Commensal microflora induce host defense and decrease bacterial translocation in burn mice through toll-like receptor 4

Abstract

Background: Major burn is associated with decreased gut barrier function and increased bacterial translocation (BT). This study is to investigate whether commensal microflora induce host defense and decrease BT in burn mice.

Methods: First, we treated Wild type (WT) mice with antibiotics in drinking water for 4 weeks to deplete gut commensal microflora. At week 3, drinking water was supplemented with lipopolysaccharide (LPS); a ligand for TLR4, to trigger TLRs in gut. The intestinal permeability, glutathione level, NF-kappaB DNA-binding activity, TLR4 expression of intestinal mucosa, BT to mesenteric lymph nodes (MLNs), and bacterial killing activity of peritoneal cells were measured after thermal injury. Second, lung of animals were harvested for MPO activity and TNFalpha mRNA expression assay. Third, WT animals were treated with oral antibiotics with or without LPS supplement after burn. At 48 hr after burn, TLR4 expression of intestinal mucosa and bacterial killing activity of cells were examined. Finally, bacterial killing activity and BT to MLNs after thermal injury in C3H/HeJ (TLR4 mutant) mice were measured.

Results: Burn induced BT to MLNs in WT mice. Commensal depletion decreased TLR4 expression as well as NF-kappaB activation of intestine, myeloperoxidase (MPO) activity as well as TNFalpha expression of lung, and bacterial killing activity of peritoneal cells. Oral LPS supplement markedly reduced 81% of burn-induced BT and increased TLR4 expression, MPO activity of lung, as well as bacterial killing activity of peritoneal cells. LPS supplement did not change BT or bacterial killing activity in C3H/HeJ mice.

Conclusions: Collectively, commensal microflora induce TLR4 expression of intestine and bacterial killing activity of inflammatory cells in burn. TLR4 ligand increases bacterial killing activity and decreases burn-induced BT. Taken together with the abolition of LPS effect in TLR4 mutant mice, we conclude that commensal microflora induce host defense and decrease bacterial translocation in burn mice through toll-like receptor 4.

Figure 1

Thermal injury induced permeability and decreased glutathione (GSH) level of intestinal mucosa. (A) Thermal injury induced intestinal permeability and commensal depletion with or without LPS supplement did not change it. ! p < 0.05, vs. sham group. (B) Thermal injury decreased GSH level of intestinal mucosa and commensal depletion with or without LPS supplement did not change it. ! p < 0.05, vs. sham group.

Figure 2

TLR4 ligand decreased thermal injury-induced bacterial translocation and increased neutrophil accumulation in lung. (A) LPS supplement in oral antibiotics decreased thermal injury-induced bacterial translocation in comparison with commensal depletion + burn group. ! p < 0.05, vs. sham group. ^# p < 0.05, vs. commensal depletion + burn group. (B) Commensal depletion significantly decreased thermal injury-induced lung myeloperoxidase (MPO) activity and LPS supplement reversed it. ! p < 0.05, vs. sham group. * p < 0.05, vs. burn group. ^# p < 0.05, vs. commensal depletion + burn group.

Figure 3

TLR4 ligand increased TNFα expression in lung and the bacterial killing activity of peritoneal cells. (A) Commensal depletion decreased TLR4 as well as TNFα mRNA expression in lung and LPS supplement reversed them. The values represent the average fold increase (± s.d.) in mRNA expression in 3 different mice of each group. ! p < 0.05, vs. sham group. * p < 0.05, vs. burn group. ^# p < 0.05, vs. commensal depletion + burn group. (B) Commensal depletion induced a significant increase of bacterial retention of peritoneal cells in burn mice compared with that of burn group. LPS supplement significantly decreased 55% of bacterial retention compared with that of antibiotics + burn group. * p < 0.05, vs. burn group. ^# p < 0.05, vs. commensal depletion + burn group.

Figure 4

TLR4 ligand increased TLR4 expression and NF-κB DNA binding activity of intestinal mucosa after thermal injury. (A) LPS supplement increased TLR4 protein expression of intestinal mucosa in commensal depleted mice. * p < 0.05, vs. burn group. ^# p < 0.05, vs. commensal depletion + burn group. (B) LPS supplement increased TLR4 mRNA expression of intestinal mucosa in commensal depleted mice. The values represent the average fold increase (± s.d.) in 3 different mice of each group. * p < 0.05, vs. burn group. ^# p < 0.05, vs. commensal depletion + burn group. (C) LPS supplement increased TLR4 immunostaining of intestinal epithelial cells. (D) Commensal depletion decreased the NF-κB DNA-binding activity of intestinal mucosa and LPS supplement increased it. (n = 4). *p < 0.05, vs. burn group. * p < 0.05, vs. commensal depletion + burn group. NS = non-specific binding.

Figure 5

Oral TLR4 ligand supplement after burn decreased bacterial translocation (BT) and increased bacterial killing activity of macrophages as well as TLR4 expression of intestinal mucosa. (A) Oral antibiotics feeding after burn (Burn+Anti) increased BT to MLN in comparison to burn group and LPS supplement reversed it. * p < 0.05, vs. burn group. ^# p < 0.05, vs. burn + antibiotics group. (B) Oral antibiotics feeding after burn induced a significant increase of bacterial retention in both peritoneal macrophages and bone marrow cells. LPS supplement (Burn+Anti+LPS) decreased bacterial retention of peritoneal macrophages and bone marrow cells in comparison with antibiotics + burn group (Burn + Anti). * p < 0.05, vs. burn group. ^# p < 0.05, vs. antibiotics + burn group. (C) Oral antibiotics feeding after burn decreased TLR4 mRNA expression of intestinal mucosa in comparison with burn group and LPS supplement increased it. * p < 0.05, vs. burn group. ^# p < 0.05, vs. burn + antibiotics group

Figure 6

TLR4 ligand did not change bacterial translocation (BT), bacterial killing activity of peritoneal cells, and TNFα mRNA expression in C3H/HeJ mice. (A) Commensal depletion with or without LPS supplement did not change BT in C3H/HeJ mice. (B) Commensal depletion with or without LPS supplement did not change bacterial retention of peritoneal cells in C3H/HeJ mice. (C) Commensal depletion with or without LPS supplement did not change TNFα mRNA expression of peritoneal cells in C3H/HeJ mice. The values represent the average fold increase (± s.d.) in mRNA expression in 3 different mice of each group. ! p < 0.05, vs. sham group.