Toll-like receptor 3 agonist protection against experimental Francisella tularensis respiratory tract infection.

We investigated whether Toll-like receptor 3 (TLR3) stimulation would protect the host from inhaled Francisella tularensis. TLR3 is expressed by respiratory epithelial cells and macrophages and can be activated by a synthetic double-stranded RNA ligand called polyinosine-polycytosine [poly(I:C)]. Thus, we evaluated poly(I:C) as a novel treatment against inhaled F. tularensis. In vivo, BALB/c mice intranasally (i.n.) treated with poly(I:C) (100 microg/mouse) 1 h before or after Schu 4 or LVS (100 CFU) i.n. challenge showed that poly(I:C) treatment significantly reduced bacterial load in the lungs (P < 0.05). Bronchoalveolar lavage from poly(I:C)-treated mice alone or combined with F. tularensis infection significantly increased cytokine secretion and enhanced neutrophil influx to lung tissues. Poly(I:C) responses were transient but significantly prolonged the survival of treated mice after i.n. F. tularensis challenge relative to mock treated animals. This prolonged survival providing a longer window for initiation of levofloxacin (LEVO) treatment (40 mg/kg). Animals treated with poly(I:C), challenged with F. tularensis, and then treated with LEVO 5 days later had 100% survival relative to 0% survival in animals receiving LEVO alone. Mechanistically, poly(I:C) given to human monocyte-derived macrophages before or after Schu 4 or LVS challenge (multiplicity of infection, 20:1) had significantly reduced intracellular bacterial replication (P < 0.05). These data suggest that poly(I:C) may represent a potential therapeutic agent against inhaled F. tularensis that prolongs survival and the opportunity to initiate standard antibiotic therapy (i.e., LEVO).

Escherichia coli isolated from a Crohn's disease patient adheres, invades, and induces inflammatory responses in polarized intestinal epithelial cells.

Inflammatory diseases of the intestinal tract are a major health concern both in the United States and around the world. Evidence now suggests that a new category of Escherichia coli, designated Adherent Invasive E. coli (AIEC) is highly prevalent in Crohn's Disease (CD) patients. AIEC strains have been shown to colonize and adhere to intestinal epithelial cells (IEC). However, the role AIEC strains play in the induction of an inflammatory response is not known. Therefore, we examined several E. coli strains (designated LF82, O83:H1, 6604 and 6655) that were isolated from CD patients for their ability to induce inflammation in two IEC, Caco-2BBe and T-84 cells. Results showed that each strain had varying abilities to adhere to and invade IEC as well as induced cytokine secretion from polarized IEC. However, E. coli O83:H1 displayed the best characteristics of AIEC strains as compared to the prototype AIEC strain LF82, inducing cytokine secretion from IEC and promoting immune cell migration through IEC. Upon further analysis, E. coli O83:H1 did not harbor virulence genes present in known pathogenic intestinal organisms. Further characterization of E. coli O83:H1 virulence determinants showed that a non-flagellated O83:H1 strain significantly decreased the organism's ability to adhere to and invade both IEC and elicit IEC cytokine secretion compared to the wild type and complemented strains. These findings demonstrate that E. coli O83:H1 possesses the characteristics of the AIEC LF82 strain that may contribute to the low-grade, chronic inflammation observed in Crohn's disease.

Differential mechanisms and development of leptin resistance in A/J versus C57BL/6J mice during diet-induced obesity.

Changes in the biological efficacy of leptin were evaluated in obesity-resistant (A/J) and obesity-prone (C57BL/6J) mice at weaning and after consuming a high-fat (HF) diet for 4 and 8 wk. There was no evidence of leptin resistance in either strain at the start of the study, but after 4 and 8 wk on the HF diet, C57BL/6J mice became unresponsive to ip leptin. C57BL/6J mice responded to intracerebroventricular leptin at these time points but developed peripheral resistance to sympathetic stimulation of retroperitoneal white adipose tissue. In contrast, intracerebroventricular leptin was fully effective in A/J mice, reproducing the complete profile of responses observed in weanling mice. A/J mice were also partially responsive to ip leptin at both time points, increasing uncoupling protein 1 mRNA expression in brown adipose tissue and decreasing leptin mRNA in white adipose tissue. The findings indicate that retention of leptin responsiveness is an important component of the ability of A/J mice to mount a robust adaptive thermogenic response and resist diet-induced obesity.

Adaptive changes in adipocyte gene expression differ in AKR/J and SWR/J mice during diet-induced obesity.

Obesity-prone (AKR/J) and obesity-resistant (SWR/J) mice were weaned onto low (LF) or high fat (HF) diets to identify adaptive changes in adipocyte gene expression that are associated with differences between the strains in fat deposition. Food consumption was monitored at weekly intervals and all mice were evaluated after consuming their respective diets for 4 wk for analysis of mRNA levels of selected metabolic genes. Despite similar food consumption, body weight and fat deposition were significantly greater in AKR/J than in SWR/J mice, and this difference was greatly accentuated by the HF diet. The HF diet produced distinct differences between strains in gene expression patterns among fat depots. In AKR/J mice, UCP1 mRNA was decreased 10-fold in interscapular brown adipose tissue (BAT) and four- to fivefold in retroperitoneal and inguinal white adipose tissue (WAT). The HF diet also decreased PGC-1 and beta(3)-adrenergic receptor mRNA by two- and ninefold in BAT from AKR/J mice. In contrast, the HF diet either increased uncoupling protein (UCP)1 in BAT or had no effect on expression of these genes in adipose tissues from SWR/J mice. UCP2 mRNA was fourfold higher in WAT from AKR/J compared with SWR/J mice and increased by an additional twofold in WAT from AKR/J mice fed the HF diet. UCP2 was unaffected by diet in SWR/J mice. These studies show that the diet-induced obesity of AKR/J mice is characterized by increased metabolic efficiency and is associated with changes in adipocyte gene expression that limit the adaptive thermogenic response to increased energy density.