The Ron Receptor Tyrosine Kinase Regulates ...

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The Ron Receptor Tyrosine Kinase Regulates Macrophage Heterogeneity and Plays a Protective Role in Diet-Induced Obesity, Atherosclerosis, and Hepatosteatosis

J Immunol 2016; 197:256-265; Prepublished online 27 May 2016; doi: 10.4049/jimmunol.1600450 http://www.jimmunol.org/content/197/1/256

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Shan Yu, Joselyn N. Allen, Adwitia Dey, Limin Zhang, Gayathri Balandaram, Mary J. Kennett, Mingcan Xia, Na Xiong, Jeffrey M. Peters, Andrew Patterson and Pamela A. Hankey-Giblin

The Journal of Immunology

The Ron Receptor Tyrosine Kinase Regulates Macrophage Heterogeneity and Plays a Protective Role in Diet-Induced Obesity, Atherosclerosis, and Hepatosteatosis Shan Yu,*,†,1 Joselyn N. Allen,*,‡,1 Adwitia Dey,*,† Limin Zhang,* Gayathri Balandaram,*,x Mary J. Kennett,* Mingcan Xia,*,‡ Na Xiong,*,‡ Jeffrey M. Peters,*,x Andrew Patterson,* and Pamela A. Hankey-Giblin*,†,‡,x

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acrophages play a central role in regulating tissue homeostasis, inflammation, and the resolution of inflammation. Many tissue-resident macrophages arise from primitive progenitors in the yolk sac and fetal liver of the developing embryo (1–4). These cells seed tissues during embryogenesis and can be maintained, independent of contribution from the bone marrow, for the lifetime of the animal. These cells have stem cell–like properties and can proliferate in situ to maintain their numbers. Tissue-resident macrophages exhibit a wide range of functions depending on the needs of the tissue in which they reside *Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, PA 16802; †Graduate Program in Physiology, Pennsylvania State University, University Park, PA 16802; ‡Graduate Program in Immunology and Infectious Disease, Pennsylvania State University, University Park, PA 16802; and x Graduate Program in Molecular Medicine, Pennsylvania State University, University Park, PA 16802 1

S.Y. and J.N.A. are cofirst authors.

ORCIDs: 0000-0003-1974-0116 (A.D.); 0000-0002-7354-7539 (M.X.); 0000-00034405-6818 (N.X.); 0000-0002-4290-8709 (P.A.H.-G.). Received for publication March 23, 2016. Accepted for publication April 13, 2016. Address correspondence and reprint requests to Dr. Pamela A. Hankey-Giblin, Department of Veterinary and Biomedical Sciences, 115 Henning Building, Pennsylvania State University, University Park, PA 16802. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: ALT, alanine aminotransferase; ApoE, apolipoprotein E; Arg1, arginase 1; AST, aspartase aminotransferase; ATM, adipose tissue macrophage; DKO, double knockout; HCD, high-cholesterol diet; HDL, high-density lipoprotein; HFD, high-fat diet; iNOS, inducible NO synthase; KO, knockout; LDL, low-density lipoprotein; M1, inflammatory macrophage; M2, alternatively activated macrophage; MSP, macrophage-stimulating protein; NC, normal control; NMR, nuclear magnetic resonance; ORO, Oil Red O; VLDL, very LDL; WAT, white adipose tissue; WT, wild-type. Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600450

(1–3, 5). These cells tend to be broadly characterized as alternatively activated macrophages (M2) or reparative macrophages, whereas in response to infection or tissue damage, inflammatory macrophages (M1) predominate (6, 7). These two phenotypes are typified by the expression of inducible NO synthase (iNOS) and arginase 1 (Arg1), which share a common substrate, L-arginine (8–10). Arg1 converts L-arginine to polyamides and prolines promoting proliferation and matrix synthesis, whereas iNOS converts L-arginine to NO, which is cytotoxic (10, 11). However, the origin and/or plasticity of these cells remain loosely defined. The Ron receptor tyrosine kinase is expressed on tissue-resident macrophages, where it inhibits pathogen- and cytokine-induced inflammatory gene expression (12–17). NF-kB and Stat1 promote an inflammatory phenotype in macrophages by coordinately inducing inflammatory gene expression. Ron inhibits the activation of NF-kB in response to LPS by targeting the ubiquitination of TNFR-associated factor 6 and the nuclear localization of NF-kB (16, 18, 19). Ron also inhibits IFN-g–induced Stat1 phosphorylation, associated with the upregulation of the inhibitors of cytokine signaling, suppressor of cytokine signaling 1 and 3 (12, 20). Rondeficient mice exhibit exacerbated inflammation in vivo in response to endotoxin, increased liver damage in response to acetaminophen, and increased lung damage in response to nickel (13, 21, 22). Alternatively, Ron promotes some hallmarks of alternatively activated (M2) macrophage activation including the induction of Arg1 in an AP-1–dependent, Stat6-independent manner, as well as the upregulation of scavenger receptor-A and IL-b receptor antagonist (17). Although Ron also induces expression of Dectin-1, it does not induce the full range of M2-associated genes and, in fact, inhibits IL-4 induction of Ym1 and Fizz1 in vitro, suggesting that these cells may be more reparative in nature (17). In vivo, the


