NLRP3 inflammasome activation is prevented by blocking the caspase 3 –dependent release of MVs.

<p><b>A.</b> Western blot analysis of caspase-3 in total extracts of HepG2 cells not transfected (C), transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. Equal loading was confirm by re- probing the same membrane with monoclonal antibody against the house-keeping β-actin. <b>B.</b> Protein quantification of preparation of MVs as obtained from control HepG2 cells (C), from HepG2 cells transfected with the non silencing RNA and then exposed to PA (NsC) and HepG2 cells silenced for caspase 3 and then exposed to PA (siCASP3). Data are expressed as μg/μl and as mean ± SD being (n = 12 for any condition). <b>C.</b> Flow cytometric analysis of internalization of PKH26-fluorescent die-MVs in HepG2 naïve cells. Color legend: i) black trace is related to HepG2 naïve cells treated with ultracentrifugated solution (also marked with PKH26-fluorescent die) derived from cells just exposed to BSA (used as control), ii) pink trace is related to HepG2 naïve cells treated with MVs derived from HepG2 cells transfected with non-silencing treated with palmitic acid 0,25mM for 24hrs; iii) green trace is related to HepG2 naïve cells treated with MVs derived from HepG2 cells transfected with specific siRNA for caspase-3 and treated with palmitic acid 0,25mM for 24hrs. <b>D.</b> Western blot analysis of NF-Kb/p65 protein levels in cytosolic and nuclear extract obtained from HepG2 naïve cells exposed or not (C) to MVs derived from HepG2 cells transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. Equal loading was confirmed by re-probing membrane with α-tubulin and Lamin-A (cytosolic and nuclear house-keeping, respectively). <b>E.</b> Western blot analysis of NLRP3 protein levels as well as of cleaved caspase-1 (p-10-CASP-1) and IL-1β in total extracts obtained by HepG2 naïve cells exposed or not (C) to MVs derived from HepG2 cells transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. LPS 1mg/ml was used as positive control. Equal loading was confirmed by re-probing membrane with β-actin.</p

In vivo time-dependent analyses of inflammasome components, inflammatory cytokines and necrosis in MCD fed mice.

<p>“In vivo” analysis by quantitative real-time PCR (qPCR) of transcripts of inflammasome components (NLRP3 and CASP-1 <b>A, C</b>) and related cytokine (IL-1β, <b>B</b>) as well as of TNF-α (<b>E</b>) in WT mice fed with MCS diet or MCD diet for 4d, 2wks, 4wks and 8wks. <b>D</b>. Analysis of serum ALT in WT mice fed with MCS diet or MCD diet for 2wks, 4wks and 8 wks. Data in graphs are expressed as means ± SEM (n = 6 mice for any experimental group); p values are indicated and referred to mice fed the MCS control diet.</p

Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis

<div><p>Non-Alcoholic Fatty Liver Disease (NAFLD) is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH), fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs) released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1β. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.</p></div

Immunohistochemistry analysis for NLRP3 and F4/80.

<p><b>A,B.</b> Immunohistochemistry analysis for NLRP3 on liver specimens from WT mice fed with MCS diet or MCD diet for 2wks, 4wks and 8wks (<b>A</b>) as well as WT mice and Casp 3^-/- knockout mice fed for 6wks (<b>B</b>). <b>C.</b> Image analysis quantification for NLRP3 staining (Fig 2B) as evaluated with Image<i>j</i> software in liver sections from WT or caspase 3 -/- mice fed a MCD diet for 6 weeks. <b>D,E.</b> Immunohistochemistry analysis for F4/80 on liver specimens from WT mice fed with MCS diet or MCD diet for 2wks, 4wks and 8wks (<b>D</b>) as well as WT mice and Casp 3^-/- knockout mice fed for 6wks (<b>E</b>). <b>F</b>. Immunohistochemistry analysis for NLRP3 and CD68 on liver specimens from human NAFLD/NASH-related advanced fibrosis (F3 and F4). Original magnification as indicated.</p

MVs activate NLRP3 inflammasome cascade.

