Bone morphogenetic protein 4 inhibits liposaccharide-induced inflammation in the airway

Inhalation of LPS increases BMP4 expression in mouse lung

Lung inflammation was induced by i.n. inhalation of LPS at 7.5 μg/mouse, and evaluated at 12, 24, and 48 h post-LPS challenge. In LPS-treated mice, remarkable increases of total cell and neutrophil counts (Fig. 1A), TNF-α (Fig. 1B), and keratinocyte chemoattractant (KC; Fig. 1C) levels in BALF were observed at all three time points. The peak increases of total cell and neutrophil counts (Fig. 1A) and KC (Fig. 1C) in BALF were observed at 24 h post-LPS challenge, whereas TNF-α was peaked at 12 h, followed by remarkable drop but remaining significant increases at 24 and 48 h (Fig. 1B).

Next, we examined pro-BMP4 and mature BMP4 protein expression in lungs from saline control and LPS-treated mice. As shown in Figure 1D and E, the levels of both pro-BMP4 and mature BMP4 in LPS-treated mouse lung were markedly increased when determined at 12, 24, and 48 h, with the maximal increases observed at 12 h, followed by reduced increases at 24 h, and further reduced increases at 48 h post-LPS challenge.

LPS and TNF-α stimulate BMP4 expression in airway epithelial cells

To understand the mechanism of increased BMP4 expression during acute lung inflammation, we examined the effects of LPS and TNF-α on BMP4 expression in airway epithelial cells that are known to be the major sites of BMP4 production in the lung 10. As shown in Figure 2A, treatment of LPS (1 or 5 μg/mL) for 24 h dose-dependently increased BMP4 mRNA expression and mature protein secretion in 16HBE cells, with significant increases seen at 5μg/mL of LPS treatment. When cells were treated with 5 μg/mL LPS for various times from 3 to 24 h, the levels of secreted mature BMP4 in 16HBE-cultured medium were found increased as early as 6 h; further incubation to 12 and 24 h did not augment the degree of LPS stimulation (Fig. 2B). Similar to the effects of LPS, treatment of TNF-α (25-100 ng/mL) for 24 h dose-dependently increased BMP4 mRNA expression and mature protein secretion in 16HBE cells, with significant increases seen at 50 ng/mL of TNF-α (Fig. 2C). When cells were treated with 50 ng/mL TNF-α for various times from 3 to 24 h, the level of secreted mature BMP4 in 16HBE-cultured medium was also found increased as early as 6 h; again, further incubation to 12 and 24 h did not raise the degree of TNF-α stimulation (Fig. 2D). In primary mouse tracheal surface epithelial (MTSE) cells, treatments with LPS (1 or 5 μg/mL) for 24 h dose-dependently increased BMP4 secretion with significant increase seen with 5μg/mL of LPS (Fig. 2E).

BMP4 mutant mice exhibit impairment in resolution of lung inflammation after LPS challenge

To determine the role of upregulated BMP4 in LPS-induced acute lung inflammation, we utilized BMP4 WT (BMP4^+/+) and heterozygous mutant (BMP4^+/−) mice. Treatment of LPS increased both pro-BMP4 and mature BMP4 protein expression in lungs from BMP4^+/+ mice; these increases were absent in BMP4^+/− mice (Fig. 3A). In conjunction with the diminished increases of BMP4 expression, BMP4^+/− mice exhibited enhanced lung inflammation as indicated by greater total cell and neutrophil counts (Fig. 3B), higher TNF-α and KC levels in BALF (Fig. 3C), and increased inflammatory cell infiltration in lung tissue (Fig. 3D) when observed at 48 h post-LPS challenge as compared with the BMP4^+/+ mice. These results suggest that BMP4 acts as an anti-inflammatory factor and plays an essential role in resolution of LPS-induced acute lung inflammation. In this study, we selected the time point of 48 h for assessments, as the LPS-induced respective changes in BMP4 WT mice were all reduced comparing to their peak changes at 12 or 24 h, which indicates the acute lung inflammation were subjected to the process of resolution at 48 h post-LPS challenge (Fig. 1).

