2.1 Animals

α7 nAChR deficient mice (C57BL/6J, B6.129S7-Chrna7tm1Bay) were from the Jackson Laboratory and housed in a pathogen-free condition. α7 nAChR floxed mice were generated in our lab with the method shown in Supporting Information S1: Figure S1. Spc-Cre⁺ mice were kindly provided as a gift from Prof. Kaifeng Xu (Chinese Academy of Medical Sciences). The surfactant protein C (SPC) is exclusively expressed in the type II AECs.¹⁹ Wildtype mice in C57BL/6J background were purchased from the Model Animal Research Center of Nanjing University. Anesthesia was induced with an intraperitoneal (i.p.) injection of a mixture of Pentobarbital sodium (50 mg/kg). The Committees on Animal Research of the Institut Pasteur of Shanghai, Chinese Academy of Sciences approved the protocols.

2.2 Unilateral vagotomy

As previously described,²⁰ right or sham cervical vagotomy was performed with the animals under anesthesia. The procedure involved a longitudinal midline incision in the ventral region of the neck. Using blunt dissection, the overlying muscles and fascia were separated until the right vagus and carotid artery were visible. The vagus was carefully stripped away from the carotid artery and lightly cut off in the vagotomy group. The vagus was kept intact in the sham group. The wound was closed and sutured. The respiration rhythm was not affected by unilateral vagotomy.

2.3 Influenza viral propagation

The viruses (PR8, mouse adapted A/Puerto Rico/8/1934(H1N1), kindly provided by Dr. Ertl HC, Wistar Institute) were propagated in 10-day-old chicken embryo from specific-pathogen-free flocks (Beijing Merial). Each egg was injected with 0.1 mL of phosphate-buffered saline (PBS) containing ~10³ infectious particles, incubated at 37°C with forced air circulation and egg rotation, and maintained at 4°C for 12 h before harvesting. The allantoic fluid was harvested separately from each egg. Those with high hemagglutinin activity were pooled, and aliquots were prepared and stored at −80°C. The hemagglutinin titer of the above subtype of influenza virus was measured with 1% chicken blood cells in a V-shaped plate. After quantification, the viruses were stored at −80°C for future use. H3N2 strain (kindly provided by Dr. Ertl HC) was also prepared in the same way.

2.4 Cell culture

Human lung carcinoma cell A549, human embryonic kidney 293T cell (HEK293T), and Madin–Darby Canine Kidney (MDCK) cell were purchased from ATCC and maintained in minimal essential medium (DMEM, Invitrogen/Ambion/Gibco/MP). All cell cultures were supplemented with 10% fetal calf serum (fetal bovine serum [FBS] qualified Australia origin, Invitrogen/Ambion/Gibco/MP), 100 U penicillin, and 100 μg streptomycin per milliliter (Invitrogen/Ambion/Gibco/MP). The cells were incubated at 37°C and 5% carbon dioxide.

2.5 Cell viral infection

For influenza virus infection, 5 × 10⁵ low-passage A549 or HEK293T cells were seeded in designated plates. At 24 h post seeding, cells were washed with PBS and infected with PR8 at a multiplicity of infection (MOI) of 0.2 in DMEM containing 0.2% BSA and 1 μg/mL TPCK for the indicated time in a 5% CO₂ incubator at 37°C. For mock infection, the same amount of allantoic fluid from noninoculated 10-day-old chicken embryos was used. The samples were collected at 24 h post infection (hpi).

2.6 Foci forming assay for determining viral load

For the cell culture study, the supernatant of PR8-infected cell culture media was collected and then plated in MDCK monolayer. After incubation, blocking, and immune staining, the plaques were quantified. The viral titer was expressed as log10-transformed Foci Forming Units (FFU) per mL. For determining lung viral load, mice were killed 6 days post infection (dpi), and the lungs were removed and homogenized in PBS containing 0.1% BSA. The supernatant was plated on confluent MDCK cells followed by the standard procedure of plaque assay. The viral titer was also expressed as FFU per mL with log10 transformation.

