Commensal microflora in the gut are reported to be important regulators for the intestinal haemostasis and the intestinal innate immunity . In the present study, we further demonstrate that gut flora are critical in enhancing lung inflammatory reaction against E.coli pneumonia. MPO system plays an important role in the microbicidal activity of neutrophils in the innate immune response to infection . However, an acute innate immune phagocytes response to bacteria in the lung has also been characterized by the infiltration of neutrophils , thus, they are necessary for this process. Our data clearly demonstrate that commensal depletion decreases E.coli intratracheal injection-induced MPO activity. This indicates that gut flora are important in maintaining neutrophils infiltration in the lung while bacteria invasion. LPS, a TLR4 ligand, supplementation with oral antibiotic pretreatment reverses commensal depletion-induced reduction of MPO activity, suggesting that oral TLR4 stimulation increases neutrophils infiltration in the lung. Moreover, oral antibiotic treatment with or without LPS supplementation does not change E.coli pneumonia-induced MPO activity in TLR4 mutant mice. At last, E.coli pneumonia induces less MPO activity in TLR4 mutant mice than in WT mice. These suggest that TLR4 signaling pathways are involved in E.coli pneumonia-induces neutrophils infiltration in the lung. Altogether, our data demonstrate that gut flora are important in enhancing lung neutrophils infiltration against E.coli pneumonia through TLR4 and depletion of TLR4 decreases bacterial challenge-induced lung inflammation.
Next, we try to clarify the effect of commensal depletion on the innate immunity in the lung. Since alveolar macrophages are pivotal to the phagocytic defense in the lung , our results indicate that gut commensal microflora are critical in maintaining the bacterial killing activity of alveolar macrophages. First, commensal depletion decreases the bacterial killing activity of alveolar macrophages in WT mice and LPS supplementation reverses its effect. Moreover, alveolar macrophages in TLR4 mutant mice demonstrate a decrease of bacterial killing activity compared with those in WT mice. Also, commensal depletion with or without LPS supplementation does not change the bacterial killing activity of alveolar macrophage in C3H/HeJ mice. This suggests that effect of commensal depletion on the alveolar macrophage is through TLR4. Together, our data indicate that gut flora play an important role in maintaining the bacterial killing activity of alveolar macrophages through TLR4 and depletion of TLR4 decreases the bacterial killing activity of alveolar macrophages.
NF-κB family members control transcriptional activity of various promoters of proinflammatory cytokines, cell surface receptors, transcription factors, and adhesion molecules that are involved in inflammation such as TNFα, ICAM, KC, and MIP-2 . Previous studies have shown that TLR4 stimulation could maintain intestinal haemostasis through the NF-κB activation of the intestinal mucosa . NF-κB activation is an essential immediate early step in innate immune cells activation . The inhibitory effect of oral antibiotic pretreatment on NF-κB DNA binding activity in the lung further corroborates the important role that commensal microflora play in inducing NF-κB signaling in the lung. Nuclear factor kappa B (NF-κB) regulates the transcription of a wide array of gene products that are involved in the molecular pathobiology of the lung . Three lung cell types, epithelial cells, macrophages and neutrophils, have been shown to be involved in the generation of lung inflammation through signaling mechanisms that are dependent on activation of the NF-κB pathway . Inflammatory signaling through the NF-κB pathway by airway epithelial cells critically regulates the innate immune response to P. aeruginosa. Our present results further suggest that commensal microflora in intestinal tract are critical in inducing the NF-κB activation and lung defense against E.coli pneumonia. Moreover, the abolition of the stimulatory effect of LPS on pulmonary NF-κB activation and bacterial killing activity of alveolar macrophages in TLR4 mutant mice further corroborates that gut flora are important in enhancing NF-κB activation in the lung through TLR4 and LPS supplementation enhances lung defense through the TLR4 and NF-κB signaling pathways.
Both polymicrobial sepsis and intratracheally lipopolysaccharide (LPS) injection can induce acute lung inflammation with elevated IL-1β, KC, MIP-2 levels and MPO activity of lung in mice . Interleukin-1β (IL-1β) has been shown to induce the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and contributes to inflammatory responses . Our data demonstrate that commensal depletion decreases MPO activity and NF-κB activation but induces IL-1β, KC, and MIP-2 expression of lung after E.coli pneumonia. Previously, mice deficient in TLR4 demonstrated a substantial delay in clearance of H. influenzae with diminished IL-1β, IL-6, TNFα, MIP-α, and MIP-2 in bronchoalveolar lavage . Similarly, our present data demonstrate that oral antibiotic pretreatment with E.coli pneumonia-induced NF-κB activation as well as IL-1β, KC, and MIP-2 expression of lung are decreased in C3H/HeJ mice. Altogether, our data suggest that commensal microflora are critical in decreasing KC, MIP-2, and IL-1β of lung in response to E.coli pneumonia.
More importantly, commensal depletion increases E.coli pneumonia-induced mortality and LPS supplementation significantly decreases it in WT mice. This further corroborates that gut commensal microflora is critical in maintaining lung defense against bacterial challenge through the increase of the bacterial killing activity of alveolar macrophage and neutrophils infiltration. Our recent work have demonstrated that commensal gut depletion by antibiotic pretreatment before E.coli pneumonia challenge in WT mice 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 . Our present data further demonstrate that E.coli pneumonia challenge induced a 30% decrease of MPO activity in the lung and a significant decrease of bacterial killing activity of alveolar macrophage in WT mice but not in TLR4 deficient mice. Altogether, our data indicate that commensal flora play an important role in maintaining lung inflammation reaction against E.coli pneumonia through TLR4. Our data also imply that early enteral feeding to restore commensal microflora or adding TLRs ligands in diet might be a feasible way to increase host defense against pneumonia in intensive care patients.