Serum-Glucocorticoid Regulated Kinase 1 Regulates Alternatively Activated Macrophage Polarization Contributing to Angiotensin II–Induced Inflammation and Cardiac Fibrosis

Antibodies and Reagents

The antibodies for phospho-STAT1, STAT1, phospho-STAT3, and STAT3 were from Cell Signaling Technology (Beverly, MA); the antibodies for F4/80, cardiac myosin heavy chain, α-smooth muscle actin (α-SMA), and SGK1 were from Abcam (Cambridge, MA); and the antibodies for Mac-2, CD206, CTGF, transforming growth factor-β (TGF-β), interleukin-10 (IL-10), IL-13, GAPDH, and IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). ChemMate TM EnVision System/DAB Detection Kits were from Dako (Glostrup, Denmark). Antibodies for PerCP/Cy5.5-conjugated CD45.2, phycoerythrin (PE)-conjugated F4/80, fluorescein isothiocyanate (FITC)-conjugated F4/80, PE-conjugated CD11b, FITC-conjugated CD206, PE-conjugated CD3, FITC-conjugated CD4, FITC-conjugated CD8, and isotype control were from Biolegend (San Diego, CA). Ang II was from Sigma (Sigma-Aldrich, St.Louis, MO). Penicillin, streptomycin, DMEM, and fetal bovine serum were from Invitrogen (Carlsbad, CA). The protein assay kit was from Bio-Rad (Hercules, CA).

Animals and Treatment

The SGK1 knockout (SGK1^−/−) mouse strain was backcrossed onto the genetic background of wild-type (WT) B6/129S mice for >10 generations, and littermates were used as controls. Mice were 10 to 12 weeks old at the beginning of the experiments and matched for age and sex with WT mice. We infused 10- to 12-week-old male mice (n=6–10 per group) for 7 days with vehicle (saline) or a pressor dose of Ang II (1500 ng.kg⁻¹.min⁻¹) by osmotic minipumps (ALZET Model 1007D, DURECT, Cupertino, CA) implanted subcutaneously. Systolic blood pressure was measured by tail-cuff plethysmography (Visitech Systems, Apex, NC) as a control for efficient Ang II infusion; in this setting, the tail-cuff method is comparable with the telemetry method and is sufficient as we and other investigators reported.^15–17

After the study period, Ang II– and saline-treated mice were anesthetized, and the LV was flushed by puncture with 20-mL saline to remove blood from systemic circulation. The hearts were removed and prepared for further histological and molecular analyses. Mouse protocols were approved by the Institutional Animal Care and Use and Committee of the Capital University of Medical Science (Beijing, China).

Echocardiography

Mice were killed with 2% to 4% isoflurane. Echocardiography involved use of a Vevo 770 High-Resolution Imaging System (VisualSonics Inc) as described.¹⁸ All measurements were averaged for 5 consecutive cardiac cycles and determined by an experienced technician blinded to treatment group.

Histology and Immunohistochemistry

Heart tissues were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 5-μm intervals. Hematoxylin-eosin and Masson trichrome staining involved standard procedures. Picrosirius red staining was used for the histological assessment of collagen types I and III accumulation by polarization microscopy. Wheat germ agglutinin staining was used to evaluate myocyte cross-sectional area. Heart sections were deparaffinized and incubated with 100-μg/mL FITC-labeled wheat germ agglutinin (Sigma) for 90 minutes. The cell area was calculated by measuring at least 200 cells per slide.

Immunohistochemistry of paraffin sections involved the Dako ChemMate TM EnVision System (Dako) with primary antibodies for SGK1 (1:200), α-SMA (1:200), CTGF (1:200), Mac-2 (1:200), TGF-β (1:300), IL-13 (1:300), and IL-10 (1:300). Negative controls were omission of the primary antibody, goat nonimmune IgG, rabbit nonimmune IgG, rat nonimmune IgG, or secondary antibody only; in all cases, negative controls showed insignificant staining. To verify the genotype and treatment, quantitative histology involved standard procedures, and results were confirmed by independent pathologists blinded to genotype or treatment group. Images were viewed and captured using a Nikon Labophot 2 microscope equipped with a Sony CCD-IRIS/RGB color video camera attached to a computerized imaging system and analyzed by Image Pro Plus 3.0 (ECLIPSE 80i/90i; Nikon, Tokyo, Japan). The expression of collagen, α-SMA, CTGF, macrophage markers, or cytokines was calculated as proportion of positive area to total tissue area for all measurements of the section.