Obesity is a chronic inflammatory disease mediated in large part by the activation of inflammatory macrophages. This chronic inflammation underlies a whole host of diseases including atherosclerosis, hepatic steatosis, insulin resistance, type 2 diabetes, and cancer, among others. Macrophages are generally classified as either inflammatory or alternatively activated. Some tissueresident macrophages are derived from yolk sac erythromyeloid progenitors and fetal liver progenitors that seed tissues during embryogenesis and have the ability to repopulate through local proliferation. These macrophages tend to be anti-inflammatory in nature and are generally involved in tissue remodeling, repair, and homeostasis. Alternatively, during chronic inflammation induced by obesity, bone marrow monocyte-derived macrophages are recruited to inflamed tissues, where they produce proinflammatory cytokines and exacerbate inflammation. The extent to which these two populations of macrophages are plastic in their phenotype remains controversial. We have demonstrated previously that the Ron receptor tyrosine kinase is expressed on tissue-resident macrophages, where it limits inflammatory macrophage activation and promotes a repair phenotype. In this study, we demonstrate that Ron is expressed in a subpopulation of macrophages during chronic inflammation induced by obesity that exhibit a repair phenotype as determined by the expression of arginase 1. In addition, we demonstrate that the Ron receptor plays a protective role in the progression of diet-induced obesity, hepatosteatosis, and atherosclerosis. These results suggest that altering macrophage heterogeneity in vivo could have the potential to alleviate obesity-associated diseases. The Journal of Immunology, 2016, 197: 256–265.


FIGURE 1. Baseline animal characteristics and lipid profile. (A) Body weights of WT and Ron KO animals maintained on an HFD for 18 wk (n = 14–28). Representative epididymal WAT gross morphology, measured epididymal WAT (Ep; n = 21–30) (B) and liver weights of HFD-fed WT and Ron KO mice (n = 6) (C). (D) Fasting blood glucose of HFD-fed mice at indicated time points (n = 13–16). (E) An i.p. glucose tolerance test performed on HFD mice after an overnight fast (n = 6). Blood glucose was plotted against the time after the i.p. injection. Measured levels of cholesterol (n = 23), triglyceride (n = 11) (F), LDL/HDL ratio (n = 11) (G), ALT (n = 11), and AST (n = 11) (H) within serum collected from HFD-fed WT and Ron KO mice. Differences were considered significant at p , 0.05 and expressed as follows: *p , 0.05, **p , 0.01, ***p , 0.001.

could alleviate the chronic inflammation associated with obesity that leads to the development of obesity-associated disease (28). In this study, we show that Ron is expressed in ATMs, liver macrophages (Kupffer cells), and macrophages from atherosclerotic lesions in obese mice and that Ron expression generally correlates with an anti-inflammatory phenotype in these tissues. Ron expression in macrophages in chronic inflammation tightly correlates with the expression of Arg1, suggesting that Ron marks a subpopulation of macrophages in chronic inflammation in vivo that retain a reparative phenotype. Finally, we demonstrate that Ron deficiency results in accelerated development of insulin-resistance, hepatic steatosis, and atherosclerosis in obese animals. These results demonstrate that Ron not only marks a subpopulation of reparative macrophages in vivo, but also plays a protective role in the progression of chronic inflammation associated with obesity.

Materials and Methods Mouse models and diets Male wild-type (WT) and apolipoprotein E knockout (ApoE KO) mice on the C57BL/6 background were purchased from The Jackson Laboratory


absence of Ron in the myeloid lineage in the tumor microenvironment inhibits tumor growth and cytotoxic CD8+ T cell activation. Isolated tumor-associated macrophages from Ron-deficient mice exhibit significantly lower levels of Arg1 and increased phosphorylation of Stat3, suggesting that Ron promotes an alternatively activated phenotype that promotes tumor growth (17, 23). Obesity and obesity-associated disease has become the leading cause of death worldwide. It has become increasingly clear that obesity promotes a chronic inflammatory state, mediated in large part by macrophages, and that this chronic inflammation underlies many of the diseases associated with obesity (24–27). In lean adipose tissue, adipose tissue macrophages (ATMs) are largely M2 or reparative in nature (28). However, upon increasing lipid accumulation, there is insufficient blood supply and the adipocytes become necrotic. This promotes the accumulation of macrophages with an inflammatory signature. The resulting imbalance between the inflammatory versus repair functions of macrophages promotes an environment of chronic inflammation (29, 30). It has been speculated that the ability to promote a shift in the development and/or maintenance of the inflammatory versus reparative macrophages

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258 (Bar Harbor, ME). C57BL/6 mice with a targeted disruption of the Ron gene (Ron KO) by which the ligand binding domain of Ron is deleted (RON LBD) were generated as previously described (31). Mice deficient in both ApoE and Ron were acquired by crossing ApoE KO mice with Ron KO mice to generate ApoE KO 3 Ron KO (double KO [DKO]) mice. The genotypes of these mice were analyzed using PCR. WT, Ron KO, ApoE KO, and DKO mice were fed ad libitum a high-fat diet (HFD; diet number F3282; Bioserv), high-cholesterol diet (HCD; diet number F6334; Bioserv), or normal chow diet for the indicated number of weeks. On a caloric basis, HFD consisted of 36% fat from lard, 35.7% carbohydrate, and 20.5% protein, whereas the HCD was fortified with 1.25% cholesterol. These animals were maintained in a temperature-controlled room on a 12-h light/dark cycle. These experimental protocols were approved by the Institutional Animal Care and Use Committee at Pennsylvania State University. Mice were euthanized by CO2 narcosis. Blood was collected, and tissues were harvested for analyses.