<p><b>A, B.</b> Quantitative real-time PCR (qPCR) analysis of inflammasome components (NLRP3 and CASP-1) in HepG2 naïve cells treated with MVs up to 24 hrs. <b>C.</b> Western blot analysis of activation of inflammasome pathway by evaluating NLRP3 protein levels as well as cleaved caspase-1 (p-10-CASP-1) and IL-1β in total extracts obtained by HepG2 naïve cells exposed to MVs for indicated times. Equal loading was confirm by re-probing the same membrane with monoclonal antibody against the house-keeping β-actin. <b>D.</b> ELISA assay to evaluate IL-1β release (pg/ml) in culture medium of HepG2 naïve cells exposed to MVs up to 16 hrs. Data in graphs are expressed as means ± SEM (*p< 0.05 and ** p<0.01 vs related control HepG2 cells) of three independent experiments.</p

In vitro experimental model of lipotoxicity.

<p><b>A.</b> Red Oil-O staining in control HepG2 cells, HepG2 cells treated with BSA 1% or HepG2 cells exposed to palmitic acid 0.25mM in BSA 1% (BSA + PA) for 24hrs. <b>B.</b> Detection of Caspase-3/7 Activity in HepG2 cells treated with BSA 1% or PA 0.25mM for indicated times, analyzed by using Apo-ONE Caspase-3/7 Homogeneous Assay. <b>C.</b> Viability of HepG2 cells treated with BSA 1% or PA 0.25mM for indicated times, evaluated by MTT assay. <b>D,E.</b> Analysis of internalization of MVs in HepG2 cells by (<b>D</b>) flow cytometry or by (<b>E</b>) confocal microscopy: nuclei (blue fluorescence), MVs (red fluorescence) and cytoskeleton (F-actin, green fluorescence).</p

MVs up-regulate NF-KB pathway.

<p><b>A.</b> Western blot analysis of activation of IKK and IKβ evaluated in total extracts of HepG2 naïve cells exposed to MVs for indicating time. Equal loading was confirmed by re-probing the same membrane with un-phosphorylated protein. <b>B</b>. Western blot analysis of NF-Kb/p65 protein levels in cytosolic and nuclear extract obtained from HepG2 naïve cells exposed to MVs up to 16 hrs. Equal loading was confirmed by re-probing membrane with α-tubulin and Lamin-A (cytosolic and nuclear house-keeping, respectively). <b>C</b>. Immunofluorescence analysis of NF-Kb/p65 nuclear translocation in control HepG2 cells or HepG2 cells exposed to MVs for 6hrs: nuclei (blue, DAPI staining), NF-Kb/p65 (green). Original magnifications are indicated.</p

Silencing of NLRP3 in target cells prevents MVs-dependent inflammasome activation.

<p><b>A.</b> Quantitative real-time PCR (qPCR) of NLRP3 mRNA in HepG2 cells transfected for 72hrs with non-silencing siRNA (NsC) or specific siRNA for NLRP3 (siNLRP3) and exposed or not to MVs derived from HepG2 cells treated with PA 0.25mM for 24hrs. LPS 1mg/ml was used as positive control. <b>B.</b> Quantitative real-time PCR (qPCR) of IL-1β mRNA in HepG2 cells not transfected (C) and transfected for 72hrs with non-silencing siRNA (NsC) or specific siRNA for NLRP3 (siNLRP3) and exposed or not for 3hrs to MVs derived from HepG2 cells treated with PA 0.25mM for 24hrs. Data in graphs are expressed as means ± SEM (*p< 0.05 and ** p<0.01 vs related control HepG2 cells; # p<0.05 vs NsC+MVs) of three independent experiments. <b>C,D.</b> Western blot analysis of NLRP3 protein levels as well as of cleaved caspase-1 (p-10-CASP-1) and IL-1β in total extracts obtained by HepG2 cells transfected for 72hrs with non-silencing siRNA (NsC) or specific siRNA for NLRP3 (siNLRP3) and exposed for indicating time to MVs derived from HepG2 cells treated with PA 0.25mM for 24hrs. Equal loading was confirmed by re-probing membrane with β-actin.</p