Cells and frozen heart sections (7 μm) were incubated with the primary antibodies for F4/80 (1:100), cardiac myosin heavy chain (1:100), SGK1 (1:200), CD206 (1:200), phospho-STAT3, and α-SMA (1:200) at 4°C overnight and then with FITC- or tetramethylrhodamine isothiocyanate–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) at room temperature for 1 hour. Sections were viewed with a confocal laser scanning microscope (TCS 4D; Leica, Heidelberg, Germany) and a Nikon Labophot 2 microscope equipped with a Sony CCD-IRIS/RGB color video camera.

Flow Cytometry

The content of inflammatory cells was quantified by flow cytometry as described.¹⁹ Briefly, heart tissues were cut into multiple small cubes and digested in an enzyme mixture containing collagenase type I (0.05 mg/mL) and type IV (0.05 mg/mL) and hyaluronidase (0.025 mg/mL), and DNase I (0.01 mg/mL) and soybean trypsin inhibitor (0.01 mg/mL) dissolved in DMEM for 45 minutes at 37°C. The cell suspension was centrifuged and preincubated with fragment crystallizable-γ block antibody (anti-mouse CD16/32; PharMingen, San Diego, CA) to prevent nonspecific binding. Cell staining involved different combinations of fluorochrome-coupled antibodies to CD45.2, F4/80, CD11b, CD206, CD3, CD4, or CD8 for 30 minutes at 4°C in the dark. Fluorescence data were collected using an EPICS XL Flow Cytometer (Beckman Coulter), and analyzed using CellQuest (Beckman). Total cell population was recorded by cell size (forward scatter) and internal complexity (side scatter), and showed even distribution in a representative cell suspension prepared from heart tissues. Leukocytes were stained with monoclonal anti-CD45.2 and gated with CD45.2 fluorescence versus side scatter. To determine the viability of leukocytes, we used double staining analysis of uptake of propidium iodide (PI, Sigma-Aldrich) in CD45⁺ cells. Heart infiltrating myeloid cells (CD11b), macrophages (F4/80 or CD206), and CD4⁺ and CD8⁺ T lymphocytes were further gated on CD45⁺ cells. Appropriate isotype controls of irrelevant specificity were included (rat IgG2a-PerCP as control for CD45.2-PerCP, rat IgG2a-PE for F4/80-PE, rat IgG2a-FITC for CD206-FITC). Fluorescence minus one controls were included to determine the level of nonspecific staining and autofluorescence associated with subsets of cells in each fluorescence channel.

Cell Culture and Peptide Gel Coculture

Bone marrow–derived macrophages (BMDMs) were isolated from tibias and femurs of 3-month-old WT and SGK1^−/− mice as described.²⁰ Briefly, BM was flushed from the femur and tibia and purified through Ficoll-Paque gradient (Amersham Biosciences, Freiburg, Germany). Cells were then incubated in complete culture medium supplemented with 50-ng/mL recombinant murine cerebral spinal fluid for 5 days.

Cardiac fibroblasts were prepared from the hearts of adult male WT B6/129S mice as described.²¹ Briefly, the LVs were isolated, minced, and placed in a solution of 100-U/mL collagenase type I and 0.1% trypsin, and underwent sequential 10-minute periods of digestion with constant stirring at 37°C. After a 30-minute attachment to uncoated culture plates, cells were rinsed and amplified by trypsinization. All studies involved cardiac fibroblasts (passages 2 through 4) grown to subconfluence in serum-containing media and then growth-arrested for 24 hours in serum-free medium before treatment.

