Cell culture

Human A549 pulmonary epithelial cells (ATCC, Manassas, VA, USA) (the cell line was characterized as being representative of the alveolar type II pneumocytes of the human lung) and CCD25 human pulmonary fibroblasts (ATCC, Manassas, VA, USA) were cultivated in DMEM (Gibco Life Technologies, Grand Island, NY, USA) and EMEM (Lonza, Alpharetta, GA, USA), respectively; both media were supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, Grand Island, NY, USA) and antibiotic-antimycotic (100×) (Gibco Life Technologies).

Stimulation of A549 cells with CSE

CSE was prepared using a modification of the method described by Liu (^{Liu, Gao & Zhang., 2010}). Briefly, one filterless commercial cigarette (^{Oldham, DeSoi, Rimmer, Wagner & Morton, 2014}) (Marlboro, Philip Morris, Richmond, VA, USA) was burned using a modified syringe-driven apparatus. Mainstream smoke was generated from one cigarette by drawing consecutive puffs into a 20 mL plastic syringe, with a stopcock connected through one port to a glass containing 25 mL of serum-free DMEM-F12 (Gibco Life Technologies).

A 20-mL puff drawn in 1 second was obtained in 10-second intervals; each puff was held for 3 seconds and then bubbled through DMEM for 5 seconds. One cigarette yielded an average of 45 puffs by this procedure. The resulting suspension was adjusted to pH 7.4 with concentrated NaOH and filtered through a 0.2-µm-pore filter to remove bacteria and large particles (Barnant, Barrington, IL, USA). The resulting solution was designated a 100% CSE solution. The CSE solutions were prepared by the same person using the same method and were used within 30 minutes of preparation. This stock was diluted to 5% in DMEM, supplemented with 1% FBS and penicillin-streptomycin (Gibco Life Technologies). Exposure of A549 epithelial cells to CSE was standardized to 6.72 × 10⁶ cells in T75 culture flasks (Merck KGaA, Darmstadt, Germany). The medium was changed every 24 h until one week was completed. A549 cells were stimulated with 5% CSE for 6 days.

CCL2 transfection in A549 cells

The overexpression of CCL2 was achieved through stable transfection with the entry vector pCMV6 (Origene, Rockville, MD, USA), which has a TrueORF with a Myc label and a DDK sequence in the carboxyl-terminal region. According to the company’s instructions, the plasmid was amplified in BL21 (DE3) chemically competent Escherichia coli cells (Merck KGaA) and then inserted using heat shock. The transfected bacteria were kanamycin resistant (Merck KGaA). The plasmid was isolated with a purification kit (QIAGEN, Germantown, MD, USA) according to the manufacturer’s instructions. The CCL2 plasmid was inserted into A549 cells with turbofectin (Origene; 90 µL) in a 3:1 ratio with DNA. Cells were incubated with antibiotic G418 (Merck KGaA; 800 µg/mL).

Knockdown of CCL2 in A549 cells

A549 cells were seeded in 12-well culture plates (Merck KGaA) with 7.5 × 10⁵ cells in F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS. The infection was done according to the instructions of the company, with a mixture of polybrene (Santa Cruz Biotechnology, Dallas, TX, USA; 5 µg/mL) and MCP-1 shRNA-(h) lentiviral particles (Santa Cruz Biotechnology; 15 µL), and this mixture was applied to A549 cells. The infection efficiency was evaluated using a positive control of lentiviral particles with a green fluorescent protein (GFP; Santa Cruz Biotechnology; 0.1 µg/µl). Serial dilutions were made to obtain stable clones that were selected with puromycin (Santa Cruz Biotechnology; 7.5 µg/mL). Plasmid-A (Santa Cruz Biotechnology; 0.1 µg/µl) was used as a negative control, and we denoted it as the scramble control. The knockdown of CCL2 in transfected cells was evaluated by real-time PCR and Western blotting using CCL2 antibody.

CCL2 recombinant Protein Stimulation to CCL2 Knockdown A549 Cells

A549 cells were seeded in 6-well culture plates (Merck KGaA) with 2 × 10⁵ cells in F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS and 3 mL of human CCL2 (100 ng/ml, R&D Systems) was added to the culture of CCL2 knockdown cells, for 12 hours.

