Hepatic inflammation is a key pathological feature of Nonalcoholic Steatohepatitis (NASH). Natural Killer T-cells (NKT) and CD8+ T-cells are known to play an important role in obesity related adipose tissue inflammation. We hypothesized that these same inflammatory phenotypes would be present in progressive NASH. We used a previously established high fat high carbohydrate (HFHC) murine obesogenic diet model of progressive NASH to investigate the role of NKT cells and CD8+ T-cells in C57Bl6/J mice. Further, to better understand the impact of these cell populations; CD1d-deficient and CD8+ T-cell depleted mice were subjected to HFHC diet for 16 weeks. C57Bl6/J mice fed HFHC diet had increased body weight, liver triglyceride content, serum alanine aminotransferase (ALT) levels and increased NKT cells and CD8+ T-cells infiltration in the liver. In addition human liver sections from patients with NASH showed increased CD8+ T-cells. In comparison, CD1d-deficient and CD8-T cell depleted mice fed HFHC had lower hepatic triglyceride content, lower ALT levels, as well reduced α-smooth muscle actin (αSMA), collagen type 1 alpha 1 (Col1a1), collagen type 1 alpha 2 (Col1a2) mRNA expression, lower activated resident macrophages and infiltrating macrophages and improved NAFLD activity scores. Further, while CD1d-deficient mice were protected against weight gain on the HFHC diet, CD8 T-cell depleted mice gained weight on the HFHC diet. Conclusion We found that NASH has an immunological signature that includes hepatic infiltrating NKT and CD8+ T-Cells. Depletion of these cells resulted in reduced NASH progression and thus presents novel therapeutic avenues for the treatment of NASH.

No abstract

Retinitis pigmentosa (RP) is a common retinal degeneration disease caused by mutation in any gene of the photo transduction cascade and results in photoreceptor dystrophy. Over decades, several animal models have been used to address the need for the elucidation of effective therapeutics and factors regulating retinal degeneration to prohibit or renew the damaged retina. However, controversies over the immune privilege of retina during cell transplantation and the role of immune modulation during RP still remain largely uninvestigated because of the lack of suitable animal models. Here, we have developed an immunocompromised mouse model, NOD.SCID-rd1, for retinitis pigmentosa (RP) by crossing CBA/J and NOD SCID mice and selecting homozygous double mutant animals for further breeding. Characterization of the newly developed RP model indicates a similar retinal degeneration pattern as CBA/J, with a decreased apoptosis rate and rhodopsin loss. It also exhibits loss of T cells, B cells and NK cells. The NOD.SCID-rd1 model is extremely useful for allogenic and xenogenic cell-based therapeutics, as indicated by the higher cell integration capacity post transplantation. We dissect the underlying role of the immune system in the progression of RP and the effect of immune deficiency on immune privilege of the eye using comparative qPCR studies of this model and the immune-competent RP model.

Magnetic sorting of cells, based on microbead conjugated Background antibodies (Abs), employs positive as well as negative immunomagnetic separation methods, for isolation of a specific cell population. These microbeads are suggested to be nontoxic, biodegradable carriers conjugated to various antibodies. Isolation of cells through positive selection involves the attachment of antibody conjugated microbeads to the cells of interest, followed by their isolation in the presence of a strong magnetic field to obtain higher purity. Negative selection involves attachment of microbead conjugated antibodies to all other cell populations except the cells of interest, which remain untagged. In the present study, we compared the two methods for their effect on functional and immunophenotypic behavior of isolated CD14+ monocytes.: Peripheral blood mononuclear cells (PBMCs) were isolated from Methods blood collected from healthy volunteers by density gradient centrifugation. Human blood derived monocytes were isolated through positive selection and negative selection, making use of the appropriate monocyte isolation kit. Monocytes were then stimulated with lipopolysaccharide (LPS) and their activation and proliferation capacity were examined. The degradation or dissociation of cell-bound microbeads was also investigated.: We observed an impaired LPS sensitivity as well as poor activation Results and proliferation capacity upon stimulation by LPS in positively sorted CD14+ monocytes as compared to negatively sorted CD14+ monocytes. The attached microbeads did not degrade and remained attached to the cells even after 6 days of culture.: Our results suggest that positively sorted CD14+ cells exhibit Conclusions hampered functionality and may result in inaccurate analysis and observations in downstream applications. However, these cells can be used for immediate analytical procedures.

