_P1020860 AW112010 124.5708844 A_51_P217463 Cxcl2 127.7905284 A_55_P2180176 Ms4a6b 129.2112969 A_51_P312485 Fpr1 138.1310649 A_52_P638459 Ccl5 150.3835205 A_52_P100926 Il1a 151.7994023 A_55_P1972872 I830012O16Rik 162.5778495 A_52_P161488 Clec4e 164.8426327 A_51_P500082 Gm14446 173.2199156 A_51_P161021 Ifit2 182.4862053 A_51_P183812 Slfn4 183.8237673 A_55_P1990633 Iigp1 187.0503688 A_55_P2086433 Oasl1 187.3353565 A_55_P2158404 Cmpk2 189.363255 A_55_P2135986 Ms4a4c 194.409108 A_52_P186937 Cmpk2 195.6308929 A_51_P136294 Ms4a4b 235.0260888 A_55_P2016459 Cxcl10 242.3157719 A_51_P212782 Il1b 266.1898037 A_51_P123625 Irg1 312.4512063 A_51_P413866 Cfb 339.1411182 A_51_P359570 Ifit3 341.6351955 A_52_P232813 Cxcl3 367.2186702 A_52_P425890 Slfn1 433.7225971 A_66_P106388 Ms4a4c 506.7490609 A_51_P327751 Ifit1 517.1138015 A_51_P181286 Cd69 619.7589112 A_66_P117933 I830012O16Rik 821.2860596 A_51_P461665 Cxcl9 916.3510601 A_55_P1953169 Saa3 994.1424644 A_55_P2016462 Cxcl10 1412.42329