VSL#3 Resets Insulin Signaling and Protects against NASH and Atherosclerosis in a Model of Genetic Dyslipidemia and Intestinal Inflammation

<div><h3>Background</h3><p>Signals generated by the inflammed intestine are thought to contribute to metabolic derangement. The intestinal microbiota contributes to instructing the immune system beyond the intestinal wall and its modulation is a potential target for treating systemic disorders.</p> <h3>Aims</h3><p>To investigate the pathogenetic role of low grade intestinal inflammation in the development of steatohepatitis and atherosclerosis in a model of genetic dyslipidemia and to test the therapeutic potential of a probiotics intervention in protecting against development of these disorders.</p> <h3>Results</h3><p>ApoE^−/− mice were randomized to receive vehicle or VSL#3, a mixture of eight probiotics, at the dose of 20×10⁹ colony-forming units/kg/day for three months alone or in combination with 0.2% of dextran sulfate sodium (DSS) in drinking water. Administering DSS to ApoE^−/− mice failed to induce signs and symptoms of colitis but increased intestinal permeability to dextran FITC and, while had no effect on serum lipids, increased the blood levels of markers of liver injury and insulin resistance. DSS administration associated with low level inflammation of intestinal and mesenteric adipose tissues, caused liver histopathology features of steatohepatitis and severe atherosclerotic lesions in the aorta. These changes were prevented by VSL#3 intervention. Specifically, VSL#3 reversed insulin resistance, prevented development of histologic features of mesenteric adipose tissue inflammation, steatohepatitis and reduced the extent of aortic plaques. Conditioned media obtained from cultured probiotics caused the direct transactivation of peroxisome proliferator-activated receptor-γ, Farnesoid-X-receptors and vitamin D receptor.</p> <h3>Conclusions</h3><p>Low grade intestinal inflammation drives a transition from steatosis to steatohepatitis and worsens the severity of atherosclerosis in a genetic model of dyslipidemia. VSL#3 intervention modulates the expression of nuclear receptors, corrects for insulin resistance in liver and adipose tissues and protects against development of steatohepatitis and atherosclerosis.</p> </div

Effect of VSL#3 administration on PPARy, VDR and FXR, activity and expression. A

<p>) Luciferase reporter assay performed in HepG2 transiently transfected with p(UAS)5-TKLUC, pSG5GAL4-PPARγ and pCMV-βgal vectors and stimulated 18 h with rosiglitazone 100 nM and condiditioned media (CM) undiluted, diluted 1∶2 (50%) and 1∶10 (10%). *p<0.05 <i>versus</i> not treated (NT); <b>B</b>) Luciferase reporter assay performed in HepG2 transiently transfected with p(UAS)5-TKLUC, pSG5GAL4-VDR and pCMV-βgal vectors and stimulated 18 h with 25-hydroxycholecalciferol, 50 nM, and CM undiluted, diluted 1∶2 (50%) or 1∶10 (10%). *p<0.05 <i>versus</i> not treated (NT); <b>C</b>) Luciferase reporter assay performed in HepG2 transiently transfected with p(hsp27)TKLUC, pSG5-FXR, pSG5-RXR, pCMV-βgal vectors and stimulated 18 h with CDCA 10 μM and CM undiluted, diluted 1∶2 (50%) and diluted 1∶10 (10%). *p<0.05 <i>versus</i> not treated (NT). (<b>Panel </b><b>D–G</b>) RT-PCR analysis of the intestinal expression of PPARγ, VDR and FXR. Data represent the mean ± SE of 5 mice per group. * p<0.05 ApoE^−/− experimental group versus naive wild type group; ** p<0.05 ApoE^−/− naïve group versus ApoE^−/− plus VSL#3 group; $ p<0.05 ApoE^−/− naive group versus ApoE^−/− plus DSS group; # p<0.05 ApoE^−/− plus DSS group versus ApoE^−/− plus DSS and VSL#3 group.</p

Effect of VSL#3 administration in mesenteric fat inflammation. A)

<p>Histopathology analysis of mesenteric fat isolated from experimental groups, original magnification 20×, H&E staining. Adipose tissue concentration of (<b>B</b>) TNFα and (<b>C</b>) RANTES. Data are mean ± SE of n = 5 mice per group. * p<0.05 ApoE^−/− experimental group versus naive wild type group; ** p<0.05 ApoE^−/− naïve group versus ApoE^−/− plus VSL#3 group; $ p<0.05 ApoE^−/− naive group versus ApoE^−/− plus DSS group; # p<0.05 ApoE^−/− plus DSS group versus ApoE^−/− plus DSS and VSL#3 group.</p

VSL#3 administration reverts insulin resistance in ApoE^−/− mice: effect on OGTT, ITT and insulin signaling.