Three-dimensional peptide gel coculture was as described.^22,23 Briefly, macrophages and cardiac fibroblasts were mixed with peptide gel suspended in PBS (K2(QL)6K2; Beijing Seajet Scientific, Beijing, China) at a final concentration of 1.3 mg peptide/mL DMEM. The mixture of peptide gel with cells distributed well at the center of the cell culture. Polymerization of the gel was initiated by adding 1 to 2 volumes of medium to the side of each well, followed by gentle agitation, then incubation at 37°C for 1 hour to allow the gel to form. Peptide gel coculture was maintained in DMEM containing 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO₂ with Ang II treatment (1 μmol/L) for 48 hours.

RNA Analysis

Total RNA was extracted by the Trizol reagent method (Invitrogen), and first strand cDNA was synthesized with SuperScript II (Invitrogen). The transcript levels of SGK1, α-SMA, and collagen I and III were detected by semiquantitative polymerase chain reaction (PCR) or real-time PCR analysis. The primer sequences were for SGK1, 5’-TCCAGAATGAGGGGAATGGTAGCGA-3’, 5’-AGATGAGTAGAAGCGAGCCCGTGGT-3’; β-actin, 5’-TCATCACTATTGGCAACGAGC-3’, 5’-AACAGTCCGCCTAGAAGCAC-3’; α-SMA, 5’-ACTCTCTTCCAGCCATCTTTCA-3’, 5’-ATAGGTGGTTTCGTGGATGC-3’; collagen I, 5’-CATGTTCAGCTTTGTGGACCT-3’, 5’-GCAGCTGACTTCAGGGATGT-3’; collagen III, 5’-TCCCCTGGAATCTGTGAATC-3’, 5’-TGAGTCGAATTGGGGAGAAT-3’; and tubulin, 5’-TCTAACCCGTTGCTATCATGC-3’, 5’-GCCATGTTCCAGGCAGTAG-3’. PCR products were electrophoresed through 1.5% agarose gels containing ethidium bromide. Quantitative real-time PCR involved the iCycler iQ system (Bio-Rad).

Western Blotting Analysis

Western blotting analysis was performed as described.²⁴ Briefly, protein extracts were obtained from LV samples using cell lysis buffer (20-mmol/L Tris, 1% Triton X-100, 0.05% SDS, 5-mg sodium deoxycholate, 150-mmol/L NaCl, and 1-mmol/L phenylmethylsulfonyl fluoride) containing protease/phosphatase inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Cell lysates were separated by electrophoresis on 8% to 10% SDS-polyacrylamide gels before transfer to nitrocellulose membranes (Bio-Rad), which were incubated with the primary antibodies phospho-STAT1 (1:1000), STAT1 (1:1000), phospho-STAT3 (1:1000), STAT3 (1:1000), and GAPDH (1:1000) at 4°C overnight, then secondary antibodies (1:5000, Alexa Fluor 680 or IRDye 800; Rockland Immunochemicals, Gilbertsville, PA) at room temperature for 1 hour. Images were obtained by the Odyssey Infrared Imaging System (LI-COR Biosciences) and Odyssey software.

STAT3 Small Interfering RNA Transfection and Lentivirus Infection

Because small interfering RNAs (siRNAs) target many genes, a rescue experiment was performed to establish specificity. BMDMs were transfected with siRNA targeting mouse STAT3 or nonsilencing siRNA according to the manufacturer’s instructions (Santa Cruz Biotechnology). The mouse coding sequence for STAT3 was inserted into pBOBI with Flag-tag fused with its N terminal. The Flag-STAT3 lentivirus was packed in 293T cells by the calcium phosphate precipitation method. At the end of siRNA transfection, cells were infected with 100 multiplicity of infection of lentivirus expressing STAT3 or green fluorescent protein as a control. At 24 hours, the medium was replaced, and cells were cultured in 3-dimensional peptide gels with Ang II (1 μmol/L) for 48 hours as described.²⁵

Cytokine Production

The amount of active and latent TGF-β in the supernatant was measured with Quantikine TGF-β ELISA Kit (MB100B; R&D Systems, Minneapolis, MN). For measurement of total TGF-β in the supernatant, latent TGF-β was converted into the activated form by acidification, followed by a neutralization step. Supernatants were assessed for content of IL-10, IL-12p70, and IL-6 using FACSCalibur with BD cytometry bead array Flex Set bead assays (BD Biosciences). Sample files were analyzed using FCAP Array (version 1.0.1 software, Soft Flow).

