Background
Viral respiratory tract infections by RNA viruses (e.g., influenza A virus [IAV], rhinovirus, coronavirus, and respiratory syncytial virus) are among the most common illnesses worldwide, and their severity widely varies from the common cold to severe respiratory tract infections [
1‐
3]. For instance, IAV causes seasonal respiratory infection, leading to half a million deaths annually. Only a few clinically effective vaccines or specific antiviral drugs are available for the prevention and treatment of viral respiratory infections. Thus, the rational mechanisms by which viruses are removed from the respiratory tract are indispensable for achieving effective viral clearance and airway host defense. Viral respiratory infections are also major causes of the acute exacerbation of chronic airway diseases, such as asthma and chronic obstructive pulmonary disease [
4,
5].
The airway epithelium acts as a frontline defense against foreign substances, including viruses, bacteria, and environmental air pollution, serving both as a physical barrier and a regulator of innate and adaptive immune responses [
6‐
9]. Mucociliary clearance is an important defense mechanism in the respiratory tract that requires coordinated ciliary activity and proper mucus production to propel airway surface liquids that traps pathogens and pollutants, permitting their clearance from the lungs [
10,
11]. Ciliated cells, with a large number of cilia on the luminal side, represent the major cell type of the airway epithelium, and they play a central role in mucociliary clearance through the coordination and modulation of ciliary beating [
10‐
12]. Ciliary beat frequency (CBF) is a major determinant of mucociliary clearance. To properly generate cilia-driven flow, the vigorous asymmetric beating of the cilia, which can be represented as effective and recovery strokes, are also required [
13‐
15]. The importance of ciliary activity and mucociliary clearance in airway host defense is well documented in several diseases, such as primary ciliary dyskinesia and cystic fibrosis, in which impaired ciliary activity and mucociliary clearance predispose patients to recurrent airway infection [
16,
17].
Previous studies revealed that extracellular components have the potential to enhance ciliary activity in the airway epithelium. Ciliated cells express bitter taste receptors. Bitter compounds (e.g., denatonium, thujone) stimulate these receptors and increase CBF in human airway epithelial cells, making ciliated cells chemosensory organelles [
12]. The proinflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β, upregulate ciliary motility in a nitric oxide-dependent manner [
18]. Arginine vasopressin, an antidiuretic hormone, rapidly increased CBF in rabbit tracheal epithelium by increasing intracellular Ca
2+ levels [
19]. In addition, CBF can be stimulated by several drugs used to treat pulmonary diseases, such as β-adrenergic agonists [
20] and macrolide antibiotics [
21].
The impact of respiratory virus infection on airway ciliary activity and mucociliary clearance remains to be elucidated. During respiratory viral infection, the interaction between viruses and airway epithelial cells through pattern-recognition receptors (PRRs) on the cell surface, such as Toll-like receptors (TLRs) [
8,
22], results in the production of antiviral substances, including types I and III interferon (IFN), β-defensin, cytokines, and chemokines [
23‐
28], which inhibit viral replication and mediate adaptive immunity responses. Recently, our group reported that IAV and polyI:C, a ligand of TLR3, increased the expression of IFN-λ and proinflammatory cytokines and chemokines (e.g., G-CSF, IL-8, IL-17C, CXCL1, CXCL5) in normal human bronchial epithelial cells [
26‐
29], indicating that IAV-mediated TLR3 signaling plays pivotal roles in initial antiviral responses in the airway. However, no report has elucidated the relationships between TLR3 signaling and ciliary activity during viral infection, which is critical given that ciliary activity is a major determinant of the ability of mucociliary clearance to eliminate noxious viruses from the respiratory tract.
To more efficiently activate airway host defense mechanisms during respiratory viral infection, ciliary activity and mucociliary clearance should be activated in the airway. Given that airway epithelial cells produce antiviral substances and cytokines via virus-mediated TLR3 activation to promote protective immune responses, it is possible that viral infection exerts significant effects on ciliary activity and mucociliary clearance via TLR3 activation in the airway epithelium. In the present study, we investigated the effects of short-term IAV infection on ciliary activity and transport using an organ culture of murine tracheal tissue, an in vitro IAV infection model, and imaging techniques to analyze ciliary activity and cilia-driven flow. We also examined the roles of TLR3 activation in ciliary activity and cilia-driven flow during IAV infection to elucidate the regulatory mechanisms of IAV-mediated ciliary activation using tracheal epithelium from TLR3-knockout (KO) mice.
Methods
Mice
BALB/c mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). TLR3-KO BALB/c mice were purchased from Oriental Bioservice, Inc. (Kyoto, Japan). All experiments involving mice followed protocols approved by the Animal Care and Use Committees of Hamamatsu University School of Medicine (license number H29-064) and were performed in accordance with the relevant guidelines and regulations.
