Jianyang Luo

CSF1/CSF1R Blockade Reprograms Tumor-Infiltrating Macrophages and Improves Response to T Cell Checkpoint Immunotherapy in Pancreatic Cancer Models.

Cancer immunotherapy generally offers limited clinical benefit without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment. Critical drivers of immune escape in the tumor microenvironment include tumor-associated macrophages and myeloid-derived suppressor cells, which not only mediate immune suppression, but also promote metastatic dissemination and impart resistance to cytotoxic therapies. Thus, strategies to ablate the effects of these myeloid cell populations may offer great therapeutic potential. In this report, we demonstrate in a mouse model of pancreatic ductal adenocarcinoma (PDAC) that inhibiting signaling by the myeloid growth factor receptor CSF1R can functionally reprogram macrophage responses that enhance antigen presentation and productive antitumor T-cell responses. Investigations of this response revealed that CSF1R blockade also upregulated T-cell checkpoint molecules, including PDL1 and CTLA4, thereby restraining beneficial therapeutic effects. We found that PD1 and CTLA4 antagonists showed limited efficacy as single agents to restrain PDAC growth, but that combining these agents with CSF1R blockade potently elicited tumor regressions, even in larger established tumors. Taken together, our findings provide a rationale to reprogram immunosuppressive myeloid cell populations in the tumor microenvironment under conditions that can significantly empower the therapeutic effects of checkpoint-based immunotherapeutics.

Decreased Hepatic Triglyceride Accumulation and Altered Fatty Acid Uptake in Mice with Deletion of the Liver Fatty Acid-binding Protein Gene

Liver fatty acid-binding protein (L-Fabp) is an abundant cytosolic lipid-binding protein with broad substrate specificity, expressed in mammalian enterocytes and hepatocytes. We have generated mice with a targeted deletion of the endogenous L-Fabp gene and have characterized their response to alterations in hepatic fatty acid flux following prolonged fasting. Chow-fed L-Fabp–/– mice were indistinguishable from wild-type littermates with regard to growth, serum and tissue lipid profiles, and fatty acid distribution within hepatic complex lipid species. In response to 48-h fasting, however, wild-type mice demonstrated a ∼10-fold increase in hepatic triglyceride content while L-Fabp–/– mice demonstrated only a 2-fold increase. Hepatic VLDL secretion was decreased in L-Fabp–/– mice suggesting that the decreased accumulation of hepatic triglyceride was not the result of increased secretion. Fatty acid oxidation, as inferred from serum β-hydroxybutyrate levels, was increased in response to fasting, although the increase in L-Fabp–/– mice was significantly reduced in comparison to wild-type controls, despite comparable induction of PPARα target genes. Studies in primary hepatocytes revealed indistinguishable initial rates of oleate uptake, but longer intervals revealed reduced rates of uptake in fasted L-Fabp–/– mice. Oleate incorporation into cellular triglyceride and diacylglycerol was reduced in L-Fabp–/– mice although incorporation into phospholipid and cholesterol ester was no different than wild-type controls. These data point to an inducible defect in fatty acid utilization in fasted L-Fabp–/– mice that involves targeting of substrate for use in triglyceride metabolism.

Protection against Western diet–induced obesity and hepatic steatosis in liver fatty acid–binding protein knockout mice†

