The roles of the TLR/NF‑κB signaling pathway in the mutual interactions between the lung and the large intestine.

The 'exterior-interior relationship between the lung and the large intestine' is a classical basic theory in Traditional Chinese Medicine. The present study aimed to investigate the roles of the toll like receptor/nuclear factor‑κB (TLR/NF‑κB) signaling pathway in the mutual interactions between the lung and the large intestine. A rat model of allergic asthma complicated with intestinal flora disorder was established by oral administration of Candida albicans and intraperitoneal injection with ovalbumin. The number of inflammatory cells and expression levels immunoglobulin (Ig)E, secretory IgA, interleukin (IL)‑4 and interferon‑γ in serum and bronchoalveolar lavage fluid were subsequently measured. Bacterial colonies and expression of 16S ribosomal DNA were studied in feces samples and pathological alterations of lung tissues were identified. Furthermore, the expression levels of genes associated with the TLR/NF‑κB signaling pathway in the lung and intestinal tissues were determined by reverse transcription‑quantitative polymerase chain reaction. The results of the present study indicated that, in the rat model of allergic asthma complicated with intestinal flora disorder, the expression levels of IL‑4 and IgE, and the numbers of inflammatory cells and C. albicans increased, and marked inflammatory cell infiltration was observed in lung tissues, suggesting that the animal model was successfully established. Furthermore, the present results revealed the mRNA expression levels of genes associated with the TLR/NF‑κB signaling (including myeloid differentiation primary response 88, TNF receptor associated factor 6 and β‑arrestin) were upregulated in both of the lung and intestinal tissues of the model group rats. Collectively, the results demonstrated that the TLR/NF‑κB signaling may serve roles in the mutual interactions between the lung and the large intestine, and TLR and NF‑κB may be potential targets for the treatment of lung diseases complicated with intestinal disorders.

Introduction

In Traditional Chinese Medicine, the ‘exterior-interior relationship between the lung and the large intestine’ is a classical basic theory, which was first postulated in the Inner Canon of Huangdi ancient Chinese medical text (1,2). This theory serves as guidance for treatment of certain pulmonary diseases combined with disorders of the large intestine (3). In addition, an increasing amount of clinical evidence demonstrated that there is a relationship between the physiology and pathology of the lung and large intestine (4,5). However, at present, systemic reports regarding molecular mechanisms underlying mutual interactions between the lung and the large intestine are lacking.

Allergic asthma is a complex chronic airway inflammatory reaction mediated by mastocytes, eosinophils and T lymphocytes (6,7). The prevalence of asthma in industrialized countries has been increasing and asthma is now the most common chronic disease of children in the United States (8). It is believed that an additional 100 million people will be suffering with asthma by 2025 (9). Therefore, allergic asthma has become a public health issue. Increasing amount of research indicated that allergic asthma is closely associated with the intestinal flora disorder and is considered a typical disease model for investigating the mutual interactions between the lung and the large intestine (1,10). Therefore, in the present study, an animal model of allergic asthma complicated with intestinal flora disorder was established in rats to elucidate the molecular mechanism of mutual interactions between the lung and the large intestine.

Materials and methods

Animals

A total of 30 male Sprague-Dawley rats (3–4 weeks old, 200±20 g) were purchased from the Dashuo Laboratory Animal Co., Ltd. (Chengdu, China; http://www.jianyang.ccoo.cn/post/zhaopin/minqi/index462587.html). The animals were housed in a temperature and humidity controlled room (temperature 22±2°C, atmosphere 40–60% CO2, and 10–12-h light/dark cycle) with food and water ad libitum. All animal experimental protocols were approved by the Ethics Committee for Laboratory Animal Experimentation of Chengdu University of Traditional Chinese Medicine.