Isolation of murine trachea and organ culture of tracheal tissue
Tracheal samples were taken from 9 to 11-week-old female wild-type (WT) and TLR3-KO BALB/c mice and placed in cold collection medium solution (DMEM with sodium pyruvate solution) kept in an ice bath. Excess fat and connective tissue debris were immediately removed from the trachea using forceps, and subsequently, the membranous portion of the trachea was excised to expose the ciliated epithelium in the trachea. After the tracheal sample was treated, organ culture of murine trachea was performed in 2 mL of culture medium in the presence or absence of IAV, TLR ligand, and/or reagents in 35-mm culture dishes at 37 °C for 5–120 min. After organ culture of tracheal tissue, tracheal epithelium was observed and analyzed at room temperature (23–28 °C) using a dedicated microscope.
Analysis of cilia-driven flow
Ciliary transport on the surface of intact trachea was analyzed after transient organ culture. To visualize cilia-driven flow, tracheal tissue was placed in a 35-mm culture dish with the luminal face down in 2 mL of culture medium containing 0.2-μm-diameter polystyrene beads (0.2-μm red fluorescent beads: Thermo Fisher Scientific, Waltham, MA, USA). Methylcellulose (M0512, Sigma-Aldrich, St. Louis, MO, USA) was added to the culture medium at a concentration of 0.5% to stabilize the movement of fluorescent beads by increasing the viscosity of the medium. The movement of beads under the tracheal epithelium was observed from the bottom of the dish using an inverted fluorescence microscope (Eclipse TE2000-U, Nikon, Tokyo, Japan) equipped with a CFI Plan Fluor objective lens (Nikon) and a CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). The optimal configuration of the lens for tracking the movement of the beads, which were 0.5–2 mm from the chamber bottom, was × 20 magnification, an NA of 0.5, and a long working distance (2.1 mm). The velocity of each bead was calculated by dividing the width of the field of view by the time each individual bead took to travel across the field. Three or more independent tracheal samples and at least 10 fields of view in each sample were analyzed (more than 30 fields for each condition, 5 or more beads in each fields). The fluid movement velocity was measured from the migration distance and the time of travel for the beads by tracking individual fluorescent beads using Aquacosmos image analysis software (Hamamatsu Photonics). The rainbow trace was depicted with a macro provided for free software, ImageJ, from Hiratsuka laboratory of JAIST (
https://www.jaist.ac.jp/ms/labs/hiratsuka/).
Analysis of the ciliary beating orientation
The analysis was performed as described previously [
13,
14] with slight modifications. After the tracheal tissue was transiently cultured in culture medium, the cilia tips of ciliated cells were labeled with Indian ink diluted with culture medium (1:100) to analyze intact cilia. The motility of ink-labeled cilia tips was recorded using HAS-L1 and HAS-U1 high-speed cameras (DITECT Co. Ltd, Tokyo, Japan) at 300 fps to reflect ciliary motility. The recording was performed at 23–28 °C. CBF was determined by subjecting the original traces to fast Fourier transform using Excel (Microsoft). The amplitude of ciliary beating, effective stroke velocity, recovery stroke velocity, and the effective stroke velocity/recovery stroke velocity ratio were calculated from the movement of the cilia tips. CBF data were presented as the median (range). The amplitude of ciliary beating, effective stroke velocity, recovery stroke velocity, and effective stroke velocity/recovery stroke velocity ratio were presented as the mean ± SEM. Three independent tracheal samples and at least 10 ink-labeled cilia in each sample were analyzed (n = more than 30 ink-labeled cilia for each condition). Kymographs of ciliary beating were depicted with a macro embedded in ImageJ.
Adenosine triphosphate (ATP) measurements
ATP concentrations were measured in culture supernatants using an ATP assay kit based on luminometric techniques (Lucifell 250 plus, Kikkoman Biochemifa, Tokyo, Japan) according to the manufacturer’s protocol. In total, 100 µL of culture medium from tracheal tissue with or without polyI:C were used. Briefly, 100 µL of the ATP extraction reagent were added to each sample, and after 20 s, luciferin-luciferase (100 µL) was added to each sample. The luminescence of each sample was measured using Lumitester C-100 (Kikkoman Biochemifa).
PolyI:C and suramin
PolyI:C and suramin were purchased from Sigma-Aldrich and used in this study at concentrations of 100 µg/mL and 100 µM, respectively.