Sugar-sweetened beverage consumption is a known independent risk factor for nonalcoholic steatohepatitis (NASH). Non-caloric sweeteners (NCS) are food additives providing sweetness without calories and are considered safe and/or not metabolized by the liver. The potential role of newer NCS in the regulation of NASH, however, remain unknown. Our study aimed to determine the impact of newer NCS including Rebaudioside A and sucralose on NASH using high fat diet induced obesity mouse model by substituting fructose and sucrose with NCS in the drinking water. We characterized the phenotype of NCS-treated obesity and investigated the alterations of hepatic function and underlying mechanisms. We found that NCS have no impact on weight gain and energy balance in high fat diet induced obesity. However, in comparison to fructose and sucrose, Rebaudioside A significantly improved liver enzymes, hepatic steatosis and hepatic fibrosis. Additionally, Rebaudioside A improved endoplasmic reticulum (ER) stress related gene expressions, fasting glucose levels, insulin sensitivity and restored pancreatic islet cell mass, neuronal innervation and microbiome composition. We concluded that Rebaudioside A significantly ameliorated murine NASH, while the underlying mechanisms requires further investigation. Current treatment strategies for nonalcoholic steatohepatitis (NASH) have focused on lifestyle management of modifiable risk factors, through a combination of diet and exercise 1-3. Potential therapeutic targets for NASH intersect with the complex pathogenesis of NASH including hepatic steatosis from the imbalance of lipogenesis and free fatty acid (FFA) promoting inflammatory response and fibrosis progression 4,5. However, despite pharmaceutical agents currently in advanced stages of clinical testing 6-8 , NASH is on track predicted to become the main reason for liver transplant in the very near future 9. Therefore, it is essential to continue to explore novel NASH therapies. Sugar sweetened beverages are now well acknowledged to have severe consequences on human health. Consequently, non-caloric sweeteners (NCS) such as aspartame, sucralose, saccharin, and Rebaudioside A have increased in popularity and usage. However, there is a limited evidence for the benefits of frequent consumption of NCS sugar substitutes. This is especially true for the most recent addition Rebaudioside A, that is an extract of the stevia leaf that provides sweetness without calories 10. Interestingly, recent literature has reported that Rebaudioside A may in fact play a role in glucose metabolism and has even been reported to improve post-prandial glucose-insulin index 11 , and its consumption may result in weight loss in mice fed a high fat diet 12. These observations suggest a potential role for Rebaudioside A on glucose metabolism in general, and on liver function and NASH in particular. The well-known interactions between human health, diet and intestinal microbiota are based on the involvement of the microbiome in metabolism and immunity, which also par...

Taken together, the data suggest that TAP1(-/-) -deficient mice displayed reduced levels of CD8 cells that have a vital role in the initiation and propagation of liver inflammation and is a casual role in the beginning of fructose-induced liver damage as well as insulin resistance in mice.

Argonaute 2 (Ago2) is the main component of the RNA-induced silencing complex. We recently showed that liver-specific Ago2-deficiency in mice (L-Ago2 knockout [KO] mice) enhances mitochondrial oxidation and alleviates obesity-associated pathophysiology. However, the precise mechanisms behind the role of hepatic Ago2 in regulating the mitochondrial oxidation associated with glucose metabolism are still unclear. Here, we show that hepatic Ago2 regulates the function of peroxisome proliferator–activated receptor α (PPARα) for oxidative metabolism. In both genetically and diet-induced severe obese conditions, L-Ago2 KO mice developed obesity and hepatic steatosis but exhibited improved glucose metabolism accompanied by lowered expression levels of pathologic microRNAs (miRNAs), including miR-802, miR-103/107, and miR-152, and enhanced expression of PPARα and its target genes regulating oxidative metabolism in the liver. We then investigated the role of hepatic Ago2 in the outcomes of vertical sleeve gastrectomy (VSG) in which PPARα plays a crucial role in a drastic transcription reprogram associated with improved glycemia post VSG. Whereas VSG reduced body weight and improved fatty liver in wild-type mice, these effects were not observed in hepatic Ago2-deficient mice. Conversely, glucose metabolism was improved in a hepatic Ago2-dependent manner post VSG. Treating Ago2-deficient primary hepatocytes with WY-14643, a PPARα agonist, showed that Ago2-deficiency enhances sensitivity to WY-14643 and increases expression of PPARα target genes and mitochondrial oxidation. Our findings suggest that hepatic Ago2 function is intrinsically associated with PPARα that links Ago2-mediated RNA silencing with mitochondrial functions for oxidation and obesity-associated pathophysiology.

Magnetic sorting of cells, based on microbead conjugated Background antibodies (Abs), employs positive as well as negative immunomagnetic separation methods, for isolation of a specific cell population. These microbeads are suggested to be nontoxic, biodegradable carriers conjugated to various antibodies. Isolation of cells through positive selection involves the attachment of antibody conjugated microbeads to the cells of interest, followed by their isolation in the presence of a strong magnetic field to obtain higher purity. Negative selection involves attachment of microbead conjugated antibodies to all other cell populations except the cells of interest, which remain untagged. In the present study, we compared the two methods for their effect on functional and immunophenotypic behavior of isolated CD14+ monocytes.: Peripheral blood mononuclear cells (PBMCs) were isolated from Methods blood collected from healthy volunteers by density gradient centrifugation. Human blood derived monocytes were isolated through positive selection and negative selection, making use of the appropriate monocyte isolation kit. Monocytes were then stimulated with lipopolysaccharide (LPS) and their activation and proliferation capacity were examined. The degradation or dissociation of cell-bound microbeads was also investigated.: We observed an impaired LPS sensitivity as well as poor activation Results and proliferation capacity upon stimulation by LPS in positively sorted CD14+ monocytes as compared to negatively sorted CD14+ monocytes. The attached microbeads did not degrade and remained attached to the cells even after 6 days of culture.: Our results suggest that positively sorted CD14+ cells exhibit Conclusions hampered functionality and may result in inaccurate analysis and observations in downstream applications. However, these cells can be used for immediate analytical procedures.