Chemicals and reagents

Cefoperazone was purchased from the North China Pharmaceutical Co., Ltd. (Shijiazhuang, China). Culture media (the selective culture media for Enterococcus, the selective culture media for enteric Bacilli, the BS culture media for Bifidobacterium, the selective culture media for Lactobacillus and the SDA culture media for Candida) for Enterococcus, enteric Bacilli, Bifidobacterium, Lactobacillus and Candida were purchased from the Qingdao Haibo Biotechnology Co., Ltd. (Qingdao, China). Ovalbumin (OVA) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Aluminum hydroxide was purchased from Chengdu Chron Chemicals Co., Ltd., (Chengdu, China). Live combined Bacillus subtilis and Enterococcus faecium granules (BEG) were purchased from the Hanmi Pharmaceutical Co., Ltd. (Beijing, China). Aminophylline (ANP) was purchased from the Southwest Pharmaceutical Co., Ltd. (Chongqing, China). Candida albicans was purchased from the Guangdong Huankai Microbial Technology Co., Ltd. (Guangzhou, China; http://huankaiye.bioon.com.cn/). TRIzol reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Rat ELISA kits for secretory(s) immunoglobulin (Ig) A (cat. no. 201411), IgE (cat. no. R141126-117a) interleukin (IL)-4 (cat. no. R141126-002a) and interferon (IFN)-γ (cat. no. R141126-101a) were purchased from the Neobioscience Technology Co., Ltd. (Shenzhen, China). HiScript 1st Strand cDNA Synthesis kit and SYBR-Green Master Mix were purchased from the Vazyme Biotech Co., Ltd. (Nanjing, China). Hematoxylin and eosin (H&E) and Wright-Giemsa kits were purchased from the Baso Biotech Co., Ltd. (Taiwan, China). All primers used in the present study were designed by Primer-Express version 3.0 (Thermo Fisher Scientific, Inc.) and synthetized by Sangon Biotech Co., Ltd. (Shanghai, China; Table I).

Table I.

Primers used in this research.

	Primer sequence (5′→3′)
Gene	Forward	Reverse
TLR-2	5′-GTTGCGTTACATCTTGGA-3′	5′-GGAATACACAGTGCTCAG-3′
TLR-4	5′-CAGCTCGTTTCTCACCCAGT-3′	5′-TGTATCGGTGGTCAGTGTGC-3′
MyD88	5′-CGACGCCTTCATCTGCTA-3′	5′-GCCGATAGTCTGTCTGTTCT-3′
TRAF6	5′-CAGTCCCCTGCACATTCAGT-3′	5′-CTGGGCCAACAGTCTCATGT-3′
β-arrestin	5′-GGGCATTTGTACTGAGCTGT-3′	5′-TGCACCTTGAGGCATCTCTG-3′
β-actin	5′-AGGGAAATCGTGCGTGACAT-3′	5′-GAACCGCTCATTGCCGATAG-3′

TLR, toll like receptor; MyD88, myeloid differentiation primary response 88; TRAF6, TNF receptor associated factor 6.

Experimental protocols and animal model preparation

A total of 30 rats were randomly divided into 3 groups (n=10): i) Normal group; ii) model group; and iii) positive treatment group (treated with live combined BEG and ANP). With the exception of the normal rats, all animals in the model and positive groups were freely administered orally Cefoperazone aqueous solution (0.5 g/l) for consecutive 5 days. Subsequently, rats of the model and positive treatment groups were orally administered 50 µl Candida albicans [109 colony forming unit (CFU)/ml] on the sixth day. OVA was used to induce allergic asthma in rats according to the previous methods (11,12) with minor modifications. Briefly, rats were immunized via intraperitoneal (IP) injection of 1 ml OVA-aluminum hydroxide mixture on days 0 and 7 (1 mg OVA and 200 mg aluminum hydroxide were dissolved in 1 ml saline). Subsequently, allergic asthma in rats was induced with 1% OVA-saline solution by aerosol inhalation in a glass box (10×10×20 cm) from day 14 to day 21 (30 min/day). Rats in the normal group were treated using the same protocol, with saline instead of OVA-aluminum hydroxide mixture. In the positive treatment group, rats were orally administered BEG (500 mg/kg) and ANP (300 mg/kg) from day 14 to day 21. On the day 21, the pulmonary functions were determined, then rats were sacrificed under pentobarbital sodium anesthesia (45 mg/kg; IP) after the blood samples and bronchoalveolar lavage fluid (BALF) were collected. Subsequently, the lung tissues, the large intestine tissues and intestinal mucous were harvested for the following biochemical assays.

