INTRODUCTION
The human intestines are colonized by trillions of microorganisms, including hundreds of different species of bacteria and viruses (1). These microbes, collectively referred to as the commensal microflora, have an important role in human nutrition and health, by promoting nutrient supply, preventing pathogen colonization, and shaping and maintaining normal mucosal immunity (2). Pneumonia is the leading cause of morbidity and mortality in the intensive care unit (3). Increased mortality during bacterial pneumonia may have resulted from a failure to control bacterial growth in the lung or to prevent inflammatory injury to the lung. The influence of the gut-lung axis on lung injury and immunity has been known for years, yet the underlying mechanism is not completely understood (4). It has previously been shown that protecting the integrity of the gut mucosa was effective in reducing idiopathic pneumonia syndrome after bone marrow transplantation (4). Furthermore, clinical trials have demonstrated that enteral feedings significantly reduced the incidence of pneumonia compared with patients fed parenterally (5). These data suggest that further defining the role of commensal microflora in host defense against pneumonia is warranted if we are to characterize the immunologic link between the gut and respiratory tract.
The host immune responses against the bacterial challenge comprised both adaptive and innate immune responses, the former consisting of T and B lymphocytes, whereas the latter was mediated through cytokines and complement and effected by phagocytic cells and cytotoxic natural killer cells (6). The innate immune system detects the invasion of microorganism through toll-like receptors (TLRs), which recognize microbial components and trigger inflammatory responses (7). Toll-like receptors comprise a family of pattern-recognition receptors that detect conserved molecular products of microorganisms, such as LPS and lipoteichoic acid, recognized by TLR4 and TLR2, respectively (8). The bacterial ligands recognized by TLRs are not unique to pathogens, but rather are shared by entire classes of bacteria, and are produced therefore by commensal microorganisms as well as pathogens. Previous work has shown that activation of TLRs by LPS administration via the oral route completely protected animals from the dextran sulfate sodium-induced inflammatory mortality, morbidity, and severe colonic bleeding seen in mice with depletion of commensal microflora (9). Also, recent findings suggest that TLR4 plays a critical role in mediating an effective innate immune response against Haemophilus influenzae in the lung (10). However, it is still not understood what the role of TLRs is in the regulatory mechanism of the gastrointestinal tract on lung immunity.
Toll-like receptors are type I integral membrane glycoproteins that contain leucine-rich repeats glanced by characteristic cysteine-rich motifs in their extracellular regions and a cytoplasmic TIR homology domain. Ligand-induced TLR dimerization permits the binding of cytoplasmic adapter proteins, MyD88, to the TLR cytoplasmic tails (11). A major downstream effect of TLR signaling is the activation of the transcription factor nuclear factor κB (NF-κB), which is required for expression of many genes related to innate immunity and inflammation (12). Previous studies have shown that inflammatory signaling through the NF-κB pathway in airway epithelial cells is critical to regulating the innate immune response against Pseudomonas aeruginosa (13). However, the influence of commensal microflora on NF-κB activation in the lung and its relationship with the host defense against pneumonia has not been examined.
Commensal microflora in the intestinal tract could play an important role in the relationship between gut-associated lymphoid tissue and lung defense to bacterial challenge. We hypothesized that commensal microflora in the gut could increase lung immunity through the stimulation of the TLRs and NF-κB DNA-binding activity in intestinal tract. We studied the effect of commensal depletion on TLR4 expression of intestinal mucosa and pulmonary bacterial counts after Escherichia coli pneumonia. Using a commensal depletion model in mice, we demonstrate that both the gut commensal microflora and TLR4 expression are involved in maintaining lung defense to bacterial challenge through TLRs. Also, we propose a novel function of gut commensal flora: as a stimulant of lung defense mechanism through the increase of the bacterial killing activity of alveolar macrophages (AMs). Adding TLR ligands in drinking water could be a promising therapeutic strategy to enhance host defense against E. coli pneumonia in critically ill patients.
MATERIALS AND METHODS
Animals
Specific pathogen-free male C57BL/6 weighing between 20 and 25 g were obtained from the National Laboratory Breeding and Research Center (Taipei, Taiwan). Mice were bred in the animal room of National Sun Yat-Sen University. They were fed standard laboratory chow and water ad libitum in the animal facility. All animal procedures were in compliance with regulations on animal used for experimental and other scientific purposes approved by the National Sun Yat-Sen University Animal Experiments Committee.
Depletion of gut commensal microflora and reconstitution of commensal-depleted animals with TLR ligands
Animals are provided ampicillin (A; 1 g/L; Bio Basic Inc, Ontario, Canada), vancomycin (V; 500 mg/L; Hospira, Inc, Lake Forest, Ill), neomycin sulfate (N: 1 g/L; Pharmacia/Upjohn, Mich), and metronidazole (M; 1 g/L; Sigma Chemicals, St. Louis, Mo) in drinking water for 4 weeks. Previously, a 4-week oral administration of vancomycin, neomycin, metronidazole, and ampicillin with the same dose described above in mice has been proved to deplete all detectable commensals (9). To those animals receiving TLR ligands, drinking water is supplemented with 10 μg/μL of purified E. coli 026:B6 LPS (Sigma) at week 3 and continued in drinking water for the duration of E. coli pneumonia. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4.
