CC16-TNF-α negative feedback loop formed between Clara cells and normal airway epithelial cells protects against diesel exhaust particles exposure-induced inflammation

Characterization of the sorted cells

Single cell suspensions were prepared by human distant normal lung tissues where Clara cells are located, and CC16⁺ and CC16^- cells were sorted directly by Fluorescence-activated cell sorting (FACS). The sorted CC16⁺ and CC16^- cells were cultured overnight, and unattached dead cells were removed from the medium. The CC16 mRNA and protein expression were subsequently detected. As shown in Figure 1A, 1B, CC16 mRNA and protein expression were sharply higher in CC16⁺ cell fraction than in CC16^- cells. We also performed immunocytochemistry staining of CC16 in sorted cells. As evidence by Figure 1C, 98% of the cells in CC16⁺ sorted fraction were positive for CC16 immunofluorescence (IF). In addition, to further confirm that the sorted CC16⁺ cells were distal airway Clara cells, the IF was performed for detection of pan-keratin expression, the epithelial marker. The results showed that all cells in the CC16⁺ sorted fraction expressed pan-keratin (Figure 1D). These data suggested that CC16-containing Clara cells are successfully isolated.

Figure 1. Characterization of sorted cells. (A, B) CC16 mRNA and protein expression in the sorted CC16⁺ and CC16^- cell fraction are detected by RT-PCR and western blot assay. (C, D) Immunofluorescence (IF) staining shows that 98% of the cells in CC16⁺ sorted fraction express CC16, and all the cells in CC16⁺ sorted fraction show positive expression of pan-keratin, the epithelial marker (× 100). ***P < 0.001 vs. CC16^- fraction.

DEP exposure induces C/EBPA hypermethylation and decreases CC16 production and secretion from CC16⁺ cells

Our previous study showed that in C22 cells, DEP decreases CC16 expression by methylation of the C/EBPα promoter [10]. But it is unknown whether DEP regulates CC16 secretion via C/EBPα hypermethylation. The level of CC16 protein released into the culture media represents CC16 secretion capacity. The CC16⁺ cells were exposed to 5 ppm SRM 1650b (the commercial DEP matter) in the presence of demethylating agent 5-aza-2’-deoxycytidine (DAC) or not for 48 h. Then cells and culture supernatants were collected for methylation-specific polymerase chain reaction (MSP), RT-PCR, western blot and ELISA detection. The results of MSP determination showed that DEP treatment induced C/EBPα promoter methylation, while DAC treatment restored C/EBPα expression by demethylation of the promoter (Figure 2A). The results of RT-PCR and western blot showed that DEP treatment decreased C/EBPα mRNA and protein expression, which was significantly alleviated by DAC treatment (Figure 2B, 2C). Changes in CC16 level were consistent with changes in C/EBPα expression: DEP treatment significantly inhibited CC16 mRNA expression and level in culture supernatants, which were abolished by DAC treatment (Figure 2D, 2E). In addition, we also detected the effects of treatment with other concentrations of SRM 1650b (0.1, 1, 3 and 10 ppm) on C/EBPα mRNA and protein expression, and CC16 mRNA expression and level in culture supernatants. This can be seen in Figure 2F–2H that a dose-dependent inhibition of C/EBPα and CC16 by DEP was observed in CC16⁺ cells. Collectively, DEP exposure induces C/EBPα hypermethylation and decreases CC16 production and secretion in CC16⁺ cells.

Figure 2. DEP exposure induces C/EBPα hypermethylation and decreases CC16 production and secretion in CC16⁺ Cells. CC16⁺ cells are exposed to 5 ppm SRM 1650b (the commercial DEP matter) in the presence of DAC or not for 48 h, and then cells and culture supernatants are collected. (A) MSP analysis is performed to detect the methylation of C/EBPα promoter. Lane U, amplified product with primers recognizing unmethylated C/EBPα sequence. Lane M, amplified product with primers recognizing methylated C/EBPα sequence. (B, C) The mRNA and protein expression of C/EBPα are determined by RT-PCR and western blot assay. (D, E) CC16 mRNA expression and level in culture supernatants are detected by RT-PCR and ELISA assay. (F–H) After treatment of CC16⁺ cells with different concentrations of SRM 1650b (0.1, 1, 3, 5 and 10 ppM), C/EBPα and CC16 mRNA expression, C/EBPα protein expression, and CC16 level in culture supernatants are determined by RT-PCR, western blot and ELISA assay. **P < 0.01 and ***P < 0.001 vs. Ctrl group; ^##P < 0.01 and ^###P < 0.001 vs. DEP group.

