The inflammatory microenvironment in epithelial ovarian cancer: a role for TLR4 and MyD88 and related proteins.

The tumor-associated inflammatory microenvironment may play a pivotal role in epithelial ovarian cancer (EOC) carcinogenesis and outcomes, but a detailed profile in patient-derived tumors is needed. Here, we investigated the expression of TLR4- and MyD88-associated markers in tumors from over 500 EOC patients using immunohistochemical staining. We demonstrate that high expression of TLR4 and MyD88 predicts poorer overall survival in patients with EOC; most likely, this is due to their association with serous histology and features of high tumor burden and aggressiveness, including stage, grade, and ascites at surgery. Combined TLR4 and MyD88 expression appears to serve as an independent risk factor for shortened survival time, even after covariate adjustment (both moderate HR 1.1 [95 % CI 0.7-1.8], both strong HR 2.1 [95 % CI 1.1-3.8], both weak as referent; p = 0.027). We reveal that in EOC tissues with elevated expression of both TLR4 and MyD88 and activated NF-κB signaling pathway, expression of hsp60, hsp70, beta 2 defensin, and HMGB1 are also enriched. In total, these results suggest that activation of TLR4/MyD88/NF-κB signaling by endogenous ligands may contribute to an inflammatory microenvironment that drives a more aggressive phenotype with poorer clinical outcome in EOC patients.

Tlr4 upregulation in the brain accompanies depression- and anxiety-like behaviors induced by a high-cholesterol diet.

An association between metabolic abnormalities, hypercholesterolemia and affective disorders is now well recognized. Less well understood are the molecular mechanisms, both in brain and in the periphery, that underpin this phenomenon. In addition to hepatic lipid accumulation and inflammation, C57BL/6J mice fed a high-cholesterol diet (0.2%) to induce non-alcoholic fatty liver disease (NAFLD), exhibited behavioral despair, anxiogenic changes, and hyperlocomotion under bright light. These abnormalities were accompanied by increased expression of transcript and protein for Toll-like receptor 4, a pathogen-associated molecular pattern (PAMP) receptor, in the prefrontal cortex and the liver. The behavioral changes and Tlr4 expression were reversed ten days after discontinuation of the high-cholesterol diet. Remarkably, the dietary fat content and body mass of experimental mice were unchanged, suggesting a specific role for cholesterol in the molecular and behavioral changes. Expression of Sert and Cox1 were unaltered. Together, our study has demonstrated for the first time that high consumption of cholesterol results in depression- and anxiety-like changes in C57BL/6J mice and that these changes are unexpectedly associated with the increased expression of TLR4, which suggests that TLR4 may have a distinct role in the CNS unrelated to pathogen recognition.

Cardiolipin Biosynthesis Genes Are Not Required for Salmonella enterica Serovar Typhimurium Pathogenesis in C57BL/6J Mice.

Salmonella enterica serovar Typhimurium is an intracellular pathogen that parasitizes macrophages from within a vacuole. The vacuolar environment prompts the bacterium to regulate the lipid composition of the outer membrane (OM), and this influences host inflammation. S. Typhimurium regulates the levels of acidic glycerophospholipids known as cardiolipins (CL) within the OM, and mitochondrial CL molecules can prime and activate host inflammasomes. However, the contribution of S. Typhimurium's CL biosynthesis genes to intracellular survival, inflammasome activation, and pathogenesis had not been examined. S. Typhimurium genes encode three CL synthases. Single, double, and triple mutants were constructed. Similar to other Enterobacteriaceae, ClsA is the primary CL synthase for S. Typhimurium during logarithmic growth, while ClsB and ClsC contribute CL production in stationary phase. It was necessary to delete all three genes to diminish the CL content of the envelope. Despite being devoid of CL molecules, ΔclsABC mutants were highly virulent during oral and systemic infection for C57BL/6J mice. In macrophages, ΔclsA, ΔclsB, ΔclsC, and ΔclsAC mutants behaved like the wild type, whereas ΔclsAB, ΔclsBC, and ΔclsABC mutants were attenuated and elicited reduced amounts of secreted interleukin-1 beta (IL-1ß), IL-18, and lactate dehydrogenase. Hence, when clsA and clsC are deleted, clsB is necessary and sufficient to promote intracellular survival and inflammasome activation. Similarly, when clsB is deleted, clsA and clsC are necessary and sufficient. Therefore, the three CL synthase genes cooperatively and redundantly influence S. Typhimurium inflammasome activation and intracellular survival in C57BL/6J mouse macrophages but are dispensable for virulence in mice. IMPORTANCE Salmonella enterica serovar Typhimurium is a pathogenic Gram-negative bacterium that regulates the cardiolipin (CL) and lipopolysaccharide (LPS) composition of the outer membrane (OM) during infection. Mitochondrial CL molecules activate the inflammasome and its effector caspase-1, which initiates an inflammatory process called pyroptosis. Purified bacterial CL molecules also influence LPS activation of Toll-like receptor 4 (Tlr4). S. Typhimurium resides within macrophage vacuoles and activates Tlr4 and the inflammasome during infection. However, the contribution of the three bacterial CL synthase genes (cls) to microbial pathogenesis and inflammation had not been tested. This study supports that the genes encoding the CL synthases work coordinately to promote intracellular survival in macrophages and to activate the inflammasome but do not influence inflammatory cytokine production downstream of Tlr4 or virulence in C57BL/6J mice. The macrophage phenotypes are not directly attributable to CL production but are caused by deleting specific combinations of cls gene products.

Insulin receptor sensitizer, dicholine succinate, prevents both Toll-like receptor 4 (TLR4) upregulation and affective changes induced by a high-cholesterol diet in mice.

BACKGROUND: High cholesterol intake in mice induces hepatic lipid dystrophy and inflammation, signs of non-alcoholic fatty liver disease (NAFLD), depressive- and anxiety-like behaviors, and the up-regulation of brain and liver Toll-like receptor 4 (Tlr4). Here, we investigated whether dicholine succinate (DS), an insulin receptor sensitizer and mitochondrial complex II substrate would interact with these effects. METHODS: C57BL/6J mice were given a 0.2%-cholesterol diet for 3 weeks, alone or along with oral DS administration, or a control feed. Outcomes included behavioral measures of anxiety/depression, and Tlr4 and peroxisome-proliferator-activated-receptor-gamma coactivator-1b (PPARGC1b) expression. RESULTS: 50mg/kg DS treatment for 3 weeks partially ameliorated the cholesterol-induced anxiety- and depressive-like changes. Mice were next treated at the higher dose (180mg/kg), either for the 3-week period of dietary intervention, or for the last two weeks. Three-week DS administration normalized behaviors in the forced swim and O-maze tests and abolished the Tlr4 up-regulation in the brain and liver. The delayed, 2-week DS treatment had similar effects on Tlr4 expression and largely rescued the above-mentioned behaviors. Suppression of PPARGC1b, a master regulator of mitochondrial biogenesis, by the high cholesterol diet, was prevented with the 3-week administration, and markedly diminished by the a 2-week administration of DS. None of treatments prevented hepatic dystrophy and triglyceride accumulation. LIMITATIONS: Other conditions have to be tested to define possible limitations of reported effects of DS. CONCLUSIONS: DS treatment did not alter the patho-morphological substrates of NAFLD syndrome in mice, but ameliorated its molecular and behavioral consequences, likely by activating mitochondrial functions and anti-inflammatory mechanisms.