Background
Antibiotic use is a crucial part of surgery and treatments such as chemotherapy. However, the antibiotic-resistant infections that are difficult to treat are becoming more and more common. The emergence of these strains is currently a global health crisis. Over 2 million people are infected with antibiotic-resistant bacteria annually in the USA alone, resulting in 23,000 deaths annually, on average [
1]. The wide spread use of antibiotics in the intensive care unit (ICU) is an important factor in the emergence of nosocomial infections caused by antibiotic-resistant Gram-negative bacteria [
2,
3]. Impairment of the host defenses is a key element in the pathogenesis of infection by these bacteria. However, the effects and the mechanisms of antibiotic-mediated changes in the host defense against bacteria invasion in critical patients are currently still unclear.
The human gastro-intestinal tract is colonized by trillions of microorganisms which encompass hundreds of different species of bacteria and viruses [
4,
5]. The intestinal epithelial cells express pattern recognition receptors that protect against antagonistic microbial invasion and maintain epithelial barriers in the presence of commensal microflora [
6]. A major downstream effect of toll-like receptor signaling is the activation of nuclear factor-κB (NF-κB) [
7]. Paneth cells also limit bacterial penetration to the small intestinal antimicrobial barrier through synthesizing different antimicrobial peptides and proteins, such as enteric α-defensins (known as cryptdins in mice), lysozyme, RegIIIβ, RegIIIγ, RELMβ (resistin-like molecules β), and CRP-ductin [
8]. The aryl hydrocarbon receptor (Ahr) is a ligand-dependent transcription factor that is best known for mediating the carcinogenicity xenobiotic ligands. Recent study suggests that Ahr also plays an important physiological role in the immune system [
9]. The expression of Ahr is critical for the maintenance, survival, and functioning of Type 3 innate lymphoid cells (ILC 3) [
10]. Ahr cooperates with Rorγt to induce the transcription of interleukin-22 (IL-22), which is essential for the clearance of
Citrobacter rodentium infection [
10]. Indole-3-aldehyde, a tryptophan catabolite produced by the microbiota, stimulates the ILCs through the aryl hydrocarbon receptor to induce the ILCs to produce IL-22 [
10]. The regulatory mechanisms of the gut-lung axis and the role of the Ahr receptors of the intestinal mucosa in innate lung immunity have remained elusive.
Interleukin-6 (IL-6) is a pleiotropic cytokine involved in both pro-inflammatory and anti-inflammatory responses via the regulation of leukocyte function and apoptosis. Overproduction of IL-6 results in immunological and hematological disorders. In addition, IL-6 induces inflammation by promoting T cell activation, adhesion molecule expression, and influencing leukocyte recruitment [
11]. The majority of patients in the ICU are treated with strong antibiotics, which can have pervasive and long-term effects on the intestinal microbiota [
12]. In an earlier study, we have demonstrated that antibiotic treatments reduced the total bacteria counts in the terminal ileum, but increased the translocation of injected pathogenic
Klebsiella pneumoniae due to a reduction in the mucosal bacterial killing activity and the expression of nondefensin proteins [
13]. Recently, we also showed that dead
L. salivarius or FOS feeding reversed antibiotic-induced lung defense impairment through the intestinal ROS/MyD88 pathways [
14]. The gut microbiota has been proven to enhance primary alveolar macrophage (AM) function and plays a protective role in the host defense against pneumococcal pneumonia [
15]. We hypothesize that Ahr ligands or dead
Lactobacillus plantarum could be used to strengthen the lung defense against
Pseudomonas aeruginosa (PA)-induced pneumonia in ICU patients through ROS production and NF-κB activation from the intestine. Here, mice were used as a model in antibiotic treatment with or without PA-induced pneumonia to study the effects and mechanisms of the antibiotic treatment on lung defense mechanisms. There are three main objectives in this study: (1) To determine the effects and mechanisms of antibiotic treatments on lung defense mechanisms. (2) To determine whether Ahr ligands could reverse the inhibitory effects of antibiotics on lung defense. (3) To examine the involvement of Ahr and IKKβ of the intestine in the regulatory mechanisms of the gut on lung defense. In ICU patients, oral supplementation of Ahr ligands to reverse lung defense impairment caused by antibiotic treatments can potentially be a useful therapeutic treatment in the future.