IAV infection
IAV strain A/Yokohama/110/2009 (H3N2) was provided by Dr. Kawakami (Yokohama City Inst. of Health, Japan). The median tissue culture infectious dose of the virus stock solution was 6 × 105. The virus stock solution was diluted with DMEM containing sodium pyruvate solution up to 100-fold. Tracheal tissue taken from WT and TLR3-KO mice was infected with 2 mL of IAV solution for 1 h, and cilia-driven flow and ciliary beating orientation were analyzed. For inactivation of IAV, IAV in 20 µL medium was treated either with UV irradiation (254 nm; HL-2000 HybriLinker, Upland, CA) for 30 min.
RNA isolation and real-time polymerase chain reaction (PCR)
After murine tracheal tissues were cultured with/without IAV for 1 h or 24 h, total RNA of the tracheae was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). For quantification of murine β-actin mRNA, reverse transcription (RT)-PCR was performed on 50 ng total RNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, Rockford, IL, USA). Quantitative real-time PCR was conducted using the THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) on the CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) according to manufacturer instruction. For quantification of IAV RNA, the one-step RT- quantitative real-time PCR method was performed in a 10 μL volume containing 1 μL of TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific), 0.7 μL of forward primer IAV MP-39-67For (10 pmol/μL), 0.7 μL of reverse primer IAV MP-183-153Rev (10 pmol/μL), 0.5 μL of TaqMan probe IAV MP-96-75Probe (1 pmol/μL), 2 μL total RNA (20 ng), and 5.1 μL RNase-free water on the CFX Connect Real-Time System (Bio-Rad). Cycling conditions were as follows: reverse transcription at 50 °C for 5 min and 95 °C for 2 min, followed by 45 cycles of PCR at 95 °C for 15 s and 60 °C for 60 s. The IAV RNA levels were normalized to the levels of murine β-actin mRNA. The results were presented as 2−(Ct of IAV RNA − Ct of β-actin) in arbitrary units. The sequences of primers and probe used in real-time RT-qPCR analysis were as follows:
murine β-actin For 5′-AGTGTGACGTTGACATCCGT-3′
murine β-actin Rev 5′-TGCTAGGAGCCAGAGCAGTA-3′
IAV MP-39–67 For 5′-CCMAGGTCGAAACGTAYGTTCTCTCTATC-3′
IAV MP-183–153 Rev 5′-TGACAGRATYGGTCTTGTCTTTAGCCAYTCCA-3′
IAV MP-96–75 Probe 5′-[FAM]-ATYTCGGCTTTGAGGGGGCCTG-[BHQ1]-3′
Statistical analysis
Data are presented as the mean ± SEM, unless otherwise noted. Comparisons between groups were performed using Student’s t-test. p ≤ 0.05 was considered statistically significant.
Discussion
In the present study, we investigated the impact of respiratory virus infection on mucociliary clearance, with a particular focus on ciliary activity and cilia-driven flow, using organ culture models of murine trachea and in vitro IAV infection models. This study provides evidence that IAV infection in the airway readily stimulates ciliary activity and ciliary transport via TLR3 activation, which promote mucociliary clearance to hasten the elimination of viruses from the respiratory tract, highlighting their important roles as initial airway defense responses.
To the best of our knowledge, this is the first report to reveal that IAV directly promotes cilia-driven flow and ciliary activity in the airway epithelium. During respiratory viral infections, airway epithelial cells play central roles in airway host defenses by producing antiviral substances through the recognition of virus nucleic acid by PRRs, including TLR3 [
8,
24‐
28]. However, the direct effects of viral infection on ciliary activity in the airway epithelium had not been studied previously. Our results illustrated that IAV infection immediately increased cilia-driven flow and ciliary activity in the airway epithelium. To minimize airway epithelium impairment during viral infection, invading viruses should be eliminated from the respiratory tract as soon as possible. Thus, it is reasonable that airway epithelial cells have the ability to promote ciliary activity immediately in response to virus invasion, which hastens the elimination of the virus from the airway as an initial host defense response. A prior study using in vivo models of IAV infection in the chinchilla Eustachian tube demonstrated that IAV infection induced a decrease of CBF in the Eustachian tube epithelium 7–14 days after infection via the impairment of cilia and ciliated cells [
33]; however, they recorded no data regarding changes of ciliary activity within 1 day after IAV infection. Moreover, no study reported the direct effects of short-term IAV infection on the ciliary activity of airway epithelial cells. The methodology used in this study, organ cultures of murine trachea with short-term IAV infection and imaging techniques to analyze ciliary motion and cilia-driven flow, enabled us to investigate the impact of short-term IAV infection on airway ciliary activity. Moreover, these findings should spur future research to clarify whether promotion of ciliary activity can be targeted using drugs to hasten to elimination of the virus from the airway.