Statistical Analysis

Data analysis involved use of GraphPad software (GraphPad Prism version 5.00 for Windows, GraphPad Software). Results are expressed as mean±SEM. Differences were analyzed by t test or ANOVA, and results were considered significant at P<0.05.

Ang II Infusion Increases SGK1 Expression in Mouse Cardiac Tissues

To investigate the role of SGK1 in hypertensive mice hearts, we examined SGK1 expression in mice in response to Ang II infusion. The mRNA level of SGK1 was time-dependently upregulated in cardiac tissue of WT mice infused with Ang II for 5 to 14 days (Figure 1A). Immunohistochemistry revealed the number of SGK1-positive cells in WT hearts greater with Ang II than saline infusion, with no change in SGK1 levels in SGK1^−/− hearts with Ang II or saline infusion (Figure 1B). Frozen sections were immunostained with the cardiomyocyte marker myosin heavy chain and anti-SGK1 antibodies to assess SGK1 expression in cardiomyocytes. SGK1 was expressed in cardiomyocytes, and its expression was markedly induced after Ang II infusion (Figure IV in the online-only Data Supplement). SGK1^−/− cardiomyocytes showed no expression of SGK1. Thus, SGK1 expression and activation may play a critical role in cardiac fibrosis in response to Ang II.

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Figure 1.

Angiotensin II (Ang II) induces serum-glucocorticoid regulated kinase 1 (SGK1) expression in mouse cardiac tissue. A, Semiquantitative polymerase chain reaction (PCR) of mRNA level of SGK1 in wild-type (WT) hearts after 5, 7, and 14 days of Ang II infusion. β-actin was a loading control. Data are mean±SEM for n=6 mice. *P<0.05, **P<0.01 vs saline control. B, Immunohistochemical analysis of SGK1 expression in heart sections of WT and SGK1 knockout (SGK1^−/−) mice with saline infusion or Ang II infusion for 7 days. (n=6 mice per group). Magnification, ×400.

SGK1^−/− Inhibits Ang II–Induced Cardiac Hypertrophy

Given that SGK1 was upregulated in the WT mouse heart after Ang II stimulation, we sought to determine the effect of SGK1 deficiency on Ang II–induced hypertension. Both WT and SGK1^−/− mice were infused with Ang II for 7 days. Ang II infusion markedly increased systolic blood pressure in both WT and SGK1^−/− mice compared with nontreated mice (Figure 2A), with no difference between the groups, so SGK1 is not involved in Ang II–induced hypertension.

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Figure 2.

Serum-glucocorticoid regulated kinase 1 (SGK1) knockout protects against angiotensin II (Ang II) infusion–induced cardiac hypertrophy in mice. A, Blood pressure in wild-type (WT) littermates and SGK1 knockout (SGK1^−/−) mice before and during 7 days of Ang II infusion. Data are mean±SEM for a group of 10 mice. NS indicates not significant. **P<0.01. B, Wheat germ agglutinin staining to evaluate tissue morphology and cardiomyocyte size in heart sections of WT and SGK1^−/− mice with saline or Ang II infusion for 7 days (magnification, ×400). Quantitative analysis of cardiomyocyte cross-sectional area. Data are mean±SEM for n=6 mice with 200 cells per animal. **P<0.01 vs WT Ang II infusion. (C, Table) Representative M-mode echocardiography of the left ventricle in WT and SGK1^−/− mice with saline infusion or Ang II infusion for 7 days. Data are mean±SEM for n=6 mice. *P<0.05, **P<0.01 vs Ang II–infused WT mice.

