Cafeteria Diet Impacts the Body Weight and Energy Expenditure of Brown Norway Rats in an Apparent Age Dependent Manner, but Has no Effect on Muscle Anabolic Sensitivity to Nutrition.

While obesity blunts the ability of muscle to mount a protein synthetic response to an amino acid infusion in older adults, it is unclear if this insensitivity to nutrition persists long term and in response to complete foods is unknown. To address this, young (2 months old) and old (17-20 months old) Brown Norway rats were randomized to receive either chow or a 12 wk diet of calorie-dense human foods. At wk 10 drinking water was supplemented with 2% heavy water, followed 2 weeks later by a flooding dose of [2H5]-phenylalanine and an oral leucine bolus, allowing the short and long-term effects of age and diet on muscle protein synthesis rates to be determined. The experimental diet increased energy intake in both young (7.4 ± 0.9%) and old (18.2 ± 1.8%) animals (P < 0.01), but only led to significant increases in body weight in the former (young: 10.2 ± 3.0% (P < 0.05) and old: 3.1 ± 5.1% (NS) vs. age-matched controls). Notably, energy expenditure in response to the cafeteria diet was increased in old animals only (chow: 5.1 ± 0.4; cafe: 8.2 ± 1.6 kcal.kg b.w-1.h-1; P < 0.05). Gastrocnemius protein fractional synthetic rates in response to either an acute leucine bolus or two weeks of feeding were equivalent across groups irrespective of age or diet. Rodents in old age appear capable of preventing weight gain in response to a calorie-dense diet by increasing energy expenditure while maintaining the anabolic sensitivity of muscle to nutrition; the mechanisms of which could have important implications for the aging obese human.

Therapeutic efficacy of CXCR3 blockade in an experimental model of severe sepsis.

INTRODUCTION: In our previous studies we demonstrated that CXC chemokine receptor 3 (CXCR3) participates in the regulation of lymphocyte trafficking during cecal ligation and puncture (CLP)-induced sepsis. In this study, we evaluated the effects of treatment with anti-CXCR3 immunoglobulin (IgG) and antibiotics on outcome during septic shock caused by CLP. METHODS: C57BL/6J mice were treated with neutralizing IgG against CXCR3 plus Primaxin either 24 hours prior to, 2 hours after or 6 hours after CLP. Control mice received nonspecific IgG plus Primaxin in the same regimen. Survival, core body temperature, bacterial clearance and systemic cytokine production were evaluated. RESULTS: Our results show that treatment with anti-CXCR3 IgG plus Primaxin significantly improved survival when administered 24 hours prior to CLP (50% vs. 10%), 2 hours after CLP (55% vs. 10%) or 6 hours after CLP (55% vs. 25%) compared with mice receiving nonspecific IgG plus Primaxin. Treatment with anti-CXCR3 plus Primaxin 24 hours prior to CLP attenuated hypothermia and IL-6 and macrophage inflammatory protein 2 (MIP-2) production but did not alter bacterial clearance. Treatment with anti-CXCR3 IgG and Primaxin 2 hours after CLP did not improve bacterial clearance and systemic cytokine production compared with mice treated with IgG and Primaxin, whereas 6 hours after CLP the bacterial clearance and IL-6 and MIP-2 concentrations, both in plasma and peritoneal lavage fluid, were significantly improved in mice receiving anti-CXCR3 IgG and Primaxin compared with mice that only received nonspecific IgG and Primaxin. CONCLUSION: The results from this study indicate that neutralization of CXCR3 prior to, 2 hours after or 6 hours after the initiation of CLP-induced septic shock improves survival and attenuates CLP-induced inflammation and physiologic dysfunction.

STAT1-deficient mice are resistant to cecal ligation and puncture-induced septic shock.