Liver fatty acid–binding protein (L‐Fabp) regulates murine hepatic fatty acid trafficking in response to fasting. In this study, we show that L‐Fabp−/− mice fed a high‐fat Western diet for up to 18 weeks are less obese and accumulate less hepatic triglyceride than C57BL/6J controls. Paradoxically, both control and L‐Fabp−/− mice manifested comparable glucose intolerance and insulin resistance when fed a Western diet. Protection against obesity in Western diet–fed L‐Fabp−/− mice was not due to discernable changes in food intake, fat malabsorption, or heat production, although intestinal lipid secretion kinetics were significantly slower in both chow‐fed and Western diet–fed L‐Fabp−/− mice. By contrast, there was a significant increase in the respiratory exchange ratio in L‐Fabp−/− mice, suggesting a shift in energy substrate use from fat to carbohydrate, findings supported by an approximately threefold increase in serum lactate. Microarray analysis revealed increased expression of genes involved in lipid synthesis (fatty acid synthase, squalene epoxidase, hydroxy‐methylglutaryl coenzyme A reductase), while genes involved in glycolysis (glucokinase and glycerol kinase) were decreased in L‐Fabp−/− mice. Fatty acid synthase expression was also increased in the skeletal muscle of L‐Fabp−/− mice. In conclusion, L‐Fabp may function as a metabolic sensor in regulating lipid homeostasis. We suggest that L‐Fabp−/− mice are protected against Western diet–induced obesity and hepatic steatosis through a series of adaptations in both hepatic and extrahepatic energy substrate use. (HEPATOLOGY 2006;44:1191–1205.)

Compensatory increase in hepatic lipogenesis in mice with conditional intestine-specific Mttp deficiency.

Microsomal TG transfer protein (MTTP) is required for the assembly and secretion of TG (TG)-rich lipoproteins from both enterocytes and hepatocytes. Liver-specific deletion of Mttp produced a dramatic reduction in plasma very low density lipoprotein-TG and virtually eliminated apolipoprotein B100 (apoB100) secretion yet caused only modest reductions in plasma apoB48 and apoB48 secretion from primary hepatocytes. These observations prompted us to examine the phenotype following intestine-specific Mttp deletion because murine, like human enterocytes, secrete virtually exclusively apoB48. We generated mice with conditional Mttp deletion in villus enterocytes (Mttp-IKO), using a tamoxifen-inducible, intestine-specific Cre transgene. Villus enterocytes from chow-fed Mttp-IKO mice contained large cytoplasmic TG droplets and no chylomicron-sized particles within the secretory pathway. Chow-fed, Mttp-IKO mice manifested steatorrhea, growth arrest, and decreased cholesterol absorption, features that collectively recapitulate the phenotype associated with abetalipoproteinemia. Chylomicron secretion was reduced dramatically in vivo, in conjunction with an ∼80% decrease in apoB48 secretion from primary enterocytes. Additionally, although plasma and hepatic cholesterol and TG content were decreased, Mttp-IKO mice demonstrated a paradoxical increase in both hepatic lipogenesis and very low density lipoprotein secretion. These findings establish distinctive features for MTTP involvement in intestinal chylomicron assembly and secretion and suggest that hepatic lipogenesis undergoes compensatory induction in the face of defective intestinal TG secretion.

Altered hepatic triglyceride content after partial hepatectomy without impaired liver regeneration in multiple murine genetic models

Liver regeneration is impaired following partial hepatectomy (PH) in mice with genetic obesity and hepatic steatosis and also in wild‐type mice fed a high‐fat diet. These findings contrast with other data showing that liver regeneration is impaired in mice in which hepatic lipid accumulation is suppressed by either pharmacologic leptin administration or by disrupted glucocorticoid signaling. These latter findings suggest that hepatic steatosis may actually be required for normal liver regeneration. We have reexamined this relationship using several murine models of altered hepatic lipid metabolism. Liver fatty acid (FA) binding protein knockout mice manifested reduced hepatic triglyceride (TG) content compared to controls, with no effect on liver regeneration or hepatocyte proliferation. Examination of early adipogenic messenger RNAs revealed comparable induction in liver from both genotypes despite reduced hepatic steatosis. Following PH, hepatic TG was reduced in intestine‐specific microsomal TG transfer protein deleter mice, which fail to absorb dietary fat, increased in peroxisome proliferator activated receptor alpha knockout mice, which exhibit defective FA oxidation, and unchanged (from wild‐type mice) in liver‐specific FA synthase knockout mice in which endogenous hepatic FA synthesis is impaired. Hepatic TG increased in the regenerating liver in all models, even in animals in which lipid accumulation is genetically constrained. However, in no model—and over a >90‐fold range of hepatic TG content—was liver regeneration significantly impaired following PH. Conclusion: Although hepatic TG content is widely variable and increases during liver regeneration, alterations in neither exogenous or endogenous lipid metabolic pathways, demonstrated to promote or diminish hepatic steatosis, influence hepatocyte proliferation. (HEPATOLOGY 2008.)

