Acute exacerbation of chronic obstructive pulmonary disease was associated with respiratory syncytial virus infection and the upregulation of TLR3

Respiratory syncytial virus (RSV) infection is known as a risk factor for chronic obstructive pulmonary disease (COPD). RSV infection induces the upregulation of Toll-like receptor 3 (TLR3). This study aimed to investigate the association of TLR3 with RSV induced acute exacerbations of chronic obstructive pulmonary disease (AECOPD). Serum/sputum samples from AECOPD patients, stable chronic obstructive pulmonary disease (SCOPD) patients, and healthy controls were collected. Nested PCR was used to detect RSV. The lung function parameters were assessed by blood gas and lung function analysis. The expression levels of inflammatory factors in sputum and serum samples were determined by enzyme-linked immunosorbent assay. BEAS-2B cell lines were infected with RSV, and the expression of TLR3 mRNA was determined by PCR and the levels of inflammatory factors were also investigated. The presence of RSV was detected in 3 SCOPD and 8 AECOPD patients, but not in healthy patients. The expression levels of TNF-α and IRF-3 in both sputum and serum samples of RSV-positive group were significantly higher than in RSV-negative group. TLR3 mRNA levels in RSV-positive group were significantly higher than those in RSVnegative group. Interestingly, the level of TLR3 mRNA expression was negatively correlated with oxygenation index and lung function parameters. Furthermore, BEAS-2B cells infected with RSV led to significant increase of the expression of TLR3 mRNA and inflammatory factors IFN-β, IL-13, IL-32, and TNF-α. Our observations indicate that AECOPD is associated with RSV infection and the upregulation of TLR3.


Introduction
Chronic obstructive pulmonary disease (COPD) is a common chronic disease with high mortality and morbidity (Ford et al., 2015). The incidence of COPD in Chinese over 40 years old was as high as 13.7% (Wang et al., 2018). Although the diagnosis and treatment of COPD have significantly improved, the survival of patients remains poor due to COPD-induced irreversible lung dysfunction (Lange et al., 2015). Airway remodeling is a typical pathological feature of COPD, and neutrophils are the main effector cells. Lymphocytes are the primary cells implicated in airway inflammation, and the percentages of CD8 + cells significantly increase (Polosukhin et al., 2017). Protease causes the destruction of elastin, the main component of connective tissues of lung parenchyma, leading to tissue damage and airway remodeling in COPD (Chotirmall et al., 2015). Harmful substances stimulate the vagus nerve and cause the release of acetylcholine, leading to excessive secretion of airway mucus, smooth muscle contraction, and airway spasm (Mirza et al., 2018;Morélot-Panzini et al., 2016). Various predisposing factors have been identified for COPD, such as respiratory syncytial virus (RSV) (Clark et al., 2014;Coultas et al., 2019). RSV is an independent risk factor for COPD patients in Intensive Care Unit (ICU) (Friedman et al., 2017;Marçôa et al., 2018;Mehta et al., 2013).
Toll-like receptors (TLRs) are key components of innate and adaptive immune system, and have been implicated in tumor progression, inflammation, and autoimmune diseases (Alexander et al., 2016). TLR3 and TLR4 recruit the adaptor protein TRIF through MyD88 independent pathway to activate interferon regulatory factor 3 (IRF-3) and induce the production of interferon-I (IFN-I), causing the secretion of interferon-β (INF-β) (Alexander et al., 2016). As a result, the expression of IRF3, a TLR3 downstream signaling factor, increases and it activates nuclear factor κB (NF-κB) (Brownell et al., 2014;Chen et al., 2015). NF-κB activation causes the upregulation of inflammatory interleukins and tumor necrosis factor-α (TNF-α) (Tsubaki et al., 2015). The excessive release of inflammatory factors can cause secondary immune injury (Hong et al., 2020). Therefore, this study aimed to investigate the association of TLR3 with RSV induced acute exacerbations of chronic obstructive pulmonary disease (AECOPD).