Abs

Flow cytometric analysis Isolated cells were washed and preblocked of FcRs using anti-CD16/32 (Fcblock). Cells were then incubated with each mAb in 4˚C for 30 min. After cells were washed with PBS containing 2% FBS, secondary Ab was added as needed. Stained cells were analyzed on a BD LSR Fortessa (BD Biosciences), and the flow cytometric output was analyzed with FlowJo software (Tree Star).

Blood serum analysis Following CO2 asphyxiation, blood was immediately collected through cardiac puncture. Serum was separated by centrifugation and assayed for triglyceride, cholesterol, aspartase aminotransferase (AST), and alanine aminotransferase (ALT) using the IDEXX VetTest Chemistry Analyzer coupled with the IDEXX VetLab Station Laboratory Information Management System available at Central Biological Laboratories, Pennsylvania State University.

FIGURE 2. Ron expression on ATMs plays a protective role in the progression of obesity. (A) Correlation between body weight and Ron expression (%) on CD45+F4/80+ cell population isolated from the epididymal WAT stromal vascular fraction of WT and Ron KO animals (n = 14). (B Correlation between the fat weightto-body weight ratio and Ron expression (%) (n = 14). (C) Flow cytometry analysis of Ron expression on CD45+F4/80+ ATMs isolated from mice maintained on an HFD for 8 and 13 wk (n = 3–5). (D) Percentage of CD11c+ ATMs in fat tissue isolated from HFD-fed animals (n = 3–8). (E) Ron expression (%) within the CD11c+ and CD11c2 ATM fractions. *p , 0.05, **p , 0.01, ***p , 0.001.

Metabolic analysis Fasting glucose levels were measured on HFD-fed mice that were deprived of food (morning fast) for 5 to 6 h. Plasma glucose was measured through acquisition of a blood sample from the tail tip using a OneTouch Ultra blood glucose monitoring system (LifeScan Wayne, PA). Glucose tolerance tests were performed on age-matched mice fasted for 18 h. Glucose (2 g/kg) was administered i.p. Tail blood was collected at the indicated time points, and blood glucose concentration was determined on the OneTouch Ultra blood glucose monitoring system (LifeScan).

ATM isolation Epididymal white adipose fat pads were excised and weighed. The abdominal white adipose tissue (WAT) was washed in 13 PBS; connective tissue and blood clots were removed using forceps. The epididymal WAT was minced into small fragments (,10-mg pieces) using a razor blade and digested in Collagenase Type II digestion buffer (DMEM supplemented with 2.5% HEPES and 0.3% [3 mg/ml] Collagenase II at .125 collagen digestion units/mg) at 37˚C in a rotating incubator for 45 min. The digested suspension was filtered through a filter (size 70 mm) to remove debris and centrifuged at a speed of 300 3 g for 8 min at 4˚C. The pelleted stromal vascular cells were washed, and present erythrocytes were lysed with treatment with ACK buffer (150 mmol NH4Cl, 10 mmol KHCO3, and 0.1 mmol EDTA). The cells were washed in 13 PBS and stained with macrophage-specific mAbs. The ATMs were assessed by flow cytometry on the BD LSR Fortessa (BD Biosciences), and the flow cytometric was analyzed with FlowJo software (Tree Star).

Nuclear magnetic resonance spectroscopy Blood serum samples were prepared as previously documented (32). Briefly, 200 ml collected serum samples were mixed with 400 ml saline solution containing 30% D2O. The samples were then vortexed, centrifuged (11,180 3 g, 10 min, 4˚C), and 550 ml sample was transferred into 5-mm nuclear magnetic resonance (NMR) tubes. [1H] NMR spectra of the prepared serum was acquired, and spectral data processing and multivariate analysis was performed as previously described (32). The low-density lipoprotein (LDL) plus very LDL (VLDL)/high-density lipoprotein (HDL) ratio was calculated by using values derived from normalizing the NMR peak area normalized to total integration for each sample.

Aortic cell isolation Aortic cells were isolated as described previously (31). Briefly, aortas were flushed with heparinized PBS, and the thoracic aorta was collected. Excess fat and connective tissue associated with the aortas were cleaned. Singlecell suspensions were prepared from the removed aorta by incubation with


For flow cytometry and FACS, CD16/32 Fc-block, PE-conjugated anti-F4/ 80, PECy7-conjugated anti-CD11c, Pacific Blue–conjugated anti-CD45, and the isotype controls for CD11c (PECy7 Armenian Hamster IgG) and F4/80 (PE rat IgG2a) were purchased from BioLegend (San Diego, CA), and PECy5–anti-CD11b was purchased from eBioscience (San Diego, CA). Anti-mouse Ron was purchased from R&D Systems (Minneapolis, MN), isotype control for Ron (goat IgG) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the secondary Ab Alexa 647–conjugated anti-goat for Ron and Texas Red live/dead fixable stain was purchased from Molecular Probes (Eugene, OR).