To further assess the role of SGK1 in regulating cardiac hypertrophy and function, we used histology with wheat germ agglutinin staining to evaluate myocyte size. WT and SGK1^−/− mice did not differ in myocyte size after saline infusion (Figure 2B). Ang II infusion significantly increased myocyte size in LV tissues of WT mice, which was largely blocked in SGK1^−/− mice (Figure 2B). On echocardiography, WT and SGK1^−/− hearts did not differ in LV wall thickness, diameter, or fractional shortening after saline infusion (Figure 2C and Table), but anterior wall thickening and LV mass were smaller in SGK1^−/− than WT hearts after Ang II infusion. Thus, SGK1 deletion may reduce Ang II–induced cardiac hypertrophy but not affect hypertension and cardiac function.

SGK1^−/− Inhibits Ang II–Induced Cardiac Fibrosis

To determine whether SGK1 activation contributes to Ang II–induced cardiac remodeling, we examined cardiac fibrosis of WT and SGK1^−/− hearts after Ang II infusion by Masson trichrome staining. WT and SGK1^−/− hearts did not differ in fibrosis in cardiac tissues with saline treatment (Figure 3A). Ang II infusion significantly increased collagen deposition in LV tissues of WT hearts, particularly in the interstitial and perivascular areas. In contrast, SGK1^−/− hearts showed attenuated cardiac fibrosis with Ang II infusion. Picrosirius red staining was used for analysis of total collagen content on bright field microscopy, and collagen fibers were detected by polarized light microscopy. WT and SGK1^−/− hearts showed little collagen deposition with saline treatment (Figure 3B). However, collagen accumulation in the interstitial and perivascular sites was greater in WT than SGK1^−/− hearts after Ang II infusion. Collagen I and III fiber content was lower in SGK1^−/− than WT hearts after Ang II infusion. Moreover, immunohistochemistry confirmed that cardiac expression of α-SMA and CTGF protein was significantly lower in SGK1^−/− than WT hearts after Ang II but not saline infusion (Figure 3C). Thus, SGK1 deletion reduces Ang II–induced cardiac fibrosis.

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Figure 3.

Serum-glucocorticoid regulated kinase 1 (SGK1) knockout reduces interstitial and perivascular fibrosis in the mouse heart. A, Collagen deposition in the heart after 7 days saline or angiotensin II (Ang II) infusion by Masson trichrome staining. Quantitative analysis of fibrotic area (Masson trichrome-stained area in light blue normalized to total myocardial area; magnification, ×100). B, Histology of collagen type I (orange) and III (green) content in wild-type (WT) and SGK1 knockout (SGK1^−/−) hearts after 7 days of saline or Ang II infusion by Sirius red staining on light microscopy (magnification, ×200) and polarized light microscopy (magnification, ×400). Data are mean±SEM for n=6 mice with 10 fields per animal. C, Immunohistochemisty of α-smooth muscle actin (SMA)- and connective tissue growth factor (CTGF)–positive cells in WT and SGK1^−/− mice after 7 days of saline or Ang II infusion (magnification, ×400). Quantitative analysis of α-SMA– and CTGF-positive cells in heart sections. Data are mean±SEM for n=6 mice with 10 fields per animal.*P<0.05, **P<0.01 vs WT Ang II infusion.

SGK1^−/− Inhibits Ang II–Induced Cardiac Inflammation

To examine whether SGK1 affects Ang II–induced cardiac inflammation, we analyzed proinflammatory cell infiltration in WT and SGK1^−/− hearts. Ang II infusion significantly increased the infiltration of proinflammatory cells in WT hearts, primarily localizing to the perivascular and interstitial areas (Figure 4A), with lower infiltration in SGK1^−/− than WT hearts (Figure 4A). Furthermore, immunohistochemical staining of myocardial sections demonstrated the expression of Mac-2 (macrophage marker) markedly lower in SGK1^−/− than WT hearts after Ang II infusion, with no significant difference between WT and SGK1^−/− hearts after saline infusion (Figure 4A).

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Figure 4.