C/EBPA regulates CC16 secretion via Munc18b in CC16⁺ cells

Our previous study demonstrated that in C22 cells, C/EBPα regulates CC16 expression [10]. Herein, we investigated whether and how C/EBPα regulates CC16 secretion. As shown in Figure 3A, 3B, C/EBPα overexpression caused the increases in CC16 mRNA and protein released into the culture medium, whereas C/EBPα knockdown resulted in the decreases of CC16 mRNA and secretion.

Figure 3. C/EBPα regulates CC16 secretion via Munc18b in CC16⁺ cells. After transfection of CC16⁺ cells with pcDNA-ctrl, pcDNA-C/EBPα, pcDNA-C/EBPα+si-Munc18b, si-ctrl, si-C/EBPα, or si-C/EBPα+ pcDNA-CC16, cells are cultured for 48 h, and then cells and culture supernatants are collected. (A, B) The effects of C/EBPα overexpression or knockdown on CC16 mRNA and protein released into the culture media are detected by RT-PCR and ELISA. (C, D) The effects of C/EBPα overexpression or knockdown on Munc18b mRNA and protein expression are detected by RT-PCR and western blot assay. (E, F) The increase of CC16 secretion induced by C/EBPα overexpression is abolished by Munc18b knockdown, while the decrease of CC16 secretion induced by C/EBPα knockdown is not affected by CC16 overexpression. **P < 0.01 and ***P < 0.001 vs. pcDNA-ctrl or si-ctrl group; ^##P < 0.01 vs. pcDNA-C/EBPα group.

Munc18b is a limiting component of the exocytic machinery of secretory epithelial and immune cells. By searching the Jaspar database, we found that the 2-kb region upstream of Munc18b gene contains potential binding motifs for transcription factor C/EBPα in different species (human, rat and mouse) (Table 1). We thus examined whether C/EBPα regulates the transcription of Munc18b. As shown in Figure 3C, 3D, overexpression of C/EBPα significantly increased the expression of Munc18b mRNA and protein. As expected, knockdown of C/EBPα significantly reduced the expression of Munc18b mRNA and protein. In addition, the increase of CC16 secretion induced by C/EBPα overexpression was abolished by Munc18b knockdown, while the decrease of CC16 secretion induced by C/EBPα knockdown was not affected by CC16 overexpression (Figure 3E, 3F). These data suggested that C/EBPα promotes CC16 secretion through upregulation of Munc18b in CC16⁺ cells.

C/EBPA can directly bind to the promoter region of Munc18b

Given the above findings that the upstream of human Munc18b gene has two putative C/EBPα binding sites (-1705 and -1405 bp), and C/EBPα promotes Munc18b expression at the transcriptional level, we performed dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay to further investigate whether C/EBPα activates Munc18b transcription by directly binding to its putative promoter region. The luciferase activity was detected in CC16⁺ cells transfected with both Munc18b promoter construct (-2000/200; Munc18b-promS) or empty pGL3-basic vector (Munc18b-vector) and C/EBPα plasmid or its corresponding empty vector for 48 h. The results showed that Munc18b promoter construct led to an obvious increase in the relative luciferase activity, which was further reinforced by C/EBPα (Figure 4A). To identify the exact position where C/EBPα could bind within this fragment, a ChIP test was performed. As shown in Figure 4B, C/EBPα could bind to two putative binding sites, site 1 and site 2 sequences. Furthermore, to further clarify the function of site 1 and site 2, Munc18b promoter with wild-type (WT) or mutation of the binding motif 1 or 2, and C/EBPα plasmid were co-transfected into CC16⁺ cells. The results of the dual-luciferase reporter assay showed that in comparison with WT group, mutation of the binding motif 1 significantly inhibited the relative luciferase activity, and mutation of the binding motif 2 had no effect on the relative luciferase activity, suggesting that the site 1 was a functional site controlling Munc18b transcription (Figure 4C). These results indicated that C/EBPα activates Munc18b transcription by direct binding to the sites -1715/-1705 in Munc18b promoter.

