Virus
Human respiratory syncytial virus type A2 (RSV A2) was supplied initially by Dr. B. Wang, Center for Disease Control and Prevention, Shenyang, China. The virus was propagated in monolayers of HEp-2 cells (ATCC). The virus titer was expressed as a 50% tissue culture infectious dose (TCID50).
Mice
All animal experiments were approved and are in accordance with the guidelines of the Institutional Animal Care and Use Committee of China Medical University.
Wide-type mice on BALB/c background were purchased from Liaoning Changsheng Biotechnology Company (Benxi, China). NMU-knockout mice on C57BL/6J background were purchased from Cyagen Biotechnology Company (Suzhou, China). Mice were bred and maintained under specific pathogen-free condition at the laboratory animal center of China Medical University. All the experimental mice were female, 8 weeks old.
Mice were anaesthetized with 1% pentobarbital sodium. For RSV infection model, mice were administered with 20μl RSV suspension (containing approximately 1×107 TCID50) via nasal mucosa, and experimental specimens were collected before infection and on day 1, 3, 5 after infection. In the NMU stimulation experiment, the BALB/c mice were treated intranasally with 20μg NMU for 4 consecutive days from 1 day before RSV infection. For anti-NMU neutralization test, mice were injected intraperitoneally with anti-NMU-IgG (10mg/kg) 1 day before RSV infection. All experimental specimens were collected on day 3 after RSV infection.
Pulmonary single cell suspensions
Mice were anesthetized with intraperitoneal injection of 1% pentobarbital sodium, heart perfusion was performed with sterile PBS. The alveolar lavage fluid was collected by alveolar flushing with 1 ml PBS. The supernatant after centrifugation was used for ELISA to detect cytokine levels. Cell precipitation was used for HE staining. Lung tissue was removed and washed with cold PBS. The lung tissue was cut up into puree and RPMI-1640 with 10%FBS was used to suspend the tissue fragments on ice. Lung tissues were digested with collagenase D (200μg/ml, Roche) and DNaseⅠ(40μg/ml, Roche) at 37°C for 90 minutes under agitation. After lysis of erythrocytes, cells were resuspended with PBS with 2% FBS and filtered through a 70μm cell strainer.
Flow cytometry
Intracellular cytokine staining was performed after incubation with PMA (25ng/ml, Sigma), Ionomycin (1μg/ml/L, Sigma) and Brefeldin A (10μg/ml, Sigma) for 4 hours. Cell fixation was conducted according to the instructions of Cytofix/Cytoperm buffer set (BD bioscience). Ki67 as the proliferation indicator was stained per the staining protocol of manufacturer with 70% Ethanol. After blocking the Fc-receptors with CD16/32 antibody (Biolegend), single cell suspensions were incubated with fluorescein-conjugated antibodies on ice for surface staining. Lineage antibody cocktail (eBioscience) components include DX5 (or NK1.1), CD3ε, CD4, CD5, CD8, CD11b, CD19, B220, Gr-1 and TCRδ. Anti-CD45 (30-F11) and anti-Ki67(16A8) were purchased from Biolegend. Anti-IL-5 (TRFK5), anti-IL-13 (eBio13A) and anti-ST2 (DIH9) were obtained from eBioscience. Anti-NMUR1 was ordered from Bioss. Flow cytometry analysis were performed using LFRFortessa (BD Biosciences). The percentage of ILC2s was gated in CD45+Lineage-ST2+cells. Data analysis was done by FlowJo_V10 software (Tristar).
Quantitative Real-time PCR
Total RNA was extracted from lung homogenates or sorted cells using TRIzol (Life Technologies). RNA concentration was determined by Nanodrop 2000 Spectrophotometer (Thermo Fisher). And then, PrimeScriptTM RT reagent kit (Takara) was applied to synthesize cDNA from isolated RNA. Quantitative Real-time PCR was performed using SYBR@ Premix Ex TaqTMⅡ (Takara). The sequences of primers are listed in Table 1.
Table 1 Primers used for Real-time PCR
Gene
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Forward (5’ -3’)
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Reverse (5’ -3’)
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NMU
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GCAGCTCGTCCCTCAACTG
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CGTTGCGTGGCCTGAATAAA
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NMUR1
|
CCTCGCTGTGTCCGATATGC
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GCTGGAACGGGTAATTTTGCT
|
IL-5
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GGCTTCCTGTCCCTACTCAT
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TCCTCGCCACACTTCTCTTT
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IL-13
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AGCATGGTATGGAGTGTGGA
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TTGCAATTGGAGATGTTGGT
|
PI3K
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CGAGAGTGTCGTCACAGTGTC
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TGTTCGCTTCCACAAACACAG
|
MEK1
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GTTGCTTTCAGGCCTCTCC
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AGTGATGGGCTCTGCTTAGG
|
NFAT
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AGGCTCATTCCAGACACCG
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GTCCTGCTCAGGTTTAGCCG
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TLR3
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GTGAGATACAACGTAGCTGACTG
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TCCTGCATCCAAGATAGCAAGT
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TLR4
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GCCTTTCAGGGAATTAAGCTCC
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GATCAACCGATGGACGTGTAAA
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TLR7
|
ATGTGGACACGGAAGAGACAA
|
GGTAAGGGTAAGATTGGTGGTG
|
β-actin
|
GGTCATCACTATTGGCAACG
|
TCCATACCCAAGAAGGAAGG
|
Reactions were run using a 7500 Real-time PCR System (Applied Biosystems) under conventional amplification conditions. Results are normalized to β-actin and presented as fold mRNA expression (2-△△CT).