STAT1 (signal transducer and activator of transcription 1) is a member of the JAK-STAT signaling family and plays a key role in facilitating gene transcription in response to activation of the types I and II interferon (IFN) receptors. TYK2 is essential for type I, but not type II, IFN-induced STAT1 activation. Previous studies show that STAT1-deficient mice are resistant to endotoxin-induced shock. The goal of the present study was to assess the response of STAT1- and TYK2-deficient mice to septic shock caused by cecal ligation and puncture (CLP). End points included survival, core temperature, organ injury, systemic cytokine production, and bacterial clearance. Results showed that survival rates were significantly higher in STAT1 knockout (STAT1KO) mice compared with wild-type controls (80% vs. 10%). The improved survival of STAT1KO mice was associated with less hypothermia, metabolic acidosis, hypoglycemia, and hepatocellular injury. Plasma interleukin 6, MIP-2, CXCL10, and IFN-α concentrations were significantly lower in STAT1KO mice than in wild-type mice. In the absence of antibiotic treatment, blood and lung bacterial counts were significantly lower in STAT1KO mice than in controls. However, treatment with antibiotics ablated that difference. A survival advantage was not observed in TYK2-deficient mice compared with control. However, CLP-induced hypothermia and systemic interleukin 6 and CXCL10 production were significantly attenuated in TYK2-deficient mice. These results indicate that STAT1 activation is an important factor in the pathogenesis of CLP-induced septic shock and is associated with the development of systemic inflammation and organ injury. TYK2 activation also appears to contribute to CLP-induced inflammation, but to a lesser extent than STAT1.

Regulation of lymphocyte trafficking by CXC chemokine receptor 3 during septic shock.

RATIONALE: Lymphocytes have been shown to facilitate systemic inflammation and physiologic dysfunction in experimental models of severe sepsis. Our previous studies show that natural killer (NK) cells migrate into the peritoneal cavity during intraabdominal sepsis, but the trafficking of NKT and T lymphocytes has not been determined. The factors that regulate lymphocyte trafficking during sepsis are currently unknown. OBJECTIVES: To ascertain the importance of CXC chemokine receptor 3 (CXCR3) as a regulator of lymphocyte trafficking during sepsis and determine the contribution of CXCR3-mediated lymphocyte trafficking to the pathogenesis of septic shock. METHODS: Lymphocyte trafficking was evaluated in control and CXCR3-deficient mice using flow cytometry during sepsis caused by cecal ligation and puncture (CLP). Survival, core temperature, cytokine production, and bacterial clearance were measured as pathobiological endpoints. MEASUREMENTS AND MAIN RESULTS: This study shows that concentrations of the CXCR3 ligands CXCL9 (monokine induced by interferon γ, MIG) and CXCL10 (interferon γ-induced protein 10, IP-10) increase in plasma and the peritoneal cavity after CLP, peak at 8 hours after infection, and are higher in the peritoneal cavity than in plasma. The numbers of CXCR3(+) NK cells progressively decreased in spleen after CLP with a concomitant increase within the peritoneal cavity, a pattern that was ablated in CXCR3-deficient mice. CXCR3-dependent recruitment of T cells was also evident at 16 hours after CLP. Treatment of mice with anti-CXCR3 significantly attenuated CLP-induced hypothermia, decreased systemic cytokine production, and improved survival. CONCLUSIONS: CXCR3 regulates NK- and T-cell trafficking during sepsis and blockade of CXCR3 attenuates the pathogenesis of septic shock.

The role of interferon-γ in the pathogenesis of acute intra-abdominal sepsis.

Several studies indicate that IFN-γ facilitates systemic inflammation during endotoxin-induced shock. However, the pathobiology of IFN-γ in clinically relevant models of septic shock, such as CLP, is not well understood. In this study, the role of IFN-γ in the pathogenesis of CLP-induced septic shock was evaluated by examining IFN-γ production at the tissue and cellular levels. The impact of IFN-γ neutralization on systemic inflammation, bacterial clearance, and survival was also determined. Following CLP, concentrations of IFN-γ in plasma and peritoneal lavage fluid were low in comparison with concentrations of IL-6 and MIP-2, as was IFN-γ mRNA expression in liver and spleen. The overall percentage of IFN-γ+ splenocytes was <5% after CLP and not statistically different from control mice. Intracellular IFN-γ was present in a large proportion of peritoneal exudate cells after CLP, primarily in infiltrating myeloid cells and NK cells. i.p. myeloid cell activation was decreased in IFN-γKO mice, and plasma concentrations of IL-6 and MIP-2 were significantly lower in IFN-γKO mice and in mice treated with anti-IFN-γ compared with controls, but bacterial clearance was not affected. IFN-γKO mice were resistant to CLP-induced mortality when treated with systemic antibiotics. However, neutralization of IFN-γ with blocking antibodies did not improve survival significantly. These studies show that IFN-γ facilitates the proinflammatory response during CLP-induced septic shock. However, neutralization of IFN-γ did not improve survival uniformly.

