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American Journal of Physiology - Lung Cellular and Molecular Physiology logoLink to American Journal of Physiology - Lung Cellular and Molecular Physiology
. 2016 Nov 11;311(6):L1222–L1233. doi: 10.1152/ajplung.00183.2016

Dung biomass smoke activates inflammatory signaling pathways in human small airway epithelial cells

Claire E McCarthy 1,3, Parker F Duffney 1,3, Robert Gelein 1, Thomas H Thatcher 2,3, Alison Elder 1,3, Richard P Phipps 1,2,3, Patricia J Sime 1,2,3,✉
PMCID: PMC5206396  PMID: 27836898

Abstract

Animal dung is a biomass fuel burned by vulnerable populations who cannot afford cleaner sources of energy, such as wood and gas, for cooking and heating their homes. Exposure to biomass smoke is the leading environmental risk for mortality, with over 4,000,000 deaths each year worldwide attributed to indoor air pollution from biomass smoke. Biomass smoke inhalation is epidemiologically associated with pulmonary diseases, including chronic obstructive pulmonary disease (COPD), lung cancer, and respiratory infections, especially in low and middle-income countries. Yet, few studies have examined the mechanisms of dung biomass smoke-induced inflammatory responses in human lung cells. Here, we tested the hypothesis that dung biomass smoke causes inflammatory responses in human lung cells through signaling pathways involved in acute and chronic lung inflammation. Primary human small airway epithelial cells (SAECs) were exposed to dung smoke at the air-liquid interface using a newly developed, automated, and reproducible dung biomass smoke generation system. The examination of inflammatory signaling showed that dung biomass smoke increased the production of several proinflammatory cytokines and enzymes in SAECs through activation of the activator protein (AP)-1 and arylhydrocarbon receptor (AhR) but not nuclear factor-κB (NF-κB) pathways. We propose that the inflammatory responses of lung cells exposed to dung biomass smoke contribute to the development of respiratory diseases.

Keywords: biomass smoke, respiratory toxicology, particulates, small airway epithelial cells, inflammatory responses

three billion people worldwide use biomass fuels, such as animal dung, wood, and crop residues, for cooking and heating their homes. Yet, biomass smoke exposure is the leading environmental risk factor for all-cause mortality, with 4.3 million premature deaths each year (13, 28, 29, 49, 50). Individuals living in poverty are often exposed to high levels of biomass smoke, which can reach up to 50 mg/m3 during peak cooking times (13, 14, 43). Furthermore, the inhalation of biomass smoke is epidemiologically correlated with inflammatory, respiratory diseases, such as COPD, lung cancer, and respiratory infections (3, 13, 14, 29, 38, 49). Thus biomass smoke exposure is a global environmental health problem that leads to inflammatory and obstructive respiratory diseases.

Many people, especially in developing countries, use animal dung as a fuel since it costs little, is easy to collect and prepare to burn, and is accessible in areas with limited vegetation. Despite these economic considerations, the burning of animal dung is of particular concern to human health. Previous studies have found that dung biomass smoke contains more fine particles per mass of sample burned, a greater oxidative capacity, and higher levels of bacterial endotoxin compared with other combustion aerosols (3, 20, 33, 40). Thus animal dung is a biomass fuel at the bottom of the “energy ladder,” meaning it has the lowest cost, lowest energy density, and highest polluting potential per unit energy released compared with cleaner sources of energy, like wood and gas. Research is needed to understand the biological impact of dung biomass smoke inhalation, a worldwide health disparity issue.

Despite strong evidence linking dung biomass smoke exposure with pulmonary diseases, few experimental studies have examined the inflammatory effects of dung biomass smoke exposure on human lung cells. The responses of small airway epithelial cells (SAECs) to dung biomass smoke are of interest since airway epithelial cells are the primary target of inhaled toxicants, such as smoke. Additionally, human studies with women and young adults show that biomass smoke exposure is related to airway obstruction and small airway abnormalities (3, 21). Here, using a novel, automated dung biomass smoke generation system, we investigate the activation of signaling pathways and the production of proinflammatory mediators, which play a role in respiratory diseases, in human primary SAECs exposed to dung biomass smoke. These findings can be used to examine potential therapeutic targets to reduce dung biomass smoke-induced small airway inflammation.

MATERIALS AND METHODS

Generation of dung biomass smoke.

Horse dung, which is burned as a cooking fuel in Mongolia, was dried overnight in an oven at 50°C (8). Although the type of animal dung used for fuel varies throughout the world, we could obtain a consistent supply of horse dung to optimize a method for generating a biomass smoke aerosol. Dried dung was ground in a food processor, passed through a sieve to obtain pieces between 850 and 420 μm, mixed with 2% glycerol, and rolled into 87-mm cigarette tubes (Zen) with a cigarette packing machine. The amount of glycerol added to the horse dung is less than that used in 1R4F reference tobacco cigarettes (2.8%) and would not increase the production of toxic by-products (9). To generate dung biomass smoke, the “cigarettes” were placed in a Baumgartner-Jaeger CSM2072i cigarette smoking machine (CH Technologies, Westwood, NJ) that was programmed for two cigarettes per cycle and one puff of 35-ml volume per min of 2-s duration for each cigarette. The dung biomass smoke was from the cigarette machine was diluted with air and pumped into a plexiglass chamber (Fig. 1A). The total particulate matter (TPM) of the smoke was determined by gravimetric sampling to be 45 + 15 mg/m3. The exposure time for experiments was 15–60 min, to mimic the short, peak levels of biomass smoke exposure during cooking times.

