B7 immune-checkpoints as targets for the treatment of neuroendocrine tumors.

The B7 family, and their receptors, the CD28 family, are major immune checkpoints that regulate T-cell activation and function. In the present study, we explore the role of two B7 immune-checkpoints: HERV-H LTR-Associating Protein 2 (HHLA2) and B7 Family Member, H4 (B7x), in the progression of gastrointestinal and pancreatic neuroendocrine tumors (GINETs and PNETs). We demonstrated that both HHLA2 and B7x were expressed to a high degree in human GINETs and PNETs. We determined that the expression of B7x and HHLA2 correlates with higher grade and higher incidence of nodal and distant spread. Furthermore, we confirmed that HIF-1α overexpression is associated with the upregulation of B7x both in our in vivo (animal model) and in vitro (cell culture) models. When grown in vitro, islet tumor ß-cells lack B7x expression, unless cultured under hypoxic conditions, which results in both hypoxia-inducible factor 1 subunit alpha (HIF-1α) and B7x upregulation. In vivo, we demonstrated that Men1/B7x double knockout (KO) mice (with loss of B7x expression) exhibited decreased islet ß-cell proliferation and tumor transformation accompanied by increased T-cell infiltration compared with Men1 single knockout mice. We have also shown that systemic administration of a B7x mAb to our Men1 KO mice with PNETs promotes an antitumor response mediated by increased T-cell infiltration. These findings suggest that B7x may be a critical mediator of tumor immunity in the tumor microenvironment of NETs. Therefore, targeting B7x offers an attractive strategy for the immunotherapy of patients suffering from NETs.

Role of LpL (Lipoprotein Lipase) in Macrophage Polarization In Vitro and In Vivo.

OBJECTIVE: Fatty acid uptake and oxidation characterize the metabolism of alternatively activated macrophage polarization in vitro, but the in vivo biology is less clear. We assessed the roles of LpL (lipoprotein lipase)-mediated lipid uptake in macrophage polarization in vitro and in several important tissues in vivo. Approach and Results: We created mice with both global and myeloid-cell specific LpL deficiency. LpL deficiency in the presence of VLDL (very low-density lipoproteins) altered gene expression of bone marrow-derived macrophages and led to reduced lipid uptake but an increase in some anti- and some proinflammatory markers. However, LpL deficiency did not alter lipid accumulation or gene expression in circulating monocytes nor did it change the ratio of Ly6Chigh/Ly6Clow. In adipose tissue, less macrophage lipid accumulation was found with global but not myeloid-specific LpL deficiency. Neither deletion affected the expression of inflammatory genes. Global LpL deficiency also reduced the numbers of elicited peritoneal macrophages. Finally, we assessed gene expression in macrophages from atherosclerotic lesions during regression; LpL deficiency did not affect the polarity of plaque macrophages. CONCLUSIONS: The phenotypic changes observed in macrophages upon deletion of Lpl in vitro is not mimicked in tissue macrophages.

Endothelial cell CD36 optimizes tissue fatty acid uptake.

Movement of circulating fatty acids (FAs) to parenchymal cells requires their transfer across the endothelial cell (EC) barrier. The multiligand receptor cluster of differentiation 36 (CD36) facilitates tissue FA uptake and is expressed in ECs and parenchymal cells such as myocytes and adipocytes. Whether tissue uptake of FAs is dependent on EC or parenchymal cell CD36, or both, is unknown. Using a cell-specific deletion approach, we show that EC, but not parenchymal cell, CD36 deletion increased fasting plasma FAs and postprandial triglycerides. EC-Cd36-KO mice had reduced uptake of radiolabeled long-chain FAs into heart, skeletal muscle, and brown adipose tissue; these uptake studies were replicated using [11C]palmitate PET scans. High-fat diet-fed EC-CD36-deficient mice had improved glucose tolerance and insulin sensitivity. Both EC and cardiomyocyte (CM) deletion of CD36 reduced heart lipid droplet accumulation after fasting, but CM deletion did not affect heart glucose or FA uptake. Expression in the heart of several genes modulating glucose metabolism and insulin action increased with EC-CD36 deletion but decreased with CM deletion. In conclusion, EC CD36 acts as a gatekeeper for parenchymal cell FA uptake, with important downstream effects on glucose utilization and insulin action.

Pupil-linked arousal modulates behavior in rats performing a whisker deflection direction discrimination task.

Non-luminance-mediated changes in pupil size have been widely used to index arousal state. Recent animal studies have demonstrated correlations between behavioral state-related pupil dynamics and sensory processing. However, the relationship between pupil-linked arousal and behavior in animals performing perceptual tasks has not been fully elucidated. In the present study, we trained head-fixed rats to discriminate between directions of whisker movements using a Go/No-Go discrimination paradigm while imaging their pupils. Reaction times in this discrimination task were significantly slower than in previously reported detection tasks with similar setup, suggesting that discrimination required an increased cognitive load. We found the pupils dilated for all trials following stimulus presentation. Interestingly, in correct rejection trials, where pupil dilations solely resulted from cognitive processing, dilations were larger for more difficult stimuli. Baseline pupil size before stimulus presentation strongly correlated with behavior, as perceptual sensitivity peaked at intermediate pupil baselines and reaction time was fastest at large baselines. We further explored these relationships by investigating to what extent pupil baseline was predictive of upcoming behavior and found that a Bayesian decoder had significantly greater-than-chance probability in correctly predicting behavioral outcomes. Moreover, the outcome of the previous trial showed a strong correlation with behavior on present trials. Animals were more liberal and faster in responding following hit trials, whereas perceptual sensitivity was greatest following correct rejection trials. Taken together, these results suggest a tight correlation between pupil dynamics, perceptual performance, and reaction time in behaving rats, all of which are modulated by fluctuating arousal state. NEW & NOTEWORTHY In this study, we for the first time demonstrated that head-fixed rats were able to discriminate different directions of whisker movement. Interestingly, we found that the pupil dilated more when discriminating more difficult stimuli, a phenomenon reported in human subjects but not in animals. Baseline pupil size before stimulus presentation was found to strongly correlate with behavior, and a Bayesian decoder had significantly greater-than-chance probability in correctly predicting behavioral outcomes based on the baseline pupil size.

Human Aldose Reductase Expression Prevents Atherosclerosis Regression in Diabetic Mice.

Guidelines to reduce cardiovascular risk in diabetes include aggressive LDL lowering, but benefits are attenuated compared with those in patients without diabetes. Consistent with this, we have reported in mice that hyperglycemia impaired atherosclerosis regression. Aldose reductase (AR) is thought to contribute to clinical complications of diabetes by directing glucose into pathways producing inflammatory metabolites. Mice have low levels of AR, thus raising them to human levels would be a more clinically relevant model to study changes in diabetes under atherosclerosis regression conditions. Donor aortae from Western diet-fed Ldlr-/- mice were transplanted into normolipidemic wild-type, Ins2Akita (Akita+/- , insulin deficient), human AR (hAR) transgenic, or Akita+/- /hAR mice. Akita+/- mice had impaired plaque regression as measured by changes in plaque size and the contents of CD68+ cells (macrophages), lipids, and collagen. Supporting synergy between hyperglycemia and hAR were the even more pronounced changes in these parameters in Akita+/- /hAR mice, which had atherosclerosis progression in spite of normolipidemia. Plaque CD68+ cells from the Akita+/- /hAR mice had increased oxidant stress and expression of inflammation-associated genes but decreased expression of anti-inflammatory genes. In summary, hAR expression amplifies impaired atherosclerosis regression in diabetic mice, likely by interfering with the expected reduction in plaque macrophage inflammation.