RON REGULATES MACROPHAGE ACTIVATION IN OBESITY

The Journal of Immunology an enzymatic mixture 20 mmol/l HEPES, 1.5% BSA, 0.1% collagenase type II (Life Technologies), 125 U/ml collagenase type XI, 450 U/ml collagenase type I, 60 U/ml hyaluronidase type I, and 60 U/ml DNase I (Sigma-Aldrich) for an hour at 37˚C with gentle shaking. The isolated cells were counted and used for analysis. For flow cytometry, cells were stained with the indicated fluorochrome-conjugated Abs. Cells were characterized on a flow cytometer BD LSR Fortessa (BD Biosciences), and analyzed flow cytometric output was analyzed with FlowJo software (Tree Star). To purify aortic macrophages, cells were stained by various combinations of macrophage-specific mAbs and sorted using the BD Influx sorter (BD Biosciences). Cells sorted from two to three aortas were pooled for realtime PCR analysis.

En face Oil Red O staining and immunohistochemistry

Kupffer cell isolation Kupffer cells were isolated by collagenase perfusion of liver as previously described (33). Livers were first perfused with Buffer I (142 mmol NaCl,

FIGURE 3. Ron activation attenuates accelerated aortic atherosclerosis in HCD-fed ApoE KO mice. (A) Serum cholesterol and triglyceride levels were measured in ApoE KO and DKO mice maintained on an HCD for the indicated number of weeks (n = 6–13). (B) NMR spectroscopy analysis of ApoE KO and DKO mice sera; n = 8. ORO-stained aortas isolated from ApoE KO and DKO mice maintained on an HCD for 13 wk (n = 6–10) (C) and 18 wk (n = 6) (D) and quantification of plaque area. Mean (6 SEM) area data are expressed as percent of total area of the thoracic aorta. (E) Representative OROstained aortic cryosections from HCD-fed ApoE KO and DKO mice (n = 7). Scale bar, 500 mm. Values are represented as mean 6 SEM and were obtained by using Student t test analysis. *p , 0.05, **p , 0.01.

6.4 mmol KCl, 9.6 mmol HEPES, and 30 mmol NaOH), followed by perfusion with the Buffer I solution supplemented with 4.76 mmol CaCl 2 and collagenase type IV (Worthington Biochemical, Lakewood, NJ) (Buffer II). The livers were then gently minced, suspended in the Buffer I solution supplemented with 1% BSA (Buffer III), and filtered through a 70-mm nylon cell strainer. Parenchymal cells were removed by low-speed centrifugation at 54 3 g for 3 min. Nonparenchymal cells were blocked with Abs against CD16/32 to decrease nonspecific binding. Next, cells were stained with a combination of macrophage-specific Abs and purified by a BD Influx sorter (BD Biosciences).

Liver histology and immunohistochemistry Liver sections were preserved in 10% neutral buffered formalin or embedded in Tissue Tek OCT compound and frozen. Cryosections (7 mm) of the liver were stained with ORO. Formalin-fixed sections were processed and stained with H&E (at Pennsylvania State University Animal Diagnostic Laboratory, University Park, PA). The unstained vacuoles were visible in H&E-stained liver sections of HCD-fed mice, and the red-stained droplets represent ORO-stained neutral fat. For analysis of liver lipid accumulation, H&E and ORO sections were examined and scored blindly by a board-certified laboratory animal veterinarian with training in pathology. Scoring of the lesions were as follows: 0, no lesions, or within normal limits; 1, very few lesions, minimally affected, a visual estimate that is ,20% of the tissue was affected; 2, mild, ∼21–40% affected; 3, moderate, ∼41–60% affected; 4, marked lesions, ∼61–80% affected; and 5, severe or extensive lesions, ∼80–100% affected. Representative images were obtained using an Olympus BH-2 light microscope (Olympus) attached to a Nikon digital camera (DXMI200F; Nikon).


Mice were euthanized, and excised thoracic aortas were used for en face staining with Oil Red O (ORO) according to the manufacturer’s instructions (American MasterTech, Lodi, CA). The aorta was thoroughly cleaned of adventitial fat and rinsed in propylene glycol. The inner surface of the aorta was stained for 2 min, rinsed in 85% propylene glycol, and returned to distilled water. The ORO-stained area was quantified by ImageJ (National Institutes of Health) analysis of digitized microscopic images. Results were expressed as a percentage of the ORO-stained lesion area of the total aortic area analyzed. Frozen cross sections of ORO-stained samples were obtained using cryomicrotome. The quantification of lesion size for each section was performed using ImageJ software (National Institutes of Health).

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260 RNA isolation and quantitative real-time PCR analysis mRNA was extracted from whole liver, whole aortas, primary Kupffer cells, primary aortic macrophages, and other mentioned cell types using TRIzol Reagent (Invitrogen) as instructed by the manufacturer. RNA was quantified by A260 absorbance. For cDNA synthesis, 2 mg RNA was reverse transcribed using the High Capacity Reverse transcription Kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed on the resultant cDNA using FAM-labeled TaqMan probes (Applied Biosystems) and the 7900HT Fast Real-Time PCR System (Applied Biosystems). All TaqMan probes used for real-time PCR were purchased from Applied Biosystems. Results were expressed as threshold cycle values normalized to Gapdh (a housekeeping gene).

