Nestin promotes pulmonary fibrosis via facilitating recycling of TGF-β receptor I

Background Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that is characterised by aberrant proliferation of activated myofibroblasts and pathological remodelling of the extracellular matrix. Previous studies have revealed that the intermediate filament protein nestin plays key roles in tissue regeneration and wound healing in different organs. Whether nestin plays a critical role in the pathogenesis of IPF needs to be clarified. Methods Nestin expression in lung tissues from bleomycin-treated mice and IPF patients was determined. Transfection with nestin short hairpin RNA vectors in vitro that regulated transcription growth factor (TGF)-β/Smad signalling was conducted. Biotinylation assays to observe plasma membrane TβRI, TβRI endocytosis and TβRI recycling after nestin knockdown were performed. Adeno-associated virus serotype (AAV)6-mediated nestin knockdown was assessed in vivo. Results We found that nestin expression was increased in a murine pulmonary fibrosis model and IPF patients, and that the upregulated protein primarily localised in lung α-smooth muscle actin-positive myofibroblasts. Mechanistically, we determined that nestin knockdown inhibited TGF-β signalling by suppressing recycling of TβRI to the cell surface and that Rab11 was required for the ability of nestin to promote TβRI recycling. In vivo, we found that intratracheal administration of AAV6-mediated nestin knockdown significantly alleviated pulmonary fibrosis in multiple experimental mice models. Conclusion Our findings reveal a pro-fibrotic function of nestin partially through facilitating Rab11-dependent recycling of TβRI and shed new light on pulmonary fibrosis treatment.

trachea was ligated with thread to retain the agarose inside the lung. The lung was excised, transferred into a tube with cultivation medium and cooled on ice for 10 min to allow gelling of the agarose. The lobes were separated and cut with a vibratome (VT1200s, Leica, Germany) to a thickness of 300 μm using a speed of 10-12 μm·s −1 , a frequency of 150 Hz and an amplitude of 1.2 mm. The 3D-LTCs were cultivated in medium supplemented with 0.1% fetal calf serum (FCS, Gibco). Individual 3D-LTCs were cultivated at 37°C in humidified conditions containing 5% (volume/ volume) CO2 in 24-well plates under submerged conditions with changes of medium every other day. 3D-LTCs obtained from mice subjected to PBS or bleomycin were stimulated either with AAV6-Scramble or AAV6-ShNES (1*10 12 vg/slice) for 72h.

Preparation of pulmospheres
Lung biopsies (approximately 6 mm × 6 mm) from IPF patients or control subjects were perfused, isolated under sterile conditions and excised into nearly 1-mm 3 fragments, washed with cold PBS solution. The tissue was then digested in DMEM supplemented with 20 mg/ml Collagenase type I (Gibco, Thermo Fisher Scientific, 9001-12-1) at 37°C for 1 hour. The tube containing tissue was then vigorously shaken for 1 min and the resulting suspension was filtered through a 100-μm strainer. The filtered cell suspension was centrifuged for 5 minutes at 300 g. The cell pellet was resuspended in DMEM and then seeded into HEMA-coated (5 mg/ml made in 95% ethanol) 96-well U-bottom plates in complete culture medium consisting of DMEM supplemented with 10% fetal bovine serum (FBS, Gibco) and penicillin-streptomycin. The cells were cultured in a humidified atmosphere of 5% CO2 at 37°C as described previously.

Real-time quantitative PCR
Total RNA was extracted from lung tissues or cells using the TRIzol reagent (Molecular Research Center, Inc.) following the manufacturer's protocol.
Quantification was performed with a NanoDrop 8000 spectrophotometer and 1 µg of total RNA was used to reverse transcription with a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1622). The obtained cDNAs were used as the template for real-time quantitative PCR (qPCR) reactions with the FastStart Essential DNA Green Master Mix (Roche, 06924204001). All samples were run in triplicate and the results were normalized to those obtained for the 18S rRNA or GAPDH. The primers designed and used for qPCR are described in Supplemental Table 4.

Histopathological evaluation
After sacrifice, mouse lung tissues were perfused with 4% paraformaldehyde and then subjected to paraffin embedding or saturation in 30% sucrose for 24 hours for frozen sections. Paraffin-embedded human IPF and mouse lung tissue sections were exposed to H&E or Masson trichrome staining to analyze collagens and inflammation, respectively, in the lung tissue sections.