Patients
This study was approved by the Ethics Committee of the Third Affiliated Hospital of Zunyi Medical University (Approval No. 20354). A total of 90 participants were enrolled, including 30 healthy controls and 60 COPD patients. The patients were hospitalized in the Department of Respiratory Medicine of the First People's Hospital of Zunyi City from October 2016 to March 2017. The inclusion and exclusion criteria were given below: Inclusion criteria a) According to GOLD COPD classification and grouping guidelines (Lee et al., 2013), we selected those participants who, after inhaling short-acting bronchodilators, had forced expiratory volume in one second (FEV 1 ) to forced vital capacity (FVC) ratio of less than 70%, percentage of predicted FEV 1 of less than 80, and the reversibility of airflow limitation of less than 15% of the baseline value and/or <200 mL. b) AECOPD group (30 cases): had any two symptoms out of exacerbated dyspnea, increased sputum and increased sputum volume, and the symptoms persisted for more than 2 days, or had any one of the above symptoms accompanied with one of these symptoms: wheezing, cough, sore throat and common cold. c) Stable chronic obstructive pulmonary disease (SCOPD) group (30 cases): were asymptomatic and not in the acute phase for more than 30 days and did not take any treatment (inhalation or oral medication) for the 14 days before enrollment. d) Healthy group (30 cases): had no COPD, pneumonia, bronchial asthma, bronchiectasis, pulmonary tuberculosis, or any other respiratory disease, nor had any serious disease of the heart, liver, kidney, and blood, nor any systemic disease. e) Exclusion criteria: patients with respiratory diseases other than COPD such as bronchial asthma, bronchiectasis, and pneumonia. Patients with tuberculosis or any serious heart, liver, kidney, blood, and systemic disease were also excluded.

Data collection
The age, gender, lung function parameters (FEV 1 , FVC, FEV 1 /FVC), arterial blood gas analysis PH value (hydrogen ion concentration), oxygenation index, carbon dioxide partial pressure, white blood cells, and neutrophils ratio of subjects were recorded. Sputum samples were collected within 24 h of admission; sputum from participants showing little or no sputum was collected by induced sputum excretion (Tables 1 and 2).

Cell culture and infection
Human bronchial epithelial cell line (BEAS-2B) (Cat # CRL-9609) and RSV standard strain (Long) (Cat #ATCC VR-26) were purchased from American Type Culture Collection, RSV Long strain was first isolated by Robert Chanock from a child with bronchopneumonia in 1956 and was subsequently passaged 11 to 13 times in HEp-2 cells. It is the "first" prototypic strain of RSV (Pandya et al., 2019). BEAS-2B cells were cultured in DMEM/F12 medium (Hyclone, USA) supplemented with 100 U/mL penicillin and 25 mg/mL gentamicin and incubated at 37°C with 5% CO 2 . BEAS-2B cells were infected with RSV at multiplicity of infection (MOI) of 1, 0.3, 0.1, and 0.01 (Liu et al., 2018). Prime Script RT reagent kits (Cat # RR0370) and One-Step TB Green PrimeScript RT-PCR Kit (Cat # RR086B) were obtained from TaKaRa (Dalian, China). PCR primers were provided by TaKaRa (the sequences were listed in Table 3).

Statistical analysis
The data were expressed as mean ± standard deviation (SD). T-test was used for the data between the two groups with normal distribution, and rank sum test was used for the data between the two groups without normal distribution. Variance analysis was used for comparison among multiple groups. Pearson correlation analysis was used, and Pearson correlation was expressed by r. Categorical variables were reported as frequency and percentage. Data were processed and analyzed using Microsoft Excel 2010 and SPSS statistical software (version 19.0, SPSS Inc., Chicago, Illinois), and P < 0.05 was considered as statistically significant.

Detection of RSV in clinical samples
Since RSV is an independent risk factor for COPD patients (Friedman et al., 2017;Marçôa et al., 2018;Mehta et al., 2013), we detected RSV. The participants included in our study were categorized into 3 groups (N = 30): healthy control group, AECOPD group, and SCOPD group. RSV was not detected in healthy group, while RSV was detected in 3 SCOPD patients (3/30) and 8 AECOPD patients (8/30).

