MyD88 and Trif signaling play distinct roles in cardiac dysfunction and mortality during endotoxin shock and polymicrobial sepsis

Abstract

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.

Fig. 1. Langendorff measurements of LV function in WT, MyD88^−/−, Trif^−/− mice during polymicrobial sepsis

Twenty-four hours after CLP or Sham surgery, the hearts were isolated and perfused in a Langendorff apparatus. Left ventricular (LV) contractile function was measured as described in Materials and Methods. A. MyD88^−/− vs. WT; B. Trif^−/− vs. WT. CLP = cecum ligation and puncture; dP/dt_max = the maximum first derivative of LVDP; dP/dt_min = the minimum first derivative of LVDP; LVDP = LV developed pressure; MyD88 = myeloid differentiation factor 88; Trif = TIR-domain-containing adaptor protein inducing interferon-β mediated transcription-factor; WT = wild type. Each data point and error bar represents the mean ± SE. The number of animals in each group: WT-Sham = 3, WT-CLP = 8, MyD88^−/−-Sham = 3, MyD88^−/−-CLP = 8, Trif^−/−-Sham = 5, Trif^−/−-CLP = 7. ** P<0.01, *** P<0.001, MyD88^−/−-CLP or WT-Sham vs. WT-CLP; # P<0.05, ## P<0.01 WT-CLP vs. WT-Sham.

Fig. 2. Mouse mortality during polymicrobial sepsis

Age-matched WT, MyD88^−/−, and Trif^−/− mice were subjected CLP or sham procedures and the mortality of the septic mice were assessed daily for up to 14 days. CLP = cecum ligation and puncture; MyD88 = myeloid differentiation factor 88; Trif = TIR-domain-containing adaptor protein inducing interferon-β mediated transcription-factor; WT = wild type. The number of animals in each group: WT-Sham = 5, WT-CLP = 5, MyD88^−/−-Sham = 5, MyD88^−/−-CLP = 6, Trif^−/−-Sham = 5, Trif^−/−-CLP = 6. * P<0.05 vs. WT-CLP.

Fig. 3. Langendorff measurements of LV function in WT, MyD88^−/−, Trif^−/− mice during endotoxin shock

Age-matched WT, MyD88^−/−, and Trif^−/− mice were administered with LPS (15 mg/kg, intra-peritoneal injection) or saline. Six hours later, the hearts were isolated and perfused in a Langendorff apparatus. Left ventricular (LV) contractile function was measured as described in Materials and Methods. A. MyD88^−/− vs. WT; B. Trif^−/− vs. WT. dP/dt_max = the maximum first derivative of LVDP; dP/dt_min = the minimum first derivative of LVDP; LPS = lipopolysaccharide; LVDP = LV developed pressure; MyD88 = myeloid differentiation factor 88; Trif = TIR-domain-containing adaptor protein inducing interferon-β mediated transcription-factor; WT = wild type. Each data point and error bar represents the mean ± SE. The number of animals in each group: WT-Saline = 6, WT-LPS = 7, MyD88^−/−-Saline = 5, MyD88^−/−-LPS = 5, Trif^−/−-Saline = 4, Trif^−/−-LPS = 4. ** P<0.01, *** P<0.001, MyD88^−/−-LPS vs. WT-LPS or WT-saline; # P<0.05, ## P<0.01, ###, P<0.001, WT-LPS vs. WT-Saline.

Fig. 4. Both MyD88^−/− and Trif^−/− mice are resistant to endotoxin shock

Age-matched WT, MyD88^−/− and Trif^−/− mice were administered with LPS (15 mg/kg, intra-peritoneal injection) or saline (data not shown). The mice were subsequently examined for left ventricular function by serial echocardiography and mortality. A. Representative M-mode echocardiograms were presented at the baseline and 6 hrs after LPS administration. B. Survival rate after LPS administration. LPS = lipopolysaccharide; MyD88 = myeloid differentiation factor 88; Trif = TIR-domain-containing adaptor protein inducing interferon-β mediated transcription-factor; WT = wild type. N = 5 in all 3 LPS groups; * P<0.05 vs. WT.

Fig. 5. MyD88^−/− mice had attenuated peritoneal lavage and serum cytokine levels during polymicrobial sepsis

WT and MyD88^−/− mice underwent sham or CLP procedures. Twenty-four hours later, peritoneal lavage and blood were collected. Six cytokines, namely IL-6, IL-10, TNFα, KC, MCP-1, and MIP-2, in the peritoneal lavage and sera were measured using a multiplex fluorescent bead-based immunoassay. A. Peritoneal lavage cytokines. B. Serum cytokines. Each error bar represents mean ± SE. The number of animals in each group: WT-Sham = 4, WT-CLP = 4, MyD88^−/−-Sham = 3, MyD88^−/−-CLP = 3. CLP = cecum ligation and puncture; IL = interleukin; TNF = tumor necrosis factor; KC = keratinocyte chemoattractant; MCP-1 = monocyte chemoattractant protein-1; MIP-2 = macrophage inflammatory protein-2; MyD88 = myeloid differentiation factor 88; Trif = TIR-domain-containing adaptor protein inducing interferon-β mediated transcription-factor; WT = wild type. * P<0.05 ; ** P<0.01; *** P<0.001.