Figure 4. C/EBPα can directly bind to the promoter region of Munc18b. (A) CC16⁺ cells are transfected with both Munc18b promoter (-2000/200; Munc18b-promS) or empty pGL3-basic vector (Munc18b-vector) and C/EBPα plasmid or its corresponding empty vector control for 48 h, and then the cells are collected for luciferase activity detection. (B) ChIP assay is conducted in CC16⁺ cells with the anti-C/EBPα antibody. Interaction sites are identified by PCR for two possible C/EBPα binding sites in the Munc18b promoter. (C) Munc18b promoter with wild-type (WT) or mutation of the binding motif 1 or 2, and C/EBPα plasmid are co-transfected into CC16⁺ cells used in the luciferase assay. ***P < 0.001 vs. pcDNA-ctrl or WT group.

DEP induces bronchial epithelial cell autophagy and inflammation

DEP has been widely reported to induce bronchial epithelial cell injury by regulation of autophagy, inflammation, oxidative stress, and apoptosis [27–30]. Here, we investigated the effects of DEP on autophagy and inflammation of BEAS-2B bronchial epithelial cells. LC3 and p62 are two proteins vital for autophagy. When autophagy is activated, soluble LC3-I is converted into lipid bound LC3-II, followed by being recruited to autophagosomal membranes, and thus the amount of LC3-II positively correlates with the number of autophagosomes. Furthermore, autophagosome formation enhances the autophagic flux, thereby accelerating p62 protein degradation. As evidenced by Figure 5A, DEP increased the ratio of LC3-II/LC3-I, and decreased p62 protein expression, which suggested that DEP treatment activated autophagy. DEP-activated autophagy was further reinforced by the autophagy promoter rapamycin (Rap), and relieved by the autophagy inhibitor 3-Methyladenine (3-MA). In addition, autophagosomes were identified as bright green dots by IF. The co-treatment group with Rap and DEP had the largest number of green spots, followed by DEP group, DEP+3-MA group, and ctrl group (Figure 5B). The effects of DEP exposure on pro-inflammatory cytokines were also detected. The results indicated that DEP exposure increased TNF-α, IL-6, and IL-8 level, which were relieved by Rap treatment, and aggravated by 3-MA treatment (Figure 5C–5E). These data suggested that enhancing autophagy can mitigate DEP-induced inflammation in BEAS-2B cells.

Figure 5. DEP induces bronchial epithelial cell autophagy and inflammation. BEAS-2B cells are pre-treated with 1.5 mM 3-MA or 10 nM Rap for 1 h, and then exposed to 5 ppm SRM 1650b. After 12 h, the cells and culture supernatants are collected. (A) Autophagy-related proteins LC3B and p62 expression in BEAS-2B cells are detected by western blot. (B) Autophagy induction is evaluated by LC3B immunostaining (× 400). Autophagosomes are identified as bright green dots. (C–E) Pro-inflammatory cytokines TNF-α, IL-6, and IL-8 level are detected by ELISA. ***P < 0.001 vs. ctrl; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. DEP group.

CC16 secretion from CC16⁺ cells protects against DEP-induced bronchial epithelial cell inflammation

