Small tumor necrosis factor receptor biologics inhibit the tumor necrosis factor-p38 signalling axis and inflammation.

Despite anti-TNF therapy advancements for inflammatory diseases such as rheumatoid arthritis, the burden of diseases remains high. An 11-mer TNF peptide, TNF70-80, is known to stimulate selective functional responses compared to the parent TNF molecule. Here, we show that TNF70-80 binds to the TNF receptor, activating p38 MAP kinase through TNF receptor-associated factor 2. Using truncated TNFR mutants, we identify the sequence in TNFRI which enables p38 activation by TNF70-80. Peptides with this TNFRI sequence, such as TNFRI206-211 bind to TNF and inhibit TNF-induced p38 activation, respiratory burst, cytokine production and adhesion receptor expression but not F-Met-Leu-Phe-induced respiratory burst in neutrophils. TNFRI206-211 does not prevent TNF binding to TNFRI or TNF-induced stimulation of ERK, JNK and NF-κB. TNFRI206-211 inhibits bacterial lipopolysaccharide-induced peritonitis, carrageenan-induced and antigen-induced paw inflammation, and respiratory syncytial virus-induced lung inflammation in mice. Our findings suggest a way of targeting TNF-p38 pathway to treat chronic inflammatory disorders.

Selective suppression of adipose tissue apoE expression impacts systemic metabolic phenotype and adipose tissue inflammation.

apoE is a multi-functional protein expressed in several cell types and in several organs. It is highly expressed in adipose tissue, where it is important for modulating adipocyte lipid flux and gene expression in isolated adipocytes. In order to investigate a potential systemic role for apoE that is produced in adipose tissue, mice were generated with selective suppression of adipose tissue apoE expression and normal circulating apoE levels. These mice had less adipose tissue with smaller adipocytes containing fewer lipids, but no change in adipocyte number compared with control mice. Adipocyte TG synthesis in the presence of apoE-containing VLDL was markedly impaired. Adipocyte caveolin and leptin gene expression were reduced, but adiponectin, PGC-1, and CPT-1 gene expression were increased. Mice with selective suppression of adipose tissue apoE had lower fasting lipid, insulin, and glucose levels, and glucose and insulin tolerance tests were consistent with increased insulin sensitivity. Lipid storage in muscle, heart, and liver was significantly reduced. Adipose tissue macrophage inflammatory activation was markedly diminished with suppression of adipose tissue apoE expression. Our results establish a novel effect of adipose tissue apoE expression, distinct from circulating apoE, on systemic substrate metabolism and adipose tissue inflammatory state.

ApoE derived from adipose tissue does not suppress atherosclerosis or correct hyperlipidemia in apoE knockout mice.

The synthesis of apoE by adipocytes has profound effects on adipose tissue lipid flux and gene expression. Using adipose tissue transplantation from wild-type (WT) to apoE knockout (EKO) mice, we show that adipose tissue also contributes to circulating apoE. Different from circulating apoE produced by bone marrow transplantation (BMT), however, adipose tissue-derived apoE does not correct hyperlipidemia or suppress atherosclerosis. ApoE secreted by macrophages has a more acidic isoform distribution, and it increases binding of reconstituted VLDL particles to hepatocytes and fibroblasts more effectively than apoE secreted by adipocytes. The incremental binding can be entirely accounted for by binding to the LDL receptor. After BMT into EKO hosts, plasma cholesterol and macrophage-derived apoE are largely within IDL/LDL- and HDL-sized particles. After adipose tissue transplantation, most cholesterol and adipocyte apoE remain in VLDL. After BMT, circulating apoE no longer demonstrates predominance of acidic isoforms compared with that circulating after fat transplantation. In conclusion, fat transplantation provides circulating apoE levels similar to those provided by bone marrow transplantation, but it does not suppress hyperlipidemia or atherosclerosis. A potential mechanism contributing to this difference is differential binding to cell surface lipoprotein receptors.

Expression of the human apoE2 isoform in adipocytes: altered cellular processing and impaired adipocyte lipogenesis.

