Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages.

Alternative (M2) activation of macrophages driven via the α-chain of the receptor for interleukin 4 (IL-4Rα) is important for immunity to parasites, wound healing, the prevention of atherosclerosis and metabolic homeostasis. M2 polarization is dependent on fatty acid oxidation (FAO), but the source of the fatty acids that support this metabolic program has not been clear. We found that the uptake of triacylglycerol substrates via the scavenger receptor CD36 and their subsequent lipolysis by lysosomal acid lipase (LAL) was important for the engagement of elevated oxidative phosphorylation, enhanced spare respiratory capacity (SRC), prolonged survival and expression of genes that together define M2 activation. Inhibition of lipolysis suppressed M2 activation during infection with a parasitic helminth and blocked protective responses to this pathogen. Our findings delineate a critical role for cell-intrinsic lysosomal lipolysis in M2 activation.

Lysosomal Acid Lipase Deficiency Controls T- and B-Regulatory Cell Homeostasis in the Lymph Nodes of Mice with Human Cancer Xenotransplants.

Utilization of proper preclinical models accelerates development of immunotherapeutics and the study of the interplay between human malignant cells and immune cells. Lysosomal acid lipase (LAL) is a critical lipid hydrolase that generates free fatty acids and cholesterol. Ablation of LAL suppresses immune rejection and allows growth of human lung cancer cells in lal-/- mice. In the lal-/- lymph nodes, the percentages of both T- and B-regulatory cells (Tregs and Bregs, respectively) are increased, with elevated expression of programmed death-ligand 1 and IL-10, and decreased expression of interferon-γ. Levels of enzymes in the glucose and glutamine metabolic pathways are elevated in Tregs and Bregs of the lal-/- lymph nodes. Pharmacologic inhibitor of pyruvate dehydrogenase, which controls the transition from glycolysis to the citric acid cycle, effectively reduces Treg and Breg elevation in the lal-/- lymph nodes. Blocking the mammalian target of rapamycin or reactivating peroxisome proliferator-activated receptor γ, an LAL downstream effector, reduces lal-/- Treg and Breg elevation and PD-L1 expression in lal-/- Tregs and Bregs, and improves human cancer cell rejection. Treatment with PD-L1 antibody also reduces Treg and Breg elevation in the lal-/- lymph nodes and improves human cancer cell rejection. These observations conclude that LAL-regulated lipid metabolism is essential to maintain antitumor immunity.

Lack of invariant natural killer T cells affects lipid metabolism in adipose tissue of diet-induced obese mice.

OBJECTIVE: Obesity promotes a chronic inflammatory condition in adipose tissue (AT). Impairment of insulin sensitivity coincides with infiltration of T cells into AT in early stages of obesity, when macrophages are not yet present. Here, we examine the role of invariant natural killer T (iNKT) cells, a subtype of T cells activated by lipid antigens, on glucose and lipid metabolism in obesity. APPROACH AND RESULTS: Jα18(-/-) mice, specifically lacking iNKT cells, and wild-type mice consumed a chow or high-fat diet for 10 weeks. One third of all T lymphocytes in the liver of wild-type mice were iNKT cells, whereas few were detected in AT. Diet-induced obesity increased blood glucose in both genotypes of mice, whereas glucose tolerance test revealed similar kinetics of glucose clearance in Jα18(-/-) and wild-type mice. Under obese conditions, expression of inflammatory cytokines in AT did not differ between the groups, although the number of T cells and macrophages was lower in Jα18(-/-) mice. Nonetheless, AT homeostasis in Jα18(-/-) mice was altered evidenced by lower AT weight, smaller adipocytes, accelerated lipogenesis, increased expression of hormone-sensitive lipase, and accelerated basal lipolysis. CONCLUSIONS: iNKT cells do not affect glucose clearance but rather modulate lipid metabolism in both liver and AT. Only few iNKT cells are found in AT under lean and obese conditions, suggesting that their effects on lipid metabolism are mainly mediated in the liver, their primary host organ.

Myeloid-specific expression of human lysosomal acid lipase corrects malformation and malfunction of myeloid-derived suppressor cells in lal-/- mice.