Experimental design
Experiment 1
To examine the influence of commensal microflora on lung innate immunity, C57BL/6 mice were divided into eight groups. Group 1 (PBS group, n = 6) received phosphate-buffered saline [PBS] intratracheal injection; group 2 (LPS + PBS group, n = 6) received LPS supplementation in drinking water for 1 week and PBS intratracheal injection; group 3 (antibiotic + PBS group, n = 6) received oral antibiotic for 4 weeks with PBS intratracheal injection; group 4 (antibiotic + LPS + PBS group, n = 6) received oral antibiotic for 4 weeks with LPS supplementation in drinking water at week 3 and PBS intratracheal injection; group 5 (E. coli group, n = 6) received E. coli intratracheal injection; group 6 (LPS + E. coli group, n = 6) received LPS supplementation in drinking water for 1 week and E. coli intratracheal injection; group 7 (antibiotic + E. coli group, n = 6) received oral antibiotic for 4 weeks and E. coli intratracheal injection at the end of the fourth week; and group 8 (antibiotic + LPS + E. coli group, n = 6) received oral antibiotic for 4 weeks with LPS supplementation at week 3 and E. coli intratracheal injection at the end of the fourth week. At 18 h after E. coli intratracheal injection, animals were killed, blood was harvested for culture, and lungs were harvested for bacterial culture and histology study. In another experiment, the myeloperoxidase (MPO) activity of the lung in animals with the same quantity and treatment was measured in anesthetized animals. Also, the intestinal mucosa of animals with the same treatment was harvested for NF-κB DNA-binding activity; TLR4 protein expression; and TNF-α, IL-6, TLR2, keratinocyte-derived chemokine (KC), TLR4, intercellular adhesion molecule (ICAM), IL-1β, CXCR2, macrophage inflammatory protein 2 (MIP-2), and β-actin mRNA expression.
Experiment 2
C57BL/6 mice were divided into four groups (n = 15 for each group). Group 1 received E. coli intratracheal injection; group 2 received LPS supplementation in drinking water for 1 week and E. coli intratracheal injection. Group 3 received oral antibiotic for 4 weeks and E. coli intratracheal injection; group 4 received oral antibiotic with oral LPS supplementation and E. coli intratracheal injection at the end of the fourth week. Animals were monitored for mortality after E. coli injection for 96 h.
Experiment 3
To examine the effect of commensal depletion on lung defense against bacterial challenge. Animals were divided into three groups each. Group 1 (E. coli group, n = 6) received E. coli intratracheal injection; group 2 (antibiotic + E. coli group, n = 6) received oral antibiotic for 4 weeks and E. coli injection at the end of the fourth week. Group 3 (antibiotic + LPS + E. coli group, n = 6) received oral antibiotic with LPS supplementation and E. coli intratracheal injection. At 8 h after E. coli intratracheal injection, lungs were harvested for NF-κB DNA-binding activity and KC, MIP-2, IL-1β, and β-actin protein as well as mRNA expression.
Induction of pneumonia
Mice were anesthetized with ketamine hydrochloride (100 mg/kg i.m.; Veterinary Laboratories, Wyeth-Ayerst Canada Inc, Mississauga, Ontario, Canada) and xylazine (5 mg/kg i.m.; Bayer Inc, Mississauga, Ontario, Canada). We have conducted a dose-dependence study, with 1.0 × 109 colony-forming units (CFUs) being the highest dose, and found that less dose did not cause pulmonary sepsis and lethality in normal immunocompetent mice. Also, a previous article suggested that intratracheal injection of 1.0 × 109 CFUs E. coli could induce significant pneumonia (14). Therefore, the trachea was surgically exposed, and 50 μL (1.0 × 109 CFUs E. coli) was instilled via an angiocatheter through the trachea as the previous article suggested (14). Concentrations of E. coli (strain 19138; American Type Culture Collection, Manassas, Va) were determined by colony counting.
Bacterial viability
Viable bacteria were quantified by colony counting. Serial dilutions from blood or organs homogenized in sterile saline were plated on blood agar, and colonies were enumerated after incubation at 37°C.
Processing of tissue samples after exposure to bacteria
Animals were killed by i.p. injection of ketamine (80 mg/kg) and xylazine (10 mg/kg) at 18 h after E. coli intratracheal injection for comparison of their bacterial clearance between different groups of mice. The whole lung was excised and washed with 10 mL of sterile cold saline. The viable bacteria counts of homogenized lung and blood were determined after an 18-h culture at 37°C in tryptic soy broth (TSB) agar plates. Data were expressed as CFUs per milliliter.