2.7 Animal infections and lung histology

Some 8–10 weeks male C57BL/6J and α7 nAChR deficient mice were used in the infection experiments. Mice were anesthetized and then given either 20 µL of PR8 (1.4 × 10⁵ FFU) or PBS intranasally. Live virus experiments were conducted in Biosafety Level II facilities obeying governmental and institutional guidelines. After mice were anesthetized and killed with 80 mg/Kg pentobarbital sodium. The lungs were collected and fixed in 4% paraformaldehyde overnight at room temperature. The tissues were embedded in paraffin; 4 μm sections were cut by a Leica manual microtome (RM2125 RTS) and stained with hematoxylin and eosin. Lung injury score was graded independently in a double-blinded manner as we described previously.²¹

2.8 Flow cytometric analysis

As described,²² harvested lungs were inflated with 3 mL of a mixture of collagenase (150 units/mL of liberase Cl, Sigma/flu/Ald) and DNaseI (10 µg/mL, Sigma/flu/Ald) in RPMI-1640 containing 5% FBS and 20 mM HEPES. Lungs were chopped in the 3 mL enzyme mix and incubated for 35 min at 37°C. In total, 10 mM ethylenediaminetetraacetic acid (EDTA) was added and any remaining pieces were further dispersed by 12 passages through a 21 G needle. Suspensions were passed through a 100 µm nylon mesh, and cells were washed multiple times in RPMI-1640 with 5% FBS and 20 mM HEPES. red blood cell lysis was performed on cell preparations for cellular analysis by flow cytometry but not on preparations for flowcytometry. Unspecific staining was minimized through preincubation for 15 min with antimouse CD16/32. The antibodies for flow cytometry analysis were CF633 α-Bungarotoxin (Biotium, Cat#00009), mouse anti-CD326 (Ep-CAM), APC antibody (Biolegend, Cat#118213), anti-ProSPC antibody (Merck/Millipore, Cat# AB3786), and anti-Influenza A Virus Nucleoprotein (FITC) antibody (Abcam, Cat# ab20921). Fluorescent cells were analyzed after the exclusion of debris and aggregates with LSRFortessa (BD Biosciences). Data were analyzed by Flowjo 7.6 (Tree Star Inc.).

2.9 Chemicals and kits

The following chemicals and kits were used: MG-132 (SIGMA, Cat# 474790), GTS-21 (Abcam, Cat# ab120560), Collagenase (SIGMA, Cat# C9891-100MG), DNaseI (SIGMA, Cat# D4527), PTP1B activity Assay Kit (SIGMA, Cat# 539736), Pierce ECL Western blot analysis Substrate (Pierce, Cat# 32109), BCA Protein Assay Kit (Pierce, Cat# 23227), Trizol Reagent (Invitrogen/Ambion/Gibco/MP, Cat# 15596026), ACK Lysing Buffer (Invitrogen/Ambion/Gibco/MP, Cat# A1049201), Protease Inhibitor Cocktail (100X) (Cell Signaling Technology, Cat# 5871 S), Phosphatase inhibitor cocktail (Santa Cruz, Cat# SC-45044), Cytofix/cytoperm with Golgi plug kit (BD Pharmingen, Cat# 555028), Fixation and Permeabilization Solution (BD Pharmingen, Cat# 554722), and Perm Wash Buffer (BD Pharmingen, Cat# 554723).