<p>ApoE^−/− mice were administered daily with VSL#3 alone or in combination with DSS for 12 weeks starting at the age of 8–9 months. The OGTTs were performed after 10 weeks of treatment. <b>A</b>) Blood glucose levels in response to OGTT. <b>B</b>) Area under the OGTT curve (AUC) from 0 to 120 min after glucose administration: all curves were adjusted by subtracting the initial glucose values from actual glucose measurements. The ITTs were performed after 11 weeks of treatment, the data were expressed as % of basal glucose values . <b>C</b>) The ITTs were performed. <b>D</b>) illustrates glucose plasma levels after 15 minutes of insulin administration. Mean ± SE is plotted; n = 8–12 mice per group. Effect of VSL#3 administration on AKT Ser(473) phosphorylation in (<b>E</b>) liver and (<b>F</b>) epididymal fat tissues. Data are means ± SE of n = 8 mice per group. * p<0.05 ApoE^−/− experimental group versus naive wild type group; ** p<0.05 ApoE^−/− naïve group versus ApoE^−/− plus VSL#3 group; $ p<0.05 ApoE^−/− naive group versus ApoE^−/− plus DSS group; # p<0.05 ApoE^−/− plus DSS group versus ApoE^−/− plus DSS and VSL#3 group.</p

Effects of VSL#3 intervention on clinical and biochemical parameters.

<p>The values are expressed as mean ± SE (n = 8–12). * p<0.05 ApoE^−/− experimental group versus naive wild type group; ** p<0.05 ApoE^−/− naïve group versus ApoE^−/− plus VSL#3 group; $ p<0.05 ApoE^−/− naive group versus ApoE^−/− plus DSS group; # p<0.05 ApoE^−/− plus DSS group versus ApoE^−/− plus DSS and VSL#3 group.</p

Effect of VSL#3 administration on atherosclerosis development in ApoE−/− mice. Panel A.

<p>Administration of DSS to ApoE^−/− for 12 weeks increases the size of aortic plaques (ratio of plaque surface area to vessel surface area) and numbers of plaques. Individual data are shown n = 7–12. <b>Panel B.</b> Representation of aortic plaques from individual animals. The images shows the plaque surface from individual animals, each one representative of a specific experimental group. The lipids in the vessel wall were staining with Sudan IV. <b>Panel C.</b> Aortic concentration of TNFα, TGF-β1, RANTES, IL-10, ICAM-1 and VCAM. Data are mean ± SE of n = 5 mice per group. <b>D</b>) VSL#3 intervention reduced the percentage of CD36 positive cells on circulating macrophages. The mean ± SE of n = 5 mice per group is shown. * p<0.05 ApoE^−/− experimental group versus naive wild type group; ** p<0.05 ApoE^−/− naïve group versus ApoE^−/− plus VSL#3 group; $ p<0.05 ApoE^−/− naive group versus ApoE^−/− plus DSS group; # p<0.05 ApoE^−/− plus DSS group versus ApoE^−/− plus DSS and VSL#3 group.</p

Effect of VSL#3 administration on the induction of T-lymphocytes IL-10-producing cells in spleen in ApoE^−/−mice.

<p>T-lymphocytes were isolated from spleens of mice and then stimulated with concanavalin A for 36 h in vitro. INFγ and IL-10 in the supernatants were assayed by ELISA. The bar indicates mean ± SE of 5 samples from 5 mice in each group. * p<0.05 ApoE^−/− experimental group versus naive wild type group; ** p<0.05 ApoE^−/− naïve group versus ApoE^−/− plus VSL#3 group; $ p<0.05 ApoE^−/− naive group versus ApoE^−/− plus DSS group;# p<0.05 ApoE^−/− plus DSS group versus ApoE^−/− plus DSS and VSL#3 group.</p

Stimulation of THP-1 cells with p17 causes reciprocal regulation of genes involved in immune function and lipid metabolism.

<p>THP-1 cells were stimulated with 1 µg/ml p17 recombinant protein for 18 hours. (A) Relative mRNA expression of proinflammatory mediators TNFα, IL1β, MCP-1, ICAM-1 and nuclear receptors FXR and PPARγ. (B) Relative mRNA expression of co-stimulatory molecules CD40, CD80 and CD86. (C) Relative mRNA expression of proatherogenic genes CD36 and ABCA1. Real-Time analysis was carried out in triplicate and the experiment was repeated twice. *P<0.05 versus not treated cells.</p

An intact STAT-1 signal is required to mediate the biological activity of p17.

<p>THP-1 cells were stimulated for 18 hours with p17 (1 µg/ml) in presence or in absence of a specific STAT-1 inhibitor fludarabine (0.5 µM). At the end of treatments cellular lysates were used for Real-Time or immunoblot analysis. (A) Immunoblot of STAT-1 protein (total and phosphorylated fraction). (B) Relative mRNA expression of MCP-1, ICAM-1, PPARγ, CD40, CD80 and CD86 was expressed relative to not treated cells. Analysis was carried out in triplicate and the experiment was repeated twice. *P<0.05 versus not treated cells. #P<0.05 versus p17 stimulated cells.</p

HIV-1 p17 regulates pro-inflammatory, co-stimulatory and pro-atherogenic molecules in CD14-derived PBMC isolated from healthy donors.

<p>CD-14 derived PBMC isolated from healthy donors were stimulated for 18 hours with 1 µg/ml p17 recombinant protein. (A and B) Exposure to p17 drives an activated phenotype in macrophages and causes macrophages adhesion. Magnification 20×. (C) Relative mRNA expression of proinflammatory cytokines TNFα, IL1β, MCP-1, ICAM-1 and nuclear receptors FXR and PPARγ. (D) Relative mRNA expression of co-stimulatory molecules CD40, CD80 and CD86. (E) Relative mRNA expression of proatherogenic genes CD36 and ABCA1. Real-Time analysis was carried out in triplicate and the experiment was repeated twice. *P<0.05 versus not treated cells.</p