Western blot
Western blot was performed with standard methods. Cleaned and shredded lung tissues or cultured cell extracts were prepared in RIPA buffer supplemented with protease inhibitor. After protein quantification by BCA, equivalent amounts of total protein per sample were fractionated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA for 90 minutes at room temperature and incubated with the primary antibodies at 4°C overnight. After washed with PBST, the membranes were incubated with corresponding secondary antibody for 1 hour at room temperature and detected by chemiluminescence. The rabbit anti-GAPDH (Affbiotech) or mouse anti-β-actin antibody (Absin) was used as an internal control. Densitometry was performed by Image J software. Protein levels were normalized to GAPDH or β-actin.
Primary antibodies: mouse anti-NMU (Santa Cruz); rabbit anti-NMUR1 (EpiGentek); rabbit anti-pPI3K/PI3K (Affbiotech); rabbit anti-pMEK1/2/MEK1/2 (Affbiotech); rabbit anti-pNFAT2/NFAT2 (Affbiotech); rabbit anti-TLR4 (Affbiotech); rabbit anti-TLR7 (Affbiotech). Secondary antibodies: goat anti-mouse IgG HRP (Affbiotech); goat anti-rabbit IgG HRP (Affbiotech).
ELISA
The protein levels of IL-5 and IL-13 in the supernatants of cultured cells were measured by ELISA kits (Thermo Fisher) according to the manufacturer’s instruction. The optical density at 450 nm was determined by Multimode Microplate Reader (Thermo Fisher).
Cell sorting of ILC2s and CD4+ T cells
RSV was administered to BALB/c mice intranasally, and pulmonary single cell suspensions were taken on day 3 after infection, unless stated otherwise. Lymphocytes were isolated according to the manufacturer’s instruction of mouse viscera tissue lymphocyte isolation KIT (TBD, China). ILC2s defined as CD45+Lineage-ST2+ cells and CD4+ Tidentified as CD45+Lineage+CD4+ cells were obtained using Beckman MoFlo Astrios EQ (Beckman Coulter Life Science). The average purity of sorted-purified cells was > 90%.
Isolation and culture of neurons
Thoracic dorsal root ganglia (DRG) were dissected from neonatal mice of 1-3 days old and digested in 0.25% trypsin for 30 minutes at 37°C. Triple volume of DMEM/F12 (Gibco) supplemented with 10% FBS, 1% glutamine and 1% streptomycin/penicillin was added to terminate digestion. DRG cell suspension was triturated with pasteur pipettes, followed by centrifugation. Then supernatant was discarded by centrifugation, and the cell pelltes was resuspended in neurobasal medium (Gibco) containing 2% B27 (Gibco), 1% glutamine, 0.5% streptomycin/penicillin and 20ng/ml nerve growth factor (NGF) (Sigma). DRG neurons were cultured on 0.01% poly-L-Lysine pre-coated 12-well culture plates in the culture medium described above for about a week to differentiate mature. DRG neurons were stimulated with RSV of increasing multiplicity of infection (MOI) for 60 minutes and RNA or protein was obtained at the indicated time for analysis.
For signal protein stimulation test, 1ug/ml LPS (TLR4 agonist, Sigma) or 2ug/ml R837 (TLR7 agonist, MCE) was used to activate DRG neurons instead of RSV for 60 minutes. As for signal blocking analysis, we pretreated the DRG neurons with 200nM TAK-242 (TLR4 inhibitor, MCE) or 5ug/ml IRS661 (TLR7 antagonist, Invitrogen) for 15 minutes before RSV infection.
In vitro stimulation
Sorted ILC2s were cultured in RPMI-1640 (supplemented with 10% FBS, 1% gulutamine and 1% streptomycin/penicillin) at 37°C. ILC2s were cultured in the presence of 10ng/ml IL-2 and 20ng/ml IL-7 (Biolegend) for 24 hours. Before use, ILC2s were gently washed to remove residual IL-2 and IL-7. ILC2s were co-cultured with PBS or 100ng/ml (unless stated otherwise) recombinant mouse NMU (Phoenix Pharmaceuticals) for 20 hours or indicated time intervals. After incubation, flow cytometry of cellular staining or RNA extraction was performed, and the cytokine levels in cultural supernatant were determined by ELISA.