Expression of apoE in adipocytes has been shown to have an important role in modulating adipocyte triglyceride (TG) metabolism and gene expression that is independent of circulating and extracellular apoE. The impact of adipocyte expression of common human apoE isoforms was evaluated using adipocytes harvested from human apoE2, -3, and -4 knock-in mice. Expression of the apoE2 isoform was associated with an increase in adipocyte apoE gene expression and apoE synthesis. Newly synthesized apoE2 was unstable in adipocytes and demonstrated increased degradation and decreased secretion. ApoE2-expressing mice were hyperlipidemic, and had increased size of gonadal fat pads and of adipocytes, compared with apoE3 mice. In isolated cells, however, expression of the apoE2 isoform produced defective lipogenesis and increased TG hydrolysis. Incubation of adipose tissue with apoE3-containing TG-rich lipoproteins resulted in a significant increase in TG in adipose tissue from apoE3 and -E4 mice, but not apoE2 mice. Reduced capacity to internalize FFA as lipogenic substrate contributed to defective lipogenesis. Newly synthesized apoE2 is unstable in adipocytes and results in decreased adipocyte TG synthesis and defective FA uptake. These changes recapitulate those observed in apoE knockout adipocytes and have implications for understanding metabolic disturbances in humans expressing the E2 isoform.

Adipose tissue depot-specific differences in adipocyte apolipoprotein E expression.

Important differences in gene expression have been documented in adipocytes derived from specific adipose tissue depots. We have previously documented an important role for adipocyte apolipoprotein E (apoE) in modulating adipocyte and adipose tissue triglyceride and lipoprotein metabolism. We now evaluate the endogenous expression of apoE in adipocytes isolated from unique adipose tissue depots in 4 different species. Adipocyte apoE expression is higher in subcutaneous fat compared with visceral fat in humans, mice, rats, and baboons. In baboons, evaluation of apoE expression in 5 adipose tissue depots (subcutaneous abdominal, subcutaneous gluteal, visceral, pericardial, epicardial) showed that, compared with subcutaneous abdominal adipocytes, the level of apoE expression is similar in subcutaneous gluteal, lower in visceral and pericardial, and higher in epicardial adipocytes. Consistent with previously demonstrated suppression of adipocyte apoE by adipose tissue inflammation, adipose tissue depots with lower apoE expression demonstrated greater infiltration of macrophages and an increased expression of tumor necrosis factor-α messenger RNA. Depot-specific differences in apoE expression were maintained after in vitro differentiation. Adipocytes isolated from depots with lower apoE expression manifested lower rates of triglyceride synthesis in the absence and presence of triglyceride-rich lipoproteins. Adenoviral-mediated increase of apoE expression in omental adipocytes increased triglyceride synthesis in these cells. Our results demonstrate significant heterogeneity in adipocyte apoE expression across adipose tissue depots in several species. Because of its role in modulating adipocyte triglyceride and lipoprotein metabolism, depot-specific differences in endogenous adipocyte apoE could have important implications for modulating the accumulation of lipid in these depots.

Effects of leptin replacement on hypothalamic-pituitary growth hormone axis function and circulating ghrelin levels in ob/ob mice.

Leptin-deficient obese mice (ob/ob) have decreased circulating growth hormone (GH) and pituitary GH and ghrelin receptor (GHS-R) mRNA levels, whereas hypothalamic GH-releasing hormone (GHRH) and somatostatin (SST) expression do not differ from lean controls. Given the fact that GH is suppressed in diet-induced obesity (a state of hyperleptinemia), it remains to be determined whether the absence of leptin contributes to changes in the GH axis of ob/ob mice. Therefore, to study the impact of leptin replacement on the hypothalamic-pituitary GH axis of ob/ob mice, leptin was infused for 7 days (sc), resulting in circulating leptin levels that were similar to wild-type controls (approximately 1 ng/ml). Leptin treatment reduced food intake, body weight, and circulating insulin while elevating circulating n-octanoyl ghrelin concentrations. Leptin treatment did not alter hypothalamic GHRH, SST, or GHS-R mRNA levels compared with vehicle-treated controls. However, leptin significantly increased pituitary GH and GHRH-R expression and tended to enhance circulating GH levels, but this latter effect did not reach statistical significance. In vitro, leptin (1 ng/ml, 24 h) did not affect pituitary GH, GHRH-R, or GHS-R mRNA but did enhance GH release. The in vivo effects of leptin on circulating hormone and pituitary mRNA levels were not replicated by pair feeding ob/ob mice to match the food intake of leptin-treated mice. However, leptin did prevent the fall in hypothalamic GHRH mRNA and circulating IGF-I levels observed in pair-fed mice. These results demonstrate that leptin replacement has positive effects on multiple levels of GH axis function in ob/ob mice.