Determination of the rat pulmonary function

Rat pulmonary function including respiratory rate and airway responsiveness were determined using a Buxco® animal pulmonary function analysis system (FinePointe Non-Invasive Airway Mechanics; DSI; Harvard Bioscience, Inc., Holliston, MA, USA; https://www.datasci.com/products/buxco-respiratory-products/finepointe-non-invasive-airway-mechanics). Airway responsiveness was evaluated using the enhanced pause values (Penh value) according to a previously reported method (13).

Blood cell counting

Blood smears were prepared with blood taken from the heart of the rats and fixed with formalin for 2–3 min at 25°C. Subsequently, Wright-Giemsa staining was performed for 15 min at 25°C and cell counting was carried out under an optical light microscope (CH20BIMF200; Olympus Corporation, Tokyo, Japan) at magnification, ×100 and ×400.

Bacterial colony counting

All experiments were carried out under sterile conditions. Briefly, 0.1 g rat feces were dissolved in saline at a dilution of 1:1010. Subsequently, the diluted feces samples were cultured in an anaerobic incubator for 48–72 h at 37°C for detection of Bifidobacterium, Lactobacillus and Candida albicans. Diluted feces samples were also cultured in an aerobic incubator for 24–48 h at 37°C for detection of Enterococcus and Enterobacterium. The colony counting was expressed as logCFU/g.

Examination of pathological alterations of the lung tissues

The histopathological examination was performed as previously described (14). Briefly, the lung tissues were collected and fixed with 4% paraformaldehyde for 24 h. The tissues were subsequently embedded in paraffin and cut into 5-µm-thick sections. The samples were deparaffinized and stained with H&E. Finally, pathological alterations of lung tissues were examined under an optical microscope (CH20BIMF200; Olympus Corporation).

ELISA assays for detection of sIgA, IgE, IL-4 and IFN-γ

The levels of sIgA in BALF and intestinal mucosa, and levels of IgE, IL-4 and IFN-γ in serum were determined using commercial ELISA kits according to the manufacturers' protocols and determined using a microplate reader at a wavelength of 450 nm (Multiskan Ascent 413MBY042078; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays

Lung and intestine tissues were collected and homogenized. Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, total RNA was used for cDNA synthesis using HiScript 1st Strand cDNA Synthesis kit, according to the manufacturer's protocol. qPCR was performed using SYBR-Green Master Mix, according to the manufacturer's protocol with primers specific to TRL-2, TRL-4, myeloid differentiation primary response 88 (MyD88), TRAF6, β-arrestin and β-actin (Table I). The thermocycling conditions for qPCR: Pre-denaturation at 95°C for 5 min and 95°C for 10 sec at 53.5°C for 30 sec, the data were recorded by fluorescent reading board, and 39 cycles were recorded. The PCR reactions were performed using CFX96TM™ Real-Time system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative mRNA expressions were determined by 2−ΔΔCq relative quantitative analysis according to the previous reported method (15).

Rat intestine 16S ribosomal DNA (16S rDNA) assay

For the determination of rat intestine 16S rDNA, 6 rats were selected from each group (n=6). Subsequently, the DNA was collected from the feces of rats. The DNA samples used in this study were isolated and purified using the QIAamp® DNA Stool isolation and purification kit (Qiagen China Co., Ltd., Shanghai, China) and the protocol is adopted following the manufacturer's protocol. Further analysis was performed using a DNA sequencer (Illumina HiSeq™ 2000; Illumina, Inc., San Diego, CA, USA) and BGI Tech Solutions Co., Ltd., (Shenzhen, China) analyzed and interpreted the sequencing data and the parameters were investigated as described previously (16,17).

Statistical analysis

Data are presented as the mean ± standard deviation, and each experiment was repeated at least 3 times. Differences between groups were determined using one-way analysis of variance followed by Dunnett's multiple comparisons test. The statistical significance of differences was analyzed using SPSS software (version 13.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Results of the determination of pulmonary function

The respiratory rate and Penh value were determined (Fig. 1). Compared with the normal rats, the respiratory rate (P<0.01) and Penh value (P<0.01) of the model rats increased significantly. However, the positive drug treatment significantly decreased the elevated respiratory rate (P<0.01) and Penh value (P<0.05) compared with the model rats. The present results revealed that a rat model of allergic asthma was successfully established.