Serum-glucocorticoid regulated kinase 1 (SGK1) knockout prevents angiotensin II (Ang II)–induced macrophage infiltration in cardiac tissues. A, Macrophage infiltration in wild-type (WT) and SGK1 knockout (SGK1^−/−) mice after saline or Ang II infusion by hematoxylin-eosin (H&E) and immunohistochemical staining of Mac-2 expression (magnification, ×200). Quantitative analysis of Mac-2–positive cells in heart sections. Data are mean±SEM for n=6 mice with 10 fields per animal. **P<0.01 vs Ang II–infused WT mice. B, Scatter plots gated on CD45⁺ cells, heart infiltrating myeloid cells (CD45⁺CD11b⁺cells), macrophages (CD45⁺CD11b⁺F4/80⁺cells), and CD4⁺ (CD45⁺CD3⁺CD4⁺cells) and CD8⁺ (CD45⁺CD3⁺CD8⁺cells) T lymphocytes analyzed in WT and SGK1^−/− hearts after 7 days of Ang II infusion. Fluorescence minus one (FMO) indicates controls, where 1 staining fluorescence is omitted to set the negative gate. Data are mean±SEM for n=6 mice. NS indicates not significant. *P<0.05, **P<0.01 vs Ang II–infused WT.

Cardiac tissue consists of several types of inflammatory cells, including macrophages, T helper cells, and cytotoxic T lymphocytes, essential for adipose tissue inflammation. Therefore, we analyzed the cell populations in collagenase-digested WT and SGK1^−/− hearts by flow cytometry. To determine the viability of leukocytes, we assayed PI uptake in CD45⁺ population cells by double staining. Gates were set on the CD45⁺ population, and the gated cell population was separated into viable leukocytes (CD45⁺PI⁻ cells) and necrotic leukocytes (CD45⁺PI⁺ cells). The number of total viable cells was stabilized to 91.71±3.80%, and CD45⁺PI⁻ cells in total leukocytes reached 81.54±2.93% in single cell suspensions of WT heart tissues after 7 days of Ang II infusion (Figure II in the online-only Data Supplement). To analyze leukocytes, we excluded PI⁺ nonviable cells and compared the relative proportions of macrophages (CD11b, F4/80), T helper cells (CD3, CD4), and cytotoxic T lymphocytes (CD3, CD8) in WT and SGK1^−/− hearts after Ang II infusion. Macrophages were identified as positive for CD11b and F4/80, gated on CD45⁺ population cells. The proportion of infiltrating CD45⁺CD11b⁺ cells, CD45⁺F4/80⁺ cells, and CD45⁺CD11b⁺F4/80⁺ cells in cardiac tissue was significantly lower in SGK1^−/− than WT hearts (57.19±3.79% versus 71.48±3.81% for CD45⁺CD11b⁺ cells; 38.03±5.37% versus 58.98±2.03% for CD45⁺F4/80⁺ cells; and 19.65±6.02% versus 55.05±3.92% for CD45⁺CD11b⁺F4/80⁺ cells, P≤ 0.05; Figure 4B). In contrast, SGK1^−/− and WT hearts did not differ in proportion of CD4⁺ or CD8⁺ T cells. Thus, SGK1 selectively promotes recruitment of macrophages to cardiac tissues.

