The influence of the gut-lung axis on the lung immunity, although appreciated, remains undefined mechanically. This study was designed to investigate whether commensal microflora in gut increase host defense against subsequent pneumonia through toll-like receptor (TLR) signaling and if oral TLR4 ligand supplementation enhances lung defense against bacterial challenge. We found that commensal gut depletion by antibiotic pretreatment before Escherichia coli pneumonia challenge induced a 15-fold and a 3-fold increase in bacterial counts in blood and lung, respectively, and a 30% increase of mortality when compared with the E. coli group. Commensal depletion also induced a suppression of cytokines expression as well as nuclear factor κB activity in intestine. Furthermore, LPS supplementation during antibiotic pretreatment reversed these effects. Commensal depletion also decreased bacterial killing activity of alveolar macrophages and increased IL-6 as well as IL-1β levels and keratinocyte-derived chemokine, macrophage inflammatory protein 2, and IL-1β expression of lung, and LPS supplementation reversed them. In conclusion, commensal gut microflora in the intestinal tract appear to be critical in inducing TLR4 expression as well as nuclear factor κB activation of intestine and lung innate defense against E. coli pneumonia.

Ischemic gut contributes to the development of sepsis and organ failure in critically ill patients. Toll-like receptors (TLRs) have been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We hypothesize that LPS, a ligand for TLR4, decreases mesenteric I/R injury-induced gut damage through tumor necrosis factor α (TNF-α) signaling. First, wild-type (WT) mice were fed with oral antibiotics for 4 weeks to deplete the intestinal commensal microflora. At week 3, drinking water was supplemented with LPS (10 μg/μL) to trigger TLRs. The intestinal mucosa was harvested for TLR4 protein, caspase 3 activity, and terminal deoxynucleotide transferase labeling assay. Second, WT and Tnfrsf1a^−/− mice received 30-min ischemia and 30-min reperfusion (30I-30R) or 30I-180R of the intestine; intestinal permeability and lipid peroxidation of the intestine were examined. Third, WT and Tnfrsf1a^−/− mice were fed with oral antibiotics with or without LPS and received 30I-180R of the intestine. The intestinal mucosa was harvested for lipid peroxidation; glutathione (GSH) level; nuclear factor κB (NF-κB) and AP-1 DNA-binding activity; Bcl-w, TNF-α, and CXCR2 mRNA expression; and HSP70 protein assay. Commensal depletion increased caspase 3 activity as well as villi apoptosis and decreased TLR4 expression of the intestinal mucosa. LPS increased TLR4 expression and decreased villi apoptosis. Commensal depletion augmented 30I-180R-induced intestine permeability as well as lipid peroxidation and decreased GSH level in WT mice but not in Tnfrsf1a^−/− mice. LPS decreased 30I-180R-induced intestinal permeability as well as lipid peroxidation and increased GSH level of the intestinal mucosa in WT mice but not in Tnfrsf1a^−/− mice. Commensal depletion with 30I-180R increased NF-κB and AP-1 DNA-binding activity, HSP70 protein expression, and decreased Bcl-w and TNF-α mRNA expression of the intestinal mucosa in WT mice but not in Tnfrsf1a^−/− mice. Collectively, commensal microflora induces TLR4 expression and decreases apoptosis of the intestinal mucosa. Commensal depletion enhances I/R-induced gut damage. LPS prevents I/R-induced intestinal permeability, lipid peroxidation, and decrease in GSH level. Given that the preventive effect of LPS on I/R-induced gut damage and NF-κB activity of the intestine is abolished in Tnfrsf1a^−/− mice, we conclude that TLR ligand decreases mesenteric I/R injury-induced gut damage through TNF-α signaling.

Background: The influence of the gut flora on lung inflammatory reaction against bacterial challenge remains undefined. This study was designed to investigate whether gut flora enhances lung defense againstE.colipneumonia through TLR4 signaling.Methods: C3H/HeN (WT) mice and C3H/HeJ (TLR4 deficient) mice were treated 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 intestinal tract. At the end of 4^thweek,E.coliwas injected to trachea to induceE.colipneumonia.Results: We found that commensal depletion by antibiotic pretreatment beforeE.colipneumonia challenge induced a 30% decrease of MPO activity in the lung, a significant decrease of bacterial killing activity of alveolar macrophage, and bacterial counts in C3H/HeN mice but not in C3H/HeJ (TLR4 deficient) mice. LPS, a TLR4 ligand, supplementation during antibiotic pretreatment reversed these effects and decreasedE.colipneumonia-induced mortality in C3H/HeN mice. Furthermore, commensal depletion induced a suppression of NF-κB DNA binding activity and an increase of KC, MIP-2, IL-1β expression in the lung in C3H/HeN mice but not in C3H/HeJ mice.Conclusions: Taken together with that commensal depletion increasedE.colipneumonia-induced mortality and LPS supplementation decreased it, we conclude that gut flora enhances bacterial clearance againstE.colipneumonia through TLR4.

Background: Although use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated. Methods: To study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKβ^△mye), IL-6^-/- to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr. WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI. Results: Our results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1β, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKβ^△mye mice as well as in IL6^-/- to WT chimeric mice. Conclusion: Given that IKKβ^△mye mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB–IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.

Prior antibiotic exposure is associated with increased mortality in Gram-negative bacteria-induced sepsis. However, how antibiotic-mediated changes of commensal bacteria promote the spread of enteric pathogenic bacteria in patients remains unclear. In this study, the effects of systemic antibiotic treatment with or without Toll-like receptor (TLR) stimulation on bacterium-killing activity, antibacterial protein expression in the intestinal mucosa, and bacterial translocation were examined in mice receiving antibiotics with or without oral supplementation of dead Escherichia coli or Staphylococcus aureus . We developed a systemic ampicillin, vancomycin, and metronidazole treatment protocol to simulate the clinical use of antibiotics. Antibiotic treatment decreased the total number of bacteria, including aerobic bacteria belonging to the family Enterobacteriaceae and the genus Enterococcus as well as organisms of the anaerobic genera Lactococcus and Bifidobacterium in the intestinal mucosa and lumen. Antibiotic treatment significantly decreased the bacterium-killing activity of the intestinal mucosa and the expression of non-defensin-family proteins, such as RegIIIβ, RegIIIγ, C-reactive protein-ductin, and RELMβ, but not the defensin-family proteins, and increased Klebsiella pneumoniae translocation. TLR stimulation after antibiotic treatment increased NF-κB DNA binding activity, nondefensin protein expression, and bacterium-killing activity in the intestinal mucosa and decreased K. pneumoniae translocation. Moreover, germfree mice showed a significant decrease in nondefensin proteins as well as intestinal defense against pathogen translocation. Since TLR stimulation induced NF-κB DNA binding activity, TLR4 expression, and mucosal bacterium-killing activity in germfree mice, we conclude that the commensal microflora is critical in maintaining intestinal nondefensin protein expression and the intestinal barrier. In turn, we suggest that TLR stimulation induces nondefensin protein expression and reverses antibiotic-induced gut defense impairment.