Determination of lung MPO activity
Lung content of MPO was determined to assess the degree of pulmonary neutrophils infiltration. Mice were anesthetized, and the thorax was opened with median sternotomy. The bilateral lungs and heart were harvested together, and the pulmonary vasculature was cleared of blood by gentle injection of 10 mL sterile saline into the right ventricle. The lungs were then blotted dry of surface blood and weighed.
Lung tissues were placed in 50 mM potassium phosphate buffer (pH 6.0) with 0.5% hexadecyltrimethylammonium bromide and homogenized. The homogenate was sonicated on ice and centrifuged for 30 min at 3,000g, 4°C. An aliquot (0.1 mL) of supernatant was added to 2.9 mL of 50 mM potassium phosphate buffer (pH 6.0) containing 0.167 mg/mL of O-dianisidine and 0.0005% hydrogen peroxide (15). The rate of change in absorbance at 460 nm was measured over 3 min. One unit of MPO activity was defined as the amount of enzyme that reduces 1 μmol of peroxide per minute, and the data were expressed as units per gram of lung tissue (U/g tissue).
Pulmonary histological studies
The lung tissue specimens harvested from different groups of animals were immersed in 4% formaldehyde solution. The tissue was embedded in paraffin wax, serially sectioned, and stained with hematoxylin-eosin. Pulmonary morphological characteristics were evaluated under light microscope.
Electrophoretic mobility shift assay for NF-κB
Nuclear extracts were prepared as described (16). Intestinal mucosa and lungs were harvested in hypotonic buffer and pelleted by centrifugation. The pellets were suspended in nuclear extract buffer. After 15 min on ice, the suspensions were centrifuged, and the supernatants were transferred to new tubes. The Bandshift kit (Promega Corp, Madison, Wis) was used according to the manufacturer's instructions. Consensus and control oligonucleotides (Santa Cruz Biotechnology Inc, Santa Cruz, Calif) were labeled by polynucleotides sequences of the NF-κB consensus (5′ to 3′) (AGTTGAGGGGAC-TTTCCCAGGC) (1.75 pmol/μL). After the oligonucleotide was radiolabeled, 5 μg of nuclear protein was incubated with 2 μg of poly (dI-dC) and 5,000 to 10,000 cpm of γ[32P]-ATP-labeled oligonucleotides. After 30 min at room temperature, the samples were analyzed on a 4% polyacrylamide gel. The gel was dried and visualized by autoradiography.
Western immunoblots
Protein levels of TLR4, IL-1β MIP-2, and KC in tissue were measured by Western immunoblotting. Tissues were homogenized in protein extract buffer (Sigma), and homogenized samples (50 μg of protein each) were subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Proteins were transferred onto PVDF membranes (Millipore, Billerica, Mass) by using a Semi-Dry Electrophoretic system (Bio-Rad, Hercules, Calif). TLR4, IL-1β MIP-2, and KC were identified by goat polyclonal antibodies (Santa Cruz Biotechnology Inc). The membranes were incubated with the secondary antibodies (biotinylated anti-rabbit and anti-goat IgG) (Perkin-Elmer Life Science, Boston, Mass) for 1 h at room temperature. Blots were developed by the ECL Western blotting detection reagents (Perkin-Elmer).
Polymerase chain reaction and quantification of polymerase chain reaction products
Total RNA was isolated from cells using TRIZOL reagent (Invitrogen Life Technologies, Grand Island, NY). Reverse transcription-generated cDNA encoding TNF-α, IL-6, KC, ICAM, IL-1β, TLR2, TLR4, CXCR2, and MIP-2 genes were amplified using polymerase chain reaction. Sets of TLR2, TLR4, CXCR2, and MIP-2 primers were designed according to those genes documented in GenBank. The sequences are 5′-CAGCCTCTTCTCATTCCTGCTTGTG-3′ (sense) and 5′-CTGGAAGACTCCTCCCAGGTATAT-3′ (antisense) for TNF-α, 5′-CGGAATTCGCCACCAGCCGCCTG-3′ (sense) and 5′-CGTCTAGACTTTCTCCGTTACTTGG-3′ (antisense) for KC, 5′-TCAGTCACCTCTACCAAGGCAG-3′ (sense) and 5′-CCACGGAGCCAATTTCTCA-3′ (antisense) for ICAM-1, 5′-AGTGGGTCAAGGAACAGAAGCA-3′ (sense) and 5′-CTTTACCAGCTCATTTCTCACC-3′ (antisense) for TLR4, 5′-TCTGGGCAGTCTTGAACATTT-3′ (sense) and 5′-AGAGTCAGGTGATGGATGTCG-3′ (antisense) for TLR2, 5′-TGTTCTTTGCCCTGACCTTGC-3′ (sense) and 5′-ACGCAGTACGACCCTCAAACG-3′ (antisense) for CXCR2, 5′-GAACAAAGGCAAGGCTAACTGA-3′ (sense) and 5′-AACATAACAACATCTGGGCAAT-3′ (antisense) for MIP-2. 5′-CAGCACGAGGCTTTTTTGTTG-3′ (sense) and 5′-TGGTGTGTGACGTTCCCATT-3′ (antisense) for IL-1β. 5′-TGCTTAGGCATAACGCACTAGGT-3′ (sense) and 5′-CTGGTGACAACCACGGCCTCCCCT-3′ (antisense) for IL-6. Meanwhile, we designed one pair of primer: 5′-GTGGGCCGCTCTAGGCACCA-3′ (sense) and 5′-CGGTTGGCCTTAGGGTTCAG-3′ (antisense) for β-actin gene as a control. To a sterile 0.2 mL tube were added 1.5 μL of 10× Ex Taq buffer, 1.2 μL of dNTP mixture (2.5 mM each), 0.2 μL each of the sense and antisense primers (0.5 mg/mL), 100 to 150 ng of the cDNA template, and an appropriate amount of water to make a total volume of 15 μL. After adding 0.075 μL of TaKaRa Ex Taq polymerase (5 U/μL), amplification was performed using a thermocycler (Bio-Rad): 5 min at 95°C before the first cycle, 1 min for denaturation at 95°C, 1 min for annealing at 58°C, and 1 min 30 s for extension at 72°C, then finally 10 min at 72°C after the last cycle. We recorded the electrophoresis by CCD camera and compared the band intensity by Kodak Digital Science TM ID Image Analysis Software (Eastman Kodak Company, Rochester, NY).