PA-infected BMP4^+/− mice have greater lung inflammation but lower bacterial load

To understand the functional significance of the increased lung BMP4 expression during LPS-induced lung inflammation, we compared the PA clearance capacity in BMP4^+/+ and BMP4^+/− mouse lungs. The pulmonary clearance of bacteria was evaluated by enumerating CFU of lung homogenates at 12 or 24 h post-PA infection. As seen in Figure 4A, inhalation of PA at 1 × 10⁵ CFU/mouse and 1 × 10⁶ CFU/mouse for 12 h resulted in approximately 1.9 × 10⁴ CFU/mouse and 4.3 × 10⁵ CFU/mouse left in BMP4^+/+ mouse lungs, indicating partial clearance of the bacterial. Comparing to BMP4^+/+ mice, the PA clearance at both dosages was greater in BMP4^+/− mice. When inhaled with 1 × 10⁵ CFU/mouse of PA, extension of the infection time to 24 h greatly reduced the CFU counts of PA to about 15% comparing to that at 12 h in BMP4^+/+ mouse lungs (Fig. 5B). At both 12 and 24 h post-PA inhalation, BMP4^+/− mice showed greater PA clearance in the lung comparing to BMP4^+/+ mice (Fig. 5B).

Next, we examined if the enhanced PA clearance in BMP4^+/− mice was associated with exaggerated lung inflammation. As shown in Figure 4C–E, infection of PA (1 × 10⁵ CFU/mouse) for 12 h caused significant inflammation in BMP4^+/+ mouse lung, which was manifested by remarkably increased total cell and neutrophil counts (Fig. 4C), elevated levels of TNF-α (Fig. 4D) and KC (Fig. 4E) in BALF. Comparing to BMP4^+/+ mice, BMP4^+/− mice exhibited enhanced inflammation in the lung with greater numbers of total cells and neutrophils, higher TNF-α and KC levels in BALF (Fig. 4C–E). These results suggest that BMP4 diminution in BMP4^+/− mice enhances bacterial clearance likely as a consequence of uncompromised innate immunity in the lung.

BMP4 activates Smad signaling and inhibits cytokine production in airway epithelial cells

Since LPS causes more severe lung inflammation in BMP4 heterozygous mutant mice, we therefore tested the possibility whether BMP4 inhibits proinflammatory cytokine expression in airway epithelial cells. Exposure to BMP4 led to rapid canonical activation of mothers against decapentaplegic homologs (Smad)/5/8 in 16HBE cells, indicating BMP4 signaling was active in bronchial epithelial cells (Fig. 5A). Exposure to BMP4-specific siRNA (siBMP4) in 16HBE cells resulted in approximately 85% reduction of BMP4 mRNA comparing to nontargeting siRNA (siNT) exposed cells, indicating efficient knockdown (Fig. 5B). In 16HBE cells exposed to siNT, LPS (5μg/mL) and TNF-α (50 ng/mL) treatment caused about 1.3- and 38.5-fold induction of IL-8 mRNA expression; knockdown of BMP4 increased the basal, LPS, and TNF-α-induced IL-8 mRNA expression for about 72, 98, and 27%, respectively, comparing to siNT-treated cells (Fig. 5D). Conversely, pretreatment with exogenous BMP4 (50 ng/mL) not only attenuated the basal expression of IL-8 mRNA, but also the one induced by LPS and TNF-α in 16HBE cells (Fig. 5E).

Moreover, we examined the effects of BMP4 on LPS and TNF-α-induced IL-8 release in primary normal human bronchial epithelial (NHBE) cells, which are considered to be more relevant to cells under physiological condition. Nucleofection delivery of siBMP4 resulted in about 83% reduction of BMP4 mRNA comparing to siNT-treated cells (Fig. 5C). Exposure to LPS and TNF-α greatly increased IL-8 releases in NHBE cells exposed to siNT; knockdown of BMP4 by siBMP4 enhanced the basal IL-8 release, as well as the IL-8 releases induced by LPS and TNF-α comparing to cells treated with siNT (Fig. 5F). In contrast to the results obtained with siBMP4, pretreatment with exogenous BMP4 (50 ng/mL) attenuated the basal IL-8 release, and the one induced by both LPS and TNF-α in NHBE cells (Fig. 5G). Similar to NHBE, we found pretreatment with BMP4 inhibited the basal and LPS-induced and TNF-α-induced KC releases in primary MTSE cells (Fig. 5H).