2.10 Western blot analysis and immunoprecipitation

Cells were scraped in lysis buffer (20 mM Tris-HCl, pH 7.6, 2.5 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% sodium deoxycholate, 10% glycerol, 1 mM Na₃VO₄, 50 mM NaF, 1 mug/mL of aprotinin, 1 mg/mL of leupeptin, and 1 mM phenylmethyl sulfonyl fluoride), were sonicated for 10 s, and were centrifuged at 4°C for 20 min at 14 000g. Lysates preabsorbed to 20 μL protein A–protein G (Invitrogen) were incubated overnight with the appropriate antibodies and were immunoprecipitated with 40 μL protein A–protein G. Immunoprecipitates were recovered by centrifugation, were washed in ice–cold wash buffer (0.1% Triton X-100 and 1 mM phenylmethyl sulfonyl fluoride in Tris-buffered saline), and were “taken up” in sample buffer (125 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 0.5 mg/mL of bromophenol blue). Denatured proteins were loaded and run on a 10% Bis-Tris gel (Invitrogen). The proteins were then transferred to a polyvinylidene difluoride membrane and incubated with primary antibodies and secondary antibodies. Images were analyzed using Tanon Gel Image System 4200 (Shanghai Tanon Technology Co., Ltd.). The primary antibodies used for Western blot were anti-Influenza A Virus NP antibody (Abcam, Cat# ab128193), anti-Influenza A Virus PB2 antibody (Pierce, Cat# PA532220), anti-α7 nAChR antibody (H302) (Santa Cruz, sc-5544), anti-GAPDH antibody (EMAR, Cat# EM32010-02), anti-ASK1 antibody (Cell Signaling Technology, Cat# 8662), anti-p-p38MAPK (Thr 180/Tyr 182) antibody (Cell Signaling Technology, Cat# 9211 S), anti-p38MAPK antibody (Cell Signaling Technology, Cat# 54470), anti-β-actin antibody (EMAR, Cat# EM32011-02), anti-Influenza NS1 antibody (Santa Cruz, Cat# sc-17596), anti-NEDD4L antibody (Santa Cruz, Cat# sc-514954), anti-p-Tyr antibody (Santa Cruz, Cat# sc-7020), anti-PTP1B antibody (Cell Signaling Technology, Cat# 5311), anti-p-PTP1B Ser378 antibody (Abcam, Cat# ab76239), anti-Myc antibody (Santa Cruz, Cat# sc-40), anti-Lamin B1 (Proteintech, Cat# 66095-1-Ig), anti-HA antibody (Santa Cruz, Cat# sc-7392), anti-CRM1 antibody (Cell Signaling Technology, Cat# 46249), and anti-Flag antibody (Sigma, Cat# F1804). The secondary antibodies were mouse anti-rabbit-HRP (Cell Signaling Technology, Cat#5127), goat polyclonal anti-rabbit-HRP (Cell Signaling Technology, Cat#7074), and goat polyclonal anti-mouse-HRP (Cell Signaling Technology Cat#7076).

2.11 Quantitative real-time polymerase chain reaction (PCR)

Total ribonucleic acid (RNA) from lungs or cultured cells was homogenized and extracted using Trizol (Invitrogen), as described by the manufacturer. The total RNA obtained was suspended in RNase-free water and stocked at −80°C. Real-time polymerase chain reaction (PCR) was performed on an ABI PRISM Step-One sequence-detection system by using SYBR Green PCR Master Mix (Applied Biosystems) after a reverse transcription reaction of 2 µg of RNA by using M-MLV reverse transcriptase (TIANGEN). The relative expression levels of corresponding genes were determined by the 2^-ΔΔCT method,²³ normalized by ribosomal subunit 18 S or GAPDH. All human and mouse primers have been summarized in Supporting Information S1: Table 1.

2.12 Bulk RNA sequencing and analysis

Total RNA was extracted from A549 cells mock-infected or infected with/without GTS-21 (50 μM) and IAV at 24 hpi using TRIzol reagent (Invitrogen) based on the manufacturer's instructions. The RNA was treated with DNase I for 30 min at 37°C to remove residual deoxyribonucleic acid (DNA). mRNA was purified with oligo (dT) beads. Then, the purified mRNA was fragmented into small pieces with fragment buffer (provided by BGI) at the appropriate temperature. The first-strand and second-strand cDNA were generated. Afterward, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments were further amplified by PCR, and the products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The amplified PCR products from the previous step were denatured and circularized by the splint oligo sequence to get the library. The single-strand circle DNA was formatted as the final library. The final library was amplified to make DNA nanoball (DNB), which had more than 300 copies of one molecular. DNBs were loaded into the patterned nanoarray, and single end 50 bases reads were generated on BGIseq500 platform (BGI). The gene expression levels were calculated in fragments per kilobase transcriptome per million mapped reads. The data were analyzed by R package DESeq2 R (1.30.0) and fgsea.