Ex vivo AM stimulation
Alveolar macrophages were harvested from adult mice by bronchoalveolar lavage with Tris-buffered saline containing 0.25 mM EDTA and EGTA. Cells were resuspended in RPMI 1640 in a final concentration of 1 × 105 cells/mL. Cells were then cultured in 96-well microtiter plates for 2 h and washed with RPMI 1640 to remove nonadherent cells (17). Adherent monolayer cells were stimulated with different doses of LPS (from E. coli O26:B6; Sigma-Aldrich) or RPMI 1640 for 4 h. Supernatants were collected and stored at −70°C until assayed for TNF-α.
Bacterial killing activity of AMs
Alveolar macrophages were washed three times with RPMI 1640 and counted using trypan blue. Alveolar macrophages were collected and resuspended in HBSS as 106 cells/mL. After 5 min of preincubation, the cell suspension was incubated with E. coli (108/mL) at 37°C for 1 h with shaking. The cells were removed as the pellet after centrifugation at 200g for 10 min, and E. coli number in the supernatant was counted (18).
Enzyme-linked immunosorbent assay
IL-6 and IL-1β levels in lung tissue were measured by a mouse enzyme-linked immunosorbent kit following the manufacturer's recommended protocol (R&D Systems, Minneapolis, Minn).
Statistical analysis
Values are expressed as means (SD) of the mean, and P < 0.05 is considered to be statistical significance. Intergroup comparisons were made using one-way ANOVA followed by Bonferroni correction. Statistical analysis was performed on Prism software (GraphPad, La Jolla, Calif). The photographs shown represent the results obtained from at least three independent experiments.
RESULTS
Antibiotic pretreatment enhanced E. coli pneumonia-induced bacteria counts in lung, and LPS supplementation alleviated it
Four-week oral antibiotic pretreatment significantly decreased bacteria in colonic fecal matter of mice as previously described (19, 20). To determine if commensal microflora were crucial in host defense against E. coli pneumonia, bacteria of lung were quantified by colony counting after E. coli intratracheal injection. Phosphate-buffered saline intratracheal injection did not increase pulmonary bacterial counts in control, LPS, commensal depletion, and commensal depletion + LPS groups (Fig. 1A). Escherichia coli injection significantly increased bacterial counts in lung compared with that of PBS injection group. LPS treatment alone did not enhance E. coli pneumonia-induced bacterial counts in lung in comparison with that of E. coli injection group. Antibiotic pretreatment with E. coli injection induced a significant 3-fold increase of bacterial growth in lung compared with that of E. coli group (22 × 104 [SD, 3 × 104] vs. 5 × 104 [SD, 1 × 104] CFUs/g tissue). LPS supplementation with antibiotic pretreatment significantly decreased 36% of E. coli injection-induced bacterial growth in lung (14 × 104 [SD, 5 × 104] CFUs/g) compared with that of commensal depletion + E. coli group.
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Fig. 1: Commensal depletion enhanced E. coli pneumonia-induced bacteremia, and oral LPS supplementation alleviated it. A, Antibiotic pretreatment for 4 weeks increased E. coli injection-induced bacterial growth in lung, and LPS supplementation alleviated it (n = 6 per group). *P < 0.05. B,Antibiotic pretreatment (Comm. depl.) increased E. coli injection-induced bacterial growth in blood, and LPS supplementation (Comm. depl. + LPS) alleviated it (n = 6 per group). *P < 0.05. C, Antibiotic pretreatment with subsequent E. coli intratracheal injection induced an increase of mortality incomparison with E. coli group. *P < 0.05 vs. E. coli control group (n = 15 per group).
