Repeated clodronate-liposome treatment results in neutrophilia and is not effective in limiting obesity-linked metabolic impairments

Abstract

Depletion of macrophages is thought to be a therapeutic option for obesity-induced inflammation and metabolic dysfunction. However, whether the therapeutic effect is a direct result of reduced macrophage-derived inflammation or secondary to decreases in fat mass is controversial, as macrophage depletion has been shown to disrupt energy homeostasis. This study was designed to determine if macrophage depletion via clodronate-liposome (CLD) treatment could serve as an effective intervention to reduce obesity-driven inflammatory and metabolic impairments independent of changes in energy intake. After 16 wk on a high-fat diet (HFD) or the AIN-76A control (low-fat) diet (LFD) ( n = 30/diet treatment), male C57BL/6J mice were assigned to a CLD- or PBS-liposome treatment ( n = 15/group) for 4 wk. Liposomes were administered biweekly via intraperitoneal injections (8 administrations in total). PBS-liposome-treated groups were pair-fed to their CLD-treated dietary counterparts. Metabolic function was assessed before and after liposome treatment. Adipose tissue, as well as the liver, was investigated for macrophage infiltration and the presence of inflammatory mediators. Additionally, a complete blood count was performed. CLD treatment reduced energy intake. When controlling for energy intake, CLD treatment was unable to regress metabolic dysfunction or nonalcoholic fatty liver disease and impaired adipose tissue insulin action. Moreover, repeated CLD treatment induced neutrophilia and anemia, increased adipose tissue mRNA expression of the proinflammatory cytokines IL-6 and IL-1β, and augmented circulating IL-6 and monocyte chemoattractant protein-1 concentrations ( P < 0.05). This study suggests that repeated intraperitoneal administration of CLD to deplete macrophages attenuates obesity by limiting energy intake. Moreover, after controlling for the benefits of weight loss, the accompanying detrimental side effects limit regular CLD treatment as an effective therapeutic strategy.

Keywords: clodronate; inflammation; macrophage; metabolism; obesity.

Fig. 1.

Body composition and metabolic data after 16 wk of dietary treatment [high-fat diet (HFD) or low-fat diet (LFD)] before start of liposome treatment. Mice were fed the HFD or LFD for 16 wk. A: body weight. B: body composition. C: fasting (5-h) blood glucose levels. D: fasting (5-h) plasma insulin levels. E: intraperitoneal glucose tolerance test (GTT) and corresponding area under the curve (AUC). F: intraperitoneal insulin tolerance test (ITT) following a 5-h fast. CLD, clodronate. Values are means ± SE; n = 9–15 mice per group. *P < 0.05, HFD vs. LFD groups. Bar graphs not sharing a common letter (a, b) are significantly different from one another (P < 0.05). %, $, #, ^P < 0.05 vs. baseline (0 min) for LFD PBS, LFD CLD, HFD PBS, and HFD CLD groups, respectively.

Fig. 2.

Body morphology following dietary [high-fat diet (HFD) or low-fat diet (LFD)] and liposome treatment. A: body weight. B: energy intake throughout the experimental protocol. C: terminal body composition analysis as assessed by dual-energy X-ray absorptiometry. D: intraperitoneal adipose tissue depot weights (epididymal, perirenal, mesenteric), and total intraperitoneal fat weight measured at euthanization. CLD, clodronate. Values are means ± SE; n = 9–15 mice per group. *Significantly different from start of liposome treatment at 16 wk. Bar graphs not sharing a common letter (a, b, c) are significantly different from one another (P < 0.05).

Fig. 3.

Treatment with clodronate (CLD) for 4 wk depletes epididymal adipose tissue macrophages in a low-fat diet (LFD), but not a high-fat diet (HFD), setting. A: epididymal adipose tissue mRNA expression of macrophage markers (F4/80, CD68, CD11c, and CD206) normalized to the most stable internal reference genes [hydroxymethylbilane synthase (HMBS), H2A histone family member V (H2AFV), β₂-microglobulin (B2M), and TATA-box binding protein (TBP)] calculated using Qbase+ software. B: representative immunofluorescence images (×40) of DAPI and F4/80 in epididymal adipose tissue of HFD-fed mice. Values are means ± SE; n = 9–15 mice per group. Bar graphs not sharing a common letter (a, b, c) are significantly different from one another (P < 0.05).

Fig. 4.

Despite not affecting epididymal adipose tissue macrophage populations in a high-fat diet (HFD) setting, clodronate (CLD) treatment augments epididymal adipose tissue inflammation and polymorphonuclear cell infiltration. A: epididymal adipose tissue mRNA expression of inflammatory mediators [monocyte chemoattractant protein-1 (MCP-1), TNF-α, IL-10, IL-6, and IL-1β] normalized to the geometric mean of the most stable internal reference genes [hydroxymethylbilane synthase (HMBS), H2A histone family member V (H2AFV), β₂-microglobulin (B2M), and TATA-box binding protein (TBP)] calculated using Qbase+ software. B: representative images (×20 and ×100) of hematoxylin-eosin-stained epididymal adipose tissue sections showing increased polymorphonuclear cell infiltration in the CLD-treated groups. LFD, low-fat diet. Values are means ± SE; n = 9–15 mice per group. Bar graphs not sharing a common letter (a, b, c) are significantly different from one another (P < 0.05).