Statistics

Results Ron is expressed primarily in resident macrophages in adipose tissue and plays a protective role in the progression of obesity and insulin resistance Chronic inflammation associated with obesity underlies many obesity-associated disorders including insulin resistance, atherosclerosis, and hepatic steatosis. The degree of inflammation at these sites correlates with the ratio of M1/M2 macrophages as broadly defined, and an imbalance of these populations results in disease progression (34–36). We have previously shown that Ron regulates the balance of inflammatory versus reparative macrophages both in vitro and in vivo (14–16). In order to determine whether Ron plays a role in the progression of obesity-associated disease, we placed 6-wk-old male mice on an HFD for up to 18 wk. Although

FIGURE 4. Functional loss of Ron stimulates proinflammatory cytokine expression in the aorta. Expression of hallmark genes that are of classically activated (Inos, Il-12b, Tnf, Il-1b, and Il-6) or alternatively activated (Arg1) macrophage phenotype was assessed in total aorta extracts (n = 5) (A) and sorted live CD45+F4/80+ aortic cells (n = 18) (B) that were isolated from ApoE KO and DKO mice maintained on either an HFD or HCD. Values are represented as mean 6 SEM and were obtained by using Student t test analysis. *p , 0.05, **p , 0.01.

there was no significant difference in body weights of WT and RON KO mice when maintained on regular chow, Ron KO mice maintained on an HFD gained significantly more weight by 5 wk, and the weights remained significantly higher through the 18-wk period when compared with WT controls (Fig. 1A). The Ron KO mice maintained on an HFD also exhibited increased adiposity at 18 wk when compared with control animals (Fig. 1B) as well as increased levels of fasting blood glucose (Fig. 1D) and reduced glucose tolerance (Fig. 1E). The liver weights of these animals were not significantly different between genotypes (Fig. 1C), and there was no significant difference in liver damage as measured by serum levels of AST and ALT (Fig. 1H). Although there were no differences in serum cholesterol or triglyceride levels (Fig. 1F), the percentage of LDL and VLDL versus HDL were significantly different, with Ron KO mice exhibiting increased VLDL and LDL, whereas WT mice exhibited higher levels of HDL (Fig. 1G, Supplemental Fig. 1A). When we compared the body weight or fat weight/body weight of Ron KO mice with the percentage of Ron+ ATMs, there was a significant negative correlation, indicating that increased body weight correlates with a decrease in Ron+ ATMs (Fig. 2A–C). CD11c is commonly used as a marker of inflammatory macrophages in adipose tissue. Consistent with other studies, we demonstrate that the number of F4/80+CD11c+ ATMs increases with increased body weight (Fig. 2D). When comparing Ron expression on F4/80+ CD11c+ inflammatory ATMs versus F4/80+CD11c2 resident ATMs, we found that Ron expression is primarily observed in the F4/80+ CD11c2 resident ATMs (Fig. 2E). We examined the percentage of Ron+ ATMs with weight gain at 8 and 13 wk on an HFD. Both F4/80+ Ron+ ATMs were reduced with increased weight over time in mice on an HFD or normal control (NC) diet; however, we observed a significant decrease in the percentage of both F4/80+ ATMs expressing Ron following 13 wk on an HFD when compared with the percentage


Values are expressed as mean 6 SEM. Statistical analysis was performed using unpaired Student t test, paired Student t test, or one-way ANOVA. Differences were considered significant at p , 0.05 (*p , 0.05, **p , 0.01, ***p , 0.001). All analysis was performed using GraphPad Prism 5.0 (GraphPad, San Diego, CA).


The Journal of Immunology of Ron+ ATMs from mice maintained on an NC (Fig. 2C). These results suggest that Ron is primarily expressed on more anti-inflammatory ATMs and that the percentage of these cells decreases with increasing obesity. Ron is expressed in reparative aortic macrophages and plays a protective role in the development of atherosclerosis

FIGURE 5. Accelerated atherosclerosis results in decreased Ron-mediated antiinflammatory response in the aorta. (A) Gating scheme of the live CD45+ cell fraction derived from ApoE KO mice maintained on either an NC or HCD for 18 wk. (B) The gating strategy used to identify Ron expression in the live CD45+F4/80+ fraction from aortic cell suspension isolated from ApoE KO mice maintained on either an NC or HCD for 18 wk. (C) Ron expression was assessed on gated F4/80+ cells of the live CD45+ population; n = 8 to 9. (D) Expression of genes involved in alternative activation (Arg1) and classical activation (Inos) in Ron-expressing (Ron+) and Ron nonexpression (Ron2) live CD45+F4/80+ macrophages that were isolated from aortas of ApoE mice maintained on an HCD (n = 4). (E and F) Flow cytometric analysis shows increased abundance of classically activated macrophages (F4/80+CD11c+) in the digested atherosclerotic lesions of HCD-fed mice compared with mice fed an NC diet (n = 9). Live C545+F4/80+CD11c+ populations showed decreased Ron expression when aortic extracts were analyzed (n = 9). Data are represented as the mean 6 SEM. *p , 0.05, ***p , 0.001. FSC, forward light scatter; SSC, side scatter.