Lysosomal acid lipase (LAL) cleaves cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. LAL deficiency causes expansion of CD11b(+)Gr-1(+) immature myeloid cells, loss of T cells, and impairment of T cell function. To test how myeloid cell LAL controls myelopoiesis and lymphopoiesis, a myeloid-specific doxycycline-inducible transgenic system was used to reintroduce human lysosomal acid lipase (hLAL) expression into LAL gene knockout (lal(-/-)) mice. Expression of hLAL in myeloid cells of lal(-/-) mice reversed abnormal myelopoiesis in the bone marrow starting at the granulocyte-monocyte progenitor stage and reduced systemic expansion of myeloid-derived suppressor cells (MDSCs). Myeloid hLAL expression inhibited reactive oxygen species production and arginase expression in CD11b(+)Gr-1(+) cells of lal(-/-) mice. Structural organization of the thymus and spleen was partially restored in association with reduced infiltration of CD11b(+)Gr-1(+) cells in these mice. In the thymus, reconstitution of myeloid cell LAL restored development of thymocytes at the double-negative DN3 stage. Myeloid cell LAL expression improved the proliferation and function of peripheral T cells. In vitro coculture experiments showed that myeloid hLAL expression in lal(-/-) mice reversed CD11b(+)Gr-1(+) myeloid cell suppression of CD4(+) T cell proliferation, T cell signaling activation, and lymphokine secretion. Blocking stat3 and NF-κB p65 signaling by small-molecule inhibitors in MDSCs achieved a similar effect. Injection of anti-Gr-1 Ab into lal(-/-) mice to deplete MDSCs restored T cell proliferation. These studies demonstrate that LAL in myeloid cells plays a critical role in maintaining normal hematopoietic cell development and balancing immunosuppression and inflammation.

Macrophage-specific transgenic expression of cholesteryl ester hydrolase attenuates hepatic lipid accumulation and also improves glucose tolerance in ob/ob mice.

Cellular cholesterol homeostasis is increasingly being recognized as an important determinant of the inflammatory status of macrophages, and a decrease in cellular cholesterol levels polarizes macrophages toward an anti-inflammatory or M2 phenotype. Cholesteryl ester hydrolase (CEH) catalyzes the hydrolysis of stored intracellular cholesteryl esters (CE) and thereby enhances free cholesterol efflux and reduces cellular CE content. We have reported earlier reduced atherosclerosis as well as lesion necrosis and improved insulin sensitivity (due to decreased adipose tissue inflammation) in macrophage-specific CEH transgenic (CEHTg) mice in the LDLR(-/-) background. In the present study, we examined the effects of reduced intracellular accumulation of CE in CEHTg macrophages in an established diabetic mouse model, namely the leptin-deficient ob/ob mouse. Macrophage-specific transgenic expression of CEH improved glucose tolerance in ob/ob-CEHTg mice significantly compared with ob/ob nontransgenic littermates, but with no apparent change in macrophage infiltration into the adipose tissue. However, there was a significant decrease in hepatic lipid accumulation in ob/ob-CEHTg mice. Consistently, decreased [(14)C]acetate incorporation into total lipids and triglycerides was noted in precision-cut liver slices from ob/ob-CEHTg mice. In the primary hepatocyte-macrophage coculture system, macrophages from CEHTg mice significantly reduced the incorporation of [(14)C]acetate into triglycerides in hepatocytes, indicating a direct effect of macrophages on hepatocyte triglyceride biosynthesis. Kupffer cells isolated from ob/ob-CEHTg mice were polarized toward an anti-inflammatory M2 (Ly6C(lo)) phenotype. Taken together, these studies demonstrate that transgenic overexpression of CEH in macrophages polarizes hepatic macrophages (Kupffer cells) to an anti-inflammatory M2 phenotype that attenuates hepatic lipid synthesis and accumulation.

Lysosomal Acid Lipase: From Cellular Lipid Handler to Immunometabolic Target.

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CEs) and triglycerides (TGs) to free cholesterol (FC) and free fatty acids (FFAs), which are then used for metabolic purposes in the cell. The process also occurs in immune cells that adapt their metabolic machinery to cope with the different energetic requirements associated with cell activation, proliferation, and polarization. LAL deficiency (LALD) causes severe lipid accumulation and affects the immunometabolic signature in animal models. In humans, LAL deficiency is associated with a peculiar clinical immune phenotype, secondary hemophagocytic lymphohistiocytosis. These observations suggest that LAL might play an important role in cellular immunometabolic modulation, and availability of an effective enzyme replacement strategy makes LAL an attractive target to rewire the metabolic machinery of immune cells beyond its role in controlling cellular lipid metabolism.

Bile salt dependent lipase promotes intestinal adaptation in rats with massive small bowel resection.