Methods
Animals
We obtained specific pathogen-free (SPF) C57BL/6J mice from the National Laboratory Breeding and Research Center (NLBRC, Taipei, Taiwan). Specific pathogen-free intestinal epithelial cell-specific, IKKβ-deficient (Vil-Cre/IkkβF/Δ) mice and control (IkkβF/Δ) mice were generated from the same back ground and transferred from Dr. Karin’s lab. (University of California, San Diego). Before the experiment, the animals were maintained in a temperature-controlled room and fed a standard diet of Purina mouse chow with water allowed ad libitum for at least 1 week before the experiment. All animal procedures were in compliance with the regulations on animals used for experimental and other scientific purposes approved by the Kaohsiung Veterans General Hospital Animal Experiments Committee.
Experimental design
Experiment 1
To simulate combined antibiotic treatment use in clinical patients, we used a broad-spectrum antibiotic treatment regimen where the mice (C57BL/6) received combined intramuscular injections (100 μl) daily. The injections are composed of a blend of different antibiotics: ampicillin (1000 mg/L; Bio Basic Inc.), vancomycin (500 mg/l; Hospira), and metronidazole (1000 mg/l; Sigma) [
13]. The mice were randomly divided into the following four groups: (1) Control, (2) Tryptophan, (3) Indole, and (4) Lactobacillus. Tryptophan (Sigma-Aldrich) or indole (Alfa Aesar) were administered to mice in drinking water to stimulate aryl hydrocarbon receptors (Ahr) in the intestine for 2 days before sacrifice in order to examine the effects of Ahr ligands on antibiotic treatment-induced lung defense impairment. To determine if increasing specific groups of intestinal commensal microbiota (i.e.,
Lactobacillus or
Bifidobacteria) can lead to the improvement and prevention by tryptophan supplementation, mice were fed dead
L. plantaris (2 × 10
8 CFU/ml) for 6 days. Other C57BL/6 mice were randomly divided into the following five groups: Group I received intramuscular injections of normal saline daily over 6 days (control group); Group II received intramuscular injections of antibiotics daily for 6 days; Group III received antibiotics injections with intramuscular injections of antibiotics daily over 6 days with tryptophan feeding daily for 2 days; Group IV received antibiotics injections over 6 days and were fed dead
Lactobacillus plantaris (2 × 10
8 CFU/ml) daily over 6 days; Group V received antibiotics injections with indole feeding daily over 2 days and antibiotics injection daily over 6 days. At the time of sacrifice, the ileum was harvested for Western Blotting, reactive oxygen species assessment, and bacterial killing activity. Phagocytic activity and tumor necrosis factor (TNF-α) production of AMs were measured in another set of animals.
Experiment 2
To investigate the effects of antibiotic treatment on PA-induced neutrophil infiltration and bacterial counts in the lung and mechanisms involved, combined antibiotics were administered to mice over 6 days. C57BL/6 mice were randomly divided into four groups as experiment 1. The mice received PA intra-tracheal instillations, and were sacrificed 8 h following the instillation. The lung tissue was harvested for PA culturing.
Experiment 3
Vil-Cre/IkkβF/Δ mice and IkkβF/Δ mice were used to investigate the involvement of IKK activity of the intestinal mucosa in PA-induced neutrophil infiltration in the lung. The mice were randomly divided into two groups: Group I received intramuscular injections of normal saline daily over 6 days (control group); Group II received intramuscular injections consisting of combined antibiotics daily over 6 days. The animals received PA intra-tracheal instillations and were sacrificed 8 h following the instillation. The lung tissue was harvested for PA culturing and myeloperioxidase activity assay.