ApoE KO and DKO mice on an HCD for 13 or 18 wk, and aortas were stained with ORO to examine the extent of lipid deposition. Although we did not see a significant difference in lipid accumulation in the aortas of ApoE KO and DKO mice following 13 wk on an HCD (Fig. 3C), we observed a significant difference in lipid deposition in the DKO mice compared with ApoE KO controls by 18 wk as measured using ImageJ (National Institutes of Health) (Fig. 3D). To further confirm these results, we examined the levels of lipid deposition in sections of aortic roots from ApoE KO and DKO mice by staining with ORO. Again, we observed a significant increase in lipid deposition in the DKO mice compared with ApoE KO animals following 18 wk on an HCD as measured by ImageJ (National Institutes of Health) (Fig. 3E). These results indicate that Ron plays a protective role in the severity of atherosclerosis at later stages of disease progression. In order to determine whether the increased progression of atherosclerosis in the DKO mice is associated with an increase in inflammatory gene expression, we isolated aortic arches from ApoE KO and DKO mice maintained on an HCD for 23 wk and examined the expression of proinflammatory mediators by realtime PCR. Consistent with the increase in progression of atherosclerosis in DKO animals, we observed a significant increase in the


In order to determine whether Ron regulates the progression of atherosclerosis and hepatic steatosis, we crossed the Ron KO animals with ApoE2/2 (ApoE KO) mice on the C57BL/6 background to generate Ron2/2 3 ApoE2/2 DKO mice. ApoE KO and DKO mice were placed on an HCD for various time points. In contrast to the single Ron KO mice, DKO mice did not exhibit increased weight gain when compared with ApoE KO animals when maintained on an HCD (data not shown). Consistent with previous results, we did not observe a significant difference in overall triglyceride or cholesterol levels (Fig. 3A). Interestingly, like the single KO animals, the DKO mice exhibited a significant increase in the levels of VLDL and LDL, whereas the ApoE KO mice exhibited higher levels of HDL (Fig. 3B). In order to determine whether the absence of Ron affects the development of atherosclerosis in ApoE KO mice, we maintained

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FIGURE 6. Protective role of Ron in reducing hepatic lipid deposition and liver injury in HCD-fed ApoE KO mice. (A) Comparison of serum transaminase levels (ALT and AST) and ratio of liver weight to body weight for ApoE KO (n = 10) and DKO (n = 10) mice fed HCD for indicated times. (B) Representative gross morphology of ApoE KO and DKO mouse livers, H&E staining, and ORO staining of liver sections. Scale bar, 100 mm. Pathological scoring of ORO (n = 5 to 6) (C) and H&E staining (D) of livers isolated from ApoE and DKO mice maintained on HCD for 18 wk (n = 5 to 6). (E) Gene expression analysis of key proinflammatory (Il-12b, Tnf, Il-1b, Il-6, and Inos) and anti-inflammatory (Arg1) genes in total liver extracts isolated from ApoE KO and DKO mice that were administered an HCD for 18 wk (n = 5 to 6). Values are expressed as mean 6 SEM and were obtained by using Student t test analysis. *p , 0.05, **p , 0.01.

LPS in the absence of MSP when compared with the MSP-treated conditioned medium (Supplemental Fig. 2C, 2D). This suggests that any contribution of endothelial cells to the overall increase in proinflammatory cytokine production is likely indirect, due to the response of these cells to macrophages in the absence of Ron. In order to examine the percentage of Ron-expressing macrophages, we maintained ApoE KO mice on an NC diet or HCD for 18 wk. Consistent with our previous results, we found that the overall percentage of F4/80+Ron+ macrophages was decreased in mice maintained on an HCD compared with a control diet. However, the overall numbers of F4/80+Ron+ cells was not significantly different, suggesting that the decrease in Ron expressing aortic macrophages in obese animals is potentially due to the influx of monocyte-derived macrophages that do not express Ron (Fig. 5A–C). In order to determine whether there are significant differences in the expression of Arg1 (a marker of reparative macrophages) in the presence or absence of Ron, we isolated F4/ 80+Ron2 and F4/80+Ron+ aortic macrophages from ApoE mice maintained on an HCD for 18 wk and examined Arg1 expression by real-time PCR (Fig. 5D). We observed a significant decrease in Arg1 expression in aortic macrophages lacking Ron when compared with Ron-expressing aortic macrophages, suggesting that Ron promotes the expression of Arg1 in aortic macrophages in vivo in


expression of iNOS, IL-12b, TNF-a, IL-1b, and IL-6 in the DKO aortas compared with ApoE KO controls (Fig. 4A). To determine whether this increase is due, in part, to an increase in expression of these inflammatory mediators in macrophages, we isolated CD45+ F4/80+ cells from the aortas of ApoE KO and DKO mice maintained on a HCD for 23 wk. Although we saw a trend in increased expression in all of the inflammatory markers, we observed a significant increase in the levels of IL-1b and TNF-a in macrophages isolated from aortas of DKO mice compared with ApoE KO controls (Fig. 4B). We also observed a trend of increased expression for IL-1b, IL-6, and TNF-a and a significant increase in iNOS expression in aortic endothelial cells isolated from DKO animals when compared with control ApoE KO animals (Supplemental Fig. 2A). To determine whether Ron could also directly regulate cytokine gene expression in endothelial cells, we stimulated the endothelial cell line MS1 with LPS directly in the presence or absence of macrophage-stimulating protein (MSP) or with conditioned medium from macrophages stimulated with LPS in the presence or absence of MSP. We found that there was no direct effect of MSP on endothelial cell expression of proinflammatory cytokines; however, there was a significant increase in endothelial proinflammatory gene expression in endothelial cells treated with conditioned medium from macrophages stimulated by


