Sex-dependent acrolein sensitivity in mice is associated with differential lung cell, protein, and transcript changes

Abstract

Acrolein is a reactive inhalation hazard. Acrolein's initial interaction, which in itself can be function-altering, is followed by time-dependent cascade of complex cellular and pulmonary responses that dictate the severity of the injury. To investigate the pathophysiological progression of sex-dependent acrolein-induced acute lung injury, C57BL/6J mice were exposed for 30 min to sublethal, but toxic, and lethal acrolein. Male mice were more sensitive than female mice. Acrolein of 50 ppm was sublethal to female but lethal to male mice, and 75 ppm was lethal to female mice. Lethal and sublethal acrolein exposure decreased bronchoalveolar lavage (BAL) total cell number at 3 h after exposure. The cell number decrease was followed by progressive total cell and neutrophil number and protein increases. The BAL total cell number in female mice exposed to a sublethal, but not lethal dose, returned to control levels at 16 h. In contrast, BAL protein content and neutrophil number were higher in mice exposed to lethal compared to sublethal acrolein. RNASeq pathway analysis identified greater increased lung neutrophil, glutathione metabolism, oxidative stress responses, and CCL7 (aka MCP-3), CXCL10 (aka IP-10), and IL6 transcripts in males than females, whereas IL10 increased more in female than male mice. Thus, the IL6:IL10 ratio, an indicator of disease severity, was greater in males than females. Further, H3.3 histone B (H3F3B) and pro-platelet basic protein (PPBP aka CXCL7), transcripts increased in acrolein exposed mouse BAL and plasma at 3 h, while H3F3B protein that is associated with neutrophil extracellular traps formation increased at 12 h. These results suggest that H3F3B and PPBP transcripts increase may contribute to extracellular H3F3B and PPBP proteins increase.

Keywords: NETs; RNAseq; acrolein; acute respiratory distress syndrome.

FIGURE 1

Survival of C57BL/6J mice following acrolein exposure. Survival of female and male C57BL/6J mice exposed to (a) 75 ppm or (b) 50 ppm acrolein for 30 min and returned to filtered air and survival monitored for up to 120 h. At either concentration, male C57BL/6J mice are more sensitive to acrolein exposure compared with female mice as determined by Kaplan–Meier log‐rank survival (p < 0.001)

FIGURE 2

Lung histology of C57BL/6J mice following acrolein exposure. Acrolein exposure increased alveolar wall thickening, lung proteinaceous deposits, and leukocyte infiltrates. Male (Left panels) and female (Right panels) mice were exposed to filtered air (Top panels), 50 ppm (Middle panels), or 75 ppm (Bottom panels) acrolein for 30 min and lungs collected 16 h after exposure for hematoxylin and eosin staining

FIGURE 3

Bronchoalveolar (BAL) protein, total cells, and neutrophils in C57BL/6J mice exposed to 50 or 75 ppm acrolein for 30 min. BAL was collected from filtered air exposed male and female mice (0), male mice exposed to 50 ppm, or female mice exposed to 50 or 75 ppm acrolein for 30 min. Following exposure, mice were returned to filtered air and BAL was collected 3 or 16 h after exposure. BAL (a) protein, (b) total cells, and (c) neutrophils were determined. Each symbol represents one mouse and midline indicates mean and bars indicate standard deviation (SD, n = 5–9 mice/group). *Values are significantly different (p < 0.01) as determined by one‐way analysis of variance with Holm–Sidak all pairwise multiple comparison procedure

FIGURE 4

Differentially expressed genes altered in mouse lungs following acrolein exposure. Mice were exposed to air (control) (n = 6/sex) or 50 ppm acrolein (n = 8/sex) for 30 min and returned to filtered air. Lungs were collected 6 h after exposure and transcripts were analyzed by RNASeq. Transcripts that (Blue) decreased or (Yellow) increased by absolute value twofold and adjusted p value < 0.5 were arranged by centroid linked hierarchical clustering using Gene Cluster 3.0 and visualized by Java TreeView. (Left panel) Each column is one mouse (n = 8 male and 8 female) and each row is one transcript (n = 1991) with enlarged cluster in which female mice had an uneven (Cluster A middle panel) decreased or (Cluster B right panel) increased response as compared to male mice

FIGURE 5

Lung cytokines altered in male compared to female mice following acrolein exposure. Mice were exposed to air (control) (n = 6/sex) or 50 ppm acrolein (n = 8/sex) for 30 min and returned to filtered air. Lungs were collected 6 h after exposure and transcripts were analyzed by RNASeq. Chemokine (C–X–C motif) ligand 7 (CCL7 aka MCP‐3), chemokine (C–C motif aka IP‐10) ligand 10 (CXCL10), interleukin 6 (IL6), increased more in male as compared to female mice. In contrast, IL10 increased more in female as compared to male mice. *Significantly different between sexes as determined by analysis of variance (ANOVA) followed by one‐way analysis of variance with Holm–Sidak all pairwise multiple comparison procedure

FIGURE 6

Lung H3.3 histone B (H3F3B) and pro‐platelet basic protein (PPBP) transcripts increased in acrolein exposed male and female C57BL/6J mice. Mice were exposed to air (control) or 50 ppm acrolein for 30 min and returned to filtered air. Lungs were collected 1, 3, 6, and 12 h after exposure. H3F3B and PPBP transcripts were analyzed by quantitative real‐time polymerase chain reaction (qRT‐PCR). H3F3B (Top panel) and PPBP (Bottom panel) transcripts increased by 3 h after exposure and remained elevated up to 12 h after exposure compared to control. (Values are Mean ± standard error of means [SEM], n = 6 mice/group). *Values are increased compared to control, ^†increased in female compared to male (p < 0.01) as determined by one‐way analysis of variance (ANOVA) with Holm–Sidak all pairwise multiple comparison procedure

FIGURE 7

Delayed H3.3 histone B (H3F3B) protein detection in bronchoalveolar lavage (BAL) of acrolein exposed male and female C57BL/6J mice. Mice were exposed to 50 ppm acrolein for 30 min and returned to filtered air. BAL (35 µl/lane) from control or acrolein exposed mice was collected 3 or 12 h after exposure and analyzed by Western blotting for H3F3B protein. Haptoglobin was included to demonstrate protein loading