Fig. 1.

Fig. 1.

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Dung biomass smoke exposure system and particle size characterization. A: schematic of dung biomass smoke exposure system using a cigarette smoking machine. B: transmission electron microscopy (TEM) images of dung biomass smoke particles at 2 and 0.5 μm, as well as TEM images of tobacco smoke particles at 2 and 0.2 μm. C: representative graph from 1 aerodynamic size measurement of dung biomass smoke particles and a table of the multiple particle path dosimetry model (MPPD) and geometric standard deviation (GSD) (means ± SD from 4 different measurements). D: estimated dung biomass smoke particle deposition in the respiratory tract by generation using MPPD modeling.

Dung biomass smoke particle size characterization and visualization.

A seven-stage cascade impactor (In-Tox Products, Albuquerque, NM) was used to examine the aerodynamic particle size distribution of dung biomass smoke. Transmission electron microscopy was performed to visualize dung biomass smoke particles.

Multiple particle path dosimetry model.

To estimate deposition of dung biomass smoke particles in the human respiratory tract, we used multiple particle path dosimetry model (MPPD; version 2.1, 2009) computer modeling (2).

Cell culture.

Primary human SAECs from six different donors (strain 1: lot-0000105938, 61-yr-old male, no tobacco smoking history; strain 2: lot-0000206158, 58-yr-old female, no tobacco smoking history; strain 3: lot-0000203964, 51-yr-old male, no tobacco smoking history; strain 4: lot-0000470903, 37-yr-old male, no tobacco smoking history; strain 5: lot-0000501937, 42-yr-old female, no tobacco smoking history; and strain 6: lot-0000419241, 61-yr-old female, no tobacco smoking history) were obtained from Lonza (Walkersville, MD) and cultured as previously described (19). For experiments, SAECs were plated onto culture inserts (1-μm pore size; Millipore), moved to the air-liquid interface (ALI) for 24 h, and exposed to air or dung biomass smoke (using particulate levels measured during peak cooking times) in a plexiglass chamber (Fig. 1A). For the air exposures, air was pumped into a plexiglass chamber containing SAECs at the same flow rate as the dilution air that was used in the dung biomass smoke exposures. After the dung smoke exposure, cells were incubated for up to 48 h in the smoke-exposed culture media to mimic the continual exposure of airway epithelial cells to combustion products (with 24-h average particulate levels of ∼1 mg/m3) in homes burning animal dung for fuel (13). In some experiments, cells were pretreated with 10 μM SP600125 (Sigma-Aldrich, St. Louis, MO) or 5 μM CH223191 (Sigma-Aldrich) for 30 min. Dimethyl sulfoxide was used as a vehicle. Trypan blue exclusion was used to assess cell viability.

For the AhR reporter assay, the H1L1.1c2 mouse hepatoma cell line with a dioxin response element-driven luciferase construct (a gift from M. S. Denison, University of California, Davis, CA) was treated with dung smoke extract. Dung smoke extract was made by burning horse dung (5 g) in a ceramic Buchner funnel and bubbling the smoke through serum-free Dulbecco's modified Eagle's medium (DMEM) using a vacuum. The crude extract was passed through a 1.2-um filter and the extract concentration was normalized by measuring its absorbance using a wavelength of 320 nm (absorbance of 1 = 1,000 units/ml) with a spectrophotometer (Bio-Rad SmartSpec 3000). This procedure is similar to that used by us in in vitro studies of tobacco-based cigarette smoke (4, 5, 26, 52).

Cytokine levels.

SAEC cell supernatants were collected 24 h postdung biomass smoke exposure and stored at −80°C. The Proteome Profiler Human Cytokine Array Kit, Panel A (R&D Systems, Minneapolis, MN) was used according to the manufacturer's instructions to examine the expression of 36 cytokines. The levels of interleukin (IL)-8 and granulocyte macrophage-colony stimulating factor (GM-CSF) were quantified by enzyme-linked immunosorbent assays (ELISAs) (R&D Systems, Minneapolis, MN).

Western blot analysis.

Cellular protein lysates were prepared 24 h postexposure, fractionated on gels, and electroblotted onto membranes as previously described (4, 27). Blots were probed with antibodies specific for Cox-2 (Cayman Chemical, Ann Arbor, MI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, MA), β-tubulin (Abcam), phospho-NF-κB p65 (Cell Signaling, Danvers, MA), NF-κB p65 (Santa Cruz Biotechnology, Dallas, TX), phospho-Jun amino terminal kinase (JNK; Cell Signaling), or JNK (Cell Signaling). Protein expression through antibody binding was visualized using enhanced chemiluminescence (PerkinElmer, Waltham, MA) and X-ray film (Laboratory Product Sales, Rochester, NY). Densitometry of protein expression was analyzed using Kodak 1D Imaging Software (Eastman Kodak, Rochester, NY).