Fibroblast stimulation

CCD25 fibroblasts were cultured (1x10⁵) in a 12-well dish and stimulated with a conditioned medium from CSE-stimulated A549, CSE-stimulated and CCL2-inhibited A549, CCL2-transfected A549 cells, and empty vector, stimulated for 48 hours.

RNA extraction and real-time PCR

The mRNA of A549 cells and CCD25 fibroblasts was extracted with Trizol (Merck KGaA), and 1 µg of RNA was reverse transcribed according to the instructions of the company, yielding a cDNA chain (Advantage RT-for PCR kit; Clontech, Palo Alto, CA, USA). For real-time PCR, the Step-One system thermocycler (Applied Biosystems, Foster City, CA, USA) was used, with a Taqman probe labeled with fluorescein amidites (FAM), the expression of CCL2 (Thermo Fisher Scientific, Waltham, MA, USA), and TGFB1 (Thermo Fisher Scientific) in A549 cells was evaluated. In fibroblasts stimulated, we evaluated the expression of IL6, ACTA2, and POLR2A (all from Thermo Fisher Scientific). All PCRs were done in a 15-µL volume containing 2 µl of cDNA and 13 µl of 2× Mastermix (Thermo Fisher Scientific). The PCR conditions were 2 minutes at 94 °C and 60 °C for 1 minute. The results are expressed as means ± SD of the 2^-ΔCT (delta of cycle threshold of the gene of interest, normalized with a housekeeping gene, POLR2A).

Proliferation analysis with WST-1

The proliferation rate was evaluated in the WST-1 assay (Merck KGaA) during 1 hour of incubation. According to the methods of the manufacturers and Van der Poel (^{Van Der Poel, 2004}) A549 cells at a concentration of 1×10³ cells/well were incubated in 96-well microplates, in a final culture medium volume of 100 µL/well. Cells were incubated for 6 days with CSE, at 37 °C and 5% CO₂. An amount of 10 µL/well of WST-1 was added and incubated for 1 hour. The absorbance was analyzed in an ELISA reader (Biotek, Winooski, VT, USA) with Gen 53.0 software at 450 nm with a reference of 620 nm. The same method was applied for transfected and inhibited A549 cells.

To verify the same number of live cells, the method with trypan blue was used, for this, A549 cells were trypsinized and mixed with trypan blue solution according to Louis and Siegel (^{Louis & Siegel, 2011}). Briefly, 0.4% trypan solution was prepared in 0.81% sodium chloride and 0.06% dibasic potassium phosphate (Merck KGaA). A 1:1 mixture was made of the cell suspension (1×10⁶ cells/mL) and the 0.4% trypan blue solution. Finally, the mixture was transferred to an automated cell counter (Invitrogen, Carlsbad, CA, USA).

Wound healing assay

A549 cells at 9 × 10⁵ cell/well density were seeded into six-well plates and incubated in DMEM with 1% FBS. After overnight culture, the A549 cells were scratched with 200 µL micropipette tips to form straight lines. The floating cells were removed using 1× PBS three times. To further reduce the risk of cell proliferation confounding the study of migration, mitomycin C (Merck KGaA; 3 µg/mL) was added every 24 h. The wound closure was monitored at 0, 24, 48, and 72 h, and it was photographed under an inverted microscope (Olympus, Richmond Hill, Ontario, Canada). The recolonized area was calculated by manually tracing the cell-free area in captured images using the ImageJ public domain software (NIH, Bethesda, MD, USA). The migration rate was expressed as the percentage of wound closure (% Wound closure = [(A_t=0h - A_t=Δh)/A_t=0h] × 100%) according to Ayman et al (^{Grada, Otero-Vinas, Prieto-Castrillo, Obagi & Falanga, 2017}).