Antibiotic pretreatment increased E. coli pneumonia-induced bacteria counts in the blood, and oral LPS supplementation reversed it
To further define the role of commensal microflora in host defense against pneumonia, bacteria of blood were quantified after E. coli intratracheal injection. Phosphate-buffered saline intratracheal injection did not induce blood bacterial counts in control, LPS treatment, commensal depletion, and commensal depletion + LPS groups (Fig. 1B). Intratracheal injection of E. coli (1 × 109 CFUs/mouse) induced an increase of bacterial counts in blood (12 × 104 [SD, 4 × 104] CFUs/mL) in comparison with PBS injection group. To evaluate the effect of TLR4 ligand on E. coli pneumonia-induced bacteria in blood, the drinking water was supplemented with LPS, TLR4 ligand. LPS supplementation in drinking water for 1 week did not increase E. coli injection-induced bacterial counts in blood compared with E. coli injection group. Commensal depletion induced a significant 15-fold increase of bacterial growth in blood compared with that of E. coli group (168 × 104 [SD, 32 × 104] vs. 12 × 104 [SD, 4 × 104] CFUs/mL) (Fig. 1B). LPS supplementation in oral antibiotic significantly decreased 29% of E. coli intratracheal injection-induced blood bacterial growth (120 × 104 [SD, 23 × 104] CFUs/mL) when compared with that of commensal depletion + E. coli group.
Antibiotic pretreatment increased mortality in E. coli pneumonia
Intratracheal injection of PBS or E. coli (1 × 109 CFUs/mouse) did not induce mortality in WT mice at 96 h after injection. Also, LPS supplementation before E. coli intratracheal injection did not induce mortality. Antibiotic pretreatment with subsequent E. coli intratracheal injection induced a significant increase of mortality (∼70%) in comparison with that of E. coli group. LPS supplementation decreased 30% of mortality compared with that of commensal depletion + E. coli group (Fig. 1C).
Antibiotic pretreatment enhanced E. coli pneumonia-induced lung edema, and LPS supplementation reversed it
To evaluate the histological change of lung after different treatments, we examined the lung tissue specimens in mice. Escherichia coli injection induced a significant increase of perivascular and interstitial inflammatory cells infiltration in comparison with that of PBS group (Fig. 2B). Oral antibiotic pretreatment with E. coli injection treatment markedly increased the interstitial edema, septal edema, and alveolar edema in lung compared with that of E. coli group. LPS supplementation in antibiotic pretreatment markedly decreased the interstitial edema, septal edema, and alveolar edema compared with that of oral antibiotic + E. coli group.
Fig. 2: Commensal depletion increased interstitial edema and decreased E. coli pneumonia-induced MPO activity in lung. A, Antibiotic pretreatment before E. coli pneumonia increased the interstitial edema as well as septal edema in lung compared with E. coli pneumonia group, and LPS supplementation reversed them (n = 3). B, Antibiotic pretreatment for 4 weeks (Comm. depl.) decreased E. coli injection-induced pulmonary MPO activity, and LPS supplementation (Comm. depl. + LPS) reversed it (n = 6 per group). *P < 0.05.
Antibiotic pretreatment decreased E. coli pneumonia-induced lung MPO activity, and LPS supplementation reversed it
Neutrophils play an essential role in the innate immune response to infection (21). To characterize the mechanism of commensal depletion on lung defense to E. coli pneumonia, we examined the MPO activity of lung in mice after E. coli injection. There was no significant difference of the lung MPO activity between control, LPS treatment, antibiotic pretreatment, and antibiotic pretreatment with LPS supplementation groups after PBS intratracheal injection (Fig. 2B). Intratracheal E. coli injection induced a significant 3-fold increase of the MPO activity of lung compared with that of PBS group (173 [SD, 18] vs. 43 [SD, 9] U/g tissue). LPS treatment with E. coli pneumonia did not change the MPO activity of lung in comparison with that E. coli injection group. Antibiotic pretreatment with E. coli pneumonia significantly decreased 30% of MPO activity in comparison with that of E. coli group (121 [SD, 26] vs. 173 [SD, 18] U/g tissue) without antibiotic pretreatment. LPS supplementation with antibiotic pretreatment significantly increased lung MPO activity by 25% in comparison to that of E. coli + commensal depletion group (151 [SD, 21] vs. 121 [SD, 26] U/g tissue) (Fig. 2A).
Antibiotic pretreatment decreased TNF-α production of AMs, and LPS supplementation restored it
To define the effect of commensal depletion on the activity of AMs, we measured TNF-α production of AM after stimulation. Antibiotic pretreatment significantly decreased TNF-α production compared with that of control group. LPS supplementation in oral antibiotic significantly increased TNF-α production compared with that of commensal depletion group (Fig. 3A).