Intestinal adaptation is important for the short bowel syndrome (SBS) patients. Growing evidence has suggested that bile salt dependent lipase (BSDL) not only has the lipolytic activity, but also the immune-modulating and pro-proliferative activities. The purpose of the present study was to investigate the effects of BSDL on intestinal adaptive growth and gut barrier function in a rat model of SBS. Twenty-four male Sprague-Dawley rats were randomly divided into three experimental groups: sham group (rats underwent bowel transection and re-anastomosis), SBS group (rats underwent 80% bowel resection), SBS-BSDL group (SBS rats orally administered BSDL). The animals were weighed daily. The intestinal morpho-histochemical changes and intestinal barrier function were determined 14 days after the operations. Meanwhile, the expressions of Wnt signaling molecules in enterocytes were also analyzed by immunohistochemistry and Western blot. The postoperative weight gain was faster in the SBS rats treated with BSDL than in the SBS/untreated group. The SBS rats treated with BSDL had significantly greater villus height, crypt depth, and enterocyte proliferation in their residual intestines, as compared with the SBS/untreated group. The recovery of intestinal barrier function was promoted and the expressions of tight-junction proteins were increased in the SBS rats treated with BSDL. Additionally, the data indicated that the proadaptive activities of BSDL might be mediated by Wnt signaling activation in the enterocytes. These observations suggested that enteral BSDL administration promoted intestinal adaptive growth and barrier repairing by activating Wnt signaling pathway in SBS rats.

Immunological identification of cholesterol ester hydrolase in the steroidogenic tissues, adrenal glands and testis.

Monoclonal antibodies were generated against the purified pancreatic cholesterol ester hydrolase (CEH, EC 3.1.1.13) to examine the expression of CEH in various bovine tissues. The presence of CEH isozyme antigenically indistinguishable from pancreatic enzyme in the steroidogenic tissues, adrenal glands and testis has been first demonstrated here using the immunoprecipitation method. These results suggest that CEH isozyme, similar to pancreatic CEH, might be involved in the cholesterol metabolism in the steroidogenic tissue.

Circulating antibodies against an exocrine pancreatic enzyme in type 1 diabetes.

In this article, we report the identification of a new autoantigen in type 1 diabetes originating from the exocrine pancreas. This antigen is a pancreatic enzyme termed bile salt-dependent lipase (BSDL). We show that antibodies present in the sera of newly diagnosed type 1 diabetic patients recognize BSDL and more specifically the COOH-terminal mucin-like region of the protein. Therefore, we engineered the COOH-terminal peptide of BSDL and demonstrated that autoreactivity was linked to specific glycosylation sites by at least two glycosyltransferases: the Core 2 beta(1-6)N-acetylglucosaminyltransferase and the alpha(1-3) fucosyltransferase FUT7. We next examined the prevalence of circulating anti-BSDL antibodies in type 1 diabetic patients and found 73.5% positivity (25 sera among 34 patients tested) at onset, whereas only 8.4% of normal individuals (7 of 83) were positive. Within a cohort of first-degree relatives of diabetic patients followed prospectively until development of diabetes, 6 of 19 (31.6%) were also positive. Interestingly, two prediabetic individuals were already positive for anti-BSDL antibodies (Abs), while islet cell cytoplasmic Abs and antibodies to GAD65, IA-2, and insulin were not detected. Anti-BSDL autoantibodies were weakly or not detected in patients suffering from pancreatitis or pancreatic adenocarcinoma or in patients with Graves' disease. Although autoreactivity to BSDL in prediabetic and newly diagnosed diabetic patients might reflect cross-reactivity, our results strongly suggest that in addition to pancreatic beta-cells, acinar cells may be also affected in type 1 diabetes.

Evaluation of the role of AMP-activated protein kinase and its downstream targets in mammalian hibernation.

Mammalian hibernation requires an extensive reorganization of metabolism that typically includes a greater than 95% reduction in metabolic rate, selective inhibition of many ATP-consuming metabolic activities and a change in fuel use to a primary dependence on the oxidation of lipid reserves. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in this reorganization. AMPK activity and the phosphorylation state of multiple downstream targets were assessed in five organs of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) comparing euthermic animals with squirrels in deep torpor. AMPK activity was increased 3-fold in white adipose tissue from hibernating ground squirrels compared with euthermic controls, but activation was not seen in liver, skeletal muscle, brown adipose tissue or brain. Immunoblotting with phospho-specific antibodies revealed an increase in phosphorylation of eukaryotic elongation factor-2 at the inactivating Thr56 site in white adipose tissue, liver and brain of hibernators, but not in other tissues. Acetyl-CoA carboxylase phosphorylation at the inactivating Ser79 site was markedly increased in brown adipose tissue from hibernators, but no change was seen in white adipose tissue. No change was seen in the level of phosphorylation of the Ser565 AMPK site of hormone-sensitive lipase in adipose tissues of hibernating animals. In conclusion, AMPK does not appear to participate in the metabolic re-organization and/or the metabolic rate depression that occurs during ground squirrel hibernation.