BMP4 inhibits LPS-induced proinflammatory cytokine production in mouse peritoneal macrophages

Besides epithelial cells, macrophages is also critical for airway innate immunity by producing proinflammatory cytokines, such as TNF-α and KC. To understand the impacts of BMP4 on macrophages-mediated inflammation, we isolated the primary peritoneal macrophages from BMP4^+/+ and BMP4^+/− littermate mice and exposed them to LPS (100 ng/mL). As shown in Figure 6A, the basal TNF-α and KC releases in macrophages from BMP4^+/+ and BMP4^+/− mice were not different. LPS exposure stimulated TNF-α and KC releases in macrophages from both strains of mice; however, their increases in cells from BMP4^+/− mice were significantly greater than that from BMP4^+/+ mice (Fig. 6A). Contrary to the above results obtained from BMP4 mutant mice in vivo, pretreatment with exogenous BMP4 (50 ng/mL) inhibited LPS-induced (100 ng/mL) TNF-α and KC releases in primary peritoneal macrophages from BMP4^+/+ mice (Fig. 6B).

BMP4 deficiency protects against chronic LPS exposure-induced lung function reduction in mice

Next, we examined if BMP4 participates in regulation of lung inflammation and function associated with chronic injury induced by repetitive LPS challenge. Treatment of LPS for 4 weeks increased pro-BMP4 and mature BMP4 protein expression in lung from both BMP4^+/+ and BMP4^+/− mice; however, the extent of increase in BMP4^+/− mice was drastically lower than that in BMP4^+/+ mice (Fig. 7A). In conjunction with these changes, chronic LPS treatment caused significant inflammation in lung from both BMP4^+/+ and BMP4^+/− mice, the inflammation in BMP4^+/− mouse lung was more severe as reflected by greater interstitial inflammatory cell infiltration in lung tissue (Fig. 7B) and greater total and neutrophil cell counts in BALF (Fig. 7C) comparing to that in BMP4^+/+ mice. In contrast, BMP4^+/+ mice treated with LPS exhibited restrictive lung function reduction manifested by increased resistance, decreased total lung capacity (TLC), forced vital capacity, inspiratory capacity, chord compliance, and forced expiratory volumes at 100 ms, whereas BMP4^+/− mice was protected against LPS-induced lung function decline (Fig. 7A–F).

Animals and LPS treatment

C57BL/6J mice (6–8 weeks old) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China). Breeders of BMP4 heterozygous mutant mice (Bmp4^+/−) with C57BL/6J background were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). The mice were housed in specific pathogen-free facilities, bred, and genotyped according to the vendor's protocols. All experimental procedures were approved by the Animal Care and Use Committee of Guangzhou Medical University. For acute challenge, BMP4 WT (BMP4^+/+) or BMP4^+/− mice (male, 6–8 weeks old) were treated with saline or LPS (7.5 μg/mouse in 50 μL saline; L3024; Sigma-Aldrich, St. Louis, MO) by i.n. inhalation, and euthanized at 12, 24, or 48 h thereafter. For chronic LPS exposure, mice (8–10 weeks old) were inhaled with saline or LPS (1.0 μg/mouse in 50 μL saline) once a week for four times, and sacrificed 24 h post the last challenge. Since homozygous mutation of BMP4 (BMP4^−/−) is lethal to the embryos, we therefore use heterozygous null (BMP4^+/−) mice for experiments. Aside from minor renal, craniofacial, and skeletal phenotypes, BMP4^+/− mice appear normal under unchallenged condition 34.

BALF collection, cell counting, and lung histological analysis

Mouse lung was lavaged with 0.8 mL saline/lung for two times. Total cells in the BALF were collected by centrifuging at 5 000 rpm for 5 min, and stained with H&E for differential cell counting. For lung histological analysis, lung lobes were infused via tracheal with 4% paraformaldehyde under 20 cm H₂O, xed for 24 h, embedded in paraffin, sectioned at 5 μm thickness, and stained with H&E.

PA infection

The PA infection and assessment of lung bacterial clearance were performed as we described previously 35. Briefly, PA strain PA01 (ATCC, Manassas, VA) was propagated in Luria broth, centrifuged, and resuspended in PBS. The mice were anesthetized by i.p. injection of 50 mg/Kg sodium pentobarbital, inoculated with 1 × 10⁵ or 1 × 10⁶ CFU of PA01 in 50 μL PBS by i.n. inhalation, and were dissected at 12 or 24 h after. The total cells in BALF were counted and the levels of TNF-α and KC in BALF were measured by ELISA. For PA clearance assay, the lungs were homogenized in 10 mL PBS. The bacterial CFU in lung were enumerated on Luria agar plates after overnight incubation under 37°C.

