H5N1 Virus Hemagglutinin Inhibition of cAMP-Dependent CFTR via TLR4-Mediated Janus Tyrosine Kinase 3 Activation Exacerbates Lung Inflammation
The host tolerance mechanisms to avian influenza virus (H5N1) infection that limit tissue injury remain unknown. Emerging evidence indicates that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent Cl− channel, modulates airway inflammation. Janus tyrosine kinase (JAK) 3, a JAK family member that plays a central role in inflammatory responses, prominently contributes to the dysregulated innate immune response upon H5N1 attachment; therefore, this study aims to elucidate whether JAK3 activation induced by H5N1 hemagglutinin (HA) inhibits cAMP-dependent CFTR channels. We performed short-circuit current, immunohistochemistry and molecular analyses of the airway epithelium in Jak3+/+ and Jak3+/− mice. We demonstrate that H5N1 HA attachment inhibits cAMP-dependent CFTR Cl− channels via JAK3-mediated adenylyl cyclase (AC) suppression, which reduces cAMP production. This inhibition leads to increased nuclear factor-kappa B (NF-κB) signaling and inflammatory responses. H5N1 HA is detected by TLR4 expressed on respiratory epithelial cells, facilitating JAK3 activation. This activation induces the interaction between TLR4 and Gαi protein, which blocks ACs. Our findings provide novel insight into the pathogenesis of acute lung injury via the inhibition of cAMP-dependent CFTR channels, indicating that the administration of cAMP-elevating agents and targeting JAK3 may activate host tolerance to infection for the management of influenza virus-induced fatal pneumonia.
Increasing evidence indicates that superinflammation, a consequence of an exacerbated innate immune response, plays a critical role (1,2) in the rapid progression to adult respiratory distress syndrome (ARDS) following H5N1 infection; however, innate immune mechanisms protect the infected host by reducing the viral burden. Thus, an optimal immune response is characterized by a balance between efficient pathogen clearance and an acceptable level of immunopathology (3,4). Tolerance to infection has recently been found to constitute a distinct strategy of host defense that limits tissue damage, facilitating a higher magnitude and duration of the immune response (5). The elucidation of the inducible tolerance mechanism in response to H5N1 infection will provide new therapeutic strategies for severe lung damage mediated by viral attack by attaining an optimal immune response.
The mechanisms that typically maintain the homeostasis of various physiological systems are likely to contribute to host tolerance to infection (6,7). Cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic adenosine monophosphate (cAMP)-dependent Cl− channel expressed at the apical membrane of epithelial cells lining the tracheobronchial tree. CFTR channels secrete Cl− and mucus simultaneously with Ca2+ activated Cl− channels (CaCCs) (8) and regulate the amiloride-sensitive epithelial Na+ channel (ENaC), constituting the pathways that restrict Na+ and mucus absorption (9). Coordinating the regulation of the secretion and absorption of NaCl and fluids and airway surface liquid (ASL) homeostasis is crucial. Changing the salt and water composition of the periciliary fluid causes isotonic dehydration of the airway surface, leading to an impairment of mucociliary clearance. In the case of the ΔF508-CFTR mutation, constitutive nuclear factor-kappa B (NF-κB) activation results in IL-8-mediated chronic neutrophilic lung disease (10,11). Several investigators in the field believe that airway inflammation in CF is secondary to persistent bacterial infection, which results from impaired mucociliary clearance; however, recent evidence supports the hypothesis that the dysregulation of the inflammatory response is an intrinsic component of the CF phenotype and that airway inflammation may occur prior to or in the absence of bacterial infection. Moreover, numerous in vitro studies using human epithelial CF and control cell lines have confirmed that CFTR is an important inflammatory regulator and that CFTR mutations are associated with both the constitutive activation of proinflammatory signaling, especially NF-κB, in the absence of any apparent microbial stimulus and the exaggerated responses to bacterial products (12,13).