To investigate the effects of CC16 secretion from CC16⁺ cells on DEP-induced bronchial epithelial cell inflammation, CC16+ cells were cultured in DMEM/F12 medium with 5 ppm SRM 1650b or not to prepare CC16⁺ medium and DEP-CC16⁺ medium, respectively. After determining CC16 level, CC16⁺ cell conditioned medium were used to culture BEAS-2B cells. The CC16 level within the medium of ctrl, DEP, DEP+CC16⁺, DEP+DEP-CC16⁺ group was 0, 0, 144.7 and 46.5 ng/ml, respectively. Following exposure to DEP, autophagy and inflammation were detected. As shown in Figure 6, DEP+CC16⁺ group has the strongest autophagy activity, followed by DEP+DEP-CC16⁺ group, DEP group, and ctrl group. As expected, DEP induced the weakest inflammatory response in DEP+CC16⁺ group, followed by DEP+DEP-CC16⁺ group, and DEP group. CC16 level was positively correlated with DEP-induced autophagy activity, and negatively correlated with DEP-induced inflammatory response, suggesting that CC16 might mitigate DEP-induced inflammation via promoting autophagy in BEAS-2B cells. In order to further confirm that CC16⁺ cell conditioned medium functions in DEP-induced autophagy and inflammation mainly through CC16, BEAS-2B cells were incubated with 5 ppm SRM 1650b, or 300 ng/ml recombinant CC16 (rCC16)+5 ppm SRM 1650b for 12 h. The results showed that rCC16 increased DEP-induced autophagy and inhibited DEP-induced inflammation (Figure 7). Collectively, these data indicated that CC16 protects against DEP-induced bronchial epithelial cell inflammation via regulation of autophagy, and DEP-inhibited CC16 secretion from Clara cells exerts a decreased protection role in DEP-induced bronchial epithelial cell inflammation.

Figure 6. CC16 secretion from CC16⁺ cells protects against DEP-induced bronchial epithelial cell inflammation via activation of autophagy. CC16⁺ cell conditioned medium containing different concentrations of CC16 is prepared, and used to culture BEAS-2B cells. The CC16 level in the medium of Ctrl, DEP, DEP+ CC16⁺, DEP+ DEP-CC16⁺ group is 0, 0, 144.7 and 46.5 ng/ml, respectively. After 12 h of culture, the cells and culture supernatants are collected. (A) LC3B and p62 protein expression in BEAS-2B cells are detected by western blot. (B) Autophagy induction is evaluated by LC3B immunostaining (× 400). Autophagosomes are identified as bright green dots. (C–E) TNF-α, IL-6, and IL-8 level in culture supernatants are detected by ELISA. ***P < 0.001 vs. ctrl; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. DEP group; and ^&P < 0.05, ^&&P < 0.01, and ^&&&P < 0.001 vs. DEP+ CC16⁺ group.

Figure 7. rCC16 relieves DEP-induced bronchial epithelial cell inflammation via activation of autophagy. BEAS-2B cells are incubated with 5 ppm SRM 1650b, or 300 ng/ml rCC16 + 5 ppm SRM 1650b for 12 h. Then the cells and culture supernatants are harvested. (A) LC3B and p62 protein expression in BEAS-2B cells are detected by western blot. (B) Autophagy induction is evaluated by LC3B immunostaining (× 400). Autophagosomes are identified as bright green dots. (C–E) TNF-α, IL-6, and IL-8 level in culture supernatants are detected by ELISA. ***P < 0.001 vs. ctrl; and #P < 0.05 and ###P < 0.001 vs. DEP group.

TNF-α released by BEAS-2B cells induces CC16 production and secretion in CC16⁺ cells

TNF-α plays a role in stimulating CC16 production [26]. Herein, we investigated whether BEAS-2B cell conditioned medium stimulates CC16 production and secretion mainly via TNF-α. As shown in Figure 8A, 8B, CC16 mRNA expression and level in culture supernatants were significantly increased when CC16⁺ cells were cultured in BEAS-2B cell conditioned medium containing 0.81 ng/ml TNF-α or the normal medium supplemented with 3 ng/ml recombinant TNF-α (rTNF-α). These data suggested that CC16 and TNF-α form a negative feedback loop: CC16 secreted by CC16⁺ cells inhibits TNF-α production in BEAS-2B cells, and TNF-α stimulates CC16 production and secretion, thereby protecting against DEP exposure-induced inflammation (Figure 8C).

Figure 8. TNF-α released by BEAS-2B cells induces CC16 production and secretion from CC16⁺ cells. BEAS-2B cell conditioned medium (DEP-BEAS-2B) is prepared, and used to culture CC16⁺ cells. The TNF-α level in the culture medium of ctrl, DEP-BEAS-2B, and rTNF-α group is 0, 0.81 and 3 ng/ml, respectively. CC16⁺ cells are cultured in these media for 48 h. Then CC16 mRNA expression (A) and level in culture supernatants (B) are detected by RT-PCR and ELISA assay. (C) CC16-TNF-α negative feedback loop between Clara cells and normal airway epithelial cells protects against DEP exposure-induced injury. Red upside down T line drawing indicates inhibitive action. Blue line with arrowhead drawing indicates driving role. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. Ctrl.