Fig. 3: Commensal depletion decreased TNF-α production and bacterial killing activity of AMs but increased IL-6 and IL-β levels in the lung. A,Commensal depletion (Comm. depl.) decreased TNF-α production of AMs after stimulation with different doses of LPS. Oral LPS supplementation (Comm. depl. + LPS) reversed it (n = 6 per group). *P < 0.05. B, Commensal depletion (Comm. depl.) decreased the bacterial killing activity of AMs, and LPS supplementation (Comm. depl. + LPS) reversed it (n = 6 per group). *P < 0.05. C, Antibiotic pretreatment before E. coli injection increased IL-6 and IL-β levels in the lung,and LPS supplementation decreased them. *P < 0.05.
Antibiotic pretreatment decreased the bacterial killing activity of AMs, and LPS supplementation restored it
To further define the mechanism of commensal depletion-induced decreasing lung defense against pneumonia, we harvested AMs from mice and examined their bacterial killing activity. Alveolar macrophages were harvested and cultured with E. coli. Bacterial killing activity of macrophages was determined by counting the E. coli number that remained. Antibiotic pretreatment significantly increased bacterial retention compared with that of control group (2,836 [SD, 370] vs. 1,916 [SD, 250] CFUs). LPS supplementation in oral antibiotic significantly decreased 31% bacterial retention compared with that of commensal depletion group (Fig. 3B).
Antibiotic pretreatment increased IL-6 and IL-1β levels of lung, and LPS supplementation reversed them
Escherichia coli injection significantly increased IL-1β levels in the lung in comparison with that of PBS injection group. Antibiotic pretreatment before E. coli injection significantly enhanced both IL-6 and IL-1β levels (Fig. 3C) in the lung tissue compared with those of E. coli group. LPS supplementation in oral antibiotic markedly decreased IL-6 and IL-1β levels compared with those of commensal depletion with E. coli injection group.
Antibiotic pretreatment decreased the NF-κB activation of intestinal mucosa, and LPS supplementation restored it
To investigate the role of commensal microflora on NF-κB activation of intestine, we examined the NF-κB activation of intestinal mucosa by determining the NF-κB DNA-binding activity using electrophoresis mobility shift assay. Antibiotic pretreatment significantly decreased the NF-κB DNA-binding activity of intestinal mucosa compared with that of control group, and LPS supplementation reversed it (Fig. 4A).
Fig. 4: Commensal depletion decreased NF-κB DNA-binding activity, TLR4 protein expression, and TNF-α, TLR4, KC, ICAM, and CXCR2 mRNA expression of intestinal mucosa, and LPS supplementation reversed them. A, Commensal depletion (Comm. depl.) decreased NF-κB DNA-binding activity of intestinal mucosa, and LPS supplementation (Comm. depl. + LPS) reversed it (n = 5). Signal intensity was determined by densitometry and normalized to nonspecific band (NS). B, Commensal depletion decreased TLR4 protein expression of intestinal mucosa, and LPS supplementation reversed it (n = 5). Signal intensity was determined by densitometry and normalized to β-actin. C, Commensal depletion decreased TLR4, TNF-α, KC, ICAM, and CXCR2 mRNA expression of intestinal mucosa, and LPS supplementation reversed them (n = 5). *P < 0.05 vs. E. coli control group.
Antibiotic pretreatment decreased TLR4, TNF-α, KC, ICAM, and CXCR2 expression in the intestinal mucosa, and LPS supplementation restored them
To evaluate the effect of commensal depletion on TLR and cytokine expression in the intestinal mucosa, we also examined TLR4 protein expression and TLR4, TLR2, TNF-α, IL-6, KC, ICAM, MIP-2, IL-1β, and CXCR2 mRNA expression in the intestinal mucosa in mice. Oral antibiotic pretreatment decreased TLR4 protein expression (Fig. 4B) and TLR4, TNF-α, KC, ICAM, and CXCR2 mRNA expression in the intestinal mucosa compared with those of control group (Fig. 4C). However, antibiotic pretreatment did not change IL-6, TLR2, MIP-2, and IL-1β mRNA expression of intestinal mucosa. Although TLR4 recognizes most forms of gram-negative bacterial LPS, TLR2 recognizes not only gram-positive bacteria but also lipopeptide, lipoarabinomannan, fungal cell wall components, and LPS of leptospirosis (22). This might be the reason why commensal depletion markedly decreased TLR4 but not TLR2 expression of intestinal mucosa. LPS supplementation in oral antibiotic restored commensal depletion-induced reduction of TLR4 protein expression (Fig. 4B) and TLR4, TNF-α, KC, ICAM, and CXCR2 mRNA expression (Fig. 4C) of intestinal mucosa.
Antibiotic pretreatment decreased E. coli pneumonia-induced NF-κB activation of lung, and LPS supplementation restored it
To define the role of NF-κB activation in the effect of oral antibiotic pretreatment on lung immunity, we examined the NF-κB DNA-binding activity of lung after E. coli intratracheal injection. Commensal depletion significantly decreased NF-κB activation of lung after E. coli pneumonia in mice compared with that of control group. LPS supplementation increased the NF-κB activation of lung in mice compared with that of commensal depletion group (Fig. 5A).