Targeted Deletion of the Murine apobec-1 Complementation Factor (acf) Gene Results in Embryonic Lethality

ABSTRACT apobec-1 complementation factor (ACF) is an hnRNP family member which functions as the obligate RNA binding subunit of the core enzyme mediating C-to-U editing of the nuclear apolipoprotein B (apoB) transcript. ACF binds to both apoB RNA and apobec-1, the catalytic cytidine deaminase, which then results in site-specific posttranscriptional editing of apoB mRNA. Targeted deletion of apobec1 eliminates C-to-U editing of apoB mRNA but is otherwise well tolerated. However, the functions and potential targets of ACF beyond apoB mRNA editing are unknown. Here we report the results of generating acf knockout mice using homologous recombination. While heterozygous acf+/ − mice were apparently healthy and fertile, no viable acf − / − mice were identified. Mutant acf − / − embryos were detectable only until the blastocyst (embryonic day 3.5 [E3.5]) stage. No acf − / − blastocysts were detectable following implantation at E4.5, and isolated acf − / − blastocysts failed to proliferate in vitro. Small interfering RNA knockdown of ACF in either rat (apobec-1-expressing) or human (apobec-1-deficient) hepatoma cells decreased ACF protein expression and induced a commensurate increase in apoptosis. Taken together, these data suggest that ACF plays a crucial role, which is independent of apobec-1 expression, in cell survival, particularly during early embryonic development.

Diet-induced alterations in intestinal and extrahepatic lipid metabolism in liver fatty acid binding protein knockout mice.

Liver fatty acid binding protein (L-FABP) is highly expressed in both enterocytes and hepatocytes and binds multiple ligands, including saturated (SFA), unsaturated fatty acids (PUFA), and cholesterol. L-fabp−/− mice were protected against obesity and hepatic steatosis on a high saturated fat (SF), high cholesterol “Western” diet and manifested a similar phenotype when fed with a high SF, low cholesterol diet. There were no significant differences in fecal fat content or food consumption between the genotypes, and fatty acid (FA) oxidation was reduced, rather than increased, in SF-fed L-fabp−/− mice as evidenced by decreased heat production and serum ketones. In contrast to mice fed with a SF diet, L-fabp−/− mice fed with a high PUFA diet were not protected against obesity and hepatic steatosis. These observations together suggest that L-fabp−/− mice exhibit a specific defect in the metabolism of SFA, possibly reflecting altered kinetics of FA utilization. In support of this possibility, microarray analysis of muscle from Western diet-fed mice revealed alterations in genes regulating glucose uptake and FA synthesis. In addition, intestinal cholesterol absorption was decreased in L-fabp−/− mice. On the other hand, and in striking contrast to other reports, female L-fabp−/− mice fed with low fat, high cholesterol diets gained slightly less weight than control mice, with minor reductions in hepatic triglyceride content. Together these data indicate a role for L-FABP in intestinal trafficking of both SFA and cholesterol.