Experiment 4
Vil-Cre/IkkβF/Δ mice and IkkβF/Δ mice were used to assess the involvement of IKKβ activity in the intestine and PA-induced neutrophil infiltration in the lung. The Vil-Cre/IkkβF/Δ mice were randomly divided into four groups as experiment 1. There were six replicates in each treatment group (n = 6). Peroxynitrite production, phagocytic activity of AMs, and plasma IL-6 levels were examined.
Tryptophan, dead Lactobacillus plantaris, and indole feeding
To investigate the effects of Ahr ligands on antibiotic treatment-induced intestinal dysbiosis and systemic defense impairment, tryptophan (i.e., trp; 2 mg in 200 μl water was administered orally by gavage the third and fourth days), indole (475 μg in 250 μl was administered orally by gavage on the third and fourth days), or dead
L. plantaris (CECT 5713, 2 × 10
8 CFU/ml in drinking water over 6 days) [
16] were administered the mice. The animals were given access to water via a bottle ad libitum. A fresh batch of water containing tryptophan, dead
L. plantaris, or indole was provided daily. The dead bacteria were completely suspended in the water where there was no precipitation of bacteria in the bottle. The control group was provided drinking water without the supplementation of trp, dead
L. plantaris, or indole.
Induction of P. aeruginosa pneumonia
Mice were anesthetized with ketamine hydrochloride (100 mg/kg intramuscularly, Veterinary Laboratories, Wyeth-Ayerst Canada Inc., Mississauga, ON, Canada) and xylazine (5 mg/kg intramuscularly, Bayer Inc., Mississauga, ON, Canada). The trachea was surgically exposed and 50 µl (1.0 × 107 CFU P. aeruginosa) were instilled via an angiocatheter through the trachea.
Bacterial counts of lung after exposure to bacteria
Animals were sacrificed by intra-peritoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg) 18 h following PA intra-tracheal injection. The bacterial clearance between the different treatments was compared. The whole lung was excised under sterile conditions and washed with 10 ml of sterile cold saline. The viable bacteria counts of homogenized lung and blood material were determined after an 18-h culturing at 37 °C on TSB–agar plates. Data were expressed as CFU/ml.
Polymerase chain reaction (PCR) and quantitative real-time PCR
Total RNAs were isolated from lung using total RNA Miniprep Purification Kits (GeneMark). Reverse transcription-generated cDNAs were amplified using PCR. Sets of ICAM and IL-6 primers were obtained from references [
17,
18], and one pair of primers for GAPDH gene as a control.
For the real-time PCR reactions, 200 ng of the cDNA template was added to 20 μl of mixture containing 12.5 μl of 2× KAPA SYBR® FAST qPCR Master Mix (Kapa Biosystems), 2.5 μl of each sense and anti-sense primers (25 μM) and 5 μl of sterile water. The amplification was performed in a StepOnePlus™ Real-Time PCR System (Applied Biosystems 7300).
Phagocytic activity of alveolar macrophages (AMs)
In the lung, alveolar macrophages (AMs) represent the first line of defense against pathogens such as
P. aeruginosa. In the presence of pathogens, the lung epithelial cells promote neutrophil sequestration which may cause injury to the lung as well as activate the immunological response [
19]. The AMs were collected and re-suspended in Hank’s Balanced Salt Solution (HBSS) (10
6 cells/ml). After 5 min of pre-incubation, the cell suspension was incubated with
P. aeruginosa (10
8 cells/ml) at 37 °C for 1 h with shaking. The culture was then centrifuged at 200×
g for 10 min. The pellet with the cells was removed and the
P. aeruginosa in the supernatant was counted [
20,
21].
Assessment of activity of AMs by ex vivo stimulation
Bronchoalveolar lavage (BAL) was conducted to harvest AMs from adult mice with Tris saline solution containing 0.25 mM EDTA and EGTA. Cells were re-suspended in RPMI 1640 medium in a final concentration of 1 × 10
5 cells/ml. Cells were then cultured in 96-well microtiter plates for 2 h and washed with RPMI 1640 to remove the nonadherent cells [
22]. The adherent monolayer of cells was stimulated with 5 µg/ml of lipopolysaccharide (LPS from
Escherichia coli O26:B6 Sigma-Aldrich) or RPMI 1640 for 4 h. The supernatants were collected and stored at − 70 °C until the TNF-α assay.