Figure 1.

Pulmonary function determined by measuring the respiratory rate and Penh value. Data are presented as the mean ± standard deviation (n=10). *P<0.05 and **P<0.01 vs. the model rats; ##P<0.01 vs. the normal rats.

Results of cell counting in blood samples

The results of cell counting (Table II) indicated that the numbers of the four types of inflammatory cells (eosinophils, neutrophils, lymphocytes and monocytes) increased in the model rats compared with the normal rats (all P<0.01). By contrast, the positive treatment decreased the number of inflammatory cells compared with the model group (P<0.01, P<0.05, P<0.05 and P<0.01, respectively for eosinophils, neutrophils, lymphocytes and monocytes).

Table II.

Blood cell count (×106/l).

	Inflammatory cell type
Group	Eosinophil	Neutrophil	Lymphocyte	Monocyte
Normal	2.66±1.2	22.50±5.5	37.00±5.0	2.00±0.36
Model	27.75±2.19[c]	43.00±1.82[c]	66.80±3.21[c]	5.33±0.88[c]
Positive	4.66±0.55[b]	32.00±2.08[a]	50.40±4.93[a]	2.88±0.26[b]

Data are presented as the mean ± standard deviation (n=10).

P<0.05

P<0.01, vs. model rats

P<0.01, vs. normal rats.

Results of bacterial colony counting in rat feces

The number of colonies of three bacterial strains including Enterococcus, Bifidobacterium and Lactobacillus significantly decreased in model rats compared with normal rats (all P<0.01; Table III), whereas the numbers of colonies of enteric Bacilli (P<0.05) and Candida albicans (P<0.01) significantly increased. By contrast, in the positive drug treatment group, the number of Enterococcus (P<0.01), Bifidobacterium (P<0.01), enteric Bacilli (P<0.05) and Lactobacillus (P<0.01) colonies significantly increased, whereas the colony numbers of Candida albicans decreased (P<0.01), compared with the model rats. The above results indicated that the model rats exhibited significant alterations of the intestinal flora.

Table III.

Intestinal flora determination (log colony forming unit/g).

	Gut microorganism
Group	Enterococcus	Enteric bacilli	Bifidobacterium	Lactobacillus	Candida albicans
Normal	6.88±0.67	6.78±0.69	10.66±1.63	9.28±0.64	0.00±000
Model	4.70±0.46[c]	7.99±0.96[b]	0.00±0.00[c]	0.00±0.00[c]	7.30±0.50[c]
Positive	5.78±0.69[a]	6.78±0.67	1.68±0.59[a]	8.86±0.55[a]	3.04±0.59[a]

Data are presented as the mean ± standard deviation (n=10).

P<0.01, vs. the model rats

P<0.05

P<0.01 vs. normal rats.

Examination of pathological alterations of the lungs

Results of the pathological examination indicated that, in the normal group, no obvious pathological alterations were observed (Fig. 2). Compared with the normal rats, marked inflammatory cell infiltration was observed in the lung tissues of the model rats, and the pulmonary septum became thick and alveolar space became narrow (Fig. 2). In addition, edema could be also observed in the blood vessel and tracheal wall. However, in the positive group, the aforementioned abnormal alterations were markedly alleviated.

Figure 2.

Histological examination of lung tissues with hematoxylin and eosin staining (top row magnification, ×100; bottom row magnification, ×400). Black arrows indicate the blood vessel; red arrows indicate the inflammatory cell infiltration; yellow arrows indicate the pulmonary alveol; green arrows indicate the pulmonary septum; blue arrows indicate the bronchus.

Results of the ELISA assays

sIgA levels in both BALF (P<0.05; Fig. 3A) and intestinal mucosa (P<0.01; Fig. 3B) decreased in the model rats compared with the normal rats, and the serum IgE levels significantly increased (P<0.01; Fig. 3C). Following treatment with BEG and ANP, the sIgA levels in the intestinal mucosa (P<0.05) increased, whereas the serum IgE levels decreased (P<0.01), compared with the model rats.

Figure 3.