SGK1^−/− Blocks M2 Macrophage Transition in Cardiac Tissue With Ang II Infusion

Because macrophages have been categorized as M1 (classically activated) and M2 (alternatively activated), we then analyzed macrophage phenotype in SGK1^−/− hearts. Immunofluorescence staining showed that Ang II infusion significantly increased the expression of macrophage marker F4/80 and SGK1 in WT hearts whereas this effect was markedly attenuated in SGK1^−/− hearts (Figure 5A). Moreover, the levels of TGF-β, IL-13, and IL-10 expression were markedly elevated in cardiac tissues of Ang II–treated WT littermates compared with saline-treated WT mice, with the increased expression significantly lower in SGK1^−/− mice than in WT littermates after Ang II infusion. The protein expression of TGF-β, IL-13, and IL-10 did not differ between WT littermates and SGK1^−/− mice with saline treatment (Figure 5B). To further establish whether SGK1 was necessary for macrophage phenotype in the heart after Ang II infusion, we assessed cell surface expression of CD206, a marker of M2 macrophages. Immunofluorescence analysis revealed double-positive F4/80 and CD206 viable macrophages strongly expressed in WT hearts but not in SGK1^−/− hearts (Figure 5C). We further analyzed macrophage phenotype in hearts by flow cytometry. Leukocytes derived from Ang II–treated WT and SGK1^−/− hearts were stained with anti-CD45.2, scatter plots were gated for CD45⁺ population cells, and number of M2 macrophages (CD45⁺F4/80⁺CD206⁺ cells) was analyzed in cardiac tissues. The proportion of infiltrating CD45⁺ cells into cardiac tissue was significantly lower in SGK1^−/− than WT hearts (1.51±0.86% versus 4.03±0.97%; P≤ 0.05; Figure 5D). Moreover, flow cytometry revealed an increased proportion of macrophages (CD45⁺F4/80⁺ cells) also positive for CD206 expression in WT mice but lower in SGK1^−/− than WT hearts (31.38±1.50% versus 55.90±2.38%). Thus, SGK1 is responsible for driving macrophages toward the M2 phenotype in cardiac tissue after Ang II infusion.

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Figure 5.

Serum-glucocorticoid regulated kinase 1 (SGK1) knockout reduces M2 macrophage infiltration in mouse cardiac tissue. A, Double immunofluorescence analysis of macrophage (anti-F4/80) and SGK1 expression in wild-type (WT) and SGK1 knockout (SGK1^−/−) hearts after 7 days of angiotensin II (Ang II) infusion. Bar=10 μm. B, Immunohistochemical analysis of cytokine expression in heart sections with saline or Ang II infusion in WT and SGK1^−/− mice. Cytokine expression detected by anti–transforming growth factor-β (TGF-β), anti–interleukin-10 (IL-10), and anti–IL-13 immunostaining. Data are mean±SEM for n=6 mice with 10 fields per animal. **P<0.01 vs Ang II–infused WT. C, Double immunofluorescence analysis of M2 macrophages (anti-F4/80 and anti-CD206) in WT and SGK1^−/− hearts after 7 days of Ang II infusion. Bar=7.5 μm. D, Leukocytes were gated with CD45 fluorescence vs side angle scatter (SS). Scatter plots are gated on CD45⁺ population cells. M2 macrophage activity (CD45⁺F4/80⁺CD206⁺ cells) was analyzed in WT and SGK1^−/− hearts after 7 days of Ang II infusion. Data are mean±SEM for n=6 mice. **P<0.01 vs Ang II–infused WT. DAPI indicates 4',6-diamidino-2-phenylindole.

SGK1^−/− Inhibits Activation of M2 Macrophages Through the STAT3 Pathway

To investigate the mechanism by which SGK1 regulates M2 macrophage activation, we analyzed the Janus kinase/STAT pathway, particularly STAT3, which plays a central role in macrophage polarization. STAT3 phosphorylation was similar in cardiac tissues of WT and SGK1^−/− mice after saline infusion (Figure 6A). However, Ang II infusion significantly enhanced STAT3 phosphorylation in WT hearts, which was greatly reduced in SGK1^−/− hearts (Figure 6A). WT and SGK1^−/− hearts did not differ in phosphorylation of STAT1 after Ang II infusion. Furthermore, immunofluorescence staining showed phosphorylated STAT3 localized primarily in the nucleus of F4/80-positive macrophages in WT hearts but mostly in cytoplasm of SGK1^−/− hearts (Figure 6B), so SGK1 can activate STAT3 in macrophages after Ang II infusion.

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Figure 6.