Luciferase reporter assays.

SAECs transduced with a lentiviral NF-κB luciferase reporter (SABiosciences, Valencia, CA) as described in Ref. 18 were cultured at the ALI and exposed to dung biomass smoke. For activator protein (AP)-1 reporter assays, SAECs were grown to confluence on 12-well Transwell inserts and transfected with a 3XAP-1 plasmid (a gift from Alexander Dent, Addgene plasmid no. 40342, Cambridge, MA) using Lipofectamine LTX with PLUS Reagent (Thermo Fisher Scientific, Rockford, IL) as per the manufacturer's instructions. After 24 h, the transfected cells were moved to the ALI for 6 h and subsequently exposed to dung biomass smoke. The ALI time was reduced to allow the cells to recover after the transfection. Both the NF-κB and AP-1 reporter SAECs were lysed 24 h after dung biomass smoke exposure.

Gene expression analysis.

RNA isolation from SAECs and quantitative PCR was performed as previously described by us (18, 19). We used primers for 18S RNA (18) and CYP1A1 (+: 5′-CTTCGTCCCCTTCACCATC-3′; −: 5′-TCAGGTAGGAACTCAGATGGG-3′).

Statistical analysis.

Results are reported as the means ± SD for data from individual SAEC strains or means ± SE for data showing the average of three to six strains as indicated. t-Tests and one-way or two-way ANOVA with multiple comparisons (Tukey or Bonferroni) was performed using GraphPad Prism version 5.0 (GraphPad Software, La Jolla CA; www.graphpad.com). P < 0.05 was considered to be statistically significant.

RESULTS

Dung biomass smoke particle characterization.

Particles generated from the combustion of animal dung are morphologically distinct from particles found in tobacco smoke. Transmission electron micrographs show that dung smoke particles are cellulose-like and fibrous, with similar morphology to cellulose nanocrystals (Fig. 1B; Ref. 54). As a comparison, we also examined the physical characteristics of tobacco smoke particles. The smoke particles from the combustion of tobacco appear small and oval shaped (Fig. 1B). Mass-based size measurements of the dung biomass smoke aerosol showed that over 90% of the particles were <2.5 μm in aerodynamic diameter and would be considered fine particulates (Fig. 1C). Over a quarter of the sample mass had a diameter <0.5 μm. The mass median aerodynamic diameter (MMAD) of the dung biomass smoke was 0.57 ± 0.07 μm, with a geometric standard deviation (GSD) of 1.7 ± 0.15 (Fig. 1C). When the dung biomass smoke particle size parameters were used in the MPPD modeling program, most of the dung smoke particles were estimated to deposit between generation 12 and generation 24 of the lung (Fig. 1D). Thus a large fraction of the dung smoke particles were predicted to deposit in the terminal bronchioles and respiratory bronchioles, which includes generation 5–19 of the respiratory tract (46). Based on this dung smoke particle deposition modeling and clinical reports of small airway abnormalities in women exposed to biomass smoke, we chose to examine the toxicological effects of dung biomass smoke in primary human SAECs (3, 21).

Inflammatory responses of SAECs to dung biomass smoke.

To investigate the impact of dung smoke in vitro, SAECs were exposed to an aerosol of dung biomass smoke at the ALI for up to 60 min to model the short, high level biomass smoke exposures that occur during cooking times (13, 43). Multiple strains of SAECs, isolated from different human donors, were used for these studies to ensure that the observed biological effects of dung biomass smoke are consistent. The rate of particulate deposition onto cell culture inserts was determined gravimetrically and averaged 2.8 μg·cm−2·h−1. To screen for inflammatory mediator production in response to dung biomass smoke, cytokine release in culture medium was measured 24 h postexposure using a cytokine array. Of 36 cytokines tested, only IL-8 and GM-CSF were increased by dung smoke (Table 1). Increased production of IL-8 and GM-CSF in response to dung biomass smoke was confirmed by ELISA in six SAEC strains from different donors (Fig. 2, A–F). Since all SAEC strains demonstrated decreased cytokine production after 60 min of dung smoke exposure, we evaluated cytotoxicity using the Alamar Blue assay. SAEC strains 1–6 showed decreased Alamar Blue reduction after 60 min of dung smoke exposure (Fig. 2, G and H, and data not shown).

Table 1.