Preparation of single-cell suspensions from human distal lung

All human lung tissues were obtained from patients who underwent pulmonary resection at the Qingdao Municipal Hospital. All patients provided informed consent, and the study was approved by the Qingdao Municipal Hospital Ethics Committee. Single cell suspensions of the human distal lung were prepared as previously described, with some modifications [39]. Briefly, human distant normal lung tissues were cut into small pieces, and incubated with DNase I (Sigma-Aldrich, St Louis, MO, USA; final concentration, 0.1 mg/ml), and collagenase (Roche Applied Science, Penzberg, Germany; final concentration, 3 mg/ml) for 1 h at 37° C with continuous agitation. Following incubation with 0.25% trypsin (Sigma-Aldrich) for 10 min, the suspensions were further disaggregated by trituration through needles (Sherwood Medical Co, St Louis, MO, USA), and filtered with a 100-μm mesh filter (BD Biosciences, San Jose, CA, USA). After washing with PBS, cells were treated with red blood cell lysis buffer (eBioscience, San Diego, CA, USA), filtered through a 40-μm mesh filter (BD Biosciences), resuspended in DMEM/10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) with 1% amino acid solution (Invitrogen), 2.5 mg/ml amphotericin B (Sigma-Aldrich), 100 U/ml penicillin, and 100 μg/ml streptomycin, and placed to recover in a humidified incubator at 37° C with 5% CO₂ for 24 h.

Flow cytometry

CC16-expressing cells (Clara cells) were obtained by FACS as previously described [39]. Briefly, the recovered cells were trypsinized, and resuspended into PBS with 3% FBS. Following incubation with an anti-rabbit CC16 antibody (Millipore, Billerica, MA, USA), an anti-rabbit-FITC secondary antibody was added and further incubated on ice for 30 min. After washing in PBS with 3% FBS, the cells were resuspended in the fresh medium. Isotype-matched rabbit IgG staining was used as a negative control, and CC16 staining with permeabilization of dissociated cells was used as a positive control. The cell fractions with minimal (CC16^- fraction) and strong (CC16⁺ fraction) expression of CC16 were obtained by a FACS Vantage SE cell sorter (Becton Dickinson, San Jose, CA, USA), and then examined by RT-PCR, western blot and IF.

Immunofluorescence

Cells were fixed in 4% paraformaldehyde for 10 min at room temperature (RT), and permeabilized with 0.1% Triton X-100 for 10 min at RT. After blocking with 5% bovine serum albumin (BSA; Sigma-Aldrich) for 30 min, immunolabelling was performed via incubation of the cells with primary antibody CC16 (1:50, Santa Cruz Biotech, Santa Cruz, CA, USA), pan-keratin (1:100, Dako, Carpinteria, CA, USA), or LC3B (1:100, Cell Signaling Technology, Beverly, MA, USA) for 1 h in a humid chamber at RT, and subsequent incubation with secondary antibodies conjugated with Alexa Fluor 488 or 546 (Invitrogen) for 1 h. After washing three times in PBS, slides were mounted with an anti-fading reagent with 4’,6-diamidine-2-phenylindole (DAPI) (Invitrogen), air dried in the dark, and then observed under a fluorescence microscope (Olympus, Hamburg, Germany).

RT-PCR

Total RNA was extracted from the cultured cells using Trizol reagent (Invitrogen) in accordance with the instructions of the manufacturer. The cDNA synthesis was performed by using Reverse Transcription Kit (Takara, Dalian, China), and then PCR was performed on SmartCycler® II System (Cepheid Inc., Sunnyvale, CA, USA). GAPDH was used as a reference. The relative quantification of transcripts was calculated by using the 2^-ΔΔCt method.

Western blot analysis

Western blot assay was conducted as previously described [40]. The primary antibodies against pan-keratin (Abcam, Cambridge, MA, USA), CC16 (Millipore), C/EBPα (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Munc18b (Abcam), LC3B (Abcam), p62 (Abcam), and GAPDH (Sigma-Aldrich), and horseradish peroxidase-conjugated secondary antibody (Abcam) were employed in this assay.