Fms-like tyrosine kinase-3 ligand alters antigen-specific responses to infections after severe burn injury.

Burn patients are susceptible to opportunistic infections partly because of decreased immune functions, especially TH1-driven antigen-specific responses, which are regulated by dendritic cells. The dendritic cell growth factor, fms-like tyrosine kinase-3 ligand (FL), has been shown to increase resistance to Pseudomonas aeruginosa, in a dendritic cell-dependent manner, in a mouse model of burn wound infection. The specific mechanisms of protection are not known. This study tested the hypothesis that FL can enhance production of P. aeruginosa-specific antibodies after burn wound infection. Mice that had been previously exposed to P. aeruginosa were infected after burn injury by wound inoculation or challenged by intraperitoneal injection of heat-killed P. aeruginosa. In response to wound infection, FL treatments enhanced bacterial clearance and induced a shift from immunoglobulin (Ig) M toward IgG and IgA. However, serum levels of neither P. aeruginosa-specific antibodies nor interferon gamma (IFN-gamma) were significantly increased by FL, possibly because of decreased systemic exposure to bacteria. After challenge with heat-killed bacteria, which ensured equal exposures, FL-treated mice produced significantly greater levels of P. aeruginosa-specific IgG2a, which correlated with an increase in serum levels of interferon gamma and enhanced opsonization capacity. IL-12, IL-10, and transforming growth factor beta were significantly increased in FL-treated mice, regardless of the type of challenge. These findings indicate that FL treatments after burn injury enhance cytokine responses to recall antigens and increase bacterial clearance. In addition, through its ability to promote TH1-associated antigen-specific responses, FL may have potential as an immunotherapy to enhance adaptive immunity after severe burn injury.

Pretreatment with the Gram-positive bacterial cell wall molecule peptidoglycan improves bacterial clearance and decreases inflammation and mortality in mice challenged with Staphylococcus aureus.

OBJECTIVE: To determine whether tolerance and enhancement of innate immune function can be induced by the Gram-positive cell wall component peptidoglycan. DESIGN: Controlled, in vivo laboratory study. SUBJECTS: Male mice, 8-12 wks (C57BL6/J; C3H/HeJ; B6.129-Tlr2/J). INTERVENTIONS: Mice were given intraperitoneal injections of 1 mg peptidoglycan on two consecutive days. Mice were then challenged with an intravenous injection of live Staphylococcus aureus (1 x 10 colony-forming units) 2 days after the second pretreatment. MEASUREMENTS AND MAIN RESULTS: Mice pretreated with peptidoglycan had diminished plasma concentrations of tumor necrosis factor-alpha and interferon-gamma in response to the bacterial challenge when compared with untreated controls. Plasma interleukin-10 after bacterial challenge was higher in peptidoglycan-pretreated mice than in controls. Clearance of bacteria after the staphylococcal challenge was improved in mice pretreated with peptidoglycan, and mortality in response to a subsequent Staphylococcus challenge was significantly attenuated. Peptidoglycan pretreatment of mice lacking intact toll-like receptor-4 signaling (C3H/HeJ) or toll-like receptor-2 signaling (toll-like receptor-2 knockouts) had similar effects on plasma cytokine balance, bacterial clearance, and mortality. CONCLUSIONS: Exposure to peptidoglycan significantly attenuated inflammation and enhanced bacterial clearance after a subsequent challenge with S. aureus. These results show that exposure to Gram-positive bacterial cell wall components can induce tolerance and enhance innate immune function and neither toll-like receptor-2 nor toll-like receptor-4 are necessary for this phenomenon. Further, although the altered cytokine balance is similar to that seen in septic patients, induced tolerance differs importantly from the clinical scenario of sepsis in that bacterial clearance and survival are improved compared with normal control animals.