Cytokine production by SAEC exposed to dung smoke

Cytokine Strain 1 Strain 5 Strain 6 Avg. of 3
GM-CSF 2.2 1.9 1.7 1.9 ± 0.3*
IL-8 1.8 2.0 2.8 2.2 ± 0.5*
CXCL1/GROα 0.9 0.7 0.7 0.8 ± 0.1
IL-1ra/IL-1F3 0.8 1.2 1.3 1.1 ± 0.3
IL-6 1.0 1.2 1.5 1.2 ± 0.3
IL-13 0.7 1.1 1.1 1.0 ± 0.2
IL-21/IL-23 2.0 N.D. N.D. N.D.
IL-27 2.0 N.D. N.D. N.D.
MIF 0.9 1.2 1.3 1.1 ± 0.2
Serpin E1/PAI-1 1.1 1.3 1.2 1.2 ± 0.1
CCL-1 N.D. N.D. N.D. N.D.
CCL-2/MCP-1 N.D. N.D. N.D. N.D.
MIP1α/MIP1β N.D. N.D. N.D. N.D.
CCL5/RANTES N.D. N.D. N.D. N.D.
CD40 ligand N.D. N.D. N.D. N.D.
CC5/CC5a N.D. N.D. N.D. N.D.
CXCL10/IP-10 N.D. N.D. N.D. N.D.
CXCL11/I-TAC N.D. N.D. N.D. N.D.
CXCL12/SDF-1 N.D. N.D. N.D. N.D.
G-CSF N.D. N.D. N.D. N.D.
ICAM-1/CD54 N.D. N.D. N.D. N.D.
IFNγ N.D. N.D. N.D. N.D.
IL-1α/IL-1F1 N.D. N.D. N.D. N.D.
IL-1β/IL-1F2 N.D. N.D. N.D. N.D.
IL-2 N.D. N.D. N.D. N.D.
IL-4 N.D. N.D. N.D. N.D.
IL-5 N.D. N.D. N.D. N.D.
IL-10 N.D. N.D. N.D. N.D.
IL-12p70 N.D. N.D. N.D. N.D.
IL-16 N.D. N.D. N.D. N.D.
IL-17A N.D. N.D. N.D. N.D.
IL-17E N.D. N.D. N.D. N.D.
IL-18 N.D. N.D. N.D. N.D.
IL-32α N.D. N.D. N.D. N.D.
TNF-α N.D. N.D. N.D. N.D.
TREM-1 N.D. N.D. N.D. N.D.
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SAECs, small airway epithelial cells; N.D., not determined.

Fig. 2.

Fig. 2.

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Dung biomass smoke induces IL-8 and granulocyte macrophage-colony stimulating factor (GM-CSF) secretion by small airway epithelial cells (SAECs). Six different SAEC strains were exposed to air or dung smoke for 15, 30, or 60 min and cell supernatants were collected 24-h postexposure. IL-8 levels in strains 1–3 (A) and strains 4–6 (B), as well as GM-CSF levels in strains 1–3 (D) and strains 4–6 (E) were measured by ELISA. Data represent means (n = 3 replicates per exposure group from a representative experiment for each SAEC strain); *P < 0.05 by one-way ANOVA (compared to air-exposed cells within an SAEC strain). The average IL-8 (C) and GM-CSF (F) production with air and dung smoke exposure across 6 different SAEC strains. Data represent means ± SE (n = 6 SAEC strains); $P < 0.05 by one-way ANOVA (compared to air-exposed cells). G: cytotoxicity in SAEC strains 1–3 exposed to dung biomass smoke was measured 24 h postexposure using the Alamar blue assay. Data represent means (n = 3 replicates per exposure group from a representative experiment for each SAEC strain), *P < 0.05 by one-way ANOVA (compared to air-exposed cells within an SAEC strain). H: average Alamar blue reduction across 6 different SAEC strains. Data represent means ± SE (n = 6 SAEC strains); $P < 0.05 by one-way ANOVA (compared to air-exposed cells).

Along with increased proinflammatory cytokine production, exposure to dung biomass smoke resulted in an upregulation of Cox-2 protein expression in six SAEC strains in an exposure-dependent manner (Fig. 3, A–C).

Fig. 3.

Fig. 3.

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SAECs upregulate Cox-2 protein expression with dung biomass smoke exposure. SAECs were exposed to air or dung biomass smoke for the indicated times and cell lysates were collected 24 h after dung smoke exposure. Protein levels were analyzed by Western blot and densitometry. A: a representative Western blot of Cox-2 protein expression in SAEC strain 6. B: Cox-2 protein levels in SAEC strains 1–6. Data represent means ± SD (n = 3 replicates per exposure group from a representative experiment for each SAEC strain); *P < 0.05 by one-way ANOVA (compared to air-exposed cells within an SAEC strain). C: average expression of Cox-2 across six different SAEC strains. Data represent means ± SE (n = 6 SAEC strains); $P < 0.05 by one-way ANOVA (compared to air-exposed cells).

To confirm that 24 h postdung smoke exposure was an appropriate time to measure inflammatory mediator production and Cox-2 protein expression in SAECS, we performed time course experiments. We found that the largest difference in IL-8 levels between air and dung smoke-exposed SAECS occurred 24 h postexposure (Fig. 4A). GM-CSF levels were increased to similar levels in dung smoke-exposed cells at both 24 and 48 h postexposure (Fig. 4B). Cox-2 protein upregulation in response to dung smoke was observed at 12 and 24 h postexposure (Fig. 4, C and D). Based on this evidence, we measured inflammatory responses at 24 h postexposure in subsequent experiments. We also compared a single exposure with two exposures 6 h apart, as a dual exposure model might better reflect human cooking exposures. However, the inflammatory response was similar whether the cells were exposed once or twice (Fig. 4E).

Fig. 4.

Fig. 4.

