Role of Hepatic Stellate and Liver Sinusoidal Endothelial Cells in a Human Primary Cell Three-Dimensional Model of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is an inflammatory and fibrotic liver disease that has reached epidemic proportions and has no approved pharmacologic therapies. Research and drug development efforts are hampered by inadequate preclinical models. This research describes a three-dimensional bioprinted liver tissue model of NASH built using primary human hepatocytes and nonparenchymal liver cells (hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells) from either healthy or NASH donors. Three-dimensional tissues bioprinted with cells sourced from diseased patients showed a NASH phenotype, including fibrosis. More importantly, this NASH phenotype occurred without the addition of disease-inducing agents. Bioprinted tissues composed entirely of healthy cells exhibited significantly less evidence of disease. The role of individual cell types in driving the NASH phenotype was examined by producing chimeric bioprinted tissues composed of healthy cells together with the addition of one or more diseased nonparenchymal cell types. These experiments reveal a role for both hepatic stellate and liver sinusoidal endothelial cells in the disease process. This model represents a fully human system with potential to detect clinically active targets and eventually therapies.

Resident microbes shape the vaginal epithelial glycan landscape.

Epithelial cells are covered in carbohydrates (glycans). This glycan coat or "glycocalyx" interfaces directly with microbes, providing a protective barrier against potential pathogens. Bacterial vaginosis (BV) is a condition associated with adverse health outcomes in which bacteria reside in direct proximity to the vaginal epithelium. Some of these bacteria, including Gardnerella, produce glycosyl hydrolase enzymes. However, glycans of the human vaginal epithelial surface have not been studied in detail. Here, we elucidate key characteristics of the "normal" vaginal epithelial glycan landscape and analyze the impact of resident microbes on the surface glycocalyx. In human BV, glycocalyx staining was visibly diminished in electron micrographs compared to controls. Biochemical and mass spectrometric analysis showed that, compared to normal vaginal epithelial cells, BV cells were depleted of sialylated N- and O-glycans, with underlying galactose residues exposed on the surface. Treatment of primary epithelial cells from BV-negative women with recombinant Gardnerella sialidases generated BV-like glycan phenotypes. Exposure of cultured VK2 vaginal epithelial cells to recombinant Gardnerella sialidase led to desialylation of glycans and induction of pathways regulating cell death, differentiation, and inflammatory responses. These data provide evidence that vaginal epithelial cells exhibit an altered glycan landscape in BV and suggest that BV-associated glycosidic enzymes may lead to changes in epithelial gene transcription that promote cell turnover and regulate responses toward the resident microbiome.

Targeting Clic1 for the treatment of obesity: A novel therapeutic strategy to reduce food intake and body weight.

OBJECTIVE: Despite great advances in obesity therapeutics in recent years, there is still a need to identify additional therapeutic targets for the treatment of this disease. We previously discovered a signature of genes, including Chloride intracellular channel 1 (Clic1), whose expression was associated with drug-induced weight gain, and in these studies, we assess the effect of Clic1 inhibition on food intake and body weight in mice. METHODS: We studied the impact of Clic1 inhibition in mouse models of binge-eating, diet-induced obese mice and genetic models of obesity (Magel2 KO mice). RESULTS: Clic1 knockout (KO) mice ate significantly less and had a lower body weight than WT littermates when either fed chow or high fat diet. Furthermore, pharmacological inhibition of Clic1 in diet-induced obese mice resulted in suppression of food intake and promoted highly efficacious weight loss. Clic1 inhibition also reduced food intake in binge-eating models and hyperphagic Magel2 KO mice. We observed that chronic obesity resulted in a significant change in subcellular localization of Clic1 with an increased ratio of Clic1 in the membrane in the obese state. These observations provide a novel therapeutic strategy to block Clic1 translocation as a potential mechanism to reduce food intake and lower body weight. CONCLUSIONS: These studies attribute a novel role of Clic1 as a driver of food intake and overconsumption. In summary, we have identified hypothalamic expression of Clic1 plays a key role in food intake, providing a novel therapeutic target to treat overconsumption that is the root cause of modern obesity.

Identification of adipose tissue transcriptomic memory of anorexia nervosa.