2.13 Plasmid constructs and transfection

FLAG-tagged NEDD4L expression plasmids were provided by Xin Ye (Institute of Microbiology, Chinese Academy of Sciences). Recombinant vectors encoding NEDD4L, PTPN1, MAPK14, and mutant MAPK14 were constructed by PCR-based amplification of cDNA from the human A549 cell line, then were subcloned into the PLKO eukaryotic expression vector (Invitrogen) through T4 DNA ligase. Transient transfection of HEK293T cells with indicated plasmids was performed routinely with polyethylenimine (PEI).

2.14 Lentivirus construction and infection

For the knockdown assay, short hairpin RNA (shRNA) sequences, synthesized by Nanjing Genewis Biotechnology, were inserted into pLKO.1 plasmid between the EcoRI and NheI restriction sites. PLKO.1 vector and packaging vectors were co-transfected into HEK293T cells with PEI reagent. For overexpression assay, the gene coding sequence was cloned into PLVX lentiviral vectors. PLVX vector and packaging vectors were co-transfected into HEK293T cells with PEI. The supernatants containing viruses were harvested after 48 h and were purified and enriched via filter and ultracentrifugation. The shRNA PLKO.1 construct was introduced into target cells via lentiviral transduction. The knockdown assay primer sequences were summarized in Supporting Information S1: Table 2.

2.15 His-pull-down assay

Myc-tagged ASK1, Flag-tagged NEDD4L, and His-tagged ubiquitin (Ubi) were co-transfected into HEK293T cells. MG-132 (10 μM) was added at 6 h before sample collection. About 48 h posttransfection, cells were harvested, washed with 1 × PBS, and lysed with lysis buffer. The lysates were incubated with nickel-nitrilotriacetic acid beads (Ni-NTA Beads, Qiagen 30210) at room temperature for 2 h. Beads were washed three times in Buffer 1 and twice in Buffer 2 (8 M urea, 100 mM Na₂HPO₄, 10 mM Tris-HCl, pH 6.3, 0.2% Triton X-100, and 10 mM imidazole) and once in Buffer 3 (100 mM NaCl, 20 mM Tris-HCl, pH 8.0, 20% glycerol, 1 mM dithiothreitol, and 10 mM imidazole). Then ubiquitination levels were detected by immunoblotting.

2.16 Immunofluorescent (IF) microscopy

A549 cells were infected with target lentiviruses. After 48 h of lentiviral infection, these cells were used for the following experiments. Following corresponding experimental treatment with PR8 infection, cells were fixed in 4% formaldehyde and permeabilized in 0.5% Triton-X 100. Cell nuclei were stained with DAPI dye and slides were imaged on a laser-scanning confocal microscope (Olympus FV-1200). Images were quantified by Image J Pro.

2.17 Statistical analysis

Statistics were done by SPSS software (SPSS Inc.) or GraphPad Prism software (GraphPad). An unpaired t test was used unless there were multiple comparisons in which case we used one-way ANOVA or two-way ANOVA. The log-rank test was used for comparing survival by GraphPad Prism software. The results are shown as mean ± SEM.