ELISA

Protein levels of CCL2 and TGF-β1 were evaluated in the conditioned medium of CCL2-overexpressing A549 cells and CCL2 knockdown A549 cells with an ELISA kit (R&D Systems, Minneapolis, MN, USA). Per the manufacturer’s directions, 200 µL of the medium was used, the ELISA plate was incubated for 2 hours at room temperature, the plate was washed four times, a conjugated antibody was added, and the mixture was incubated for 1 hour at room temperature (the antibody is polyclonal, binds specifically to CCL2, and is coupled to horseradish peroxidase [HRP]). A 200-µL volume of substrate solution was used, which contained hydrogen peroxide and a stabilizing chromogen (tetramethylbenzidine); this solution was incubated for 30 minutes, and the reaction was stopped with sulfuric acid, 2 N. The samples were read in a spectrophotometer at 450 nm (Biotek plate reader), using the Gen 5 software (Biotek). We added 300 ng of CCL2 recombinant protein (R&D Systems) to knockdown CCL2 in A549 cells, and then TGF-β1 protein levels were evaluated.

Western blot

Conditioned medium from A549 epithelial cells not expressing CCL2 was concentrated with filtration units (Merck KGaA). The proteins obtained were quantified using the Bradford method (Bradford assay reagent; Bio-Rad Laboratories, Hercules, CA, USA), adjusting to 30 µg. Equal amounts of protein from each sample and a molecular weight marker (Precision Plus Protein Dual Xtra Standards, Bio-Rad) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was exposed to a blocking solution overnight at 8 °C. An anti-CCL2 monoclonal antibody (R&D Systems) was used at a 1:1000 dilution. The production of TGF-β1 (Cell Signaling Danvers, MA, USA) was detected at a 1:1000 dilution after incubation for 1 h 30 minutes. This primary antibody incubation was followed by incubation with HRP-conjugated anti-rabbit or anti-mouse antibody (both from Invitrogen, Carlsbad, CA, USA; used at 1:20,000 dilution) as the secondary antibody for 1 hour at room temperature. The chemiluminescence was detected using the Super Signal West Femto maximum sensitivity substrate developer solution (Thermo Fisher Scientific) and read on a transilluminator ChemiDoc XRS+ (Bio-Rad). To process the images, we used Image Lab software (Bio-Rad). Ponceau S solution was used as a loading control (Merck KGaA).

Cell migration and invasion assay

The fibroblast transmigration assay was performed in QCM Haptotaxis cell migration assay-collagen I, 24 -well, colorimetric plates (Merck KGaA). A density of 2×10⁵ cells/ well was treated with an anti-CCL2 antibody (R&D Systems; 5 µg/mL) or without any treatment and suspended in 200 µL of serum-free DMEM containing 5% bovine serum albumin (BSA) and placed onto the top of chambers (Merck KGaA), and the lower chambers were filled with 600 µL of medium from A549 cells that overexpress CCL2 in DMEM with 5% BSA, or medium from A549 cells that had empty vector or platelet-derived growth factor (PDGF; PeproTech, Cranbury, NJ, USA; 50 ng/mL) as a positive control (is a migration inducer) . After 12 h, non migratory CCD25 cells were removed with a cotton swab from the top surface of the chamber, and the lower filter of the chamber was stained with crystal violet solution for 20 min at 37 °C. The upper insert was dipped into a beaker of water several times to rinse. The dry stained inserts were transferred in a clean well containing 200 µL of extraction buffer (acetic acid, 1 N) for 15 min at room temperature. Finally, 100 µL of the dye mixture was transferred to a 96- well microtiter plate suitable for colorimetric measurement. The optical density was read in a BioTek plate reader at 560 nm.

Statistical analysis

Three tests were performed independently, each in triplicate. Data from all experiments were analyzed by using Prism 8.3.0 software (GraphPad, San Diego, CA, USA) and are shown as the arithmetic means ± SD. The multiple two-tailed Student’s t-test was performed to assess the differences between experimental groups and controls over time. One-way analysis of variance (ANOVA) followed by a Tukey post hoc test was used to analyze differences between groups for multiple comparisons. The level of significance was set at p<0.05, and a high level of significance was set at p<0.01.