Expression of biologically active hormone-sensitive lipase in mammalian (COS) cells.

cDNAs encoding rat adipose tissue hormone-sensitive lipase were expressed in COS cells, under the control of the SV40 promoter to half the level in rat adipocytes, the richest native source of the enzyme. A cDNA lacking most of the long 5'-untranslated region of the full-length rat hormone-sensitive lipase cDNA was, with regard to the lipase activity, on the average 70% more efficiently expressed that the full-length cDNA. The recombinant protein was almost identical to hormone-sensitive lipase of rat adipose tissue with respect to specific activity, susceptibility to inhibitors, molecular size, phosphorylation and activation by cyclic AMP-dependent protein kinase. The described eukaryotic expression system will allow analysis of effects of amino acid substitutions introduced into the lipase molecule by site-directed mutagenesis.

Use of protein G for preparation and characterization of rabbit antibodies against rat adipose tissue hormone-sensitive lipase.

The newly described immunoglobulin G-binding streptococcal surface protein, protein G, was used to prepare and characterize rabbit antibodies. The antibodies were directed against rat hormone-sensitive lipase, the rate-limiting enzyme in the hydrolysis of the triacylglycerols stored in adipose tissue. Antiserum was obtained after two injections with 20 micrograms enzyme protein, and the immunoglobulin fraction was obtained using a protein G-based solid-phase radioimmunoassay. The hydrolysis of acylglycerols by the enzyme was inhibited by the antibodies, and the enzyme could be efficiently removed from a solution using the antibodies and heat-killed streptococci expressing surface protein G. By Western blot and detection with 125I-protein G, the antibodies were found to selectively bind to hormone-sensitive lipase and to a smaller extent to two minor contaminants, possibly proteolytic fragments of the lipase. The amount of 125I-labelled protein G bound to the lipase on the blot was quantitatively related to the amount of enzyme protein down to the detection limit 10 ng.

Specific induction by glucocorticoids of steroid esterase in rat hepatic microsomes and its release into serum.

Steroid esterase hydrolysing methylprednisolone 21-hemisuccinate was induced specifically and markedly in hepatic microsomes and serum of rats by various glucocorticoids. Among the glucocorticoids examined, dexamethasone and betamethasone showed the highest potency to induce the hepatic steroid esterase, the induction ratio being 32 and 33 times higher than the basal level (about 160 mU/g liver), respectively. Steroid esterase in the serum was induced greatly by fluocinolone acetonide and betamethasone to 92 and 79 times of the basal level of about 16 mU/mL, respectively, followed by dexamethasone and methylprednisolone. When dexamethasone was given to rats, the enzyme in other tissues except for duodenum and small intestine (of which activity was lowered to 50% of the basal level) was also elevated, but the induction ratio was much lower than that in the liver and serum. The induction of the steroid esterase is probably due to stimulation of de novo synthesis of the enzyme by glucocorticoids, because the elevation of esterase activity was inhibited by treatment with cycloheximide (a translation inhibitor) and actinomycin D (a transcription inhibitor), and about 4- and 10-hr lag time was observed before the elevation of esterase activity in liver and serum, respectively. Coupled with these observations the following results indicate that the steroid esterase in serum is probably synthesized in the liver and subsequently released into the blood via the Golgi apparatus: (1) when the liver of rats treated with dexamethasone was subjected to perfusion with a recycling system, significant amounts of steroid esterase were released into the perfusate; (2) anti-hepatic esterase antibody inhibited the steroid esterase activity not only in the liver but also in serum; and (3) monensin, which prevents the secretion of various kinds of secretory proteins by disrupting the function of the Golgi apparatus, inhibited the elevation of the steroid esterase activity in serum by dexamethasone but did not affect the induction in liver.

Hormone-sensitive lipase in brown adipose tissue: identification and effect of cold exposure.