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The maximal effects of dung smoke-induced inflammatory responses in SAECs often occur 24 h after exposure. SAECs were exposed to air or dung smoke for 30 min and cell supernatants and lysates were collected between 6–48 h postexposure as indicated. IL-8 levels (strain 6 shown) (A) and GM-CSF levels (B) (strain 5 shown) from air and dung smoke exposed SAECs were measured by ELISA. A representative Western blot of Cox-2 protein expression (C) and densitometry (D) in SAEC strain 6 after air or dung smoke exposure. E: SAECs were exposed to dung biomass smoke for 30 min, 2 × 15 min (6 h apart), or 2 × 30 min (6 h apart) and cell culture media were changed after each smoke exposure to reduce cytotoxicity. Cell supernatants were collected 24 h after the single or second dung smoke exposure and IL-8 was measured by ELISA. Data represent means ± SD (n = 3 replicates per exposure group from a representative experiment for each SAEC strain); *P < 0.05 by t-test (A and B) or one-way ANOVA (D and E) compared with air-exposed cells.

Impact of dung biomass smoke on NF-κB activity.

The proinflammatory mediators that were increased at 24 h with dung biomass smoke exposure contain NF-κB binding sites in their promoters. Hence, we hypothesized that dung biomass smoke exposure would increase NF-κB p65 activity. However, we observed no differences in NF-κB luciferase activity between air and dung biomass smoke exposed SAECs containing a lentiviral NF-κB reporter (Fig. 5, A and B). The NF-κB luciferase assay showed that dung smoke did not increase NF-κB activity between the dung smoke exposure through a later time point of 24 h. To examine a shorter time-point of NF-κB p65 activation, we collected cell lysates 15 and 30 min postexposure. Similar to the reporter assay, there was no increase in phospho-NF-κB p65 in SAECs exposed to dung biomass smoke. In fact, there was a trend toward decreased phospho-p65, which would be consistent with a decrease, rather than increase, in NF-κB transcriptional activity (Fig. 5, C–E).

Fig. 5.

Fig. 5.

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Dung biomass smoke does not activate NF-κB in SAECs. A: luciferase activity was measured in SAECs (strains 1–3) transduced with a NF-κB luciferase reporter 24 h postdung biomass smoke exposure. Based on a previous study, polyinosinic:polycytidylic acid (poly I:C, 0.5 μg/ml) was used as a positive control (18). Data represent means ± SD (n = 3 replicates per exposure group from a representative experiment for each SAEC strain); *P < 0.05 by one-way ANOVA (compared to air-exposed cells within an SAEC strain). B: average NF-κB luciferase activity across three different SAEC strains. Data represent means ± SE (n = 3 SAEC strains), $P < 0.05 by one-way ANOVA (compared to air-exposed cells). B: phospho-NF-κB p65 and total NF-κB protein expression were determined by Western blot in SAECs 15 and 30 min after a 30 min dung smoke exposure. A representative Western blot is shown for strain 2. C: densitometry was performed on Western blots from SAEC strains 1–3 with n = 3 replicates per condition. Data are expressed as means ± SD (n = 3 or 6 (air-exposed) replicates per group from a representative experiment with each SAEC strain); *P < 0.05 by one-way ANOVA (compared to air-exposed cells within an SAEC strain). D: average phospho-NF-κB p65 expression across 3 different SAEC strains. Data represent means ± SE (n = 3 SAEC strains); $P < 0.05 by one-way ANOVA (compared to air-exposed cells).

Effects of dung biomass smoke on JNK-AP-1 signaling.

Another key pathway involved in the regulation of inflammatory responses is JNK-AP-1. We first assessed protein levels of phospho-JNK, the activated form of the enzyme, at 15 and 30 min postexposure. These time points were chosen based on previous studies showing that airway epithelial cells treated with tobacco and dung smoke extracts have increased protein levels of phospho-JNK after 10, 30, and 60 min of treatment (23, 25, 31). Here, we found that dung biomass smoke exposure resulted in increased phosphorylation of JNK at both 15 and 30 min (Fig. 6, A–C). Similarly, SAECs transfected with an AP-1 luciferase reporter showed greater AP-1 luciferase activity after exposure to 15 or 30 min of dung biomass smoke (Fig. 6, D and E). To confirm that the AP-1 pathway is responsible for upregulation of proinflammatory cytokines after dung smoke exposure, we used the small molecule JNK inhibitor SP600125. Pretreatment with SP600125 significantly attenuated both background and dung smoke-stimulated production of IL-8 in SAECs (Fig. 7A). Interestingly, increased protein levels of Cox-2 observed with dung biomass smoke exposure were not changed with JNK inhibition (Fig. 7, B and C).

Fig. 6.

Fig. 6.