The Journal of Immunology obese animals. Consistent with previous studies, we also observed a significant increase in infiltrating F4/80+CD11c+ cells in the aortas of mice maintained on an HCD for 18 wk when compared with mice receiving a control diet (Fig. 5E, 5F). Although the percentage of CD11c+ cells expressing Ron is much lower in this population of macrophages (∼10% in CD11c+ macrophages versus ∼30% in the total macrophage population), the percentage of CD11c+Ron+ macrophages decreased in mice maintained on an HCD for 18 wk (Fig. 5F). Ron is expressed on reparative macrophages in the liver and plays a protective role in the progression of hepatic steatosis

FIGURE 7. Hepatocellular Ron expression attenuates inflammation in HCD-fed ApoE KO mice. (A) Representative gating scheme for flow cytometric sorting of CD45+F4/80+ populations from livers WT mice fed the HCD for 18 wk. (B) Ron-expressing (Ron+) and nonexpressing (Ron2) CD45+F4/80+ sorted cells were assayed for Arg1 and Inos expression by quantitative real-time PCR. Data are represented as mean 6 SEM (n = 4). Student t test. (C) Analysis of gene expression in sorted CD11c2CD45+ F4/80+ or CD11c+CD45+F4/80 macrophages from livers in HCD-fed mice. Values are shown as mean 6 SEM (n = 9), and statistical analysis was performed using a paired t test. *p , 0.05, ***p , 0.001.

ApoE KO mice on an HCD for 18 wk and isolated F4/80+ Kupffer cells (Fig. 7A). These cells were then divided into F4/80+Ron+ and F4/80+Ron2 fractions, and the expression of Arg1 and iNOS was assessed by real-time PCR. We observed a significant increase in Arg1 expression in the F4/80+Ron+ Kupffer cells isolated from the liver of ApoE KO mice, whereas the F4/80+Ron2 fraction of Kupffer cells expressed significantly elevated levels of iNOS when compared with Ron-expressing Kupffer cells (Fig. 7B). Using CD11c as a marker of inflammatory macrophages, we compared the expression of iNOS, Arg1, and Ron in F4/80+ macrophages isolated from ApoE mice maintained on an HCD for 18 wk. We observed a significant correlation between Ron expression in F4/ 80+CD11c2 compared with F4/80+CD11c+ Kupffer cells (Fig. 7C). Similarly, there was a significant correlation between Arg1 expression and the F4/80+CD11c2 cells, whereas there was a significant inverse correlation between the expression of iNOS and the F4/80+CD11c2 resident Kupffer cell population. These results indicate that Ron is expressed largely on Kupffer cells with a reparative phenotype in obese animals.

Discussion Recent studies suggest that macrophages play an important role in regulating many chronic inflammatory diseases associated with obesity. In lean and healthy individuals, resident macrophages present in tissues exist in a resting state or exhibit an M2-like reparative phenotype and are important in maintaining immune function and tissue homeostasis (34). In response to accumulation of lipids due to dietary intake, tissue-resident macrophages or infiltrating monocyte-derived macrophages are activated and exhibit a proinflammatory phenotype, producing inflammatory cytokines and cytotoxic molecules (37, 38). Consequently, it has been suggested that a switch from a proinflammatory phenotype to an antiinflammatory M2-like phenotype could alleviate the progression of disorders associated with obesity (37, 39, 40).


In order to determine whether Ron plays a role in the progression of hepatic steatosis, we maintained ApoE KO and DKO mice on an HCD for 18 wk. At 18 wk, but not before, we observed a significant difference in the serum levels of ALT (a marker of liver damage) and in overall liver weight when compared with total body weight (Fig. 6A). The livers from the DKO mice were also paler in appearance and exhibited increased liver damage by H&E staining and increased lipid deposition as assessed by ORO staining when compared with ApoE KO livers (Fig. 6B–D). In order to determine whether this increase in liver damage in the absence of Ron is associated with increased inflammation, the expression of proinflammatory cytokines was assessed in total liver from ApoE KO and DKO mice maintained on an HCD for 18 wk. We observed significant increases in IL-12b and TNF-a expression in the livers from DKO mice when compared with ApoE control animals (Fig. 6E). We also observed a significant decrease in the expression of Arg1 in livers from DKO mice compared with ApoE KO control animals (Fig. 6E); however, this could also be due to differences in Arg1 expression by hepatocytes. These results indicate that Ron plays a protective role in the progression of hepatic steatosis in obese animals. In order to determine whether Ron expression in Kupffer cells in the liver is associated with reparative macrophages, we maintained

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embryogenesis from a distinct progenitor that develops independently of hematopoietic stem cells. It will also be important to determine whether Ron expression in resident macrophages could promote the resolution of inflammation and tissue repair. In the studies described in this paper, we have focused primarily on Ron expression and function in macrophages. However, Ron is expressed on other cells including endothelial cells and hepatocytes. It will be important to determine whether Ron expression in these cells types contributes to the protective role of Ron in atherosclerosis and hepatic steatosis. It is likely there is extensive interplay between macrophages and other cell types expressing Ron during both the progression and resolution of disease. It will also be essential to determine whether Ron plays a similar role in human tissue-resident macrophages. All of these questions remain unanswered and will be important next steps in determining whether targeting Ron expression and/or function in macrophages might be a viable therapeutic approach to limit chronic inflammation and promote tissue repair.

Disclosures The authors have no financial conflicts of interest.

