Toll-like Receptor 4 Is Essential to Preserving Cardiac Function and Survival in Low-grade Polymicrobial Sepsis

Background: Toll-like receptor 4 (TLR4), the receptor for endotoxin, mediates hyperinflammatory response and contributes to high mortality during both endotoxin shock and severe sepsis. However, little is known about the role of TLR4 in the pathogenesis of low-grade polymicrobial sepsis, which is often associated with immunosuppression. Methods: Low-grade polymicrobial sepsis was generated by cecum ligation and puncture. Mortality was monitored in wild- type (C57BL/10ScSn) and TLR4def (C57BL/10ScCr) mice. Ex vivo heart and individual cardiomyocyte function were assessed in Langendorff (Hugo Sachs Elektronik; Harvard Apparatus, Holliston, MA) and IonOptix systems (IonOptix, Milton, MA), respectively. Serum chemistry was tested for liver and kidney injury. Cytokines were examined using a multiplex immunoassay. Neutrophil migratory and phagocytic functions were assessed using flow cytometry. Reactive oxygen species were measured using redox-sensitive dichlorodihydrofluorescein dye. Results: Following cecum ligation and puncture, wild-type mice developed bacterial peritonitis with mild cardiac dysfunction (n = 3 in sham and n = 8 in cecum ligation and puncture) and a mortality of 23% within 14 days (n = 22). In comparison, septic TLR4def mice had deleterious cardiac dysfunction (n = 6 in sham and n = 10 in cecum ligation and puncture), kidney and liver injury (n = 7), and much higher mortality at 81% (n = 21). The deleterious effects observed in septic TLR4def mice were associated with increased local and systemic cytokine response, reduced neutrophil migratory and phagocytic function, increased reactive oxygen species generation in leukocytes, and impaired bacterial clearance. Conclusion: TLR4 plays an essential role in host defense against low-grade polymicrobial sepsis by mediating neutrophil migratory/phagocytic functions, attenuating inflammation, reducing reactive oxygen species generation, and enhanced bacterial clearance.

MyD88 and Trif Signaling Play Distinct Roles in Cardiac Dysfunction and Mortality during Endotoxin Shock and Polymicrobial Sepsis

Background Toll-like receptors (TLRs) such as TLR2, TLR4, and TLR9 contribute to the pathogenesis of polymicrobial sepsis. These TLRs signal via the common myeloid differentiation factor 88 (MyD88)-dependent pathways. TLR4 also signals through MyD88-independent but TIR domain-containing adaptor inducing interferon-β-mediated transcription factor (Trif)-dependent pathway. The role of the two signaling pathways in cardiac dysfunction during polymicrobial sepsis and endotoxin shock is unknown. Methods Sepsis was generated by cecum ligation and puncture. Mice were divided into sham and cecum ligation and puncture groups or subjected to saline or endotoxin. Left ventricular function was assessed in a Langendorff apparatus or by echocardiography. Cytokines were examined using a multiplex immunoassay. Neutrophil migratory and phagocytic functions were assessed using flow cytometry. Results In comparison with wild-type mice, MyD88(-/-) but not Trif(-/-) mice had markedly improved cardiac function and survival after cecum ligation and puncture. In comparison, both MyD88(-/-) and Trif(-/-) mice were protected from cardiac depression and mortality during endotoxin shock. Septic MyD88(-/-) but not Trif(-/-) mice had diminished cytokine production in serum and in peritoneal space in comparison with wild-type mice after cecum ligation and puncture. In contrast, both MyD88(-/-) and Trif(-/-) mice had attenuated serum cytokines in comparison with wild-type mice after endotoxin challenge. Neither MyD88(-/-) nor Trif(-/-) signaling had any effect on neutrophil phagocytic function or bacterial clearance at 24 h of polymicrobial sepsis. Conclusions These studies establish that MyD88 but not Trif signaling plays a critical role in mediating cardiac dysfunction, systemic inflammation, and mortality during polymicrobial sepsis. Both MyD88 and Trif are essential for cardiac depression and mortality during endotoxin shock.