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The JNK-activator protein (AP)-1 pathway is activated in SAECs exposed to dung biomass smoke. Phospho- and total JNK were measured by Western blot in SAEC strains 1–3 at 15 and 30 min postdung biomass smoke exposure (30 min). A: a representative Western blot is shown for strain 3. B: densitometry was performed on Western blots from SAEC strains 1–3 with n = 3 replicates per condition. Data are expressed as mean ± SD by one-way ANOVA (compared to air-exposed cells within an SAEC strain). C: average phospho-JNK expression across 3 different SAEC strains. Data represent means ± SE (n = 3 SAEC strains), $P < 0.05 by one-way ANOVA (compared to air-exposed cells). C: SAEC strains 1–3 were transfected with an AP-1 luciferase reporter, exposed to dung biomass smoke for the indicated period of time, and luciferase activity was measured 24 h later. Phorbol myristate acetate (PMA; 50 nM) was included as a positive control. Data represent means ± SD (n = 3 replicates per group from a representative experiment); *P < 0.05 by one-way ANOVA (compared to air-exposed cells within an SAEC strain). D: average AP-1 luciferase activity across 3 different SAEC strains. Data represent means ± SE (n = 3 SAEC strains); $P < 0.05 by one-way ANOVA (compared to air-exposed cells).

Fig. 7.

Fig. 7.

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JNK inhibition attenuates dung biomass smoke-induced secretion of IL-8 in SAECs. SAEC strain 3 was pretreated for 30 min with vehicle or SP600125 (10 μM) and exposed to dung biomass smoke for the indicated times. Cell supernatants and lysates were collected after a further 24 h. A: IL-8 levels in culture medium were measured by ELISA. B: Cox-2 protein expression was measured by Western blot after 24 h. A representative Western blot with 1 replicate per condition is shown. C: densitometry was performed on a total of 3 replicates per condition and normalized to β-tubulin. Representative data from 1 of 2 independent experiments are shown. Data are expressed as means ± SD (n = 3 replicates per group from a representative experiment); *P < 0.05 (air compared with 15 min of dung), **P < 0.05 (air and 15 min compared with 30 min of dung), and #P < 0.05 (vehicle compared with SP600125) by two-way ANOVA.

AhR activity in response to dung biomass smoke.

One important proinflammatory signaling pathway activated by tobacco smoke is the aryl hydrocarbon receptor (4, 26, 52). To investigate whether dung smoke might activate this pathway, we first used a well-characterized Hepa1c1c7 mouse liver cell line containing an AhR luciferase reporter to screen for the presence of AhR ligands in dung biomass smoke (55). Since liver cells, unlike airway epithelial cells, would not physiologically be exposed to whole dung biomass smoke, we used an aqueous dung biomass smoke extract for this assay. The reporter cell line exhibited a dose-dependent increase in luciferase activity when treated with dung biomass smoke extract (Fig. 8A). Since the AhR reporter assay indicated that dung biomass smoke contains AhR ligands, we examined AhR signaling in SAECS exposed to whole dung smoke at the ALI. SAECs had reduced levels of total AhR protein after being exposed to dung biomass smoke (Fig. 8, B–D), which is consistent with reports by us and others that activated AhR translocates to the nucleus and is subsequently degraded (26, 36). Similarly, mRNA expression of cytochrome P450 (CYP)1A1, an AhR-regulated gene, was increased in SAECs exposed to dung biomass smoke (Fig. 8, E and F).

Fig. 8.

Fig. 8.

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Dung biomass smoke activates the arylhydrocarbon receptor (AhR) in SAECs. A: H1L1.1c7 mouse hepatoma cells with an AhR luciferase reporter were treated with the indicated concentrations of dung smoke extract or a positive control of FICZ (1 μM) for 4 h. Data represent means ± SD (n = 6 replicates per group from a representative experiment), *P < 0.05 by one-way ANOVA compared with vehicle. B: SAECs (strains 1–3) were exposed to dung biomass smoke for the indicated times and cell lysates were collected 24 h postexposure. Representative Western blot (of reassembled noncontiguous lanes from a single blot of SAEC strain 1) showing 2 out of 3 replicates per condition are shown. C: densitometry of AhR protein expression was performed on a total of 3 replicates per exposure group in strain 1–3 SAECs. Data represent means ± SD (n = 3 replicates per exposure group from a representative experiment for each SAEC strain); *P < 0.05 by one-way ANOVA (compared to air-exposed cells within an SAEC strain). D: average AhR protein expression across 3 different SAEC strains. Data represent means ± SE (n = 3 SAEC strains); $P < 0.05 by one-way ANOVA (compared to air-exposed cells). E: mRNA was isolated from SAECs (strains 1–3) 6 h after a 30 min dung smoke exposure. CYP1A1 and 18S RNA gene expression were assessed by qPCR. Data represent means ± SD (n = 3 replicates per exposure group from a representative experiment for each SAEC strain); *P < 0.05 by t-test (compared to air-exposed cells within an SAEC strain). F: average CYP1A1 mRNA levels across 3 different SAEC strains. Data represent means ± SE (n = 3 SAEC strains); $P < 0.05 by t-test (compared to air-exposed cells).

When SAECs were pretreated with a CH223191, a small molecule antagonist of the AhR, the production of IL-8 in response to dung smoke was attenuated (Fig. 9A). However, similar to inhibition of JNK, antagonism of the AhR did not change the dung smoke-induced upregulation of Cox-2 protein expression in SAECs (Fig. 9, B and C). This suggests that SAECs may differentially regulate the expression of Cox-2 and IL-8 in response to dung biomass smoke.

