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.

with a 12-h light/dark photoperiod and allowed food and water ad libitum. All procedures were carried out in compliance with the National Institutes of Health-adopted Guide for Care and Use of Laboratory Animals (3) and were approved by the Bioethics Committee of State Key Laboratory of Respiratory Disease, Guangzhou Medical University. Mice were randomly divided into 8 groups as indicated in the figure legends (n = 5 in each). After anaesthetised with pentobarbital sodium (50 mg/kg), mice were intratracheally inoculated with HA (1 mg/kg) or HA (0.5 mg/kg) in the presence or absence of pretreatment with JAK3 inhibitor VI (JAK3inh, 0.15mg/kg, Calbiochem, Darmstadt, Germany), forskorlin(FSK,10mg/kg, Sigma-Aldrich, St Louis, MO, USA) and gliebenclamide (Gli,10mg/kg, Sigma-Aldrich) respectively by intraperitoneal injection. The control group received an equal volume of PBS. Lung tissues were harvested at 12 h after HA inoculation.

Measurements of CFTR-Dependent Short-Circuit Current
The tracheas of Jak3 +/and Jak3 +/+ mice that had or had not received JAK3 inhibitor VI (0.15mg/kg) and TLR4 inhibitor (TLR4inh, candesartan, 100mg/kg, 3B Scientific Corporation, Wuhan, China) respectively by intraperitoneal injection, were removed, fixed on a sample clamp with exposure of apical membranes (ex-posed surface area of 0.04 cm 2 ) to HA or saline for ~10-20 min, and then mounted into an Ussing chamber bathed in both sides with Krebs-Henseleit (K-H) at 37°C. The transepithelial PD were clamped at 0 mV, then the short circuit current was recorded with VCC MC6 voltage-current clamp amplifier (VCC MC6, Physiologic Instruments, San Diego, USA), and simultaneously displayed via a signal collection and analysis system (Acquire & Analyze Rev II, San Diego, USA). Forskolin (10 μM apical and basal), an adenylate cyclase activator known to activate CFTR, was used to induce anion secretion via an increase in cAMP. To inhibit electrically conductive Na + transport, amiloride (100 μM) was added to the apical solution in all studies.The K-H solution contained (in mM): 117 NaCl, 4.7 KCl, 1.2 KH 2 PO 4 , 1.2 MgSO 4 , 24.8 NaHCO 3 , 2.56 CaCl 2 and 11.1 glucose. The solution was bubbled with 95% O 2 /5% CO 2 to maintain the pH at 7.4 (4). In the Cl --free perfusion solution, chloride was substituted by gluconate.

Cell Culture and Treatment
16HBE and calu-3 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% FCS/FBS (Gibco, NY, USA), 5 mg/ml penicillin and 10 mg/ml streptomycin. Prior to treatment, cells were cultured for 12 h in tissue culture-treated plates, and fresh culture medium was added to the cells

H5N1 Virus Hemagglutinin Inhibition of cAMP-Dependent CFTR via TLR4-Mediated Janus Tyrosine Kinase 3 Activation Exacerbates Lung Inflammation
Ke Cao, * Minhui Chen, * Xiang Jie, Yansheng Wang, Qiasheng Li, and Jun Xu Online address: http://www.molmed.org 1 h before the treatments. Prior to HA stimulation, the cells were treated without or with JAK3 inhibitor VI (760 nM), TLR4 inhibitor (candesartan, 5μM, 3B Scientific Corporation, Wuhan, China), forskolin (10 μM, Sigma-Aldrich) and glibemcalmide (500 uM, Sigma-Aldrich) for 30 min. The supernatants of the cell culture and the cells were subjected to different experiments.To test Gαi-mediated inhibition of AC, 16HBE cells were pretreated with IBMX(1 mM Sigma-Aldrich) for 15 min and then treated with forskolin for 30 min prior to HA stimulation for 30 min. In some instances, cells were pretreated with pertussis toxin (PTx, 100 ng/ml) for 16 h or a TLR4 inhibitor or JAK3 inhibitor VI for 30 min. Cells were lysed and intracellular cAMP levels were assayed.

Intracellular cAMP and Adenylyl Cyclase (AC) Assay
cAMP and AC levels were measured in cells using a cAMP assay kit (Assay Designs, Inc., Ann Arbor, 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.

Lung Histology
Lungs isolated from mice were fixed in buffered 4% paraformaldehyde (pH 7.4) for 36 h and embedded in paraffin. Tissue sections (5μm) were stained respectively with haematoxylin and eosin (H&E) for morphological analysis. Lung injury was scored as described previously (1). Two investigators blinded to the group assignments analysed the samples and determined the level of lung injury according to the semiquantitative scoring outlined below. All lung fields were examined for each sample at 20× magnification. The assessment of histological lung injury was performed as follows: 0, normal; 1, 25% the lung section exhibits interstitial congestion and inflammatory cell infiltration; 2, 25-50% the lung section exhibits interstitial congestion and inflammatory cell infiltra-tion; 3, 50-75% the lung section exhibits consolidation and inflammatory cell infiltration. Scores were expressed as mean ± SD and Kruskal-Wallis tests were used to compare the pathological damage score of the groups.

Luminex Assay
The quantification of multiple cytokines/ chemokines in the supernatants of cell culture was performed using the Luminex assay LiquidChip system (Panomics, CA, USA) according to the manufacturer's instructions as previously described (1), which is a beadbased system for immunoassays that allows for the simultaneous assaying of multiple analytes in a single sample (5). The cytokines/chemokines included ICAM-1, IL-18, IP-10, MCP-1, macrophage inflammatory protein 3 alpha (MIP-3α), and MMP-7.

Statistical Analysis
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.