CSE increases CCL2 expression and proliferation in A549 pulmonary AECs

It has been described that A549 alveolar epithelial cells are metabolically very active, which is reflected in a high production of cytokines and growth factors. For this reason, we decided to evaluate if the expression of CCL2 is increased when stimulated with CSE.

An increase in the expression and production of CCL2 was confirmed under CSE exposure from the second day of treatment (Figure 1A-B). CCL2 levels were up-regulated on day 4. On day 6, there was a high expression of CCL2. Simultaneously, we evaluated the cytotoxic effect of CSE on the proliferation of A549 cells in a WST-1 assay (Figure 1C), we observe that on day 6 under the stimulus of CSE there was an increase in proliferation.

Figure 1 Effects of CSE on CCL2 expression and synthesis in A549 cells. A) Real- time PCR showing CCL2 gene expression in A549 lung epithelial cells stimulated with 5% CSE for 2, 4, and 6 days. B) CCL2 levels in the supernatant were quantified by ELISA. C) The WST-1 assay, in which we evaluated the proliferation of control A549 cells and cells stimulated with 5% CSE (** p˂0.01 against the control without treatment). 

CCL2 modulates the migration of A549 cells

We examined if the expression of CCL2 has any role in the migration of AECs. Real-time PCR and ELISA (Figure 2A-B) revealed that CCL2 was effectively overexpressed in A549 cells and was increased compared with empty vector and control A549 cells. Then, the efficiency of the inhibition of CCL2 was confirmed; notable decreases in the expression and synthesis of CCL2 were observed compared to the control cells and the scramble (negative) control (Figure 2C-D). The scramble control allows the comparison of knockdown with no transduced cells with a huge variable (virus infection), and this scramble will inevitably induce transcriptional programs that differ from no transduced cells. This explains why scramble cells overexpress the CCL2 gene and protein synthesis.

Figure 2 Modulation of CCL2 expression. A) Real-time PCR in cells that overexpress CCL2. B) The content in the supernatant shows the transfection efficiency quantified by ELISA. C) Real-time PCR of expression by CCL2 knockdown A549 cells. D) CCL2 content in CCL2 knockdown A549 cell supernatant. Ctrl: Control; CCL2: Cells that overexpress CCL2; EV: Empty vector; shCCL2: CCL2 knockdown A549 cells; Scr: scramble control (** p˂0.01 against no-treatment control). 

Subsequently, the wound assay showed that control cells and cells with an empty vector moved slightly, while overexpression of CCL2 induced a significant increase in the migration rate: cells reached near the middle of the scratch, indicating that CCL2 caused more mesenchymal-like characteristics and markedly promoted A549 cell mobility (Figure 3A-B). Additionally, the wound assay showed that CCL2 inhibition is associated with the rate of tissue repair being slower.

Figure 3 Evaluation of the rate of wound closure in A549 cells. A) Wound-healing assay in A549 cells that overexpress CCL2 and in CCL2 knockdown A549 cells. B) Graphical representation of the area covered by cells in the wound assay (** p˂0.01). 

The expression of CCL2 affects the proliferation of human AECs in A549 cells

To determine the role of CCL2 in cell proliferation, an assay with WST-1 was performed where cells that overexpress CCL2 (or not) were evaluated. These experiments revealed that proliferation is partially mediated by CCL2. Figure 4A details the 50% increase in the proliferation of CCL2-overexpressing A549 cells, and this effect remained until day 6 (compared to all conditions). For A549 CCL2 knockdown cells, on day 4, an increase in cell proliferation was observed compared to control cells and scramble cells, and there was a significant decrease compared to cells that overexpress CCL2, which was maintained until day 6.

Figure 4 Assessment of proliferation and expression of TGFB1 in CCL2-transfected A549 cells. A) WST-1 assay that shows epithelial cell proliferation. B) Real-time PCR showing TGFB1 expression in A549 cells that overexpress CCL2. C) ELISA of total TGF-β1 in A549 cells that overexpress CCL2. D) Western blot showing the production of TGF-β1 and CCL2 in conditioned medium of CCL2 knockdown A549 cells; a red ponceau blot (RPB) was used as a loading control. E) Real-time PCR denoting TGFB1 expression in CCL2 knockdown A549 cells. F) ELISA of TGF-β1 in A549 cells (** p˂0.01). Ctrl: Control, CCL2: Cells that overexpress CCL2; EV: Empty vector; shCCL22: CCL2 knockdown A549 cells; ShCCL2 + RecCCL2: Addition of recombinant CCL2 protein to CCL2 knockdown A549 cells; Scr: Scramble cells as a negative control. 