Peroxynitrite production of AMs (123-DHR oxidation assay of AMs)
Peroxynitrite is a potent macrophage-derived oxidizing cytotoxin that damages and kills invading pathogens. The collected AMs were adjusted to 3.0 × 10
6 ml
−1 in RPMI + 10% fetal bovine serum, and 100 μl of HBSS (without phenol red) containing 25 μM of 1,2,3-dihydrorhodamine (Invitrogen, Eugene, OR), a peroxynitrite-detecting dye was added. The cells were stimulated with 1 μg/ml of
E. coli LPS (Sigma). Peroxynitrite levels were measured using a fluorescence plate reader (Synergy HT Biotek, Winooski, VT) every 15 min for 75 min using excitation and emission wavelengths of 485 nm and 530 nm, respectively. The fluorescence due to auto-oxidation of 123-DHR was subtracted from the original measurements [
23].
Western immunoblots
The Ahr, RELMβ, and CRP-ductin were identified by mouse monoclonal antibodies (R&D Systems); the CRP-ductin were identified by mouse monoclonal, rabbit polyclonal and goat polyclonal antibodies, respectively (Santa Cruz Biotechnology Inc.).
Electrophoretic mobility shift assay for NF-κB
The intestinal mucosa was harvested by centrifugation and was used to prepare the nuclear extract as described previously [
24]. The consensus and control oligonucleotides (Santa Cruz Biotechnology Inc.) were labeled by polynucleotide kinase, the NF-κB consensus sequence was 5′AGTTGAGGGGAC-TTTCCCAGGC3′ (1.75 pmol/l). The samples were analyzed on a 4% polyacrylamide gel and the gel was dried and visualized by using autoradiography.
ROS levels in the intestinal mucosa
The levels of ROS in the intestinal mucosa were analyzed by 10 mM DCFDA fluorescent dye (Sigma), which was added into the suspension of intestinal mucosa for cultivation. DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2ʹ,7ʹ-dichlorofluorescein (DCF). DCF is detected by fluorescence spectroscopy with excitation and emission spectra of 495 nm and 529 nm, respectively [
25].
Enzyme-linked immunosorbent assay (ELISA)
The mouse ELISA kit (eBioscience) was used for IL-6 assay. The blood was centrifuged at 1000×g, 4 °C for 15 min and the serum was collected for use. The ELISA plates were coated with 100 μl capture antibody per well at 4 °C overnight. After appropriate wash, 200 μl of assay dilution buffer was added per well for blocking at room temperature for 1 h. The sample and serial dilutions of standards were added to the plate and incubated at 4 °C overnight. After coating with detection antibody, avidin-HRP was added and incubated at room temperature for 30 min. The substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was added and incubated for 15 min. Finally, 2 N H2SO4 was added to stop the reaction and absorbance at 450 nm was measured using an ELISA reader.
Neutrophil infiltration in the lungs
Lung myeloperoxidase (MPO) activity has been used as a marker of lung neutrophil infiltration [
26]. Lung tissues were weighed and homogenized in 50 mM potassium phosphate buffer (pH 6.0) with 0.5% hexadecyltrimethyl-ammonium bromide. Homogenates were centrifuged at 9500×
g, 4 °C for 10 min. An aliquot (60 μl) of supernatants was added to 939 μl of potassium phosphate buffer with 16.7 mg/ml of O-dianisidine and 0.5% hydrogen peroxide. The rate of change in absorbance at 460 nm was measured over 2 min. One unit of MPO activity is defined as the amount of enzyme that reduces 1 μmol of peroxide per min and the data were expressed as units per gram of lung tissue (Units/g tissue).