CD4+ T cells were cultured in a 1ug/ml anti-CD3 (Biolegend) pre-coated plate and in the presence of 1ug/ml anti-CD28, 20ng/ml IL-2 and 50ng/ml IL-4 (Biolegend) for 3 days. As for stimulation, T cells were co-cultured with PBS or 100ng/ml NMU (Phoenix Pharmaceuticals) for 20 hours followed by RNA isolation.
Cell signaling
Purified ILC2s were cultured with 10%FBS-RPMI-1640 with IL-2 and IL-7 for 24 hours as mentioned above. To analyze the effect of PI3K, MEK and NFAT, ILC2s were incubated with their respective inhibitors for 1 hour and then stimulated with 100ng/ml NMU for 12 hours before the analysis of cytokines and signal proteins. PI3K inhibitor: LY294002 (50μM, MCE); MEK inhibitor: U0126-EtOH (50μM, MCE); NFAT inhibitor: 11R-VIVIT (10μM, MCE).
HE staning
The left lung tissue was fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. The slices were cut into 4μm sections and stained with hematoxylin and eosin to detect inflammatory infiltrates. Photomicrographs were captured by biomicroscope (Zeiss).
Immunofluorescence microscopy
Samples from lungs were fixed with 4% PFA at 4°C overnight and then embedded in OCT to obtain frozen section. 10μm tissue slices were blocked and permeabilized with 0.5% Triton X-100 and 5%BSA, and then incubated at 4°C overnight in wetting box with the following antibodies: Syrian hamster monoclonal anti-KLRG1 (2F1, Santa CruZ), mouse monoclonal anti-NMU (A-5, Santa CruZ) and rabbit anti-SNAP-25 (Abcam).
For cell staining, sorted ILC2s were stabilized for 24 hours on poly-L-Lysine pre-coated glass slides, and then cultured with or without NMU for 4 hours as described above. After stimulation, cells were fixed with 4% PFA for 20 minutes at room temperature. Slides were permeabilized with 0.3% Triton X-100 for 20 minutes and blocked with 5%BSA for 30 minutes at room temperature. Cells were stained with Syrian hamster monoclonal anti-KLRG1 (2F1, Santa CruZ) and rabbit anti-NMUR1 (EpiGentek) for 1 hour at room temperature.
Respective secondary antibodies were used subsequently at room temperature in the dark for 1 hour and nuclei was counterstained with DAPI (Abcam). Images were captured with confocal fluorescence microscopy (Olympus). Mean fluorescence intensity (MFI) was measured by ImageJ software. Secondary antibodies were as follows: AF488 goat anti-Syrian hamster, AF647 goat anti-rabbit, AF647 goat anti-mouse, AF647 goat anti-Syrian hamster, AF488 goat anti-rabbit.
Customization of neutralizing antibodies
Rabbit anti-NMU-IgG antibody was synthesized by ProbeGene (Jiangsu, China). The mouse NMU protein was synthesized (MSRAAGHRPGLSAGQLAAATASPLLSLLLLLACCADACKGVPISPQRLQPEQELQLWNEIHEACASFLSIDSRPQASVALRELCRIVMEISQKPQEQSEKDNTKRFLFHYSKTQKLGNSNVVSSVVHPLLQLVPQLHERRMKRFKAEYQSPSVGQSKGYFLFRPRNGKRSTSFI) and New Zealand white rabbits were immunized with this protein for 4 times to obtain anti-NMU-IgG, which was purified by affinity chromatography.
Rabbit anti-NMUR1-IgG antibody was synthesized by ProbeGene (Jiangsu, China). The mouse NMUR1 peptide was synthesized (MTPPCLNCSIFPGALSPNASRSPLVCNISEFKWPYQPEDLNLTDEALRLKYLGPQQMKQFVPICVTYLLIFVVGTLGNGLTCTVILRNKTMRTPTNFYLFSLAVSDMLVLLVGLPLELYEMQQNYPFQLGASACYFRILLLETVCLASVLNVTALSVERYVAVVRPLQAKSVMTRAHVRRMVGAIWVLATLFSLPNTSLHGLSQLTVPCRGPVPDSAICSLVGPMDFYKLVVLTTALLFFCLPMVTISVLYLLIGLRLRRERMLLQVEVKGRKTAATQETSHRRIQLQDRGRRQVTKMLFALVVVFGICWAPFHADRIMWSLVYGHSTEGLHLAYQCVHIASGIFFYLGSAANPVLYSLMSTRFRETFLQALGLGTQCCHRRQPYHGSHNHIRLTTGSTLCDVGHRNSRDEPLAVNEDPGCQQETDPS) and New Zealand white rabbits were immunized with this peptide fragment for 4 times to obtain anti-NMUR1-IgG, which was purified by affinity chromatography.
Statistical analysis
Graphpad Prism 8 software was used to perform two-tailed and ordinary one-way or two-way ANOVA with Tukey’s multiple comparisons test. Results are shown as mean ± SEM. (*P<0.05; **P<0.001; ns, not significant).