G protein-coupled receptor 35 attenuates nonalcoholic steatohepatitis by reprogramming cholesterol homeostasis in hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. Fat accumulation "sensitizes" the liver to insult and leads to nonalcoholic steatohepatitis (NASH). G protein-coupled receptor 35 (GPR35) is involved in metabolic stresses, but its role in NAFLD is unknown. We report that hepatocyte GPR35 mitigates NASH by regulating hepatic cholesterol homeostasis. Specifically, we found that GPR35 overexpression in hepatocytes protected against high-fat/cholesterol/fructose (HFCF) diet-induced steatohepatitis, whereas loss of GPR35 had the opposite effect. Administration of the GPR35 agonist kynurenic acid (Kyna) suppressed HFCF diet-induced steatohepatitis in mice. Kyna/GPR35 induced expression of StAR-related lipid transfer protein 4 (STARD4) through the ERK1/2 signaling pathway, ultimately resulting in hepatic cholesterol esterification and bile acid synthesis (BAS). The overexpression of STARD4 increased the expression of the BAS rate-limiting enzymes cytochrome P450 family 7 subfamily A member 1 (CYP7A1) and CYP8B1, promoting the conversion of cholesterol to bile acid. The protective effect induced by GPR35 overexpression in hepatocytes disappeared in hepatocyte STARD4-knockdown mice. STARD4 overexpression in hepatocytes reversed the aggravation of HFCF diet-induced steatohepatitis caused by the loss of GPR35 expression in hepatocytes in mice. Our findings indicate that the GPR35-STARD4 axis is a promising therapeutic target for NAFLD.

Regulatory mucosa-associated invariant T cells controlled by ß1 adrenergic receptor signaling contribute to hepatocellular carcinoma progression.

BACKGROUND AND AIMS: The innate-like mucosa-associated invariant T (MAIT) cells are enriched in human liver and have been linked to human HCC. However, their contributions to the progression of HCC are controversial due to the heterogeneity of MAIT cells, and new MAIT cell subsets remain to be explored. APPROACH AND RESULTS: Combining single cell RNA sequencing (scRNA-seq) and flow cytometry analysis, we performed phenotypic and functional studies and found that FOXP3 + CXCR3 + MAIT cells in HCC patients were regulatory MAIT cells (MAITregs) with high immunosuppressive potential. These MAITregs were induced under Treg-inducing condition and predominantly from FOXP3 - CXCR3 + MAIT cells, which displayed mild Treg-related features and represented a pre-MAITreg reservoir. In addition, the induction and function of MAITregs were promoted by ß1 adrenergic receptor signaling in pre-MAITregs and MAITregs, respectively. In HCC patients, high proportion of the intratumoral MAITregs inhibited antitumor immune responses and was associated with poor clinical outcomes. CONCLUSIONS: Together, we reveal an immunosuppressive subset of MAIT cells in HCC patients that contributes to HCC progression, and propose a control through neuroimmune crosstalk.

Decrease of peripheral blood mucosal-associated invariant T cells and impaired serum Granzyme-B production in patients with gastric cancer.

Mucosal-associated invariant T (MAIT) cells are an invariant T cell subset, which have been reported to play an antimicrobial role in infectious diseases. However, little is known about it in malignant diseases and tumors, especially in gastric cancer (GC). So in this study, we aim to examine the frequency, phenotype, partial functional capacity and clinical relevance of this cells from GC patients' peripheral blood by flow cytometry. It was shown that the frequency of peripheral blood MAIT cells was negatively correlated with their increasing age in healthy adults. Importantly, comparing to the healthy controls (HC), the frequency and the absolute number of MAIT cells from GC patients' peripheral blood with or without chemotherapy were both significantly lower than those. For the phenotype, the proportion of CD4-MAIT cell subset in GC patients without chemotherapy was lower than in HC, but higher than in GC patients with chemotherapy. Whereas, the proportion of CD4-CD8+MAIT cell subset in GC patients without chemotherapy was significantly lower than that in HC. Finally, the level of Granzyme-B (GrB), a molecule associated with MAIT cells was markedly lower in GC patients. But the correlation between the serum levels of GC-associated tumor antigens and the percentages of MAIT cells in GC patients was not observed. In conclusion, our study shows the decreased frequency, changed phenotypes and partial potentially impaired function of MAIT cells in GC patients, suggesting a possible MAIT cell-based immunological surveillance of GC.

Impaired lipid biosynthesis hinders anti-tumor efficacy of intratumoral iNKT cells.

Dysfunction of invariant natural killer T (iNKT) cells in tumor microenvironment hinders their anti-tumor efficacy, and the underlying mechanisms remain unclear. Here we report that iNKT cells increase lipid biosynthesis after activation, and that is promoted by PPARγ and PLZF synergically through enhancing transcription of Srebf1. Among those lipids, cholesterol is required for the optimal IFN-γ production from iNKT cells. Lactic acid in tumor microenvironment reduces expression of PPARγ in intratumoral iNKT cells and consequently diminishes their cholesterol synthesis and IFN-γ production. Importantly, PPARγ agonist pioglitazone, a thiazolidinedione drug for type 2 diabetes, successfully restores IFN-γ production in tumor-infiltrating iNKT cells from both human patients and mouse models. Combination of pioglitazone and alpha-galactosylceramide treatments significantly enhances iNKT cell-mediated anti-tumor immune responses and prolongs survival of tumor-bearing mice. Our studies provide a strategy to augment the anti-tumor efficacy of iNKT cell-based immunotherapies via promoting their lipid biosynthesis.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated kif15 mutations accelerate axonal outgrowth during neuronal development and regeneration in zebrafish.