ELISA

The cells were cultured under the indicated conditions, and culture supernatants were collected used for IL-6, IL-8, TNF-α and CC16 level determination. The levels of the cytokines IL-6, IL-8, and TNF-α were determined by the ELISA kits purchased from Bender Medsystems (Burlingame, CA, USA), and CC16 level was detected by the ELISA kit obtained from BioVendor (Brno, Czech Republic).

Methylation-specific polymerase chain reaction

Genomic DNA was isolated from cultured cells using standard methods. Then DNA was modified with bisulfite by using CpGenome DNA modification kit (Intergen, Burlington, MA, USA), and then bisulfite-modified DNA (50 ng) was amplified in duplicate by MSP as described previously [41, 42]. The primer sequences for amplification were as follows: the methylated C/EBPα, sense primer: 5’-GTCGGGTATAAAAGTTGGGTCGGC-3’, and antisense primer: 5’-ATTCTCCCGACATAACGAACCTCG-3’; and unmethylated C/EBPα: sense primer: 5’-TTGTTGGGTATAAAAGTTGGGTTGGT-3’, and antisense primer: 5’-AAAATTCTCCCAACATAACAAACCTCA-3’. PCR products were subsequently separated by gel electrophoresis and visualized by ethidium bromide (EB; Sigma-Aldrich) staining.

CC16⁺ cell culture and treatment

Isolated CC16⁺ cells were cultured in the serum-free DMEM/F12 medium supplemented with 20 ng/ml EGF (Invitrogen), bFGF (Invitrogen) and gentamicin (Sigma-Aldrich), 1 μl/ml bovine hypothalamus extract (PromoCell, Corning, NY, USA), 5 μg/ml transferrin (Roche Applied Science, Indianapolis, IN, USA) and insulin (RIA, Linco Research, St Charles, MO, USA), 1 mM retinol (Sigma-Aldrich) and hydrocortisone (Sigma-Aldrich), 1% penicillin-streptomycin (Invitrogen), and 1 ng/ml amphotericin B.

To assess whether DEP regulates CC16 production and secretion in CC16⁺ cells via induction of C/EBPα hypermethylation, CC16⁺ cells were treated with 5 ppm SRM 1650b (the National Institute of Standards and Technology, Gaithersburg, MD, USA) in the presence of DAC (Sigma-Aldrich) or not for 48 h. Then MSP, RT-PCR, western blot and ELISA were performed to detect the methylation of C/EBPα, the mRNA and protein expression of C/EBPα, CC16 mRNA expression, and CC16 level in culture supernatants, respectively. Additionally, we also determined the effects of treatment with other concentrations of SRM 1650b (0.1, 1, 3 and 10 ppm) on C/EBPα mRNA and protein expression, CC16 mRNA expression and level in culture supernatants.

To further investigate whether C/EBPα regulates the secretion of CC16 from CC16⁺ cells via Munc18b, CC16⁺ cells were transfected with the specific small-interfering RNAs targeting C/EBPα (si-C/EBPα), si-Munc18b, pcDNA-C/EBPα, pcDNA-CC16 or negative control alone or in combination using Lipofectamine 2000 (Invitrogen). The sequence fragment of C/EBPα or CC16 was amplified, sequenced, inserted into pcDNA3.1 vector (Invitrogen), and finally named as pcDNA-C/EBPα or pcDNA-CC16. si-C/EBPα, si-Munc18b and scrambled siRNA were synthesized by Sangon Biotechnology, Co., Ltd. (Shanghai, China). After transfection, the cells were collected used for determination of Munc18b mRNA and protein expression, and CC16 mRNA expression and secretion via RT-PCR, western blot and ELISA.

To investigate the effects of BEAS-2B cells treated with DEP on CC16 secretion from CC16⁺ cells, a BEAS-2B cell conditioned medium was prepared used for CC16⁺ cell culture. In addition, to determinate whether the BEAS-2B cell conditioned medium functions via TNF-α, CC16⁺ cells were treated with human rTNF-α (PeproTech, Rocky Hill, NJ, USA) for 48 h. Then CC16 mRNA expression in the cells and protein level in culture supernatants were determined.