Hormone-sensitive lipase (HSL) in brown adipose tissue from mice was identified through immunoprecipitation with a polyclonal antibody (anti-HSL) towards rat white fat HSL and Western blotting. An 82 kDa polypeptide, slightly smaller than the rat white fat HSL 84 kDa subunit, was detected and its identity as HSL verified by inhibition properties. The HSL concentration per g tissue was several-fold higher in the mouse brown adipose tissue than in the rat white adipose tissue, but the specific activities per mg protein were similar. Cold-exposure (4 degrees C) of the mice for 24 h approximately doubled the HSL concentration but this increase parallelled the overall protein increase and did not reflect a specific effect on the HSL.

[Cloning, determination of primary structure, and expression of the C-terminal segment of human cholesterol-esterase/lipase, containing the antigenic determinant of the protein, in Escherichia coli]. / Klonirovanie, opredelenie pervichnoi struktury i ékspressiia v Excherichia coli C-kontsevogo uchastka chelovecheskoi kholesterol-ésterazy/lipazy, soderzhashchego antigennuiu determinantu belka.

Clone pHICE0.9 was selected from human insulinoma cDNA library by immunoscreening with antibodies against total human insulinoma proteins. This clone contains a 0.9 kb cDNA insert and expresses a fusion protein with beta-galactosidase. Nucleotide sequences of 5'- and 3'-terminal regions of this cDNA insert show that clone pHICE0.9 expresses a protein which is identical to the C-terminal fragment (amino acids 483 to 745) of human pancreatic cholesterol esterase and Homo sapiens bile-acid-salt-stimulated lipase from milk. It is concluded that the protein fragment contains the antigenic determinant of human cholesterol esterase/lipase, and can be used for lipase determination in blood.

Bile salt-stimulated lipase plays an unexpected role in arthritis development in rodents.

OBJECTIVE: The present study aimed to explore the hypothesis that bile salt-stimulated lipase (BSSL), in addition to being a key enzyme in dietary fat digestion during early infancy, plays an important role in inflammation, notably arthritis. METHODS: Collagen-induced arthritis (CIA) and pristane-induced arthritis (PIA) in rodents are commonly used experimental models that reproduce many of the pathogenic mechanisms of human rheumatoid arthritis, i.e. increased cellular infiltration, synovial hyperplasia, pannus formation, and erosion of cartilage and bone in the distal joints. We used the CIA model to compare the response in BSSL wild type (BSSL-WT) mice with BSSL-deficient 'knock-out' (BSSL-KO) and BSSL-heterozygous (BSSL-HET) littermates. We also investigated if intraperitoneal injection of BSSL-neutralizing antibodies affected the development or severity of CIA and PIA in mice and rats, respectively. RESULTS: In two consecutive studies, we found that BSSL-KO male mice, in contrast to BSSL-WT littermates, were significantly protected from developing arthritis. We also found that BSSL-HET mice were less prone to develop disease compared to BSSL-WT mice, but not as resistant as BSSL-KO mice, suggesting a gene-dose effect. Moreover, we found that BSSL-neutralizing antibody injection reduced both the incidence and severity of CIA and PIA in rodents. CONCLUSION: Our data strongly support BSSL as a key player in the inflammatory process, at least in rodents. It also suggests the possibility that BSSL-neutralizing agents could serve as a therapeutic model to reduce the inflammatory response in humans.

Wolman disease/cholesteryl ester storage disease: efficacy of plant-produced human lysosomal acid lipase in mice.

Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. Genetic LAL mutations lead to Wolman disease (WD) and cholesteryl ester storage disease (CESD). An LAL-null (lal(-/-)) mouse model resembles human WD/CESD with storage of CEs and TGs in multiple organs. Human LAL (hLAL) was expressed in Nicotiana benthamiana using the GENEWARE expression system (G-hLAL). Purified G-hLAL showed mannose receptor-dependent uptake into macrophage cell lines (J774E). Intraperitoneal injection of G-hLAL produced peak activities in plasma at 60 min and in the liver and spleen at 240 min. The t(1/2) values were: approximately 90 min (plasma), approximately 14 h (liver), and approximately 32 h (spleen), with return to baseline by approximately 150 h in liver and approximately 200 h in spleen. Ten injections of G-hLAL (every 3 days) into lal(-/-) mice produced normalization of hepatic color, decreases in hepatic cholesterol and TG contents, and diminished foamy macrophages in liver, spleen, and intestinal villi. All injected lal(-/-) mice developed anti-hLAL protein antibodies, but suffered no adverse events. These studies demonstrate the feasibility of using plant-expressed, recombinant hLAL for the enzyme therapy of human WD/CESD with general implications for other lysosomal storage diseases.