Fig. 5: Commensal depletion decreased NF-κB DNA-binding activity and increased IL-1β, KC, and MIP-2 expression in the lung, and LPS supplementation restored them. A, Commensal depletion (Comm. depl.) decreased the NF-κB activation of lung after E. coli pneumonia, and LPS supplementation (Comm. depl. + LPS) restored it (n = 5). Signal intensity was determined by densitometry and normalized to nonspecific band (NS). *P < 0.05 vs. control group. B, Commensal depletion increased IL-1β, KC, and MIP-2 protein expression in the lung after E. coli pneumonia and LPS supplementation decreased them (n = 5). Signal intensity was determined by densitometry and normalized to β-actin. *P < 0.05 vs. control group. C, Commensal depletion increased IL-1β, KC, and MIP-2 mRNA expression in the lung after E. coli pneumonia, and LPS supplementation alleviated them. Signal intensity was determined by densitometry and normalized to β-actin. *P < 0.05 vs. E. coli control group.
Antibiotic pretreatment enhanced IL-1β, KC, and MIP-2 expression of lung, and LPS supplementation reversed them
We were particularly interested in IL-1β expression in lung, because it is suggested to play an important role in the inflammatory signaling, and its signaling pathway is critical to the activation of the proinflammatory response of inflammatory cells (23). We examined IL-1β, KC, and MIP-2 protein and mRNA expression in the lung of mice. Antibiotic pretreatment before E. coli pneumonia significantly increased IL-1β, KC, and MIP-2 protein (Fig. 5B) and mRNA expression (Fig. 5C) in the lung compared with those of E. coli group. LPS supplementation markedly decreased E. coli pneumonia-induced IL-1β, KC, and MIP-2 protein as well as mRNA expression in the lung compared with those of commensal depletion group.
DISCUSSION
The gut contains our largest collection of resident microorganisms. Commensal microflora in the gut are reported to be important regulators for the intestinal hemostasis and the intestinal innate immunity (9). Previous studies have alluded to a decreased lung defense to bacterial challenge accompanying parenteral nutrition (24). In the present study, we provide evidence demonstrating the critical role of commensal microflora in maintaining lung defense to bacterial challenge. First, using an E. coli pneumonia model in mice, we clearly demonstrate that commensal depletion significantly increases E. coli intratracheal injection-induced bacteria burden in the lung and blood. Commensal depletion also increases the mortality in WT mice after E. coli intratracheal challenge. This indicates that maintenance of gut commensal microflora is critical in enhancing lung defense to bacterial challenge. Second, commensal depletion significantly decreases activity as well as the bacterial killing activity of AMs. Because AMs are pivotal to the phagocytic defense in the lung (25), our results indicate that gut commensal microflora are critical in maintaining activity as well as the bacterial killing activity of AMs. Third, commensal depletion significantly decreases E. coli pneumonia-induced neutrophils infiltration in the lung. Myeloperoxidase system plays an important role in the microbicidal activity of phagocytes and neutrophils in the innate immune response to infection (21). Myeloperoxidase, released by neutrophils, may attack normal tissue and thus contribute to the pathogenesis of lung injury. However, an acute innate immune response to bacteria in the lung has also been characterized by the infiltration of neutrophils (26); thus, they are necessary for this process. Fourth, commensal depletion significantly decreases NF-κB DNA-binding activity of lung. Nuclear factor κB activation is an essential immediate early step in innate immune cell activation (27). Our data suggest that commensal microflora are important in inducing NF-κB DNA-binding activity of both the intestine and lung. Finally, antibiotic pretreatment before E. coli pneumonia significantly increased IL-1 β, KC, and MIP-2 protein expression in the lung, suggesting that commensal microflora in the gut are important in decreasing lung inflammatory signaling. Altogether, these data suggest that commensal microflora are critical in enhancing lung defense E. coli pneumonia. Early enteral feeding to restore commensal microflora or adding probiotics in the diet might be a feasible way to increase host defense against pneumonia in intensive care patients.
The second important conclusion to be derived from the present results is that oral TLRs ligand supplementation increased lung defense against pneumonia. LPS is a well-studied TLR4 and MD-2 ligand. LPS is a component of the gram-negative bacterial cell wall that activates B cells resulting in marked production of polyclonal antibodies. Endotoxin also stimulates secretion of proinflammatory cytokines such as TNF-α and IL-1β from macrophages, suggesting the involvement in LPS-induced inflammation (28). Previously, oral LPS supplementation in commensal-depleted mice has been shown to significantly decrease dextran sulfate sodium-induced mortality and morbidity (9). Here, our data demonstrate that LPS supplementation in commensal-depleted animals could induce the lung defense against E. coli pneumonia. First, our data demonstrate that LPS supplementation in oral antibiotic effectively increases TLR4 expression of intestinal mucosa and reverses the effect of oral antibiotic pretreatment on enhancing E. coli pneumonia-induced bacterial counts in the lung and blood. Second, LPS supplementation increases pulmonary neutrophil infiltration after E. coli pneumonia. Third, LPS supplementation increases activity as well as the bacterial killing activity of AMs in lung. Our data suggest that oral TLR4 ligand supplementation could enhance the lung defense against bacterial challenge through the increase of the bacterial killing activity of AMs. Although LPS supplementation to commensal bacterial depleted mice enhances the intestinal cytokine production and pulmonary neutrophil recruitment to control levels, it does not bring bacterial loads and survival rate to that of control mice. Antibiotics treatment reduces both gram-positive and gram-negative bacteria in the intestinal tract. However, LPS supplementation restores only the TLR4 stimulation of the intestinal tract. This might be the reason why LPS supplementation does not bring bacterial loads and survival to that of control of mice. It is tempting to speculate that a new TLR4 ligand that is based on LPS structure, but without its toxicity, could be developed to enhance lung defense through the increase of the bacterial killing activity of AMs.