3.1 Deficiency of vagal-α7 nAChR signaling alleviates influenza viral disease severity and limits the infection of AECs

To figure out whether α7 nAChR signaling is involved in influenza viral infection, we first tested the effect of α7 nAChR knockout on IAV infection by infecting Chrna7^+/+ (the coding gene of α7 nAChR) and Chrna7^−/− mice with influenza virus A H1N1/PR8 (Figure 1A). The results showed that the survival of Chrna7^−/− mice was significantly higher compared to Chrna7^+/+ mice (Figure 1B), and Chrna7^−/− mice had less body weight loss than Chrna7^+/+ mice post PR8 infection (Figure 1C). Consistently, lung injury and inflammatory responses were also decreased in Chrna7^−/− mice compared to Chrna7^+/+ mice, manifested by reduced lung injury score (Figure 1D,E), lower protein levels and cell number in bronchoalveolar lavage fluid (BALF) (Figure 1F,G), and lower expression levels of inflammatory genes (Supporting Information S1: Figure S1A) at 6 dpi. Interestingly, we further identified a decreased PR8 viral titer in the lung of Chrna7^−/− mice compared to Chrna7^+/+ mice at 6 dpi (Figure 1H). Consistently, the levels of PR8 M gene were also dramatically decreased in Chrna7^−/− mice compared to Chrna7^+/+ mice at 6 dpi (Figure 1I). Additionally, we confirmed that Chrna7 gene was deleted in Chrna7^−/− mice (Figure 1J). Notably, the levels of Chrna7 gene in the lung were significantly increased after PR8 infection (Figure 1J), supporting a possible role of α7 nAChR in regulating IAV infection. Given that AECs are major targets of IAV in the lung, we disassociated the lungs into single cells by enzyme digestion and detected the PR8-infected AECs by flow cytometry. The results showed that the infected AECs of Chrna7^−/− mice were significantly decreased compared to Chrna7^+/+ mice post PR8 infection (Figure 1K,L), consistent with the results of viral titer and viral gene expression. These results of α7 nAChR knockout mice indicate that deficiency of α7 nAChR dampens IAV infection in AECs of the lung.

3.2 Specific deletion of Chrna7 in type II AECs attenuates influenza viral infection

Given that α7 nAChR is ubiquitously expressed in many cell types and tissues, the conventional knockout mice are unspecific and insufficient to justify the role of α7 nAChR in AECs during influenza viral infection. Therefore, we further generated conditional Chrna7 floxed (Chrna7^f/f) mice by CRISPR-Cas9 technology (Supporting Information S1: Figure S1B,C). By crossing with Sftpc^Cre mice, Chrna7 gene was specifically deleted in type II AECs in the Sftpc^CreChrna7^f/f mice as we previously showed.¹³ After PR8 infection, we found that Sftpc^CreChrna7^f/f mice had improved survival and body weight loss compared to control littermates (Figure 1M–O). Similar to the results of whole-body knockout mice, Sftpc^CreChrna7^f/f mice showed decreased viral loads in the lung compared to control mice (Figure 1P–S). In addition, PR8-induced inflammatory responses were also reduced in Sftpc^CreChrna7^f/f mice compared to Chrna7^f/f mice (Supporting Information S1: Figure S1D), consistent with the results of whole-body knockout mice (Supporting Information S1: Figure S1A). These results indicate that a deficiency of α7 nAChR in AECs is sufficient to render protection by reducing IAV infection in the lung.

Moreover, to test the role of vagal circuits in mediating α7 nAChR signaling during IAV infection, we performed unilateral vagotomy in the mice and then infected these mice with PR8 (Supporting Information S1: Figure S2A). However, the survival and lung viral load at 6 dpi had no significant differences between sham and vagotomized mice (data not shown), which may be due to the complexed effect on the body function after vagotomy. Interestingly, we found that the viral loads, BALF cell number, and infected AECs in the lung of vagotomized mice were significantly lower compared to those of sham mice at 2 dpi (Supporting Information S1: Figure S2B–E), indicating that loss of vagal circuits dampens influenza infection in AECs. Taken together, these findings support that deficiency of vagal-α7 nAChR signaling attenuates influenza infection in lung AECs and suggest that α7 nAChR activation in AECs may promote influenza infection.