The present study also revealed the potential roles of TLR3 signaling as a regulator of ciliary activity in the airway epithelium. The regulatory effects of TLR signaling activation on ciliary activity and mucociliary clearance in the respiratory tract had not been clarified. In the present study, we mainly focused on TLR3 using IAV infection models and polyI:C stimulation, since we previously observed that TLR3 signaling played pivotal roles in initial antiviral responses to induce the expression of IFN-λ and proinflammatory cytokines and chemokines (e.g., G-CSF, IL-8, IL-17C, CXCL1, CXCL5) in normal human bronchial epithelial cells [
26‐
29]. Our results in this study revealed that TLR3 activation increased ciliary activity and cilia-driven flow in the airway epithelium. The involvement of TLR3 signaling in IAV- and polyI:C-mediated increases of ciliary activity was confirmed using TLR3-KO tracheal culture. Given that TLR signaling plays a vital role in the initiation of host defense responses in the airway epithelium, it is understandable that TLR signaling increases ciliary activity and cilia-driven flow. A former study using an experimental murine model of IAV infection demonstrated that TLR3-KO mice had a higher IAV amount in their lung as compared to WT mice [
34], suggesting that TLR3 signaling was involved in viral clearance. In addition, Our findings provide insight into the novel functions of TLR3 signaling as a regulator of mucociliary clearance, a primary defense mechanism for protecting the airways against harmful viral and bacterial pathogens.
Few studies have examined the effects of TLRs, excluding TLR3, on ciliary function in airway epithelial cells. Alpizar et al. reported that lipopolysaccharides (LPS), which are ligands of TLR4, increased CBF in mouse tracheobronchial epithelial cells by activating transient receptor potential vanilloid 4 (TRPV4) cation channels in a process that was independent of the TLR4 signaling pathway [
35]. They also mentioned that LPS-induced TRPV4 activation occurred more rapidly than activation of the canonical TLR4 immune pathway. In the present study, we first found that IAV- or polyI:C-mediated increases of ciliary activity were dependent on TLR3 signaling. In addition, 1 h of IAV exposure or 30 min of polyI:C stimulation were sufficient to increase CBF and cilia-driven flow, confirming that this TLR3-mediated ciliary activation occurred more rapidly than the TLR3-mediated upregulation of antiviral components (e.g., IFN-λ, IFN-β, IL-8, IL-17C, G-CSF) reported in our previous studies [
26‐
28]. Moreover, TLR3 activation readily provoked extracellular ATP release, which resulted in subsequent increases in ciliary function via the ATP-P2R pathway. Taken together, TLR3 expressed in airway epithelial cells rapidly recognizes respiratory viruses and mediates antiviral host defense responses through a variety of mechanisms (e.g., ATP release, increases of ciliary activity and gene expression) to suppress viral invasion and replication.
It has been reported that airway epithelial cells can release exocytic ATP under cellular deformation and mechanical stimulations [
36‐
39], which in turn increased ciliary activity via the autocrine activation of P2Rs, including P2Y receptors. Contrarily, only a few studies have mentioned the activation of TLR3-triggered ATP release in epithelial cells [
40,
41]. It also remains unclear whether TLR3-mediated ATP release is involved in the activation of ciliary function in airway epithelial cells. Our present study demonstrated that TLR3 activation in the tracheal epithelium increased the extracellular ATP concentration, accompanied by increases of cilia-driven flow and CBF in WT cultures but not in TLR3 KO cultures. In addition, blockade of P2R signaling using suramin suppressed polyI:C-mediated increases of cilia-driven flow and CBF, confirming the important role of TLR3-mediated ATP release in the dynamic regulation of airway ciliary function. It is hard to rule out the possibility that multiple pathways may play roles in TLR3-mediated increase of cilia-driven flow and CBF; however, extracellular ATP release and autocrine ATP-P2R loop may be major mechanisms, since blockade of ATP-P2R binding by suramin suppressed polyI:C-promoted cilia-driven flow and CBF to the control levels.
The intracellular mechanisms underlying extracellular ATP release induced by TLR3 activation in the airway epithelium have not been fully elucidated. Pannexins and connexins are structural components of gap junctions that form hemichannels, which permit the passage of ion and other small molecules including ATP, between the intra- and extracellular compartments [
42‐
44]. In the airway epithelium, extracellular ATP can be released by a conductive mechanism mediated by pannexins and connexins [
39,
45,
46], which results in increased CBF via P2R activation. Recently, calcium homeostasis modulator 1 (CALHM1), a transmembrane protein that shares structural features with connexins and pannexins, is known to participate in ATP release and the modulation of ciliary activity following mechanical stimulation in airway epithelial cells [
47]. These transmembrane proteins (e.g., pannexins, connexins, CALHM1) may be involved in the extracellular ATP release driven by TLR3 activation, which will be of great interest in future studies. Further research is warranted to elucidate the detailed molecular mechanisms of TLR3-mediated ATP release in airway epithelial cells.
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