BACKGROUND: Anorexia nervosa (AN) is a complex debilitating disease characterized by intense fear of weight gain and excessive exercise. It is the deadliest of any psychiatric disorder with a high rate of recidivism, yet its pathophysiology is unclear. The Activity-Based Anorexia (ABA) paradigm is a widely accepted mouse model of AN that recapitulates hypophagia and hyperactivity despite reduced body weight, however, not the chronicity. METHODS: Here, we modified the prototypical ABA paradigm to increase the time to lose 25% of baseline body weight from less than 7 days to more than 2 weeks. We used this paradigm to identify persistently altered genes after weight restoration that represent a transcriptomic memory of under-nutrition and may contribute to AN relapse using RNA sequencing. We focused on adipose tissue as it was identified as a major location of transcriptomic memory of over-nutririon. RESULTS: We identified 300 dysregulated genes that were refractory to weight restroration after ABA, including Calm2 and Vps13d, which could be potential global regulators of transcriptomic memory in both chronic over- and under-nutrition. CONCLUSION: We demonstrated the presence of peristent changes in the adipose tissue transcriptome in the ABA mice after weight restoration. Despite being on the opposite spectrum of weight perturbations, majority of the transcriptomic memory genes of under- and over-nutrition did not overlap, suggestive of the different mechanisms involved in these extreme nutritional statuses.

Cell-type-specific responses to the microbiota across all tissues of the larval zebrafish.

Animal development proceeds in the presence of intimate microbial associations, but the extent to which different host cells across the body respond to resident microbes remains to be fully explored. Using the vertebrate model organism, the larval zebrafish, we assessed transcriptional responses to the microbiota across the entire body at single-cell resolution. We find that cell types across the body, not limited to tissues at host-microbe interfaces, respond to the microbiota. Responses are cell-type-specific, but across many tissues the microbiota enhances cell proliferation, increases metabolism, and stimulates a diversity of cellular activities, revealing roles for the microbiota in promoting developmental plasticity. This work provides a resource for exploring transcriptional responses to the microbiota across all cell types of the vertebrate body and generating new hypotheses about the interactions between vertebrate hosts and their microbiota.

Mapping the common gene networks that underlie related diseases.

A longstanding goal of biomedicine is to understand how alterations in molecular and cellular networks give rise to the spectrum of human diseases. For diseases with shared etiology, understanding the common causes allows for improved diagnosis of each disease, development of new therapies and more comprehensive identification of disease genes. Accordingly, this protocol describes how to evaluate the extent to which two diseases, each characterized by a set of mapped genes, are colocalized in a reference gene interaction network. This procedure uses network propagation to measure the network 'distance' between gene sets. For colocalized diseases, the network can be further analyzed to extract common gene communities at progressive granularities. In particular, we show how to: (1) obtain input gene sets and a reference gene interaction network; (2) identify common subnetworks of genes that encompass or are in close proximity to all gene sets; (3) use multiscale community detection to identify systems and pathways represented by each common subnetwork to generate a network colocalized systems map; (4) validate identified genes and systems using a mouse variant database; and (5) visualize and further investigate select genes, interactions and systems for relevance to phenotype(s) of interest. We demonstrate the utility of this approach by identifying shared biological mechanisms underlying autism and congenital heart disease. However, this protocol is general and can be applied to any gene sets attributed to diseases or other phenotypes with suspected joint association. A typical NetColoc run takes less than an hour. Software and documentation are available at https://github.com/ucsd-ccbb/NetColoc .

Interleukin-18 signaling promotes activation of hepatic stellate cells in mouse liver fibrosis.

BACKGROUND AND AIMS: Nucleotide-binding oligomerization domain-like receptor-family pyrin domain-containing 3 (NLRP3) inflammasome activation has been shown to result in liver fibrosis. Mechanisms and downstream signaling remain incompletely understood. Here, we studied the role of IL-18 in hepatic stellate cells (HSCs), and its impact on liver fibrosis. APPROACH AND RESULTS: We observed significantly increased serum levels of IL-18 (128.4 pg/ml vs. 74.9 pg/ml) and IL-18 binding protein (BP; 46.50 ng/ml vs. 15.35 ng/ml) in patients with liver cirrhosis compared with healthy controls. Single cell RNA sequencing data showed that an immunoregulatory subset of murine HSCs highly expresses Il18 and Il18r1 . Treatment of cultured primary murine HSC with recombinant mouse IL-18 accelerated their transdifferentiation into myofibroblasts. In vivo , IL-18 receptor-deficient mice had reduced liver fibrosis in a model of fibrosis induced by HSC-specific NLRP3 overactivation. Whole liver RNA sequencing analysis from a murine model of severe NASH-induced fibrosis by feeding a choline-deficient, L-amino acid-defined, high fat diet showed that genes related to IL-18 and its downstream signaling were significantly upregulated, and Il18-/- mice receiving this diet for 10 weeks showed protection from fibrotic changes with decreased number of alpha smooth muscle actin-positive cells and collagen deposition. HSC activation triggered by NLRP3 inflammasome activation was abrogated when IL-18 signaling was blocked by its naturally occurring antagonist IL-18BP. Accordingly, we observed that the severe inflammatory phenotype associated with myeloid cell-specific NLRP3 gain-of-function was rescued by IL-18BP. CONCLUSIONS: Our study highlights the role of IL-18 in the development of liver fibrosis by its direct effect on HSC activation identifying IL-18 as a target to treat liver fibrosis.