3.3 Activation of α7 nAChR enhances influenza viral infection in vitro

Next, to test our hypothesis that α7 nAChR activation in AECs promotes influenza infection, we utilized A549 cells, a commonly used cell line of type II AECs, for the in vitro infection study. We first confirmed the expression of α7 nAChR in A549 cells by flow cytometry and staining with fluorescence-labeled α-Bungarotoxin (Figure 2A,B), a snake neurotoxin and competitively binds and blocks α7 nAChR. Furthermore, we treated A549 cell with GTS-21, a specific α7 nAChR agonist. The specificity of this agonist was previously validated through knockout mice in our study.²⁴ Before IAV infection, A549 cells were treated with GTS-21 for 30 min to set up a context of α7 nAChR activation before the infection (Figure 2C). Furthermore, the results of Western blot showed that treatment of GTS-21 increased the amount of intracellular viral protein in A549 cells (Figure 2D,E), which was further confirmed by analysis of PR8 M gene expression in cells and viral titers in the supernatant (Figure 2F,G). Consistently, flow cytometry analysis of PR8 NP⁺ cells also supported the role of α7 nAChR activation in promoting IAV infection (Figure 2H,I). Additionally, treatment with a second α7 nAChR specific agonist, PHA568487, yielded similar effects in enhancing IAV infection (Figure 2J). On the contrary, when we treated A549 cells with a specific α7 nAChR antagonist, methyllycaconitine citrate, to antagonize its effect (Figure 2K), IAV infection was significantly inhibited (Figure 2L,M). Taken together, these results support our hypothesis regarding the function of α7 nAChR activation in promoting IAV infection.

3.4 Inactivation of ASK1-p38MAPK signaling mediated by α7 nAChR activation promotes influenza viral infection in vitro

To explore the underlying mechanism, we performed bulk RNA sequencing (RNA-seq), including three groups: 1. PBS-PBS: treated with vehicle and mock infection; 2. PBS-IAV: treated with vehicle and IAV infection; 3. GTS-IAV: treated with GTS-21 and IAV infection (Figure 2N). Influenza infection and activation of α7 nAChR caused a significant alteration of the epithelial transcriptome (Figure 2O; Supporting Information S1: Figure S3A,B). Probing deeper, we found that influenza infection induced markedly higher expression of inflammatory genes compared to noninfected cells (Supporting Information S1: Figure S3B), indicating an efficient establishment of IAV infection. By performing the pathway enrichment analysis of the differentially expressed genes (DEGs) induced by IAV, we identified a variety of virus- and infection-related pathways (Supporting Information S1: Figure S3C). Previous studies showed that in vitro influenza infection induced activation of p38 mitogen-activated protein kinase (p38MAPK), which functions as an important host factor for viral infection.²⁵ Also, our results showed that multiple MAPK cascade-related pathways were enriched with the DEGs under influenza infection (Supporting Information S1: Figure S3C). Interestingly, during influenza infection, α7 nAChR activation further influenced MAPK cascade-related pathways (Figure 2P). Furthermore, gene set enrichment analysis showed that influenza infection induced significant activation of MAPK pathway compared to noninfected condition (Figure 2Q). Conversely, α7 nAChR activation suppressed MAPK pathway during influenza infection (Figure 2R). Taken together, these results indicate that MAPK cascade-related pathways are activated by influenza infection but suppressed by α7 nAChR activation, suggesting a potential role of MAPK pathways in α7 nAChR signaling-regulated influenza infection.

Next, to determine the role of p38MAPK, we first measured the changes of p-p38MAPK. In line with our RNA-seq data, the findings revealed that PR8 infection heightened p-p38MAPK levels in A549 cells, which were subsequently downregulated by GTS-21 treatment, without altering the total levels of p38MAPK (Figure 3A–C). To specifically identify the role of p38MAPK in influenza infection, we performed lentivirus-mediated knockdown of MAPK14 (the encoding gene of p38αMAPK, which is a major subtype of p38MAPK family) in A549 cells. The results showed that knockdown of MAPK14 significantly increased the levels of IAV infection (Figure 3E,F). On the contrary, overexpression of MAPK14 resulted in inhibition of the infection; however, this inhibitory effect was reversed when overexpressing MAPK14 with mutations at phosphorylation sites Thr180 and Tyr182, which are critical for p38MAPK activity (Figure 3G,H). These findings support the role of p38MAPK signaling mediated by α7 nAChR in regulating IAV infection. Next, we identified that α7 nAChR activation dose-dependently decreased the total amount of ASK1 (Figure 3A,D), which can mediate phosphorylation and activation of p38MAPK (Figure 3I). Furthermore, the knockdown of ASK1 gene reduced IAV-induced p38MAPK signaling (Figure 3J–L) and simultaneously enhanced IAV infection (Figure 3J,M), consistent with the results of MAPK14 knockdown. The effects of ASK1 and p38MAPK on IAV infection were further validated by IF antibody staining and flow cytometry measurement of IAV infected cells (Supporting Information S1: Figure S4A–D). Additionally, we detected the pattern of ASK1-p38MAPK signaling in vivo by measuring the protein levels in IAV-infected lung. Consistent with in vitro results, Chrna7^−/− mice showed increased ASK1-p38MAPK signaling in the lung compared to Chrna7^+/+ mice, accompanied by reduced IAV PB2 protein (Supporting Information S1: Figure S4E–I). Taken together, these results indicate that impaired ASK1-p38MAPK signaling mediated by α7 nAChR activation contributes to promoting influenza viral infection in AECs.

