High fat-induced expression of inflammatory cytokines and chemokines is attenuated in caspase-1 knockout mice.

<p>Inflammatory cytokine/chemokine production in the liver is associated the early stages of NASH. mRNA expression for Tnfα (A), Mcp-1 (B) and F4/80 (C) in the liver of wild-type mice on control or high fat diets was performed by RT-PCR. Values represent means ± SEM. Values with different superscripts are significantly different from one another (<i>p</i><0.05). n = 4 control, n = 6 high fat.</p

Caspase-1 and IL-1β expression is increased after high fat feeding.

<p>Livers of wild-type mice on the control or high fat diet were analyzed for pro-caspase-1 and IL-1β expression by immunoblot (A). Densitometric analysis was done for each protein normalized to HSC70 (B and C). mRNA for IL-1 expression in the liver of mice on the control or high fat diet was analyzed by RT-PCR (D). IL-1 was assessed by ELISA in whole liver tissue (E). Values with different superscripts are significantly different from one another (<i>p</i><0.05). n = 4 control, n = 6 high fat.</p

Histopathological analysis of mice on the high fat diet.

<p>Pathologist: BPG. Data are represented as Mean ± SEM.</p><p>H&E slides of the livers of mice on the control and high fat diets were analyzed for steatosis, inflammation, and ballooning. H&E sections were scored in a blinded fashion by BPG. Values represent means ± SEM. n = 4 control, n = 6 high fat.</p

Adiposity is increased in caspase-1 knockout mice.

<p>Mice on the control and high fat diets were analyzed for body adiposity. Fat content of subcutaneous (B) and visceral (C) depots were calculated from three images. Total adipose volume was calculated from subcutaneous and visceral depots (D). Adiposity was normalized to body weight and expressed as% adipose (E). Lean mass was calculated based on the density of adipose tissue and normalized to body weight and expressed as% lean mass (F). Values represent means ± SEM. Values with different superscripts are significantly different from one another (<i>p</i><0.05). n = 5 C57BL/6, n = 4 <i>Casp1^-/-</i>.</p

High fat-induced expression of lipogenesis-related genes is attenuated in caspase-1 knockout mice.

<p>Expression of mRNA for Pparγ, Srebp1c, Acc1, Fas and Scd1 were analyzed in the livers of wild-type and <i>Casp1^-/-</i> mice on high fat diets and normalized to 18S. Hepatic expression of phospho-ACC and total ACC were analyzed by western blot (A). Expression of Pparα, Aox, L-fabp, Cpt1α, Pgc1β, Lcad and Mcad were analyzed in the livers of wild-type and Casp1-/- mice on high fat diet and normalized to 18S (B). Apob and Mttp mRNA were measured (C) in the liver of wild-type compared to and Casp1-/- mice on control or high fat diets and normalized to 18S (C). Values represent means ± SEM. Values with + different superscripts are significantly different from wild-type mice on control diet one another (p<0.05). Values with * are significantly different from wild-type mice on high fat diet (p<0.05). n = 4 control, n = 6 high fat.</p

Caspase-1 knockout mice are protected from high fat-induced hepatic steatosis.

<p>Representative images of H&E stained livers in wild-type and <i>Casp1^-/-</i> mice on control or high fat diet (10x) (A). NAFLD activity score of wild-type and <i>Casp1^-/-</i> mice on the high fat diet was assessed by histopathology of H&E stained livers in a blinded-fashion by BPG (B). Hepatic triglyceride (TG) levels were measured biochemically from mice on the control or high fat diets (C). Plasma alanine aminotransferase (ALT) levels were analyzed from mice on the control or high fat diets (D). Plasma cholesterol (E), FFA (F) and TG levels (G) were measured biochemically as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056100#s2" target="_blank">methods</a> section. Values represent means ± SEM. Values with different superscripts are significantly different from one another (<i>p</i><0.05). n = 4 control, n = 6 high fat.</p

Obesity is associated with increased levels of circulating MPs and increased adipose activity of Rho associated kinase and caspase 3

<p>Annexin V positive MPs from (A) ob control or ob/ob mice. Blood was collected by cardiac puncture and PFP was obtained as detailed in methods section. Annexin V positive MPs were analyzed by flow cytometry (n = 5 each group). (B) Dynamic light scattering analysis and Transmission Electron Microscopy of isolated circulating MPs. (C) Western blot analysis of perilipin A levels in MPs isolated from mouse PFP in WT mice on a regular chow diet and ob/ob mice (n = 3 each group). Perilipin A abundance in MPs isolated from mouse PFP correlated to: (D) total mouse body weights and (E) weight of extracted mouse epididymal fat pads. (F) Western blots of p-MYPT1, total MYPT1/2, cleaved (active) caspase 3, total caspase 3, and actin levels in subcutaneous and epididymal adipose tissue lysates from ob control or ob/ob mice. Quantification of Western blots of (G) cleaved caspase 3 and (H) p-MYPT in ob control and ob/ob mice adipose tissues normalized to actin levels. Values represent mean ± S.E.M. *P < 0.05; ***P < 0.001 compared to respective controls.</p

Microparticles Release by Adipocytes Act as “Find-Me” Signals to Promote Macrophage Migration

<div><p>Macrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical “find-me” signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel “find me” signals that contributes to macrophage infiltration associated with obesity.</p></div

Adipocyte-derived MPs mediates attraction of macrophages in vitro and in vivo.

<p>(A) In vitro chemotaxis assay of MPs-free supernatant and MPs from adipocytes treated with palmitic acid. Values represent mean ± S.D. ***P < 0.001 compared to controls. (B-F) C57BL/6 mice were injected intraperitoneally with 1x10⁶ adipocyte-derived MPs (isolated from 3T3-L1 adipocytes treated with 0.5 mM palmitic acid or without palmitic acid) or controls (n = 3 each group). (B) Four days post injection infiltrated cells were isolated from peritoneal cavity by lavage and counted. (C-F) flow cytometry analysis of infiltrated cells. The infiltrated cells were stained by CD45 (leukocyte common antigen) (C), CD11b (monocytes) (D), F4/80 (macrophages) (E), or Ly6G (neutrophils) (F). Values represent mean ± S.E.M. (D) In vivo macrophages migration. C57BL/6 mice were injected intraperitoneally with 1x10⁶ palmitic acid-derived MPs or vehicle alone (n = 5 per group). Three days post injection; macrophages were isolated from peritoneal cavity by lavage. Number of macrophages present in peritoneal cavity was counted. Values represent mean ± S.E.M.</p

Transplanted circulating MPs from ob/ob mouse lead to monocyte activation in the blood and macrophage infiltration in the adipose tissue.

<p>(A) Scheme of transplanted experiment; ob/ob platelet free plasma was collected, circulating MPs were purified and injected into the WT mouse. (B) Dot plot analysis of the entire leukocyte population in blood resulting from the mock, ob ctrl (control) MP, and ob/ob MP injections, respectively. X-axis indicates CD11b intensity and Y-axis indicates Ly6C intensity. (C-D) Flow cytometry analysis of monocyte (CD11b⁺-Ly6C^high) percentage (C) and activated monocyte (CD11b⁺-Ly6C^high-CD204⁺) percentage (D) in blood resulting from the mock, ob ctrl MP, or ob/ob MP injections (n = 4 each group). (E) Flow cytometry analysis of infiltrated monocytes (CD11b⁺-Ly6C^high) percentage in epididymal (Epi) adipose tissue from the mock, ob ctrl MP, or ob/ob MP injections. P<0.4. Values represent mean ± S.E.M. *P < 0.05; **P < 0.01 compared to ob ctrl MPs as a control.</p