We previously demonstrated that exposure to hemagglutinin (HA), the surface glycoprotein of H5N1 that is indispensable for viral receptor binding, fusion, transmission, virulence and pathology (14, 15, 16), induced the activation of IFN-independent JAK/STAT and NF-κB in pulmonary epithelial cells. This activation was accompanied by the elevated secretion of chemokines/cytokines, including IP-10, IL-6, IL-8, MCP-1, MIP-1α, MIP-1β and RENTES. Furthermore, Janus tyrosine kinase (JAK) 3, a member of the JAK family of tyrosine kinases involved in cytokine receptor-mediated intracellular signal transduction, is a molecular determinant in the H5N1 HA-induced dysregulated innate immune response. The inhibition of JAK3 activation causes the negative regulation of NF-κB signaling (17); therefore, we speculated that H5N1 HA-stimulated NF-κB signaling may be, at least in part, attributed to the disturbance of epithelial CFTR inflammatory regulation, a host tolerance mechanism to infection, via a JAK3 activationdependent response. The present study reveals for the first time that H5N1 HA attachment to the airway epithelia stimulates the JAK3 signaling cascade, leading to the suppression of CFTR inflammatory regulation and exaggerating innate immune responses.
Materials and Methods
Preparation of the HA Protein
The recombinant HA protein from H5N1 (A/chicken/Guangdong/191/04, GenBank: AY737289) was generated as described previously (17).
B6129S4-Jak3tm1Lj (JAK3−/−) mice and wild-type B6129SF2/J (JAK3+/+) mice (6 to 8 wks of age) are all from the C57BL/6 mouse genetic background and were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). JAK3 heterozygous knockout (JAK3+/−) mice were generated by crossing JAK3−/− mice with JAK3+/+ mice. Then, the genotypes were identified via PCR. All mice were housed at a constant temperature (20°C) with a 12-h light-dark photoperiod and allowed food and water ad libitum. All procedures were performed in compliance with the National Institutes of Health-adopted Guide for Care and Use of Laboratory Animals (18) and were approved by the Bioethics Committee of State Key Laboratory of Respiratory Disease, Guangzhou Medical University. JAK3+/+ mice were intratracheally administered saline; HA (1 mg/kg) with or without pretreatment with either forskolin (10 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) or JAK3 inhibitor VI (0.15 mg/kg, Calbiochem, Darmstadt, Germany) by intraperitoneal (IP) injection; or HA (0.5 mg/kg) with or without pretreatment with glibenclamide IP injection (10 mg/kg, Sigma-Aldrich). JAK3+/− mice were administered HA at a dose of 1 mg/kg. Pulmonary histopathology and immunostaining were performed 12 h after the preceding treatment.
Cell Culture and Treatment
Cultured 16HBE and calu-3 cells were treated with saline, JAK3 inhibitor VI (760 nmol/L), TLR4 inhibitor (candesartan, 5 µmol/L, 3B Scientific Corporation, Wuhan, China), forskolin (10 µmol/L) or glibenclamide (500 µmol/L) for 30 min prior to HA stimulation and then subjected to different experiments. To test Gαi-mediated inhibition of AC, 16HBE cells were pretreated with IBMX (1 mmol/L, Sigma-Aldrich) and forskolin and either HA alone or HA in combination with IL-2 (100 UI/mL). In some experiments, cells were pretreated with pertussis toxin (100 ng/mL, Sigma-Aldrich) for 16 h or a TLR4 inhibitor or JAK3 inhibitor VI for 30 min.
CFTR-Dependent Short-Circuit Current Measurements
Tracheas of JAK3+/− or JAK3+/+ mice that had or had not received JAK3 inhibitor VI and TLR4 inhibitor (100 mg/kg) were pretreated with exposure of apical membranes to H5N1 HA, and then mounted in an Ussing chamber bathed in Krebs-Henseleit (K-H) solution to perform short-circuit current assay by using a VCC MC6 voltage-current clamp amplifier (VCC MC6, Physiologic Instrument, San Diego, CA, USA). The data were displayed on a signal collection and analysis system (Acquire & Analyze Rev II, San Diego, CA, USA) (19). Forskolin (10 µmol/L) was used to induce anion secretion via an increase in cAMP levels. Cl− free KH solution was employed to examine the effects on Cl− current. To inhibit electrically conductive Na+ transport, amiloride (100 µmol/L) was added in all studies.
Intracellular cAMP and Adenylyl Cyclase (AC) Assays
cAMP and AC levels were measured in cells using a cAMP assay kit (Assay Designs Inc., Ann Arbor, MI, USA) and an AC assay kit (Uscn Life Science Inc., Wuhan, China), respectively. The levels were corrected to the total protein levels, and the data were expressed as picomoles per milligram of protein.