3.5 Activation of α7 nAChR facilitates NEDD4L-mediated ubiquitination and degradation of ASK1

Because the transcripts of MAPK14 and ASK1 were not affected by α7 nAChR activation during IAV infection (Supporting Information S1: Figure S5A,B), to further test how α7 nAChR regulates ASK1 levels, we tested the role of ubiquitin-proteasome pathway in posttranslational ASK1 degradation. We found that treatment of MG-132 (a proteasome inhibitor) reversed α7 nAChR-induced ASK1 degradation (Figure 4A), suggesting that activation of α7 nAChR may result in ASK1 ubiquitination. Of note, ASK1 possesses the “PPxY” motif (Supporting Information S1: Figure S5C), a catalytic target of E3 ubiquitination ligase NEDD4L (encoded by NEDD4L gene) (Supporting Information S1: Figure S5D).²⁶ Furthermore, we observed that knockdown and overexpression of NEDD4L regulated ASK1 and p-p38MAPK in a dose-dependent manner (Figure 4B,C), making it possible that α7 nAChR may regulate ASK1-p38MAPK signaling through NEDD4L during IAV infection. Moreover, knockdown of NEDD4L enhanced infection-related ASK1-p38MAPK signaling and reduced IAV infection (Figure 4D–G); on the contrary, overexpression of NEDD4L attenuated ASK1-p38MAPK signaling but boosted IAV infection (Figure 4H–K). IF antibody staining and flow cytometry analysis confirmed the role of NEDD4L in IAV infection (Supporting Information S1: Figure S5E–H). In sum, these findings support the important role of NEDD4L in IAV infection by regulating ASK1-p38MAPK signaling. By performing co-immunoprecipitation (co-IP) in A549 cells, we confirmed the endogenous interaction between NEDD4L and ASK1 (Figure 4L). Furthermore, we overexpressed NEDD4L and ASK1 in 293 T cells and then performed co-IP. Similarly, the results supported the interaction between NEDD4L and ASK1 (Figure 4M). By his-pull-down analysis, we confirmed that NEDD4L can mediate significant ASK1 ubiquitination (Figure 4N) but not p38MAPK ubiquitination (Supporting Information S1: Figure S5I), supporting the role of NEDD4L in ASK1 ubiquitination and degradation. Of note, NEDD4L-mediated ASK1 ubiquitination was further enhanced by α7 nAChR activation (Figure 4N), indicating that α7 nAChR activation upregulates NEDD4L activity. Taken together, these results support the hypothesis that activation of α7 nAChR enhances NEDD4L-mediated ASK1 ubiquitination and degradation, thereby promoting IAV infection by limiting p-p38MAPK signaling.