The levels of TLR3 and inflammatory factors in clinical samples
The expression of TLR3 mRNA in the sputum of COPD patients increased, and the levels in the SCOPD group were higher than those in healthy group. TLR3 mRNA levels in the AECOPD group were significantly higher than the corresponding levels in the SCOPD group. Furthermore, RSV positive group showed significantly higher TLR3 mRNA levels than RSV negative group (Fig. 1A). TLR3  mRNA expression levels in blood were similar to those in the sputum (Fig. 1B). Pearson correlation analysis showed that increased expression of TLR3 mRNA in the sputum of RSVpositive patients was negatively correlated with FEV1 (Fig. 1C, r = −0.8281) and oxygenation index (Fig. 1D, r = −0.8032) (All *P < 0.05).

Discussion
In present study, sputum samples from 90 patients were tested for RSV by nested PCR. RSV was not detected in 30 control subjects; RSV was detected in 11 of 60 COPD patients (18.3%). This percentage was lower compared to previous study (Anderson et al., 2016), and the variation may be due to the difference in regional characteristics. Among the 30  SCOPD patients, only three were RSV-positive (10%), while eight out of 30 AECOPD patients were RSV-positive (26.6%). The main cause of AECOPD attacks may be the presence of latent and colonized RSV in the airway of COPD patients. TLR3 plays a regulatory role by recognizing doublestranded RNA (dsRNA) produced after viral infection (Wang et al., 2016). The dsRNA is a replication intermediate of most RNA viruses such as RSV, and it is able to induce the upregulation of TLR3 expression (Liu et al., 2018). In this study we performed PCR and found that TLR3 mRNA levels in the sputum of COPD patients were significantly higher than the levels in the sputum of the normal group, while TLR3 mRNA levels of AECOPD group patients were significantly higher than those of SCOPD group. Meanwhile, the expression levels of TLR3 mRNA in RSV-positive group were significantly higher than the levels in RSV-negative group. The expression levels of TLR3 mRNA in blood showed a similar trend. The expression of TLR3 mRNA in the sputum samples of RSV positive patients showed a negative correlation with FEV1 (r = 0.8281) and oxygenation index (r = 0.8032). A recent study based on TLR3-deficient mice reported that viral infection led to the upregulation of TLR3 in monocyte-derived dendritic cells, which would drive type 2 immune response to cause chronic inflammatory diseases of the lung such as COPD (Wang et al., 2021). These results suggest that TLR3 is required to mediate RSV induced COPD and its expression level is related to the severity of COPD.
Next, we used ELISA to detect the expression levels of IFN-β, IRF3, TNF-α, IL-13, and IL-32 in the blood, and found that COPD group had higher expression levels of IFN-β, IRF3, TNF-α, IL-13, and IL-32 than the control group. The expression levels of IRF3, TNF-α, IL-13, and IL-32 in RSV-positive group were significantly higher than the levels in RSV-negative group (P < 0.05). Previous studies have shown that the expression levels of inflammatory factors CCL3 and CCL5 in the bronchoalveolar lavage fluid were significantly increased in RSV-infected mouse models (Salimi et al., 2017). Our study found that with the increase in RSV infection time and virus titer, the expression of inflammatory factors IFN-β, TNF-α, IL-13, and IL-32 significantly increased. These observations suggest that in COPD patients, especially those with RSV infection, increased levels of inflammatory factors cause a series of inflammatory reactions and inducing immune injury (Foronjy et al., 2014;Na et al., 2020). In addition, TLR3 mediates the production of various inflammatory factors, inducing a series of inflammatory responses and aggravating the immune injury to the lungs. A recent study showed that TLR3 inhibitor could reduce mRNA and protein expression of various inflammatory cytokines and attenuate cigarette smoke induced airway inflammation (Jiang et al., 2021). Therefore, we speculate that RSV induced upregulation of TLR3 may contribute to airway inflammation and remodeling in COPD.
In summary, our results suggest that inflammatory response induced by RSV is related to the upregulation of TLR3, and it may cause secondary lung immune injury in AECOPD patients. The levels of inflammatory factors in serum and sputum can indicate the severity of COPD and are helpful for treatment evaluation and prognosis judgment. Availability of Data and Materials: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.