Immunofluorescent analysis of MAPK signaling in the lungs of biomass or smoke exposed mice.

<p>Immunofluorescence (FITC-green) for p-ERK, p-JNK and p-p38 was conducted on lung tissue sections from control mice and mice exposed to one week of biomass or cigarette smoke. DAPI staining was used to visualize nuclei.</p

Schematic of the <i>in vitro</i> and <i>in vivo</i> exposure models.

<p>Dung biomass from the village studied in North India was combusted on a barbecue grill. The smoke was channeled to either create biomass smoke extract (BSE) for the <i>in vitro</i> studies or it was directed to the mouse chamber for <i>in vivo</i> exposure studies. The total particulate matter concentration within the chamber was maintained at 200 mg/m³ for the duration of the exposure.</p

The effect of MAPK signaling on cytokine and protease induction by BSE, CSE and naphthalene in SAE cells.

<p>(A) Westerns for active and total JNK, ERK and p38 were conducted on cell lysate protein from SAE cells treated with 2–5% of BSE or CSE or 50–100 μM of naphthalene. Time point was 30 minutes. Actin was used as a loading control. (B) Densitometric analyses were conducted comparing the ratio of active to total levels of ERK, JNK and p38 in the above treated cells. (C) Westerns for total JNK, ERK and p38 were conducted on cells pre-treated with siRNA for each of these MAPK proteins. Actin was used as a loading control. (D) SAE cells were treated with control siRNA or siRNA for p38, JNK or ERK for 6 hours. These SAE cells were then treated for 24 hours with 2% BSE, CSE or 100 μM of naphthalene. IL-8 levels were determined in the media using multiplex analysis. N = 6 per group. Data is reported as mean ± standard error of measurement. (E) MMP-3 (left panel) and MMP-9 (right panel) were measured via multiplex analysis in SAE cells that had been treated with siRNA and then exposed to 2% BSE, CSE or 100 μM of naphthalene for 24 hours. N = 6 per group. Data is reported as mean ± standard error of measurement.</p

Effects of biomass or cigarette smoke exposure on cell signaling and cytokine and protease levels in mice.

<p>(A) Lung lavage (BALF) levels of IL-1β, IL-6, KC, IL-12p40, IL-12p70, IL-17, TNF-α, G-CSF and GM-CSF were measured via multiplex analysis in control mice and mice exposed to one-week of biomass or cigarette smoke. N = 10 in each group. (B) Lung lavage (BALF) levels of MMP-9, MMP-12 and TIMP-1 were measured via multiplex analysis in control mice and mice exposed to one-week of biomass or cigarette smoke. N = 10 in each group. (C) Western blot analysis for active and total p38, ERK and JNK were determined on lung lysate protein from the room air, cigarette smoke or biomass-exposed mice. (D) Densitometric analyses were conducted comparing the ratio of active to total levels of ERK, JNK and p38 in the lungs of the above treated mice.</p

The effect of biomass exposure on lung inflammation, MAPK signaling and protease expression in mice.

<p>(A) Lung bronchoalveolar lavage (BAL) cellularity (left panel) was measured in C57BL/6 mice exposed to room air, biomass smoke or cigarette smoke for one week. (B) Quik-diff analysis of the lung lavage cellular pellet was used to calculate the total number of macrophages (top left panel) or neutrophils (top right panel) in the lung lavage. N = 8 in each group. (C) H&E stained lung tissue sections of control (left panels), biomass-exposed (center panels) or cigarette smoke-exposed mice (right panels). In exposed mice, biomass material (see arrows) can be seen within the airway epithelium (top center panel) and alveolar region (middle center panel). The bottom panels demonstrate that macrophages from biomass-exposed mice (bottom center panel) were larger and were full of vacuoles that were not present in the control macrophages (bottom left panel) (scale bar = 20 µM). (D) Biomass-exposed mice (center left panel) had intense, lymphocytic, perivascular inflammation. These changes were not observed in control mice (top left panel) and were less notable in cigarette smoke-exposed mice (bottom left panel) (scale bar = 20 µM). Perivascular inflammation scores were calculated for control, biomass-exposed and cigarette smoke-exposed mice (right panel). N = 8 in each group. Data is expressed as mean ± standard error of measurement.</p

The effect of BSE and CSE on TIMP-1, MMP-1 and IL-8 expression in human SAE cells.

<p>(A) TIMP-1 levels were measured from the media of SAE cells treated with 2% or 5% of BSE or CSE for 24 hours via ELISA. N = 6 per group. Data is reported as mean ± standard error of measurement. (B) Endotoxin levels were measured in phosphate buffered saline (PBS) and biomass smoke extract (BSE) and cigarette smoke extract (CSE) before and after endotoxin removal. N = 4 in each group. Data is reported as mean ± standard error of measurement. (C) MMP-1 (left panel) and IL-8 (right panel) levels were measured from the media of SAE cells treated for 24 hours with 2% BSE, CSE or 5% BSE, CSE with and without endotoxin removal and 1 µg/ml LPS for 24 hours via ELISA. N = 6 per group. Data is reported as mean ± standard error of measurement. *Indicates endotoxin removal.</p