3.6 Activation of α7 nAChR promotes the tyrosine phosphorylation of NEDD4L by inhibiting PTP1B

Levels of tyrosine (Tyr) phosphorylation of NEDD4L reflect its ubiquitin ligase activity.²⁷ Detection of Tyr phosphorylation in immunoprecipitants of NEDD4L showed that activation of α7 nAChR increased Tyr phosphorylation of NEDD4L (Figure 5A), suggesting a role of α7 nAChR in modulating NEDD4L activity by promoting its Tyr phosphorylation. Activation of α7 nAChR in fibroblasts enhanced TGF-β signaling by regulating PTP1B (encoded by PTPN1 gene),²⁴ an important negative regulator of tyrosine phosphorylation and interferon signaling pathway during influenza infection.^{28, 29} Moreover, we identified that activation of α7 nAChR dose-dependently increased phosphorylation levels of PTP1B at Ser378 site (Figure 5B,C), which is inversely correlated with PTP1B activity.³⁰ PTP1B activity assays confirmed the inhibitory effect of α7 nAChR activation on its tyrosine phosphatase activity (Figure 5D). In line with the previous results, knockdown of PTPN1 decreased ASK1-p38MAPK signaling (Figure 5E–G) but increased IAV infection (Figure 5E,H), supporting the important role of PTP1B in mediating α7 nAChR and ASK1-p38MAPK signaling during IAV infection. Additionally, IF antibody staining and flow cytometry analysis confirmed the role of PTP1B in α7 nAChR signaling and IAV infection (Supporting Information S1: Figure S6A–D). Interestingly, we found that knockdown of PTPN1 significantly increased interferon signaling in response to IAV infection (Supporting Information S1: Figure S6E), consistent with previous findings.²⁹ Of note, the boosted antiviral interferon signaling induced by PTPN1 knockdown failed to limit IAV infection (Figure 5E,H), further supporting the importance of ASK1-p38MAPK signaling in controlling IAV infection. By performing Co-IP assays, we confirmed the interaction between α7 nAChR and PTP1B (Figure 5I) and the interaction between PTP1B and NEDD4L (Figure 5J) in A549 cells. Notably, activation of α7 nAChR attenuated the interaction between PTP1B and NEDD4L (Figure 5J), further validating our hypothesis that α7 nAChR regulates NEDD4L activity through PTP1B. Additionally, knockdown and overexpression of PTPN1 significantly altered the Tyr phosphorylation levels of NEDD4L (Figure 5K), supporting the role of PTP1B in modulating NEDD4L activity. Taken together, these results suggest that activation of α7 nAChR promotes tyrosine phosphorylation of NEDD4L through inhibiting PTP1B activity.

3.7 Activation of α7 nAChR facilitates export of influenza viral ribonucleoproteins (vRNPs) by reducing p-p38MAPK levels in the nucleus

Our RNA-seq data supported the role of α7 nAChR in regulating the process of nuclear export (Figure 2P). Importantly, the export of IAV viral ribonucleoproteins (vRNP) complex from nucleus is a key step of influenza viral life cycle. To test whether p38MAPK signaling is involved in vRNP export, we first analyzed p-p38MAPK levels in the nucleus. The results confirmed the presence of p-p38MAPK in the nucleus by knockdown and overexpression of MAPK14 (Figure 6A). Furthermore, co-IP analysis revealed the interaction between p-38MAPK and IAV NP protein (Figure 6B), suggesting a possible role of p38-MAPK in regulating nuclear export of IAV vRNP complex. CRM1 (encoded by XPO1 gene) is critical for influenza viral vRNP nuclear transport and completing viral life cycle.³¹ Co-IP analysis confirmed the interaction between NP and CRM1 in nucleus (Figure 6C). To figure out the effect of p38MAPK on the interaction of NP and CRM1, we co-overexpressed p38MAPK, IAV NP, and CRM1 in 293 T cells followed by co-IP analysis of NP. We found that NP was able to interact with CRM1; however, overexpression of p38MAPK reduced the interaction of NP and CRM1 (Figure 6D), supporting the role of p38MAPK in reducing the NP-CRM1 interaction and nuclear export of IAV vRNP complex. Additionally, by comparing the effect of MAPK14 knockdown and overexpression, we confirmed the role of p38MAPK in negatively regulating the interaction of NP and CRM1 (Figure 6E). Finally, we showed that activation of α7 nAChR by GTS-21 and MAPK14 knockdown contributed to nuclear export of vRNPs staining in A549 cells; on the contrary, MAPK14 overexpression limited this process (Figure 6F,G). Taken together, these results support our hypothesis that activation of α7 nAChR contributes to the nuclear export of influenza vRNP complex by reducing p-p38MAPK signaling, which eventually results in increased influenza viral infection in AECs.