Fig. 6. Trif deficiency has no impact on peritoneal and serum cytokine levels during polymicrobial sepsis

WT and Trif^−/− mice underwent sham or CLP procedures. Twenty-four hours later, peritoneal lavage and blood were collected. Peritoneal lavage and serum IL-6, IL-10, TNFα, KC, MCP-1, and MIP-2 were measured with a multiplex fluorescent bead-based immunoassay. A. Peritoneal cytokines. B. Serum cytokines. Each error bar represents mean ± SE. The number of animals in each group: WT-Sham = 3, WT-CLP = 4, Trif^−/−-Sham = 3, Trif^−/−-CLP = 6. CLP = cecum ligation and puncture; IL = interleukin; KC = keratinocyte chemoattractant; MCP-1 = monocyte chemoattractant protein-1; MIP-2 = macrophage inflammatory protein-2; MyD88 = myeloid differentiation factor 88; TNF = tumor necrosis factor; Trif = TIR-domain-containing adaptor protein inducing interferon-β mediated transcription-factor; WT = wild type.

Fig. 7. Effect of MyD88- and Trif-deficiency on endotoxin-induced cytokine production in serum

WT, MyD88^−/−, and Trif^−/− mice were administered with LPS (15 mg/kg, intra-peritoneal injection) or saline. Six hours later, blood was collected. Serum IL-6, TNFα, and IL-1β were measured with a multiplex fluorescent bead-based immunoassay. Each error bar represents mean ± SE. The number of animals in each group: WT-Saline = 5, WT-LPS = 7, MyD88^−/−-Saline = 5, MyD88^−/−-LPS = 5, Trif^−/−-Saline = 3, Trif^−/−-LPS = 6. * P<0.05, **P<0.01, *** P<0.001. IL = interleukin; LPS = lipopolysaccharide; MyD88 = myeloid differentiation factor 88; TNF = tumor necrosis factor; Trif = TIR-domain-containing adaptor protein inducing interferon-β mediated transcription-factor; WT = wild type.

Fig. 8. Impact of MyD88- and Trif-deficiency on neutrophil migratory and phagocytic functions during polymicrobial sepsis

Twenty-four hours after CLP procedures, peritoneal cells were harvested from lavage fluid and manually counted. 5 × 10⁵ cells from each mouse were loaded for flow cytometry analysis of Gr-1⁺ neutrophils or for phagocytosis analysis. A. Total Gr-1⁺ neutrophils in the peritoneum. Neutrophil numbers were calculated based on the total cells numbers manually counted multiplied by the percentage of neutrophils as measured by flow cytometry gated on Gr-1⁺. The horizontal bars represent the mean values in each mouse group. The number of animals in each group: WT-CLP = 18, MyD88^−/−-CLP = 18, Trif^−/−-CLP = 7. B. Percentage of phagocytic neutrophils in the peritoneal space. Peritoneal cells were incubated with opsonized fluorescent microspheres at 37 °C for 30 min and analyzed with flow cytometry. Percentages of phagocytic (FITC-stained) neutrophils (Gr-1⁺) were presented. The number of animals in each group: WT-CLP = 17, MyD88^−/−-CLP = 15, Trif^−/−-CLP = 6. * P<0.05; *** P<0.001. CLP = cecum ligation and puncture; FITC = Fluorescein isothiocyanate; MyD88 = myeloid differentiation factor 88; Trif = TIR-domain-containing adaptor protein inducing interferon-β mediated transcription-factor; WT = wild type;

Fig. 9. Bacterial load in the blood and the peritoneal cavity in WT, MyD88^−/−, and Trif^−/− mice following CLP

Both the blood and the peritoneal lavage were collected 24 h after CLP surgery. After serially dilutions, the samples were incubated at 37 °C for 14-16 hours and bacterial colony forming units (CFU) were counted. Each data point represents the CFU from one mouse and was plotted in a Log₁₀ scale. A. CFU in the blood. B. CFU in the peritoneal lavage. CLP = cecum ligation and puncture; MyD88 = myeloid differentiation factor 88; Trif = TIR-domain-containing adaptor protein inducing interferon-β mediated transcription-factor; WT = wild type. The horizontal bars represent the mean values in each mouse group. The number of animals in each group: WT-CLP = 17, MyD88^−/−-CLP = 15, Trif^−/−-CLP = 7.