Serum-glucocorticoid regulated kinase 1 (SGK1)–induced macrophage polarization is mediated by signal transducer and activator of transcription 3 (STAT3) pathway. A, Western blotting analysis of phosphorylation levels of STAT1 and STAT3 in wild-type (WT) and SGK1 knockout (SGK1^−/−) hearts. Data are mean±SEM for n=6 mice. NS indicates not significant. *P<0.05 vs Ang II–infused WT. B, Double immunofluorescence analyses of STAT3 activation of macrophages (anti-F4/80) in WT and SGK1^−/− hearts after 7 days of angiotensin II (Ang II) infusion. Bar=10 μm. C, Effect of STAT3 on macrophage polarization in 3-dimensional culture. WT bone marrow–derived macrophages (BMDMs) were transiently transfected with STAT3-specific or control small interfering RNA (siRNA) before infection with lentivirus expressing STAT3 or green fluorescent protein (GFP) as indicated. BMDMs were then seeded in 3-dimensional nanogels with or without Ang II for 48 hours. Western blotting analysis was used to verify siRNA-mediated STAT3 knockdown and lentivirus infection for rescue experiments in macrophages. ELISA was used to analyze active and latent transforming growth factor-β (TGF-β) and flow cytometry for levels of interleukin-10 (IL-10), IL-12, and IL-6 with a Cytometric Bead Array Flex Set Kit. **P<0.01; *P<0.05 vs STAT3 siRNA-treated. DAPI indicates 4',6-diamidino-2-phenylindole.

To further confirm that SGK1 plays an important role in macrophage polarization via the STAT3 pathway, STAT3-specific siRNA was used to knock down endogenous STAT3 expression in BMDMs, and we performed recombinant STAT3 lentivirus-mediated rescue experiments to measure cytokine production by BMDMs. Western blotting analysis revealed that STAT3 siRNA reduced STAT3 protein levels by ≈75%, with STAT3 lentivirus leading to significant upregulation of STAT3 protein levels. Ang II treatment significantly increased levels of TGF-β and IL-10 (M2-type cytokines), and STAT3-specific siRNA markedly attenuated these levels (Figure 6C). Moreover, infection of recombinant lentivirus rescued production of TGF-β and IL-10 by reexpression of STAT3. To determine whether SGK1-activated STAT3 pathway is linked to M1 polarization of macrophages, we measured secretion of IL-12 or IL-6. Levels of IL-12 and IL-6 did not differ with alteration of STAT3 activation (Figure 6C). Therefore, STAT3 activation is necessary for SGK1-mediated M2-type polarization of macrophages.

SGK1-Deficient Macrophages Inhibit Cardiac Fibroblast Transformation

To investigate the role of SGK1 activation of macrophages in cardiac fibrosis, we cocultured BMDMs from WT and SGK1^−/− mice with mouse cardiac fibroblasts in 3-dimensional peptide gels, as an in vitro model of macrophage-mediated fibroblast-to-myofibroblast differentiation. Immunofluorescence analysis revealed the level of α-SMA, a major marker for myofibroblasts, strongly expressed by fibroblasts after coculture with WT BMDMs in response to Ang II (Figure 7A). In contrast, the protein level of α-SMA was markedly suppressed on coculture with SGK1^−/− BMDMs in response to Ang II (Figure 7A). Real-time PCR further confirmed that coculture of fibroblasts with WT BMDMs significantly unregulated the mRNA expression of α-SMA and collagen I and III, whereas incubation with SGK1^−/− macrophages attenuated this effect (Figure 7B). Thus, SGK1^−/− macrophages show reduced myofibroblast differentiation, thereby inhibiting cardiac fibrosis.

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Figure 7.

Serum/glucocorticoid-regulated kinase 1 knockout (SGK1^−/−) macrophages inhibit cardiac fibroblast-to-myofibroblast differentiation. A, Wild-type (WT) or SGK1^−/− macrophages were cocultured with cardiac fibroblasts in nanogels with or without angiotensin II (Ang II), then underwent immunofluorescence analysis of myofibroblast differentiation by staining with α-smooth muscle actin (SMA) antibody. Bar=10 μm. B, Quantitative real-time polymerase chain reaction (PCR) analysis of the mRNA expression of α-SMA and collagen I and III in 3-dimensional coculture. Tubulin was a normalization control. Data are mean±SEM of 3 independent experiments. *P<0.05 vs coculture with WT bone marrow–derived macrophages (BMDMs). CF indicates cystic fibrosis; NS not significant.