Frozen lung tissue sections and cells cultured on glass coverslips were subjected to immunofluorescent staining for evaluation of CFTR expression or NF-κB/JAK3 activation using primary antibodies against CFTR (Abcam, Cambridge, MA, USA), phosphorylated NF-κBp65 (Cell Signaling Technology Inc., Beverly, MA, USA) and phosphorylated JAK3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively. The slides were then incubated with species-matched fluorescent secondary antibodies and DAPI (for nuclear staining). Images were captured using a Nikon C1 Si confocal system (Nikon Corporation, Tokyo, Japan).
Lung histology was performed as previously described (17).
Cytokine and chemokine production levels in supernatants were analyzed using a Luminex assay LiquidChip system (Panomics, Santa Clara, CA, USA) as previously described (17).
Western blotting for examination of JAK3 activity or CFTR expression was performed as previously described (17). The intensities of the relevant bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Quantitative Reverse-Transcription PCR (RT-PCR) Analysis
CFTR forward primer, 5′-TTAAAGCTGTCAAGCCGTGTTC-3′;
CFTR reverse primer, 5′-GCCAATGCAAGTCCTTCATCA-3′;
TLR4 forward primer, 5′-AGAACCTGGACCTGAGCTTTAATC-3′;
TLR4 reverse primer, 5′-GAGGTGGCTTAGGCTCTGATATG-3′;
β-actin forward primer, 5′-CCTGGCACCCAGCACAAT-3′;
β-actin reverse primer, 5′-GCTGATCCACATCTGCTGGAA-3′.
All of the experimental data shown are expressed as the means ± S.D. and were repeated at least three times, unless otherwise indicated. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Student t test, and p < 0.05 was considered to be significant.
All supplementary materials are available online at https://doi.org/www.molmed.org.
H5N1 HA-Induced Activation of JAK3 Inhibits cAMP-Dependent CFTR in the Airway Epithelium by Blocking AC
JAK3 Activation and the Suppression of cAMP-Dependent CFTR Expression Contribute to Acute Lung Injury Following H5N1 HA Challenge
Inhibition of cAMP-Dependent CFTR Is Mediated by JAK3 Activation and Enhances NF-κB Signaling
H5N1 HA Is Detected by TLR4 Expressed on Airway Epithelial Cells, which Facilitates JAK3 Activation
Immunofluorescence and Western blot analyses revealed that the HA-stimulated activation of JAK3 in bronchial epithelial cells was blocked by the addition of either a TLR4 or JAK3 inhibitor (Figures 6B, C). A 1.5-fold and 1.8-fold increase in TLR4 gene transcription was observed at 12 h and 24 h after HA exposure, respectively, and this increase was accompanied by a significant decrease in CFTR gene expression at 12 h (Figure 6D)
H5N1 HA Interaction with TLR4 Activates the Heterotrimeric Gαi/o Protein in Airway Epithelial Cells via JAK3 and Decreases cAMP Production
Our findings provide novel insight into the pathogenesis of H5N1-induced acute lung injury via the inhibition of airway epithelium cAMP-dependent CFTR Cl− channels, which is associated with host tolerance to infection. We report that H5N1 HA-triggered JAK3 activation inhibits AC, the enzyme exclusively responsible for cAMP production (23). This inhibition induces a defect in cAMP production, thereby blocking cAMP-dependent CFTR channels.
A reduction in barrier function due to the impairment of CFTR-mediated trafficking caused by H5N1 HA exposure may facilitate the entry of viral or bacterial toxins into the submucosa, increasing the susceptibility of the host to pathogen infection (24, 25, 26, 27).
Previous studies reported that an increase in cAMP due to the administration of forskolin or dibutyryl-cAMP reduced JAK3 expression, and this reduction resulted in impaired IL-2-dependent signal transduction and inhibition of T-cell activation (28). Consistently, targeting to JAK3 by the administration of JAK3 inhibitor VI alleviated acute lung injury (17). Current evidence provides strong support for the hypothesis that JAK3 activation via H5N1 HA attachment, which is associated with reduced cAMP production and suppression of cAMP-dependent CFTR Cl− channels in the airway epithelium, leads to an enhanced inflammatory reaction.