Influence of laparoscopy and laparotomy on gasometry, leukocytes and cytokines in a rat abdominal sepsis model

PURPOSE: Laparoscopic surgery is associated with reduced surgical trauma, and less acute phase response, as compared with open surgery. Cytokines are important regulators of the biological response to surgical and anesthetic stress. The aim of this study was to determine if CO2 pneumoperitoneum would change cytokine expression, gas parameters and leukocyte count in septic rats. METHODS: Wistar rats were randomly assigned to five groups: control (anesthesia only), laparotomy, CO2 pneumoperitoneum, cecum ligation and puncture by laparotomy, and laparoscopic cecum ligation and puncture. After 30 min of the procedures, arterial blood samples were obtained to determine leukocytes subpopulations by hemocytometer. TNFalpha, IL-1beta, IL-6 were determined in intraperitoneal fluid (by ELISA). Gas parameters were measured on arterial blood, intraperitoneal and subperitoneal exsudates. RESULTS: Peritoneal TNFalpha, IL-1beta and IL-6 concentrations were lower in pneumoperitoneum rats than in all other groups (p<0.05). TNFalpha, IL-1beta and IL-6 expression was lower in the laparoscopic than in laparotomic sepsis (p<0.05). Rats from laparoscopic cecum ligation and puncture group developed significant hypercarbic acidosis in blood and subperitoneal fluid when compared to open procedure group. Total white blood cells and lymphocytes were significantly lower in laparoscopic cecum ligation and puncture rats than in the laparotomic (p<0.01). Nevertheless, the laparotomic cecum ligation rats had a significant increase in blood neutrophils and eosinophils when compared with controls (p<0.05). CONCLUSIONS: This study demonstrates that the CO2 pneumoperitoneum reduced the inflammatory response in an animal model of peritonitis with respect to intraperitoneal cytokines, white blood cell count and clinical correlates of sepsis. The pneumoperitoneum produced hypercarbic acidosis in septic animals.

Role of Liver NK Cells and Peritoneal Macrophages in Gamma Interferon and Interleukin-10 Production in Experimental Bacterial Peritonitis in Mice

ABSTRACT Gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) production by liver, spleen, lung, peripheral blood mononuclear cells (MNC), and peritoneal exudate cells (PEC) in experimental bacterial peritonitis was examined by cecum ligation and puncture (CLP) (with an 18-gauge needle) of BALB/c mice. MNC of organs were cultured for 18 h, and cytokine levels in supernatants were examined. Cytokines contained in peritoneal lavage fluid were regarded as those produced by PEC. Only liver MNC and PEC produced substantial amounts of IFN-γ, and PEC were the main source of IL-10, especially 12 h after CLP. As reflected by the cytokine production by liver MNC and PEC, serum IFN-γ and IL-10 levels were elevated after CLP. C57BL/6 (B6) mice and BALB/c nude mice showed a similar pattern of cytokine production. TNF-α levels in culture supernatants, peritoneal lavage fluid, and sera were not significantly elevated compared to those of sham-operated mice. In vivo depletion of NK cells of B6 mice with anti-asialo GM1 or anti-NK1.1 antibody greatly decreased IFN-γ levels in liver MNC culture supernatants and sera, suggesting that liver NK cells are IFN-γ producers. On the other hand, plastic-adherent PEC macrophages are the major IL-10 producers. Mice subjected to a cecum ligation and cut procedure (which have a more severe peritonitis) showed much higher IFN-γ and IL-10 levels than those subjected to CLP, while mice subjected to CLP with a smaller (22-gauge) needle showed low levels of these cytokines. These findings show that liver NK cells and PEC macrophages are important for the production of proinflammatory and anti-inflammatory cytokines in bacterial peritonitis.