Statistical analysis
All data are analyzed by one-way analysis of variance or T-test analysis of variance (ANOVA), followed by Turkey’s Multiple Comparison Test. All values in the figures and text are expressed as mean ± standard error of the mean. P values of less than 0.05 are considered to be statistically significant.
Discussion
The widespread use of antibiotics increased the emergence of nosocomial infections caused by antibiotic-resistant Gram-negative bacteria in ICU patients, through undefined mechanisms [
2,
3]. Here we demonstrated that combined antibiotic treatments led to the impairment of the innate lung immune defenses against infection by
P. aeruginosa. There are multiple effects that account for this impairment. First, the antibiotic treatment increased the
P. aeruginosa pneumonia-induced bacterial counts in the lung tissue. Second, the antibiotic treatment induced the reduction of phagocytic activity, physiological activity, and peroxynitrite production of the AMs. Third, the antibiotic treatment increased ICAM and IL-6 expression in the lung. Altogether, our data suggest that antibiotic treatments reduces lung defense against pneumonia caused by PA through the reduction of phagocytic activity, physiological activity, and peroxynitrite production of the AMs. Our results highlight the possibility that broad-spectrum antibiotic treatment increases
P. aeruginosa pneumonia in critically ill patients by reducing lung immune defense mechanisms.
Recent breakthroughs in the understanding of the importance of the gut microbiome in both the maintenance of health and disease etiology have critical implications for respiratory and critical care medicine [
15]. Intestinal microbes not only contribute to the local host defenses against infections, but also modulate systemic immune responses (especially the lung) [
27]. Aryl hydrocarbon receptor (Ahr) regulates both innate and adaptive immune responses by detecting a variety of small synthetic and natural chemicals, which act as its ligands. Tryptophan metabolites feeding stimulates intestinal mucosa immunity via the aryl hydrocarbon receptors (Ahr) and IL-22 production [
10]. Our results suggest that Ahr ligand supplementation enhanced the lung immune defense mechanism by increasing intestinal ROS and peroxynitrite production of AMs. The Ahr ligands supplementation has three main effects. First, it reverses the antibiotic treatment-induced reduction in Ahr expression of the intestinal mucosa. Second, it increased the peroxynitrite production, physiological activity, and phagocytic activity of the AMs. Third, it increased the ROS production of the intestinal mucosa. Moreover, it also reversed the antibiotic-enhanced ICAM and IL-6 expression and PA pneumonia-induced bacterial counts of the lung. Finally, IKKβ depletion in the gut abolished the stimulatory effects of Ahr ligands and dead
L. plantarum feeding on Ahr expression of the intestine and peoxynitrite production of AMs. These results suggest that Ahr ligand supplementation reverses the antibiotic treatment-induced lung immune defense impairment through activating IKKβ/NF-κB in the intestine.
ROS levels in the gut have been suggested to be closely related to the intestinal barrier function [
28]. Our finding of decreased DCFDA levels in the intestinal mucosa in the antibiotic treatment only group suggests that antibiotic treatment decreased the ROS levels in the gut. Tryptophan, indole, and dead
L. plantarum feeding in mice receiving antibiotic treatment significantly increased the DCFDA levels in the intestinal mucosa. Superoxide and nitric oxide are essential components in the synthesis of microbicidal compounds (e.g., peroxynitrite), in macrophages [
23]. Our finding of decreased peroxynitrite production of AMs in the antibiotic only treatment group suggests that antibiotic treatment reduces phagocytic activity of AMs by decreasing the peroxynitrite production of the AMs. Taken altogether, antibiotic treatment impairs lung immune defense through decreasing ROS production in the gut mucosa and the peroxynitrite production of AMs, and through the subsequent reduction of the phagocytic activity of the AMs. Moreover, oral feeding with tryptophan, indole, and dead
L. plantarum in mice receiving antibiotic treatment significantly increased the peroxynitrite production and phagocytic activity of the AMs. These results indicate that the Ahr ligands enhance the lung immune defense mechanism by increasing the intestinal ROS production and peroxynitrite production of the AMs. Our study demonstrates clearly a gut–lung axis in the antibiotic model, and establishes a likely mechanism for pulmonary immunomodulation through the intestinal mucosa-mediated signaling pathways. The increasing ROS production of intestine, peroxynitrite production, activity, and phagocytic activity of the AMs brought on by Ahr ligands supplementation further provides support for the regulatory mechanism of the gut on the immune defense system in the lung.