Conditional Intestinal Lipotoxicity in Apobec-1-/- Mttp-IKO Mice A SURVIVAL ADVANTAGE FOR MAMMALIAN INTESTINAL APOLIPOPROTEIN B mRNA EDITING

Mammalian small intestinal lipid absorption requires the coordinated interactions of apolipoprotein B (apoB) and the microsomal triglyceride transfer protein (Mttp). The observation that apoB100 displays greater dependence on Mttp availability than does apoB48 prompted us to examine the phenotype of Mttp deletion in an Apobec-1-/- background (i.e. apoB100 Mttp-IKO). 20% apoB100 Mttp-IKO mice died on a chow diet, and >90% died following high fat feeding (versus 0 and 11% apoB48 Mttp-IKO mice, respectively). Intestinal adaptation occurred in apoB48 Mttp-IKO mice in response to high fat feeding, evidenced by increased bromodeoxyuridine incorporation and villus lengthening, changes that did not occur in apoB100 Mttp-IKO mice. There was an exaggerated unfolded protein response (UPR), which became more pronounced in apoB100 Mttp-IKO mice. To examine the role of endoplasmic reticulum stress and the UPR in the lipotoxic effects of Mttp deletion, we administered tauroursodeoxycholate to apoB100 Mttp-IKO mice upon initiation of high fat feeding. Tauroursodeoxycholate administration abrogated the UPR but produced an unexpected acceleration in the onset of lethality in apoB100 Mttp-IKO mice. The findings demonstrate that there is activation of the UPR with lethal lipotoxicity in conditional intestinal apoB100 Mttp-IKO mice. Together the data provide the first plausible biological evidence for a survival advantage for mammalian intestinal apoB mRNA editing.

Decreased body weight and hepatic steatosis with altered fatty acid ethanolamide metabolism in aged L-Fabp -/- mice.

The tissue-specific sources and regulated production of physiological signals that modulate food intake are incompletely understood. Previous work showed that L-Fabp−/− mice are protected against obesity and hepatic steatosis induced by a high-fat diet, findings at odds with an apparent obesity phenotype in a distinct line of aged L-Fabp−/− mice. Here we show that the lean phenotype in L-Fabp−/− mice is recapitulated in aged, chow-fed mice and correlates with alterations in hepatic, but not intestinal, fatty acid amide metabolism. L-Fabp−/− mice exhibited short-term changes in feeding behavior with decreased food intake, which was associated with reduced abundance of key signaling fatty acid ethanolamides, including oleoylethanolamide (OEA, an agonist of PPARα) and anandamide (AEA, an agonist of cannabinoid receptors), in the liver. These reductions were associated with increased expression and activity of hepatic fatty acid amide hydrolase-1, the enzyme that degrades both OEA and AEA. Moreover, L-Fabp−/− mice demonstrated attenuated responses to OEA administration, which was completely reversed with an enhanced response after administration of a nonhydrolyzable OEA analog. These findings demonstrate a role for L-Fabp in attenuating obesity and hepatic steatosis, and they suggest that hepatic fatty acid amide metabolism is altered in L-Fabp−/− mice.

Dextran sodium sulfate inhibition of real‐time polymerase chain reaction amplification: A poly‐A purification solution

Background: Dextran sulfate sodium (DSS) induces experimental colitis and promotes colitis‐associated cancer in rodents. Here we document potent inhibition of real‐time quantitative polymerase chain reaction (qPCR) using cDNA from DSS‐exposed mouse tissues, which complicates gene expression analysis. Methods: We characterize DSS inhibition of qPCR in‐vitro and in a wide array of murine tissues following ingestion of DSS. We examine different approaches to RNA purification prior to cDNA synthesis in order to optimize real‐time polymerase chain reaction amplification and gene expression analysis. Results: DSS inhibits qPCR amplification of cDNA between 1 and 10 nM. Orally administered DSS interferes with qPCR amplification of cDNA derived from multiple tissues. Poly‐A purification of DSS‐exposed RNA allows reliable and cost‐effective gene expression analysis in DSS‐exposed tissue. Conclusions: DSS is a potent inhibitor of real‐time qPCR amplification and interferes with tissue‐specific gene expression analysis in DSS‐exposed mice. Poly‐A purification of tissue‐derived RNA results in reliable and cost‐effective gene expression analysis in DSS‐exposed mice. (Inflamm Bowel Dis 2011;)