Airway epithelial cell culture and treatments

Human bronchial epithelial cells 16HBE were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), propagated and plated in 6-well culture plates in DMEM supplemented with 10% fetus bovine serum (FBS), 100 U/mL penicillin, and 100ug/mL streptomycin at 37°C.

Primary NHBE cells were purchased from Lonza, Inc. (Allendale, NJ), propagated in BEGM (Lonza, Inc.), and seeded at 2.5 × 10⁵ cells/insert on 12 mm Millicell insert membrane (EMD Millipore, Billerica, MA) coated with human placenta collagen type IV (Sigma). The Millicells were put in 24-well plate containing air–liquid interface (ALI) culture medium (Lonza, Inc.), and switched to ALI culture when transepithelial electrical resistance reached greater than 1 000 Ω cm² measured with an epithelial Ohm-voltmeter (MER-S00002, Millipore).

MTSE were isolated and cultured as we previously described 36. Briefly, the tracheas from C57BL/6J mice (8–10 weeks old) were removed and infused with 0.5% type XIV pronase (Sigma) to release MTSE cells. Approximately 1 × 10⁵ cells/well were plated on human placenta collagen type IV coated 12 mm Millicell inserts, cultured with DMEM/F-12 medium containing 10 μg/mL insulin, 5 μg/mL transferrin, 25 ng/mL epidermal growth factor, 10⁻⁷ M hydrocortisone, 0.1 μg/mL cholera toxin, 1% bovine pituitary extract, 5 × 10⁻⁸ M all trans-retinoic acid, 5% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin. Twenty-four hours after plating, FBS in this medium was replaced with 0.3% BSA. Media were changed every 2 days until the transmembrane resistance reached >1 000 Ω cm². Media in the apical chamber was then removed to establish an ALI, and the cells were fed only from the bottom chamber.

For drug treatments, the cells were serum starved for 24 h, incubated with LPS (1 or 5 μg/mL; Sigma), TNF-α (50 ng/mL; Sigma), or BMP4 (50 ng/mL; R&D systems, Minneapolis, MN) for indicated time, and then the cell culture medium or cell lysate was collected for subsequent analysis.

Isolation and culture of mouse peritoneal macrophages

Peritoneal macrophages (PMs) from BMP4^+/+ and BMP4^+/− mice were harvested under aseptic conditions at 3 days after i.p. injection with 3% thioglycollate (Sigma-Aldrich). Red blood cells in PM preparation were lysed in erythrocyte lysis buffer (10 mM Tris-HCl [pH 7.2] containing 150 mM NH₄Cl). The resultant PM was seeded in 6-well tissue culture plates at 2.5 × 10⁵ cells/well in DMEM medium containing 10% FBS and the nonadherent cells were removed 24 h after. Then, the cells were cultured for further 24 h in DMEM medium containing 1% FBS before being subjected to 4 h LPS treatment (100 ng/mL). For determining the effects of BMP4 on KC and TNF-α release of PM, PM cells from C57BL/6J mice were treated with LPS (100 ng/mL) for 12 h with or without pretreatment of BMP4 (50 ng/mL) for 1 h.

Small interference RNA transfection

For siRNA treatment, 16HBE cells seeded in 6-well dish were cultured for 24 h, transfected with 1.5 μg BMP4 siRNA (siBMP4; forward: 5′-GGA CUA ACA UGC GGG AUC UUT T-3′; reverse: 5′-AAG AUC CCG CAU GUA GUC CTT-3′) or a siNT using Hiperfect Transfection Reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. NHBE cells were transfected with siBMP4 or siNT at 1.0 μg siRNA/10⁶ cells using Amaxa NHBE Nucleofector Kit (Lonza, Inc.). Cells treated with siRNA for 48 h were subjected to LPS (5 μg/mL) or TNF-α (50 ng/mL) treatment for 6 h, and then followed by IL-8 or KC measurements.