A novel beta-oxa polyunsaturated fatty acid downregulates the activation of the IkappaB kinase/nuclear factor kappaB pathway, inhibits expression of endothelial cell adhesion molecules, and depresses inflammation.

Several novel polyunsaturated fatty acids (PUFAs) that contain either an oxygen or sulfur atom in the beta-position were found to exhibit more selective antiinflammatory properties than their natural PUFA counterparts. One of these, beta-oxa-23:4n-6, unlike natural PUFAs, lacked ability to stimulate oxygen radical production in neutrophils but caused marked inhibition of agonist-induced upregulation of leukocyte adhesion to cultured human umbilical vein endothelial cells (HUVEC) and E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 expression. In addition, beta-oxa-23:4n-6 inhibited acute and chronic inflammatory responses in mice as well as the upregulation of adhesion molecule expression in arterial endothelium. This action of beta-oxa-23:4n-6 required a functional 12- but not 5-lipoxygenase or cyclooxygenases, consistent with its metabolism via the 12-lipoxygenase pathway. Whereas beta-oxa-23:4n-6 did not affect the activation of mitogen-activated protein kinases by tumor necrosis factor, activation of the IkappaB kinase/nuclear factor kappaB pathway was selectively inhibited. These novel PUFAs could form the basis for a potential new class of pharmaceuticals for treating inflammatory diseases, including atherosclerosis.

Distinct cellular loci for the ABCA1-dependent and ABCA1-independent lipid efflux mediated by endogenous apolipoprotein E expression.

OBJECTIVE: Macrophage expression of both apolipoprotein E (apoE) and ABCA1 have been shown to modulate lipid efflux from these cells and to play an important atheroprotective role in vivo. We evaluated the relationship between apoE and ABCA1 for regulating cellular sterol efflux. METHODS AND RESULTS: ApoE-mediated, but ABCA1-independent, lipid efflux was demonstrated in 3 model systems. First, adenoviral-mediated expression of apoE in dermal fibroblasts isolated from ABCA1(-/-) mice significantly increased both sterol and phospholipid efflux. Second, expression of human apoE in a macrophage cell line increased sterol efflux, and this increment in efflux was not reduced by suppressing ABCA1 expression. Third, reduction of apoE expression using an apoE small interfering RNA significantly reduced sterol efflux from ABCA1(-/-) mouse peritoneal macrophages. ApoE-mediated, but ABCA1-independent, lipid efflux could be differentiated from lipid efflux that was dependent on the extracellular accumulation of secreted apoE, because exogenous cell-derived apoE stimulated efflux only from cells expressing ABCA1. Sterol efflux was usually highest in cells expressing both ABCA1 and apoE, likely representing a summation of the ABCA1-dependent and -independent pathways for apoE-mediated sterol efflux. CONCLUSIONS: ABCA1 expression is required for apoE-mediated efflux when endogenously synthesized apoE accumulates extracellularly. Our results, however, establish the existence of an ABCA1-independent pathway for lipid efflux that requires the intracellular synthesis and/or transport of apoE.

Unique effect of arachidonic acid on human neutrophil TNF receptor expression: up-regulation involving protein kinase C, extracellular signal-regulated kinase, and phospholipase A2.

Arachidonic acid (AA) regulates the function of many cell types, including neutrophils. Although much emphasis has been placed on agonist-induced down-regulation of TNFR, our data show that AA caused a rapid (10-20 min) and dose-dependent (0.5-30 micro M) increase in the surface expression of both classes of TNFR (TNFR1 and TNFR2) on human neutrophils. This increased TNFR expression correlated with an increase in TNF-induced superoxide production. In contrast, the omega3 fatty acids eicosapentaenoic acid, docosahexaenoic acid, and linolenic acid failed to stimulate TNFR expression. Although fMLP and LPS reduced the neutrophil expression of TNFR, when pretreated with AA, fMLP caused an increase in TNFR expression. Consistent with this result was the finding that AA prevented the fMLP-induced receptor release in neutrophil cultures. AA also caused an increase in TNFR expression in matured HL-60 cells (neutrophil-like cells), but a decrease in nonmatured cells and HUVEC. The AA effects were independent of the lipoxygenase and cyclooxygenase pathways, but dependent on protein kinase C, the extracellular signal-regulated kinases 1 and 2, and cytosolic phospholipase A(2). The data demonstrate a unique effect of AA in the inflammatory reaction, through its action on neutrophil TNFR expression, and suggest that AA may regulate the response of neutrophils to TNF by altering its receptor number.