Dual-luciferase reporter assay

To investigate whether C/EBPα regulates Munc18b expression via binding to the promoter of Munc18b, the upstream regions (-2000/200) of Munc18b gene with WT or mutation of the binding motifs 1 or 2 with C/EBPα were amplified and subcloned into the pGL3-Basic vector by GenePharma (Shanghai, China) to generate Munc18b promoter constructs. The cells were co-transfected with Munc18b promoter construct or empty pGL3-basic vector and C/EBPα plasmid or its corresponding empty vector. The cells were then lysed and analyzed with Dual luciferase reporter assay system (Promega, WI, USA).

Preparation of CC16⁺ or BEAS-2B cell conditioned medium

CC16⁺ cells were cultured in DMEM/F12 medium containing 5 ppm SRM 1650b or not for 48 h. Then the medium was harvested, cleared by centrifugation, diluted 1:1 with F12 medium, stored at 4° C and named as CC16⁺ medium and DEP-CC16⁺ medium. As well, the BEAS-2B cell conditioned medium was prepared.

BEAS-2B cell culture and treatment

A human bronchial epithelial cell line BEAS-2B (American Type Culture Collection, Manassas, VA, USA) was cultured in hormonally defined Ham’s F12 medium (HD-F12) supplemented with 1% penicillin-streptomycin, 5 μg/ml insulin and transferrin, 25 ng/ml EGF, 15 μg/ml endothelial cell growth supplement (Collaborative Research, Inc., Bedford, MA, USA), 200 pM triiodothyronine (Gibco, Carlsbad, CA, USA), and 1 mM hydrocortisone.

To investigate whether DEP induces bronchial epithelial cell inflammation via autophagy, BEAS-2B cells were pre-treated with 1.5 mM 3-MA (Sigma-Aldrich) or 10 nM Rap (Sigma-Aldrich) for 1 h, and then exposed to 5 ppm SRM 1650b for 12 h. Western blot, IF, and ELISA were then applied to detect the autophagy-related proteins LC3B and p62 expression, and pro-inflammatory cytokines TNF-a, IL-6, and IL-8 level in culture supernatants.

To investigate whether CC16 secretion from CC16⁺ cells protects against DEP-induced bronchial epithelial cell inflammation, BEAS-2B cells were divided into four groups and cultured with different medium for 12 h, including ctrl group (equiproportional mixing of fresh DMEM/F12 and HD-F12), DEP group (the same medium with ctrl group but containing 5 ppm SRM 1650b), DEP+CC16⁺ group (CC16⁺ medium containing 5 ppm SRM 1650b), and DEP+DEP-CC16⁺ group (DEP-CC16⁺ medium containing 5 ppm SRM 1650b). Next, the expression of p62 and LC3B protein, and the levels of TNF-a, IL-6, and IL-8 level were determined by western blot, IF, and ELISA, respectively.

For further confirmation the protection role of CC16 against DEP-induced bronchial epithelial cell inflammation, BEAS-2B cells were exposed to 5 ppm SRM 1650b in the presence of rCC16 (prepared by our laboratory) or not for 12 h. Then autophagy and inflammation were determined.

Chromatin immunoprecipitation

ChIP assay was conducted by using a ChIP assay Kit (Upstate Biotechnology, Billerica, MA, USA) as per the manufacturer’s instructions. Briefly, the cells were harvested, fixed with 1% formaldehyde for 10 min at RT, and resuspended in lysis buffer containing a protease inhibitor cocktail. Following sonication, the soluble chromatin was diluted with ChIP-dilution buffer and immunoprecipitated with C/EBPα antibody or IgG as a negative control overnight at 4° C. Then the chromatin-antibody complexes were eluted with the elution buffer. After reversion of the cross-link, the DNA was purified by Qiaquick PCR product Purification Kit (QIAGEN Inc, Chatsworth, CA, USA). End-point PCR was conducted to amplify the site 1 and 2 (two potential binding motifs for C/EBPα) by using primers spanning the C/EBPα binding elements in the promoter region of the Munc18b gene.

Statistical analysis

All statistical analyses were performed with GraphPad Prism 8. The data were presented as the mean ± standard deviation (SD) from at least three independent experiments. The comparisons between groups were done using the Student’s t test or ANOVA, and P < 0.05 was considered to be statistically significant.