Nuclear factor κB family members control transcriptional activity of various promoters of proinflammatory cytokines, cell surface receptors, transcription factors, and adhesion molecules that are involved in intestinal inflammation such as TNF-α, ICAM, KC, and MIP-2 (29). Previous studies have shown that TLR4 stimulation could maintain intestinal hemostasis through the NF-κB activation of the intestinal mucosa (9). The inhibitory effect of oral antibiotic pretreatment on NF-κB activation and TLR4, TNF-α, KC, ICAM, and CXCR2 mRNA expression of intestinal mucosa further corroborates the important role that commensal microflora play in inducing cytokine expression of intestinal mucosa through TLRs and downstream NF-κB signaling. Nuclear factor κB regulates the transcription of a wide array of gene products that are involved in the molecular pathobiology of the lung (30). 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 (30). Inflammatory signaling through the NF-κB pathway by airway epithelial cells critically regulates the innate immune response to P. aeruginosa (13). Our present results further suggest that commensal microflora in intestinal tract are critical in the NF-κB activation and host defense in lung.
Both polymicrobial sepsis and intratracheal LPS injection can induce acute lung inflammation with elevated IL-1β, KC, and MIP-2 levels and MPO activity of lung in mice (31). IL-1β has been shown to induce the expression of ICAM-1 on airway epithelial cells and contributes to inflammatory responses (32). 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. Moreover, LPS supplementation increases TLR4 expression and NF-κB activation in intestinal mucosa but decreases IL-1β, KC, and MIP-2 protein and mRNA levels in the lung. Previously, mice deficient in TLR4 demonstrated a substantial delay in clearance of H. influenzae with diminished IL-1β, IL-6, TNF-α, MIP-α, and MIP-2 in bronchoalveolar lavage (10). On the contrary, our present data demonstrate that depletion of TLR4 stimulation in gut with commensal depletion enhanced E. coli pneumonia-induced IL-1β, IL-6, TNF-α, MIP-α, and MIP-2 expression in the lung. Altogether, our data suggest that commensal microflora are critical in decreasing the inflammatory signaling especially IL-6, KC, MIP-2, and IL-1β of lung in response to E. coli pneumonia. The role of gut injury and inflammation in major trauma-induced systemic inflammatory response syndrome and multiple organ dysfunction syndrome has been known for decades (33). Recently, Deitch et al. (34) found out that the egress of gut-derived factors leading to lung injury and systemic inflammation is carried via the intestinal lymphatics. Our data further suggest that commensal microflora play an important role in inducing TNF-α, KC, CXCR2, and ICAM expression of intestinal mucosa and decreasing IL-1β, KC, MIP-2, and IL-6 levels in the lung through TLRs.
Although upregulation of tissue TLR2/4 has been implicated to mediate the proinflammatory response and pathophysiology of polymicrobial sepsis (35), our present findings suggest that commensal microflora play beneficial roles to the host defense to E. coli pneumonia through the stimulation of TLR4 expression and NF-κB DNA-binding activity in intestinal tract. Although TLRs of those macrophages that were resident in the lamina propria of the intestine have been implicated in the potent inflammatory response, intestinal inflammation, and corresponding injury (36), our data further suggest that commensal microflora induce host defense to E. coli pneumonia through the enhancement of activity as well as the bacterial killing activity of AMs. Therefore, we propose a novel function of TLRs is to increase bacterial clearance ability of AMs and enhance host defense against E. coli pneumonia.
From our present results, the mechanism by which commensal microflora affect lung defense against E. coli pneumonia has been described (Fig. 6). Commensal microflora first induce TLR4 expression, NF-κB DNA-binding activity, and TNF-α, KC, CXCR2, and ICAM expression of intestinal mucosa. Next, those signaling pathways enhance the lung immunity and reduce E. coli pneumonia-induced inflammation in the lung through the induction of the bacterial killing activity of AMs.
Fig. 6: The regulatory mechanism of commensal microflora on the lung defense against E. coli pneumonia. Commensal microflora induced TLR4 expression, NF-κB DNA-binding activity, and TNF-α, KC, CXCR2, andICAM expression of intestinal mucosa. Also, commensal microflora increased the bacterial killing activity of AMs and lung defense against E. coli pneumonia.
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