ELISA assays. (A) sIgA in bronchoalveolar lavage fluid. (B) sIgA in intestinal mucosa. (C) IgE in serum. Data are presented as the mean ± standard deviation (n=10); *P<0.05 and **P<0.01 vs. the model rats; #P<0.05 and ##P<0.01 vs. the normal rats. S, secretory; Ig, immunoglobulin.

Following activation with OVA, the IL-4 levels (P<0.01) of the model rats significantly increased compared with the normal group, whereas the IFN-γ levels significantly decreased (P<0.05; Fig. 4). By contrast, in the positive treatment group, the levels of IL-4 significantly decreased (P<0.01; Fig. 4A) and the levels of IFN-γ (P<0.05; Fig. 4B) significantly increased compared with the model rats.

Figure 4.

ELISA assays in BALF. Expression levels of (A) IL-4 and (B) IFN-γ in BALF. Data are presented as the mean ± standard deviation (n=10). *P<0.05 and **P<0.01 vs. the model rats; #P<0.05 and ##P<0.01 vs. the normal rats. BALF, bronchoalveolar lavage fluid; IL-4, interleukin 4; IFN-γ, interferon-γ.

Results of the 16S rDNA assays of the rat intestine

Following discarding the low-quality sequencing reads using an inner program (BGI Tech Solutions, Co., Ltd.) to generate clean data, a detailed result for each sample was obtained. The operational taxonomic units (OTUs) analysis revealed the OUT numbers of the model rats were lower than that of normal and positive-treated rats, indicating the species richness of the model rats was lower than that of the normal and positive-treated rats (Table IV). Furthermore, the number of bacteria constituting normal intestinal flora (including Bacteroidetes and Prevotella) decreased compared with the normal and positive groups (Fig. 5). By contrast, the number of Butyricimonas increased in the model group compared with the normal and positive rats. These results demonstrated that intestinal flora disorder was observed in the model rats. All of the above results indicated that a rat model of allergic asthma complicated with intestinal flora disorder was successfully established, and was subsequently used for the further investigation of the underlying molecular mechanisms.

Table IV.

Results of the OTU analysis of samples.

Group	Tag number	OTU number	OTU number (removing singletons)
Normal	14.751	828	369
Control	18.346	593	346
Positive	16.844	718	385

OTU, operational taxonomic unit.

Figure 5.

Composition of the intestinal flora in the normal, model and positive groups.

Results of the RT-qPCR assays

Following successful establishment of the rat model of allergic asthma complicated with intestinal flora disorder, the potential molecular mechanisms of mutual interactions between the lung and the large intestine were investigated using RT-qPCR assays. The mRNA expression levels of TRL-2, TRL-4, MyD88, TRAF6 and β-arrestin were significantly upregulated in both the lung and intestinal tissues of the model rats, compared with the normal rats (all P<0.01; Fig. 6). By contrast, treatment with BEG and ANP significantly decreased the upregulated mRNA expression levels of TRL-2, TRL-4, MyD88, TRAF6 and β-arrestin (all P<0.01; Fig. 6) in both the lung and intestinal tissues, compared with the model group.

Figure 6.

Results of reverse transcription-quantitative polymerase chain reaction analysis of expression levels of TLR-2, TLR-4, MyD88, TRAF6 and β-arrestin. Data are presented as the mean ± standard deviation (n=10). **P<0.01 vs. the model rats; ##P<0.01 vs. the normal rats. TLR, toll like receptor; MyD88, myeloid differentiation primary response 88; TRAF6, TNF receptor associated factor 6.

Discussion

In the present study, using a rat model of allergic asthma complicated with intestinal flora disorder, the roles of the TLR/NF-κB signaling pathway in the mutual interactions between the lung and the large intestine were studied. The results of the present investigation indicated that allergic asthma was associated with the intestinal flora disorder and the TLR/NF-κB signaling pathway may serve a role in this association.