Fig. 9.

Fig. 9.

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CH223191, an AhR antagonist, attenuates IL-8 production by SAECS exposed to dung biomass smoke. A: SAECs were pretreated with vehicle or CH223191 (5 μM) for 30 min, exposed to dung biomass smoke for the indicated length of time, and IL-8 was measured in the culture medium after a further 24 h. B: SAECs (strain 1) were pretreated with vehicle or CH223191 and exposed to air or dung biomass smoke for the indicated time, and Cox-2 protein expression was measured by Western blot after 24 h. A representative Western blot with 2 replicates per condition is shown. C: densitometry was performed on a total of 3 replicates per condition and normalized to β-tubulin. Representative data from 1 of 2 independent experiments are shown. Data are expressed as means ± SD (n = 3 replicates per group from a representative experiment); *P < 0.05 (air compared with dung) and #P < 0.05 (vehicle compared with CH223191) by two-way ANOVA.

DISCUSSION

Over 4,000,000 deaths every year are caused by biomass smoke inhalation (49). Animal dung is a biomass fuel that is found at the bottom of the energy ladder, meaning it is one of the least efficient types of fuel that is used by people with a low socioeconomic status who cannot afford cleaner sources of energy. Epidemiological evidence associates biomass smoke exposure with pulmonary diseases, including COPD, lung cancer, and respiratory infections (3, 13, 14, 29, 38, 49). However, there are currently few experimental models to investigate the effects of whole dung biomass smoke in vitro and little evidence on the mechanisms of dung biomass smoke-induced inflammatory responses.

We have developed an automated system using a modified cigarette smoking machine that reproducibly generates dung biomass smoke to test the ability of dung smoke to induce various proinflammatory mediators in vitro. Throughout the world, the type of dung used as a fuel varies widely depending on regional geography, climate, and agriculture, which means there is no standard type of dung to use for experiments. Therefore, we chose to use horse dung for our experiments, since it is used for fuel in Mongolia and we could ensure a consistent, supply from a local farm (8). Additionally, horses are hindgut fermenters, similar to elephants, donkeys, and zebras whose dung is used as fuel in some parts of the world (1, 8, 42, 48). Although this model may differ from the burning of dung patties in cooking fires, by packing the horse dung into cigarette tubes, we can take advantage of the automated cigarette smoking machines that are used world-wide to generate smoke for in vivo and in vitro exposures under reproducible and controllable conditions. In addition, the MMAD of the dung biomass smoke aerosol in our system (0.57 ± 0.7 μm) is similar to that previously reported for the burning of dung biomass in different types of cookstoves (MMAD 0.6–0.8 μm) (33, 44).

We found that the physical appearance of dung smoke particles is distinct from combustion particles in tobacco smoke (Fig. 1B) and diesel exhaust (16). Dung smoke particles appear to be longer, less dense, and more fibrous-like than other combustion-derived particulates. The physical differences of dung biomass smoke particles may lead to different biological responses by the human respiratory tract epithelium. Previous studies have found that the physical characteristics of particulate matter contribute to their toxicological properties (15). The morphological properties of dung biomass smoke particles may contribute to their interactions with lung cells.

In silico modeling of the deposition of dung biomass smoke particles in the human respiratory tract estimated that a large fraction of the sample mass would deposit in the small airways (Fig. 1D). Clinical evidence shows that individuals chronically exposed to biomass smoke often develop airway obstruction and small airway abnormalities (3, 21). Therefore, the predicted deposition of a large fraction of dung biomass smoke particles in this region of the lung may be contributing to inflammatory lung diseases related to biomass fuel use. Additionally, the predicted site of deposition supports the use of SAECs as in vitro targets.

Dung biomass smoke induced multiple strains of SAECs to secrete proinflammatory cytokines, which play a role in chronic inflammatory diseases (Table 1; Fig. 2). We found a dung biomass smoke exposure-dependent increase in the supernatant levels of IL-8, a potent neutrophil chemoattractant that plays a role in the pathogenesis of COPD and cancer (12, 45). This relates to individuals who cook with biomass fuels, who were found to have increased levels of neutrophils and IL-8 in their blood and sputum (6, 12). Additionally, dung biomass smoke may have unique inflammatory effects compared with other combustion products. For example, in contrast to our observation that dung smoke exposure increased production of GM-CSF, previous work found that tobacco smoke extract did not induce production of GM-CSF in a human alveolar epithelial cell line (30). Furthermore, these data correspond to a study that found increased levels of GM-CSF in the BALF of mice exposed to dung biomass smoke (31). Additionally, we found that the greatest increase of cytokine production by SAECs in response to dung biomass smoke occurred 24 h after the exposure (Fig. 4, A and B). These kinetics are earlier than those observed in lung epithelial cells treated with combustion particles, which have increased cytokine levels 24 h after treatment but a peak response at the 48-h time point (7, 10). Nevertheless, we found increased inflammatory responses in SAECs exposed to dung biomass smoke; however, other mediators involved in small airway obstruction and fibrosis, including matrix metalloproteinase activity, transforming growth factor-β production, and mucin expression, can be examined in future studies.