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We and others have previously shown that activation of Ron by its ligand, MSP, tips the balance of macrophage activation by inhibiting proinflammatory macrophage activation and inducing some hallmarks of an M2-like or reparative phenotype in vitro (16). In vivo, Ron attenuates inflammation and tissue damage in a number of animal models of M1-mediated inflammation. In response to LPS, Ron-deficient animals exhibit elevated production of IL-12p40 in the spleen and enhanced levels of IFN-g in the circulation (14). We have shown that the increased susceptibility of Ron-deficient mice to endotoxic shock is ameliorated when the mice are crossed with IFN-gR KO mice, suggesting that increased susceptibility to septic shock in the absence of Ron is mediated in part by elevated levels of IFN-g. Furthermore, deletion of Ron specifically in the myeloid lineage resulted in increased lung damage in response to LPS, associated with elevated levels of TNF-a (22). Ron has also been shown to play a protective role in the development of dextran sulfate sodium–induced colitis, and the absence of Ron is associated with enhanced inflammatory cytokine production in this model (41). Ron is also protective in a murine model of multiple sclerosis, and CNS levels of TNF-a are elevated in the absence of Ron (42). In this study, we demonstrate that the absence of Ron results in increased weight gain and reduced glucose tolerance when maintained on an HFD. We also demonstrate that Ron plays a protective role in the progression of atherosclerosis and hepatic steatosis in these animals and that the absence of Ron is associated with enhanced proinflammatory cytokine expression. Taken together, there is mounting evidence that Ron is a critical regulator of inflammatory responses in vivo in a wide range of tissues. Conversely, a recent study has found that the deletion of Ron results in reduced weight gain and decreased liver damage in mice when maintained on an HFD. Although other studies performed in these two mouse models are largely confirmatory, the reason for this discrepancy is unclear. These contrasting results could be due to differing strains of mice (C57BL/6 versus FVB). The differences in phenotype could also be due to the way in which Ron was targeted in these two mouse models. In the mice used in this study, the ligand binding domain of Ron is deleted consequently macrophages from these mice fail to respond to MSP, whereas in the previously published study, the kinase domain of Ron is deleted (RON KD). The only known KO of the entire Ron gene results in embryonic lethality. The results shown in this study are consistent with the phenotype of mice with a targeted deletion of the ligand for Ron, MSP, which develop hepatic steatosis. In addition, a number of recent reports point to a beneficial role for MSP in regulating hepatic lipid and glucose homeostasis, and a potential therapeutic use of MSP in treating metabolic syndrome has been suggested (43). There is extensive cross-talk between Ron and the closely related Met receptor and its ligand, hepatocyte growth factor, which plays a critical role in the liver (44). It would be interesting to determine whether the differences in the hepatic phenotypes of these two models are due to indirect effects of these two targeting strategies on the expression or function of MET in hepatocytes. Although we know that Ron is expressed in tissue-resident macrophages, but not monocyte-derived macrophages in vitro, it is unclear during chronic inflammation whether these cells can phenotypically switch. In studies of acute inflammation, although both Kupffer cells and monocyte-derived macrophages contribute to tissue repair, it has been shown that infiltrating monocyte-derived macrophages do not ultimately replace resident Kupffer cells following liver recovery. It will be interesting to determine whether Ron expression is unregulated in monocyte-derived macrophages during inflammation and/or repair in vivo or whether Ron expression is restricted to the resident cells that are generated during



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Supplemental Figure 1

A Ron KO

WT

B ApoE KO DKO

duration of feeding (wk)

Supplemental Figure 1 Baseline metabolic analysis and NMR output in DIO and atherosclerosis (A) NMR analysis of blood serum collected from WT and Ron KO mice maintained on a HFD for 18 weeks (n=10-11). (B) Measured body weights of ApoE KO and DKO mice fed a HCD for 18 weeks (n=7-11).

Supplemental Figure 2 A

B

MS1

(CD45-CD31+) aortic endothelial cells

Anti-Ron

Ron

Count

DAPI Isotype Unstained

Ron

C

Il12b

Il1b

Il6

Tnf

Inos

MSPMSP+

D

Il12b

Il1b

Il6

Tnf

Inos

MSPMSP+

Supplemental Figure 2 Ron expression regulates inflammatory response in endothelial cells (A) Live CD45-CD31+ endothelial cells were sorted from collagenase digested aortas of ApoE KO (n=6) and DKO (n=8) mice that were fed a HCD for 18 weeks. Endothelial expression of inflammatory genes Il1b, Il6, Tnfα, and Inos was analyzed using quantitative real-time PCR. (B) Ron expression on primary aortic endothelial cells and Miles Sven 1 (MS1) endothelial cells. (C) MS1 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum stained with anti-Ron and counterstained with DAPI. (D) Mature quiescent macrophages were isolated from the peritoneal cavity of mice by intraperitoneal lavage and cultured under 5% CO2 at 37оC for 2-3 hours in DMEM supplemented with 10% fetal bovine serum prior to stimulation. Primary peritoneal macrophages were stimulated with or without 100 ng/ml MSP overnight before stimulation of 100 ng/ml LPS for 12 hours. Macrophages were incubated with serum free media for 42 h before the media was collected. Endothelial cells were treated with macrophage-conditioned media for 4 h before RNA collection. Gene expression was analyzed by quantitative real-time PCR. Data C-D represents results of three independent experiments.