CCL2 modulates TGF-β1 production in human AECs

Several studies have identified CCL2 as a potent profibrotic mediator (^{Yang et al., 2020}); however, it is unknown how it induces these effects. To evaluate this, the expression of TGF-β1 mRNA and protein were measured in CCL2-overexpressing A549 cells (Figure 4B-C), and significant increases in its expression and synthesis were registered. Adecrease in TGF-β1 protein and mRNA levels in A549 CCL2 knockdown cell supernatants compared to control cells and scramble was detected (Fig. 4D-E). The ELISA showed that when recombinant CCL2 protein was added to the culture of CCL2 knockdown cells, there was a significant induction of TGF-β1 synthesis compared to cells that do not express CCL2 (Figure 4F).

Conditioned medium from CCL2-overexpressingA549cells modulates the expression of profibrotic genes in pulmonary fibroblasts

Epithelial and interstitial cells are important in the lung microenvironment, particularly fibroblasts, which are involved in the production of extracellular matrix components (^{Kuhn & Mcdonald, 1991}). Pulmonary fibroblasts stimulated with supernatants of A549 cells stimulated with CSE, or CCL2-overexpressing A549 cells showed a significant increase in the expression of TGFB1 compared with the control and the empty vector (Figure 5A). The ACTA2 gene, a myofibroblast marker, also indicated marked overexpression under these conditions compared to the control and the empty vector (Figure 5B).

Figure 5 The response of pulmonary fibroblasts to the conditioned medium of A549 epithelial cells exposed to CSE or transfected with CCL2. A) Real-time PCR of TGFB1 in fibroblasts. B) Real-time PCR of ACTA2 in fibroblasts. C) Real-time PCR of IL6 in fibroblasts (** p˂0.01). Ctrl: Control cells; Fib + A549: Fibroblasts stimulated with conditioned medium from A549 cells exposed to CSE; Fib + shCCL2: Fibroblasts stimulated with conditioned medium from A549 cells that do not express CCL2 and that were exposed to CSE; Fib + A549 CCL2: Fibroblasts stimulated with conditioned medium from A549 cells that overexpress CCL2; Fib + EV: Fibroblasts exposed to conditioned medium of cells A549 transfected with the empty vector. 

In contrast, on stimulation with the supernatants of A549 CCL2 knockdown and CSE cells, there was no increase in the expression of TGFB1 and ACTA2. Regarding the expression of IL6 (Figure 5C), an increase in its expression was observed in fibroblasts stimulated with A549 medium, which overexpress CCL2. We also observed a decrease in fibroblasts stimulated with medium from A549 cells that do not express CCL2, even when stimulated with CSE.

Pulmonary epithelial cells modulate the migratory fibroblast phenotype through CCL2

To examine the migratory capacity of pulmonary fibroblasts in response to CCL2 from lung epithelial cells, fibroblasts were treated with a medium from A549 cells that overexpress CCL2. Compared with the control and the empty vector, a significant increase in migration was detected (Figure 6). In contrast, anti-CCL2 antibody treatment caused decreased fibroblast migration, compared to cells stimulated with empty vector medium and control fibroblasts.

Figure 6 Assay in Boyden chambers in pulmonary fibroblasts (** p˂0.01). Ctrl: Control cells; Fib + AbCCL2: Fibroblasts stimulated with the antibody against CCL2; Fib + A549 CCL2: Fibroblasts stimulated with conditioned medium from A549 cells that overexpress CCL2; Fib + EV: Fibroblasts exposed to conditioned medium of cells A549 transfected with the empty vector; PDGF: Fibroblasts stimulated with PDGF as positive control.