Fig. 5.

Clodronate (CLD) treatment increases circulating and epididymal adipose tissue neutrophils. A: circulating neutrophils determined in whole blood by a VetScan HM5. B: Western blot analysis of lymphocyte antigen 6G (Ly6G, a neutrophil marker) in epididymal adipose tissue normalized to total protein stain (amido black). IOD, integrated optical density. C: 2 representative images of immunohistochemistry staining (×100) of Ly6G in epididymal adipose tissue. HFD, high-fat diet; LFD, low-fat diet. Values are means ± SE; n = 9–15 mice per group. *P < 0.05. Bar graphs not sharing a common letter (a, b) are significantly different from one another (P < 0.05).

Fig. 6.

Treatment with clodronate (CLD) for 4 wk depletes macrophages in the perirenal fat pad. A: perirenal adipose tissue mRNA expression of macrophage markers (F4/80, CD68, CD11c, and CD206) normalized to the geometric mean of the most stable internal reference genes [hydroxymethylbilane synthase (HMBS) and hypoxanthine phosphoribosyltransferase (HPRT)] using QBase+ software. HFD, high-fat diet; LFD, low-fat diet. B: representative immunofluorescence images (×40) of DAPI and F4/80 in perirenal adipose tissue of HFD-fed mice. Values are means ± SE; n = 9–15 mice per group. Bar graphs not sharing a common letter are significantly different from one another (P < 0.05).

Fig. 7.

An increase in perirenal adipose tissue inflammation is paired with an increase in infiltrating neutrophils. A: perirenal adipose tissue mRNA expression of inflammatory mediators [TNF-α, monocyte chemoattractant protein-1 (MCP-1), IL-1β, and IL-6] normalized to the geometric mean of the most stable internal reference genes [hydroxymethylbilane synthase (HMBS) and hypoxanthine phosphoribosyltransferase (HPRT)] using QBase+ software. HFD, high-fat diet; LFD, low-fat diet. B: 2 representative images of immunohistochemistry staining (×100) of lymphocyte antigen 6G (Ly6G) in perirenal adipose tissue of HFD-fed groups. C: Western blot analysis of Ly6G in epididymal adipose tissue normalized to total protein stain (amido black). IOD, integrated optical density. Values are means ± SE; n = 9–15 mice per group. *P < 0.05. Bar graphs not sharing a common letter (a, b, c, d) are significantly different from one another (P < 0.05).

Fig. 8.

Clodronate (CLD) treatment does not affect regression of early-stage nonalcoholic fatty liver disease development. A: liver weight following euthanization. HFD, high-fat diet; LFD, low-fat diet. B: hepatic lipid accumulation. C: plasma alanine transaminase (ALT) activity. D: representative hepatic hematoxylin-eosin-stained (×20) images. E: hepatic gene expression of F4/80. F: representative F4/80 staining (×60, arrows indicate examples of positive staining). G: mRNA expression of hepatic inflammatory mediators [monocyte chemoattractant protein-1 (MCP-1), TNF-α, IL-6, and IL-1β] normalized to the geometric mean of the most stable internal reference genes [β₂-microglobulin (B2M) and TATA-box binding protein (TBP)] using QBase+ software. ME, main effect. Values are means ± SE; n = 9–15 mice per group. Bar graphs not sharing a common letter (a, b, c) are significantly different from one another (P < 0.05).

Fig. 9.

Clodronate (CLD) treatment significantly increases circulating proinflammatory cytokine concentrations. After 20 wk of diet/4 wk of CLD-liposome treatment, plasma was assessed for circulating proinflammatory cytokines. A: IL-6. B: monocyte chemoattractant protein-1 (MCP-1). Because of limitations in the amount of plasma, only the plasma of high-fat-diet (HFD)-treated groups was assessed for concentration of circulating MCP-1. LFD, low-fat diet. Values are means ± SE; n = 7–14 mice per group. Bar graphs not sharing a common letter (a, b) are significantly different from one another (P < 0.05).

Fig. 10.

Clodronate (CLD) treatment does not rescue impaired glucose metabolism or insulin resistance and exacerbates adipose tissue insulin action. After 20 wk of diet/4 wk of CLD-liposome treatment, metabolic outcomes were assessed. A: fasting (5-h) blood glucose levels. B: fasting (5-h) plasma insulin levels. C: intraperitoneal glucose tolerance test (GTT) and corresponding area under the curve (AUC). D: intraperitoneal insulin tolerance test (ITT) following a 5-h fast. E and F: serum free fatty acid concentration following a 5-h fast measured at 0 and 30 min of the ITT. G: change in serum free fatty acids from 0 to 30 min of the ITT. HFD, high-fat diet; LFD, low-fat diet; ME, mixed effects. Values are means ± SE; n = 9–15 mice per group. Bar graphs not sharing a common letter (a, b, c) are significantly different from one another (P < 0.05). %, $, #, ^P < 0.05 vs. baseline (0 min) for LFD PBS, LFD CLD, HFD PBS, and HFD CLD groups, respectively.