We observed that antibiotic treatment significantly increased the plasma IL-6 levels as compared to that in the control group. Tryptophan, dead
L. plantarum, and indole feeding in mice receiving antibiotic treatment significantly decreased the IL-6 levels in the plasma. Previously, we have found that combined antibiotic treatment after thermal injury induced a substantial tenfold increase of IL-6 levels in the blood compared with the burn group. Oral supplementation with dead
E. coli or
S. aureus in the antibiotic treatment group significantly decreased IL-6 levels in the blood compared with thermal injury with antibiotic treatment group [
29]. These results suggest that IL-6 plays an important role in the regulatory mechanisms of gut on lung immunity. Antibiotics treatment increased plasma IL-6 levels in
IkkβF/Δ mice. However, antibiotics only, antibiotic with tryptophan, and antibiotic with dead
L. plantarum treatments did not change plasma IL-6 levels in
Vil-
Cre/IkkβF/Δ mice. These results indicate that the IKKβ of the intestine is critical in inducing plasma IL-6 levels during antibiotics treatment.
The involvements of the intestinal IKK activity/NF-κB activation on the lung defense and its related mechanisms have not been well characterized. We used
Vil-
Cre/IkkβF/Δ mice in antibiotic treatment experiment to examine the involvement of intestinal IKK activity/NF-κB activation in the lung defense mechanism. Previously, we have demonstrated that germ-free mice showed a significant decrease of NF-κB binding activity of intestinal mucosa [
13]. Our present data showed that antibiotic treatment significantly decreased binding activity of NF-κB and Ahr expression of the intestinal mucosa. Both results suggested that binding activity of NF-κB of the intestinal mucosa is closely related with microbiota in the intestine. IKKβ depletion in the gut abolished the stimulatory effects of Ahr ligands on Ahr expression of the intestine. These results suggest that IKKβ/NF-κB activation plays an important role in microbiota-induced Ahr expression of the intestinal mucosa.
Vil-
Cre/IkkβF/Δ mice demonstrated a significant increase in PA pneumonia-induced bacterial counts in the lung, and a significant decrease of the phagocytic activity of AMs compared to
IkkβF/Δ mice. Moreover, the antibiotics treatment significantly decreased the peroxynitrite production of the AMs in
IkkβF/Δ mice but not in
Vil-
Cre/IkkβF/Δ mice. These results indicate that the intestinal IKK activity/NF-κB activation of the intestine are crucial for the maintenance of peroxynitrite production, phagocytic activity of AMs, and lung defense against bacterial infection. Ahr can bind the p65 subunit of nuclear factor kappa light chain enhancer of activated β cells (NF-κB), thereby activating the expression of NF-κB [
30]. The antibiotics only, antibiotics with tryptophan, or antibiotics with dead
L. plantarum treatments did not change the protein expression of Ahr and RELMβ of the intestinal mucosa, or the peroxynitrite production of AMs in
Vil-
Cre/IkkβF/Δ. These results suggest that antibiotic treatment reduces Ahr expression of the intestine and subsequently the lung defense mechanism through the IKKβ of the intestine. Ahr ligands increased peroxynitrite production of the AMs and lung defense against
P. aeruginosa infection through IKKβ/NF-κB activation in the intestine. Altogether, we demonstrated that NF-κB activation in the intestine plays an important role in the lung immune defense mechanisms. Stimulation of IKKβ/NF-κB activation of the intestine with Ahr ligands could be a new therapeutic strategy in enhancing lung defense mechanisms in patients receiving antibiotic treatments.
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