Establishing a suitable and reliable animal model is the crucial initial step for investigating the mutual interactions between the lung and the large intestine (18,19). The present investigation successfully constructed a rat model of allergic asthma complicated with intestinal flora disorder. The results indicated that the rats in the model group exhibited obvious characteristics of allergic asthma and intestinal flora disorder, which were demonstrated by the pathological alterations, ELISA results (sIgA, IgE, IFN-γ and IL-4 levels), blood cell count and bacterial count in feces, as well as the 16S rDNA assay of the rat intestine. In the model rats, the levels of IL-4 and IgE, and the number of inflammatory cells and Candida albicans increased, and obvious inflammatory cell infiltration was observed in the lung tissues compared with the normal rats.

The sIgA is a marker for the intestinal flora disorder and damage of intestinal mucosa, and a previous report revealed that the number of intestinal Bifidobacteria is closely associated with the level of sIgA (20). In the model group the sIgA expression levels decreased in both of the intestinal mucosa and BALF, which supports the ‘exterior-interior relationship between the lung and the large intestine’ theory. TLRs, pattern recognition receptors expressed in the cytomembrane, are closely associated with the body immunocompetence (21). TLRs can recognize specific conserved molecular components of microorganisms and transfer the signals into the cell, leading to the activation of NF-κB (21,22). Furthermore, activation of NF-κB can induce the excessive release of pro-inflammatory cytokines including IL-1, IL-6, TNF-α and IL-12 (23). The characteristic pathological alterations associated with asthma include airway inflammatory reactions and airway remodeling (12). TLRs serve roles in the development of airway inflammatory reactions and further activate the MyD88- IRAK-TRAF6-IKK-NF-κB signaling pathway, resulting in excessive release of pro-inflammatory cytokines and inflammation (24–26). In addition, TLRs promote the maturation and differentiation of immune cells but also the transdifferentiation of CD4+ T cells into T regulatory cells (Tregs). Therefore, the TLR/NF-κB signaling pathway could further regulate the balance of type 1 T helper/type 2 T helper cells via Tregs (27). Recently, it has been reported that the TLR/NF-κB signaling serves roles in the intestinal defense against pathogens and maintenance of immune system homeostasis and intestinal flora balance (28). A previous study indicated that TLR-2 and TLR-4 are closely associated with the recognition of peptidoglycan and lipopolysaccharide (29), respectively. Additionally, TLR-2 and TLR-4 serve roles in the development of allergic asthma and intestinal flora-associated diseases (30,31). In addition, β-arrestin was upregulated in patients with allergic asthma and served a role in the development of chronic intestinal inflammation (32–34). Therefore, β-arrestin may serve a role in the development of allergic asthma and intestinal diseases. In the present study, mRNA expression levels of TRL-2, TRL-4, MyD88, TRAF6 and β-arrestin significantly increased in both the lung and intestinal tissues of the model rats compared with the normal rats. By contrast, treatment with BEG and ANP could decrease the up-regulated mRNA expression levels of these genes in both the lung and intestinal tissues of the model rats. The results if the present study indicated that the TLR/NF-κB signaling is a potential link between asthma and intestinal disorders in the rat models and may also be a molecular mechanism of the ‘exterior-interior relationship between the lung and the large intestine’. Furthermore the present study demonstrated, the upregulated TLR/NF-κB signaling is an important molecular mechanism for the development of lung diseases complicated with intestinal disorders.

‘Exterior-interior relationship between the lung and the large intestine’ is a classical basic theory in Traditional Chinese Medicine and the present study did not prove the direct mutual interaction between the lung and the large intestine. However, future studies should aim to investigate the direct mutual interactions between the two organs in animal models. The present study indicated that the TLR/NF-κB signaling may serve a role in the mutual interactions between the lung and the large intestine, however, alternative signal transduction mechanisms have not been investigated. Furthermore, the present study analyzed the alterations of intestinal flora by sequencing 16S rDNA, however, flora metagenomics analysis of both the lung and intestine would be an improved strategy for investigating the molecular mechanisms of the two organs the association between them.

In conclusion, the present experimental results revealed that the TLR/NF-κB signaling may serve roles in the mutual interactions between the lung and the large intestine. The results also support the Traditional Chinese Medicine theory of ‘exterior-interior relationship between the lung and the large intestine’. Furthermore, the results of the present study suggested that the TLR/NF-κB signaling is a potential target for the clinical treatment of lung diseases complicated intestinal disorders.