Endotoxin pretreatment improves bacterial clearance and decreases mortality in mice challenged with Staphylococcus aureus.

We studied the effects of tolerance induced by Escherichia coli-derived LPS on the innate immune response to a subsequent Staphylococcus aureus bacterial challenge. LPS tolerance was induced in wild-type mice by either intraperitoneal or intravenous injection of 2 microg of LPS on 2 consecutive days. Mice were challenged with an intravenous injection of live S. aureus (5 x 10(8) colony-forming units) 2 days after the second LPS dose. LPS-tolerant mice had a diminished serum interferon-gamma response to the bacterial challenge. Bacterial counts in liver and spleen tissues were decreased, and survival was improved after the Staphylococcus challenge in LPS-tolerant mice compared with saline-pretreated control mice. LPS pretreatment by the intravenous route was also associated with a decreased number of bacterial colonies in lung tissue in addition to liver and spleen, suggesting that induction of LPS tolerance was somewhat compartmentalized after intraperitoneal LPS pretreatment. Induction of tolerance seemed to be due to LPS-specific signaling because LPS pretreatment of LPS-nonresponsive C3H/HeJ mice did not provide similar effects after bacterial challenge. Flow cytometric analysis of spleens from LPS-tolerant mice revealed an increase in phagocytic cells (neutrophiles and macrophages) compared with control mice. Ex vivo culture of splenocytes from LPS-tolerant mice demonstrated increased uptake of fluorescein isothiocyanate-tagged ovalbumin, but no difference in either phagocytosis of fluorescein isothiocyanate-labeled Staphylococcus or bactericidal activity could be demonstrated on a per-cell basis. These results show that attenuation of inflammation and mortality during LPS tolerance extends to gram-positive bacterial organisms and suggests that LPS-induced enhancement of the innate immune response may be attributed to increased numbers of phagocytic cells.

Improved bacterial clearance and decreased mortality can be induced by LPS tolerance and is not dependent upon IFN-gamma.

Endotoxin (LPS) tolerance is induced by exposure to sublethal doses of LPS, resulting in a suppressed proinflammatory response and an improved survival rate after challenge with a normally lethal dose of LPS. We studied the effects of tolerance induced by either Escherichia coli-derived LPS or Pseudomonas aeruginosa-derived LPS on the innate immune response to a subsequent P. aeruginosa bacterial challenge and determined if the induction of tolerance was dependent on interferon gamma (IFN-gamma) activity. LPS tolerance was induced in wild-type (WT) and IFN-gamma knockout mice by i.p. injection of 1 microg of LPS on 2 consecutive days. Mice were challenged with an i.p. injection of live P. aeruginosa (1 x 10(8) colony-forming units) 2 days after the second LPS dose. LPS tolerance in WT mice was associated with diminished serum IFN-gamma and IL-12 and increased serum IL-10 responses to the Pseudomonas challenge. Both clearance of the bacterial challenge and survival were improved in WT animals pretreated with either E. coli LPS or P. aeruginosa LPS compared with saline-pretreated control mice. Similarly, IFN-gamma knockout mice exposed to LPS before the Pseudomonas challenge also had improved bacterial clearance of the challenge and an improved survival rate. In separate experiments, priming with IFN-gamma at a dose that approximated the serum concentration induced by LPS priming did not alter cytokine production or bacterial clearance after a Pseudomonas challenge. Finally, administration of IFN-gamma at the time of Pseudomonas challenge amplified cytokine production in LPS-tolerant animals but did not affect bacterial clearance. These results suggest that IFN-gamma is not necessary for the induction of LPS tolerance. Furthermore, IFN-gamma seems to play a role in propagating the inflammatory cytokine response to Pseudomonas challenge, but it did not seem to have any role in bacterial clearance.