Activation of toll-like receptor 4 is necessary for trauma hemorrhagic shock-induced gut injury and polymorphonuclear neutrophil priming

Abstract

Interactions of toll-like receptors (TLRs) with nonmicrobial factors play a major role in the pathogenesis of early trauma-hemorrhagic shock (T/HS)-induced organ injury and inflammation. Thus, we tested the hypothesis that TLR4 mutant (TLR4 mut) mice would be more resistant to T/HS-induced gut injury and polymorphonuclear neutrophil (PMN) priming than their wild-type littermates and found that both were significantly reduced in the TLR4 mut mice. In addition, the in vivo and ex vivo PMN priming effect of T/HS intestinal lymph observed in the wild-type mice was abrogated in TLR4 mut mice as well the TRIF mut-deficient mice and partially attenuated in Myd88 mice, suggesting that TRIF activation played a more predominant role than MyD88 in T/HS lymph-induced PMN priming. Polymorphonuclear neutrophil depletion studies showed that T/HS lymph-induced acute lung injury was PMN dependent, because lung injury was totally abrogated in PMN-depleted animals. Because the lymph samples were sterile and devoid of endotoxin or bacterial DNA, we investigated whether the effects of T/HS lymph was related to endogenous nonmicrobial TLR4 ligands. High-mobility group box 1 protein 1, heat shock protein 70, heat shock protein 27, and hyaluronic acid all have been implicated in ischemia-reperfusion-induced tissue injury. None of these "danger" proteins appeared to be involved, because their levels were similar between the sham and shock lymph samples. In conclusion, TLR4 activation is important in T/HS-induced gut injury and in T/HS lymph-induced PMN priming and lung injury. However, the T/HS-associated effects of TLR4 on gut barrier dysfunction can be uncoupled from the T/HS lymph-associated effects of TLR4 on PMN priming.

Figure1. TLR4 deficiency attenuates T/HS-induced intestinal permeability and PMN priming

WT and TLR4 ^mut mice were subjected to T/HS or T/SS for 60 min and 3 hr reperfusion. A, In vivo intestinal permeability was assessed by quantifying FD4 concentration (μg/ml) in the serum. In B, the effect of T/HS-induced PMN priming in lysed whole blood samples was measured in a respiratory burst flow cytometric-based assay. A and B, Data expressed as Mean ± SEM with n=4-6 mice per group.

Figure 2. T/HS lymph increases PMN priming via TLR4

A, Lysed whole blood samples from naïve WT or TLR4^mut mice were incubated with pig T/HS or T/SS lymph (10%v/v) for 5 min. After PMA activation, the effect of T/HS or T/SS lymph on PMN priming was assessed in a respiratory burst assay. B, Lysed whole blood samples from WT and TLR4^mut mice infused with porcine T/HS or T/SS lymph for 3 hr were tested for PMN priming in a respiratory burst assay. C, The percentage of villous injury in WT and TLR4^mut mice infused with porcine T/HS lymph for 3 hr was quantified in hematoxylin and eosin stained distal ileal sections. By t-test (but not ANOVA) analysis, the T/HS lymph-injected mice had more gut injury that the T/SS lymph-injected mice. A-C, Data expressed as Mean ± SEM with n=4-7 mice per group.

Figure 3. TRIF and Myd88 deficiency fully and partially prevent T/HS lymph-induced PMN priming

Lysed whole blood samples were collected from WT, MyD88^-/- and TRIF^mut mice infused with porcine T/HS or T/SS lymph for 3 hr. PMN priming was measured in a respiratory burst assay. Data expressed as Mean ± SEM with n=5-8 mice per group.

Figure 4. PMN depletion attenuated T/HS lymph induced lung permeability and MPO levels

CD1 mice were administered rabbit anti-mouse antibody or normal rabbit serum 48 and 24 hr before infusion with rat or porcine T/HS or T/SS lymph for 3 hr. A, The number of peripheral blood PMNs from mice administered anti- antibody, normal rabbit serum or no antibody was quantified in Wright-Giemsa stained blood smears using a hemacytometer. Data expressed as Mean ± SEM with n=10-29 mice per group. B, Lung permeability to EBD was performed in mice infused with porcine T/HS or T/SS lymph after the administration of anti- or serum control. % EBD in BALF refers to the percent of Evans blue dye in the bronchoalveolar lavage fluid. Data expressed as mean ± SE (n= 3-6 mice/group). C, Lung permeability to EBD and D, MPO levels (U/g) measured in lung homogenates was performed in mice infused with rat T/HS or T/SS lymph after the administration of anti- or serum control MPO levels (U/g) were measured in lung homogenates. C and D, Data expressed as mean ± SE (n= 3-6 mice/group).

Figure 5. HMGB1 and hyaluronic acid are not endogenous TLR4 ligands in T/HS lymph

A. Rat plasma HMGB1 levels in rats subjected to T/HS or T/SS with and without lymph diversion (LDL) was measured by western blot analysis (n= 3 rats per group). Each lane represents individual lymph. B, Hyaluronic acid levels in rat T/HS or T/SS lymph or plasma samples were measured using an ELISA (mean value of n=2 rats per group for each time point).