NF-κB is an important transcription factor that is activated during inflammatory responses and upregulates mediators, including IL-8 and GM-CSF. Our earlier, in vitro work shows that classical NF-κB signaling in lung cells is activated by combustion products (5, 27). Furthermore, mice exposed to dung smoke particulate matter displayed greater NF-κB p65 activity in the lung (40). We expected that dung biomass smoke would activate NF-κB p65 but found that human SAECs exposed to dung biomass smoke did not have increased NF-κB signaling (Fig. 5). Interestingly, these data suggest that dung biomass smoke upregulates proinflammatory mediators in SAECs through NF-κB-independent mechanisms.

A new finding is that SAECs exposed to whole dung biomass smoke had increased activation of JNK-AP-1 signaling (Fig. 6), another important pathway involved in inflammatory responses. Similar to studies with smoke extracts, we observed significantly increased protein expression of phospho-JNK at 30 min postdung biomass smoke exposure (Fig. 6, A and B; Refs. 23, 25, 31). Furthermore, inhibition of JNK attenuated production of IL-8 by SAECs in response to dung biomass smoke (Fig. 7A). Conversely, studies with human airway epithelial cells and other combustion products found no effect with a JNK inhibitor on exposure-induced cytokine production (22, 41). This suggests that JNK-AP-1 activation plays a role in small airway inflammatory responses to dung biomass smoke.

An important transcription factor involved in toxicant metabolism and pulmonary immune responses is the AhR. Activation of the AhR is increased in human lung cells exposed to combustion products (26, 34, 41). Correspondingly, we found that dung biomass smoke activates the AhR in SAECs (Fig. 8). Furthermore, dung smoke-induced IL-8 production could be attenuated with an AhR antagonist (Fig. 9A). The regulation of IL-8 by the AhR can occur through direct binding of this transcription factor to the IL-8 promoter, as well as indirectly through the upregulation of AP-1 family members (35, 47). Dung biomass smoke promotes some inflammatory responses in SAECs through the AhR.

Along with increases in soluble mediators, we found that the proinflammatory enzyme of Cox-2 was upregulated by dung smoke in SAECs at 12 and 24 h after the exposure (Figs. 3 and 4, C and D). Cox-2 is closely linked with airway inflammation and increased in the lung tissue and exhaled breath condensate of people with inflammatory lung diseases (32, 37, 51). Additionally, Cox-2 is increased in airway cells exposed to outdoor air particulates as early as 6 h after treatment, with peak levels observed between 12 and 24 h posttreatment (24, 39, 53). However, the upregulation of Cox-2 in SAECs with dung biomass smoke exposure was neither changed with JNK inhibition (Fig. 7B) nor an AhR antagonist (Fig. 9B). JNK inhibition was similarly found to have no effect on the induction of Cox-2 protein expression in cells treated with tobacco smoke extract or a chemical initiator of oxidative stress (11, 17). Although diesel exhaust-induced Cox-2 levels were inhibited with an AhR antagonist in bronchial epithelial cells, lung fibroblasts treated with the AhR antagonist of CH223191 were found to have increased Cox-2 protein expression in response to tobacco smoke extract (41, 52). Whole dung biomass smoke, which may have similar components to tobacco smoke, could activate multiple transcription factors and posttranscriptional regulators of Cox-2. For example, cow dung smoke extract activated ERK and p38 mitogen-activated protein kinases in SAECs, which are known to bind to the of Cox-2 promoter (11, 31, 41). Also, the cytoplasmic expression of a protein involved in the stabilization of Cox-2 mRNA was found to be enhanced with AhR inhibition (52). Therefore, whole dung biomass smoke may activate additional pathways to JNK-AP-1 and the AhR in SAECs, contributing to the inflammatory response.

In conclusion, we have shown that human primary SAECs exposed to whole dung biomass smoke increase inflammatory mediator production and upregulate Cox-2 through NF-κB independent pathways, including AP-1 and AhR signaling. We propose that the acute inflammatory responses observed in SAECs exposed to dung biomass smoke that occur chronically with daily cooking and heating contribute to the development of respiratory diseases. In future studies, therapeutic agents targeting AP-1 and the AhR can be assessed for efficacy in reducing small airway inflammation caused by dung biomass smoke inhalation.

GRANTS

This work was supported by the National Institutes of Health Grants HL-088325, HL-120908, T32-ES-007026, P30-ES-001247, and T32-HL-066988.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

C.E.M., T.H.T., A.C.E., R.P.P., and P.J.S. conception and design of research; C.E.M., P.F.D., and R.G. performed experiments; C.E.M., P.F.D., and R.G. analyzed data; C.E.M., P.F.D., T.H.T., A.C.E., R.P.P., and P.J.S. interpreted results of experiments; C.E.M. prepared figures; C.E.M. drafted manuscript; C.E.M., P.F.D., T.H.T., A.C.E., R.P.P., and P.J.S. edited and revised manuscript; C.E.M., P.F.D., R.G., T.H.T., A.C.E., R.P.P., and P.J.S. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Karen de Mesy Bentley, the director of the Electron Microscope Research Core at the University of Rochester, for assistance with the visualization of smoke particles.

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