Immunohistochemistry (IHC) and Immunofluorescent staining (IF)
Paraffin-embedded human IPF and mouse lung tissue sections were subjected to immunostaining using an UltraSensitiveTM SP (Mouse/Rabbit) IHC Kit (MXB, KIT-9710). Following deparaffinization and antigen retrieval, the IPF lung tissue specimens were incubated overnight with corresponding antibodies listed in Supplemental Table 5. Signal amplification and detection were performed using a DAB system according to the manufacturer's instructions (MXB, MAX-001).
Immunofluorescent staining of human IPF and mouse lung tissue sections was performed using primary antibodies listed in Supplemental Table 5

Lung Tissue Dissociations
Single-cell suspensions of lung cells were prepared by mincing and enzymatic digestion. Briefly, lungs were rinsed in sterile phosphate-buffered saline (PBS) following removal of tracheas, and were finely minced with sterile scissors and incubated in 3 mg/ml collagenase type I (#17018029, ThermoFisher) (Diluted with HBSS, in supplement with 0.1% BSA) in a volume of 2 ml per lung for 60 minutes at 37°C in a shaking incubator. The resulting cell suspension was further filtered through a 40 µm sieve successively to avoid the cell aggregates, and washed twice in PBS supplemented with 1% FBS, 1 mM EDTA (#A100105, Sangon Biotech) by centrifugation (1,100 rpm, 5 minutes, room temperature).
Then, prior to the next step, all the tissues were washed (a 5-min centrifugation at 1100rpm, room temperature) twice with FACS buffer, which was PBS in supplemented with 1% FBS, 1 mM EDTA (#A100105, Sangon Biotech).

Flow Cytometric Assay
Single-cell suspensions of lung cells where stained using corresponding antibodies listed in Supplemental Table 5, thereafter fixed with 4% PFA, permeabilized using PBS buffer (0.1% saponin in PBS with 0.1% BSA) for 90min at room temperature. Cells were then stained with secondary antibody conjugated with Alexa Flour 647 in the dark for 45min on ice. Cells were then washed twice with FACS buffer and run on CytoFLEX (Beckman Coulter, USA).
Data were analyzed using the Flow Jo software (Tree Star Inc., Ashland, Oregon).

Quantification of cell surface TβRI using FACS analysis
The cells were seeded at 2×10 5 cells per well of six-well plates, harvested from plates and washed with FACS buffer (1% bovine serum albumin in PBS containing 0.5 mM EDTA) twice. Cells were stained with anti-mouse TβRI-APC antibody or rat IgG2A APC isotype control and then incubated in the dark for 30 min (on ice). Cells were then washed twice with FACS buffer and run on CytoFLEX (Beckman Coulter, USA). Data were analyzed using the Flow Jo software (Tree Star Inc., Ashland, Oregon). were measured by immunoblotting performed as previously described [5].

Adeno-associated virus (AAV) delivery
Antibodies are listed in Supplemental Table 5. Immunoprecipitation was assessed in cells using a previously described method [5]. Bands from at least three independent blots were quantified using Image J software.

Gene targeting by the CRISPR/Cas9 system
The plasimds used for CRISPR/Cas9 KO in MRC5 was constructed in our previous study according to the established cloning protocol [8,9], and T7E1 assay was also performed for sgRNA screening as previously described [5].

Hydroxyproline assay
Frozen lung tissue samples from mice were incubated in 250 μl PBS, after which 250 μl of 12 N HCl was added into samples for pellet hydrolysis at 110°C were overnight. Samples were neutralized by the addition of 10 N NaOH. 100 μl of each sample was mixed with 400 μl oxidizing solution that contained 1.4% chloramine-T, 10% N-propanol, and 80% citrate-acetate buffer in PBS. Lung samples were then incubated for 20 minutes. Samples were finally incubated for 30 minutes at 65°C shortly after adding Ehrlich's solution (Sigma-Aldrich).
The reaction absorbance was measured at 550 nm. Sample concentrations were determined from the standard curve generated using trans-4-hydroxy-lproline (Sigma-Aldrich). A standard curve was generated using trans-4hydroxy-l-proline (Sigma-Aldrich). Hydroxyproline levels were expressed as micrograms hydroxyproline per microgram of lung tissue samples.

Public bulk and single-cell RNA-sequencing dataset acquisition
The gene expression and cell type annotation of bulk and single-cell RNAsequencing data from lungs of normal and IPF human (GSE124685, GSE132771), of control and bleomycin-treated mice (GSE110533, GSE132771) were downloaded from the Gene Expression Omnibus.