Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization

Abstract

Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.

Keywords: ARG1, arginase 1; BMDM, bone marrow-derived macrophages; CCL, chemokine (C-C motif) ligand; CD, chow diet; CHIL3/CHI3L3, chitinase-like 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GPT, glutamic pyruvic transaminase, soluble; HFD, high-fat diet; IFNG, interferon gamma; IL, interleukin; Kupffer cells; LPS, lipopolysaccharide; MAP1LC3/LC3B, microtubule-associated protein 1 light chain 3 β; MAPK, mitogen-activated protein kinase; MGL2, macrophage galactose N-acetyl-galactosamine specific lectin 2; NOS2, nitric oxide synthase 2, inducible; PBS, phosphate-buffered saline; PTGS2, prostaglandin-endoperoxide synthase 2; RETNLA, resistin like α;; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling; WAT, white adipose tissue; autophagy; innate immunity; lipopolysaccharide; macrophage; obesity; polarization; qRT-PCR, quantitative real-time PCR; steatohepatitis.

Figure 1.

HFD-induced obesity inhibits macrophage autophagy. (A) Immunoblots of total protein from BMDM from wild-type C57 BL/6 mice fed CD or HFD for 16 or 20 wk. Some cells were treated with ammonium chloride and leupeptin (AC/Leup) for 2 h. Proteins were probed for LC3B and tubulin, and LC3B-I and LC3B-II bands are indicated by arrows. (B) Quantification of the ratio of LC3B-II in ammonium chloride- and leupeptin-treated cells to untreated cells from CD- and HFD-fed BMDM from 20-wk-old animals by densitometric scanning of immunoblots (n = 4). Data presented as the mean ± SEM. *P < 0.003 as compared to control. (C) Western blots of total protein from BMDM from CD- or HFD-fed mice infected with GFP-LC3 adenovirus that were untreated or treated with LPS and IFNG for 8 h probed for GFP and tubulin. The GFP-LC3 and free GFP bands are highlighted with arrows. (D) Confocal images of GFP-LC3 puncta in BMDM from CD- and HFD-fed mice. (E to G) Quantification of GFP-LC3 puncta number, area and intensity in the BMDM from these mice (n = 9 to 13). (H) Western blots of total protein from peritoneal macrophages isolated from CD- and HFD-fed mice untreated or ammonium chloride and leupeptin-treated for 2 h.

Figure 2.

Loss of ATG5-dependent macrophage autophagy increases M1 polarization. ((A)to E) Relative mRNA levels of Nos2, Ptgs2, Tnf, Ccl5, and Il6 in BMDM from littermate control (Con) and atg5^myeΔ knockout (KO) mice 12 h after treatment with LPS and IFNG (n = 4). (F) Immunoblots of total protein isolates from control and knockout BMDM treated with LPS and IFNG for the indicated number of hours and probed for the total and phosphorylated (P-) proteins shown. (G to K) Levels of TNF, CCL5, IL6, CCL2, and IL1B secreted into the culture medium by control and knockout BMDM with no treatment (No Tx) or stimulated with LPS and IFNG (M1) for 24 h (n = 6 to 8). All data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01 versus control cells with the same treatment.

Figure 3.

Atg5-deficient macrophages undergo decreased M2 polarization. (A to D) Relative mRNA levels of Retnla, Arg1, Chil3, and Mgl2 in BMDM from littermate control (Con) and atg5^myeΔ knockout (KO) mice 12 h after treatment with IL4 and IL13 (n = 4). (E to H) Relative mRNA levels of Retnla, Arg1, Chil3, and Mgl2 in control and knockout BMDM 24 h after treatment with IL4 and IL13 (n = 6 to 12). All data are presented as the mean ± SEM. *P < 0.01 and **P < 0.00001 vs. control BMDM. (I) Immunoblots of total protein isolates from control and knockout BMDM treated with IL4 and IL13 for the number of hours shown and probed for the indicated total and phosphorylated (P-) proteins.

Figure 4.

Metabolic effects of the knockout of Atg5 in macrophages with HFD feeding and LPS. (A) Body weights of littermate control (Con) and atg5^myeΔ knockout (KO) mice after 12 wk of CD or HFD feeding (n = 21 to 26). (B) Change in body weight after 2 wk of phosphate-buffered saline (PBS) or LPS injections (n = 9 to 13). (C) Caloric intake in the different groups of mice (n = 4 or 5). (D) Serum β-hydroxybutyrate levels (n = 3 to 7). (E) Serum glucose levels (n = 9 to 13). (F) Histological grades of steatosis (n = 5 to 8). (G) Liver triglyceride content (n = 9 to 13). All data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01 versus CD-fed, PBS-treated littermate control mice.^##P < 0.01 vs. littermate control mice with the same diet and treatment.

Figure 5.

atg5-knockout mice exhibit evidence of systemic inflammation. (A) Serum white blood cell (WBC) counts (n = 4 to 6). (B) Serum monocyte counts (n = 4 to 6). (C to I) Serum levels of TNF, IL6, CCL2, IL1B, IL4, IL10 and IL13 (n = 6 to 10). All data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01 versus CD-fed, PBS-treated littermate control mice.^#P < 0.05 vs. littermate control mice with the same diet and treatment.

Figure 6.

HFD-fed, LPS-injected knockout mice have increased hepatic inflammation. (A, B) Relative levels of Emr1 and Ly6 g mRNA in the livers of CD- and HFD-fed mice treated with PBS or LPS (n = 4 to 6). (C) Immunofluorescence staining for CD68 in livers (200 × magnification). Enlarged images are shown in the inserts. (D) Numbers of CD68⁺ cells per field (n = 3). (E to I) Relative hepatic mRNA levels for Tnf, Ccl2, Ifng, Nos2, and Il1b (n = 4 to 6). All data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01 versus CD-fed, PBS-treated littermate control mice. ^#P < 0.05 and ^##P < 0.01 vs. littermate control mice with the same diet and treatment.

Figure 7.

Autophagy regulates Kupffer cell M1 and M2 polarization. (A to D) Relative mRNA levels in Kupffer cells from control (Con) and knockout (KO) mice for Nos2, Ptgs2, Tnf and Ccl5 in response to LPS and IFNG treatment for 12 h. (E to G) mRNA levels 12 h after IL4 and IL13-induced M2 polarization for Retnla, Arg1, and Chil3 (n = 3). All data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 versus control mice.

Figure 8.

HFD-fed, LPS-treated knockout mice develop liver injury. (A) Serum GPTs in CD- and HFD-fed mice treated with PBS or LPS. (B) Histological grades of liver injury in the same mice. (C) Histological grades of hepatic inflammation. (D) Numbers of TUNEL-positive cells per field (400× magnification) (n = 6 to 8). All data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01 vs. CD-fed, PBS-treated littermate control mice.^##P < 0.01 versus littermate control mice with the same diet and treatment. (E) Representative TUNEL staining (400× magnification). (F) Immunoblots of total hepatic protein from CD- and HFD-fed control and knockout mice that were PBS- (-LPS) or LPS (+LPS) treated. Immunoblots were probed with antibodies for CASP3/caspase 3, CASP7/caspase 7 and tubulin. Arrows indicate the procaspase (Pro), the cleaved CASP3 (p17) and the cleaved CASP7 (p19) forms. The images with the cleaved forms are longer exposures of the procaspase immunoblots.