Lung function measurements

Mouse lung function was measured using Forced Pulmonary Maneuver System (Buxco Research Systems, Wilmington, NC, USA) following the manufacturer's protocols. Briefly, mice challenged with saline or LPS were anesthetized by i.p. injection of pentobarbital (50 mg/Kg), tracheostomized, and placed in the body chamber of the system. The average breathing frequency was set to 140 breaths/min. To measure the TLC, inspiratory capacity and forced vital capacity, quasi-static pressure volume maneuver was performed, which inflates the lungs to a standard pressure of +30 cm H₂O and then slowly exhales until a negative pressure of −30 cm H₂O is reached. The quasi-static compliance was defined as the volume/pressure ratio at 50% of the expiration (Cchord50). With the fast flow volume maneuver, lungs were first inflated to +30 cm H₂O (TLC) and immediately afterwards connected to a highly negative pressure in order to enforce expiration until residual volume (RV) at −30 cm H₂O. Forced expiratory volumes at 100 ms were recorded during this maneuver. For each test in every single mouse, at least three acceptable maneuvers were conducted to obtain a reliable mean for all numeric parameters.

RNA isolation and quantitative real-time PCR

Total RNA was extracted from lung tissue or cells using Trizol reagent (Invitrogen, Carlsbad, CA). DNA contamination in RNA preparations was removed by TURBO DNA-Free DNase treatments (Ambion, Austin, TX). Reverse-transcription was performed in a 20 μL reaction mixture containing 1 000 ng total RNA using Takara cDNA synthesis kit (Dalian, Liaoning, China). BMP4 cDNA was quantified by real-time PCR on CFX96–C1000 system (Bio-Rad, Hercules, CA) using QuantiTect SYBR Green kit (Qiagen). The primers were: 5′-TGA GCC TTT CCA GCA AGT TT-3′ (forward) and 5′-CTT CCC CGT CTC AGG CAT CA-3′ (reverse) for human BMP4; 5'-AAC TTC TCC ACA ACC CTC TG-3′ (forward) and 5′-TTG GCA GCC TTC CTG ATT TC-3′ (reverse) for human IL-8; and 5′-GCA ATT ATT CCC CAT GAA CG-3′ (forward) and 5′-GGC CTC ACT AAA CCA TCC AA-3′ (reverse) for 18S as internal control. Relative concentration of each transcript was calculated using the Pfaffl method 37. Efficiency for each gene was determined from 5-point serial dilutions of an unknown cDNA sample. The expression of BMP4 or IL-8 was normalized to 18S in the same sample.

Western blotting

Mouse lung tissues and cells were homogenized in RIPA lysis buffer containing 1% protease inhibitor cocktail (Sigma-Aldrich), 5 μM EDTA, and 200 μM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride to extract the total protein. Equal amount of protein homogenates in samples were separated on SDS-PAGE (Bio-Rad); transferred onto polyvinylidene difluoride membranes (pore size 0.45 μM, Bio-Rad); blotted with mouse monoclonal antibody against BMP4 (1:1 000, EMD Millipore), P-Smad1/5/8, or T-Smad1 (1:500, Cell Signaling Technology, Inc., Danvers, MA) or β-actin (1:3 000, Sigma-Aldrich); and probed with corresponding peroxidase-conjugated secondary antibodies (KPL, Gaithersburg, MD). Signal of bound antibodies was developed using Immun-Star HRP Chemiluminescent kit (Bio-Rad).

ELISA

The protein levels of TNF-α and KC in mouse BALF, IL-8, and secreted BMP4 in cell-cultured media were measured by ELISA. The TNF-α and IL-8 ELISA kits were purchased from eBioscience (San Diego, CA). The capture and detection antibodies for KC measurement were obtained from R&D Systems, Inc. (Minneapolis, MN). The primary and secondary antibodies for BMP4 detection were from EMD Millipore and KPL, respectively. Signal was developed using TMB Substrate Reagent Set (BD Biosciences).

Materials and drugs

Unless otherwise specified, all reagents were obtained from Sigma-Aldrich. Recombinant human BMP4 stock solutions at 50 μg/mL were made in 4 mM HCl containing 0.1% BSA.

Statistical analysis

All experiments were repeated three times and each measurement was performed in triplicates. Statistics were analyzed with ANOVA or two-tailed Student's t-test. If significant F ratios were obtained with ANOVA, pairwise comparison of means was conducted with Bonferroni tests. Data were presented as mean ± SEM. N equals the number of treatment wells or animals. p < 0.05 was considered as significantly different.