KIF15, the vertebrate kinesin-12, is best known as a mitotic motor protein, but continues to be expressed in neurons. Like KIF11 (the vertebrate kinesin-5), KIF15 interacts with microtubules in the axon to limit their sliding relative to one another. Unlike KIF11, KIF15 also regulates interactions between microtubules and actin filaments at sites of axonal branch formation and in growth cones. Our original work on these motors was done on cultured rat neurons, but we are now using zebrafish to extend these studies to an in vivo model. We previously studied kif15 in zebrafish by injecting splice-blocking morpholinos injected into embryos. Consistent with the cell culture work, these studies demonstrated that axons grow faster and longer when KIF15 levels are reduced. In the present study, we applied CRISPR/Cas9-based knockout technology to create kif15 mutants and labeled neurons with Tg(mnx1:GFP) transgene or transient expression of elavl3:EGFP-alpha tubulin. We then compared by live imaging the homozygotic, heterozygotic mutants to their wildtype siblings to ascertain the effects of depletion of kif15 during Caudal primary motor neuron and Rohon-Beard (R-B) sensory neuron development. The results showed, compared to the kif15 wildtype, the number of branches was reduced while axon outgrowth was accelerated in kif15 homozygotic and heterozygotic mutants. In R-B sensory neurons, after laser irradiation, injured axons with loss of kif15 displayed significantly greater regenerative velocity. Given these results and the fact that kif15 drugs are currently under development, we posit kif15 as a novel target for therapeutically augmenting regeneration of injured axons.

Insm1a Regulates Motor Neuron Development in Zebrafish.

Insulinoma-associated1a (insm1a) is a zinc-finger transcription factor playing a series of functions in cell formation and differentiation of vertebrate central and peripheral nervous systems and neuroendocrine system. However, its roles on the development of motor neuron have still remained uncovered. Here, we provided evidences that insm1a was a vital regulator of motor neuron development, and provided a mechanistic understanding of how it contributes to this process. Firstly, we showed the localization of insm1a in spinal cord, and primary motor neurons (PMNs) of zebrafish embryos by in situ hybridization, and imaging analysis of transgenic reporter line Tg(insm1a: mCherry)ntu805 . Then we demonstrated that the deficiency of insm1a in zebrafish larvae lead to the defects of PMNs development, including the reduction of caudal primary motor neurons (CaP), and middle primary motor neurons (MiP), the excessive branching of motor axons, and the disorganized distance between adjacent CaPs. Additionally, knockout of insm1 impaired motor neuron differentiation in the spinal cord. Locomotion analysis showed that swimming activity was significantly reduced in the insm1a-null zebrafish. Furthermore, we showed that the insm1a loss of function significantly decreased the transcript levels of both olig2 and nkx6.1. Microinjection of olig2 and nkx6.1 mRNA rescued the motor neuron defects in insm1a deficient embryos. Taken together, these data indicated that insm1a regulated the motor neuron development, at least in part, through modulation of the expressions of olig2 and nkx6.1.

Generation of a mef2aa:EGFP transgenic zebrafish line that expresses EGFP in muscle cells.

Transgenesis is an important tool for exploring gene expression and function. The myocyte enhancer factor 2a (mef2a) gene encodes a member of the Mef2 protein family that is involved in vertebrate skeletal, cardiac, and smooth muscle development and differentiation during myogenesis. According to studies on human and animal models, mef2a is highly expressed in the heart and somites. To explore the potential of mef2a as a tool for selective labeling of muscle cells in living zebrafish embryos, we constructed a transgene mef2aa:EGFP to induce the expression of green fluorescent protein (GFP) under the control of mef2a promoter. A ~2-kb DNA fragment, upstream of the translational start site of mef2aa, was identified to drive muscle-specific expression of EGFP in zebrafish embryos. Interestingly, the cranial muscles, abductor muscle, and adductor muscle were clearly labeled with EGFP in the established line Tg(mef2aa:EGFP) ntu803 . In addition, we showed that mef2aa mRNA was highly present in adult zebrafish heart, but not the skeleton muscle, whereas it was expressed in both embryonic heart and myotome, suggesting that mef2a is vital to the function of adult heart in vertebrates.

Critical role of active repression by E2F and Rb proteins in endoreplication during Drosophila development.

E2F transcription factors can activate or actively repress transcription of their target genes. The role of active repression during normal development has not been analyzed in detail. dE2F1(su89) is a novel allele of dE2F1 that disrupts dE2F1's association with RBF [the Drosophila retinoblastoma protein (Rb) homolog] but retains its transcription activation function. Interestingly, the dE2F1(su89) mutant, which has E2F activation by dE2F1(su89) and active repression by dE2F2, is viable and fertile with no gross developmental defects. In contrast, complete removal of active repression in de2f2;dE2F1(su89) mutants results in severe developmental defects in tissues with extensive endocycles but not in tissues derived from mitotic cycles. We show that the endoreplication defect resulted from a failure to downregulate the level of cyclin E during the gap phase of the endocycling cells. Importantly, reducing the gene dosage of cyclin E partially suppressed all the phenotypes associated with the endoreplication defect. These observations point to an important role for E2F-Rb complexes in the downregulation of cyclin E during the gap phase of endocycling cells in Drosophila development.