Recent observational data reported in an investigation of primary lower airway epithelial cells (AECs) in children with CF suggest a possible nexus between human rhinovirus (HRV) infection and AEC-initiated inflammatory cell recruitment and activation, thus leading to early airway inflammation (29). In the present study, we demonstrated an exaggerated inflammatory response in the lung tissue of HA-challenged wild-type mice following pretreatment with a CFTR channel inhibitor. These results are consistent with previous studies suggesting slower viral clearance, increased severity of infection and a prolonged inflammatory response to respiratory viral infection in CF (30,31).
Accumulating evidence has indicated that alterations in intracellular Cl− concentrations play an important role in a variety of physiological and pathological processes (32, 33, 34). Yang et al. recently reported that a decrease in the intracellular Cl− concentration promotes endothelial cell inflammation by activating the NF-κB pathway (35). Consistent with this result, our findings show that the attenuation of the CFTR Cl− channel by glibenclamide exacerbated H5N1 HA-induced acute lung injury, which was associated with enhanced NF-κB translocation. These data indicate that a reduction in CFTR channel-dependent Cl− transport underlies, at least in part, the HA-induced superinflammation. By contrast, in the presence of forskolin, which is commonly used to open CFTR channels by increasing cAMP levels, or a JAK3 inhibitor, bronchial epithelial calu-3 cells display reduced activation of NF-κB p65 induced by HA challenge. Therefore, the CFTR Cl− channel may play an essential role in the regulation of the innate immune inflammatory response by dynamically modulating Cl− movement across the plasma membrane.
NF-κB plays a crucial role in inducing the expression of a plethora of inflammatory and immune mediators; thus, NF-κB is one of the master regulators of the immune response and a key target for antiinflammatory drug design. A number of fundamental molecular mechanisms that contribute to the overall inhibitory action of cAMP on NF-κB function are well established (36). In the presence of forskolin and a JAK3 inhibitor, both wild-type mice and bronchial epithelial cells counteract NF-κB activation and inflammatory damage in response to HA challenge, indicating that interactions between cAMP and the NF-κB signaling cascade modulate the outcome of inflammation-associated NF-κB activation, which are modulated by JAK3 expression.
The activity of AC is modulated by G-protein subunits. Specifically, Gαs stimulates AC, inducing it to catalyze the formation of cAMP from ATP, whereas the Gαi protein exerts an inhibitory effect on AC. Recent studies have shown that TLR ligands activate Gai proteins in endothelial cells (22,37). The authors speculated that TLR2, 3 and 4 also interact directly with Gαi via their intracellular domains due to a consensus motif for Gαi/o binding. Consistently, our results indicate that cytoplasmic JAK3 activation evokes the interaction between TLR4 and Gai via their intracellular binding domains, leading to the inhibition of AC and reducing cAMP production. JAK3 activation, which is after activation of TLR4 by H5N1 HA, results in impaired cAMP-dependent CFTR regulation and CFTR gene expression, contributing to the exacerbation of immune inflammation. Therefore, we suggest that airway epithelial cAMP-dependent CFTR regulation represents an important host tolerance mechanism for limiting tissue damage to prevent severe immunopathology and that cAMP levels and CFTR function are closely modulated by JAK3 activity, which exerts an inhibitory effect on AC by activating Gai G-protein subunits. Hence, cAMP-modulating therapeutic strategies potentially hold promise for treating ARDS induced by avian influenza virus infection; however, further investigation is required.
In conclusion, we suggest that the inhibition of cAMP-dependent CFTR channels by TLR4-stimulated JAK3 activation, which blocks AC-mediated cAMP production, disturbs the host tolerance to H5N1 infection, thereby enabling the exacerbation of innate immune inflammatory responses. Increasing the intracellular cAMP levels via the administration of cAMP-elevating agents and overcoming the attenuation of cAMP production by selective JAK3 inhibition (JAK3 inhibitor VI) may represent an effective therapeutic strategy for the management of influenza virus-induced severe pneumonia.
The authors declare that they have no competing interests as defined by Molecular Medicine or other interests that might be perceived to influence the results and discussion reported in this paper.
The authors would like to thank Wenliang Zhou (School of Life Science, Sun Yat-sen University, Guangzhou, China) for help with experiments. This work was supported by grants from the National Key Basic Research Program of China (973 Program; 2009CB522104), the National Natural Science Foundation of China (30900576), the China Postdoctoral Science Foundation (20100480734), the Yangcheng Scholars Research Program of Guangzhou Municipal Universities (10A024G) and the Program for Tackling Key Problems in Science and Technology of the Local Government in Guangzhou (2014), China.
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