Sex-specific IL-6-associated signaling activation in ozone-induced lung inflammation - PubMed (original) (raw)

Sex-specific IL-6-associated signaling activation in ozone-induced lung inflammation

Vikas Mishra et al. Biol Sex Differ. 2016.

Abstract

Background: Acute ozone (O3) exposure has known deleterious effects on the respiratory system and has been linked with respiratory disease and infection. Inflammatory lung disease induced by air pollution has demonstrated greater severity and poorer prognosis in women vs. men. Both severe damage to the bronchial-alveolar epithelium and malfunctioning of bronchial-blood barrier have been largely attributed to the pathobiology of O3-induced inflammatory response, but the associated mechanisms in the male and female lung remain unknown.

Methods: Here, we investigated sex-based differential regulation of lung interleukin-6 (IL-6) and its downstream signaling pathways JAK2/STAT3 and AKT1/NF-κB in response to O3 exposure in a mouse model. We exposed male and female mice (in different stages of the estrous cycle) to 2 ppm of O3 or filtered air (FA) for 3 h, and we harvested lung tissue for protein expression analysis by Western blot.

Results: We found significant up-regulation of IL-6 and IL-6R in females and IL-6 in males in response to O3 vs. FA. Ozone exposure induced a significant increase in STAT3-Y705 phosphorylation in both females and males. Males exposed to O3 had decreased levels of JAK2, but increased JAK2 (Y1007+Y1008) phosphorylation, while females exposed to O3 showed significant up-regulation of both proteins. Both NF-κB (p105/p50) and AKT1 protein levels were significantly increased only in females exposed to O3. In addition, females exposed to O3 during proestrus displayed increased expression of selected genes when compared to females exposed to O3 in other estrous cycle stages.

Conclusions: Together, our observations indicate a sex-based and estrous cycle-dependent differential lung inflammatory response to O3 and involvement of two converging JAK2/STAT3 and AKT1/NF-κB pathways. To our knowledge, this is the first study specifically addressing the impact of the estrous cycle in O3-associated lung inflammatory pathways.

Keywords: Estrous cycle; Gender differences; Inflammation; Lung disease; Oxidative stress.

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Figures

Fig. 1

Fig. 1

IL6 and IL6R expression and effect of ozone exposure. Left panels: a Representative Western blot images of IL6 and IL6R expression in males and females with filter air and O3 exposure; univariate analysis of IL6 (b) and IL6R (e), expression in males and females, with FA and O3 exposure; two-way ANOVA interaction effect of sex (c) and exposure (d), for IL6 expression and sex (f) and exposure (g), for IL6R expression. Right panels: h Representative Western blot images of IL6 and IL6R expression in estrous cycle stages of females, with filter air and O3 exposure; univariate analysis of IL6 (i) and IL6R (l), expression in estrous cycle stages of females, with FA and O3 exposure. Two-way ANOVA interaction effect of exposure (j) and estrous cycle stages (k), for IL6 expression and exposure (m) and estrous cycle stages (n), for IL6R expression. Univariate analysis data expressed as Ranks-Kruskal-Wallis test of densitometric analysis; the values are depicted as mean with SD, where *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 are the levels of statistical significance compared to controls (n = 6–8 per group). Two-way ANOVA for IL6 and IL6R analysis is given in Tables 1 and 3, respectively

Fig. 2

Fig. 2

STAT3, STAT3 Serine 727, and STAT3 Tyrosine 705 phosphorylation and effect of ozone exposure. Left panels: a Representative Western blot images of STAT3, STAT3 Serine 727 and STAT3 Tyrosine 705 phosphorylation expression in males and females with filter air and O3 exposure; univariate analysis of STAT3 (b), STAT3 Serine 727 (e), and STAT3 Tyrosine 705 (h), expression in males and females, with FA and O3 exposure; Two-way ANOVA interaction effect of sex (c) and exposure (d), for STAT3 expression, sex (f) and exposure (g), for STAT3 Serine 727 expression and sex (i) and exposure (j), for STAT3 Tyrosine 705 expression. Right panels: k Representative Western blot images of STAT3, STAT3 Serine 727, and STAT3 Tyrosine 705 phosphorylation expression in estrous cycle stages of females, with filter air and O3 exposure; univariate analysis of STAT3 (l), STAT3 Serine 727 (o), and STAT3 Tyrosine 705 (r), expression in estrous cycle stages of females, with FA and O3 exposure. Two-way ANOVA interaction effect of exposure (m) and estrous cycle stages (n), for STAT3 expression, exposure (p) and estrous cycle stages (q), for STAT3 Serine 727 expression, and exposure (s) and estrous cycle stages (t), for STAT3 Tyrosine 705 expression. Univariate analysis data expressed as Ranks-Kruskal-Wallis test of densitometric analysis; the values are depicted as mean with SD, where *p ≤ 0.05 and **p ≤ 0.01 are the levels of statistical significance compared to controls (n = 6–8 per group). Two-way ANOVA for STAT3, STAT3 Serine 727, and STAT3 Tyrosine 705 phosphorylation expression analysis is given in Tables 4, 5, and 6, respectively

Fig. 3

Fig. 3

JAK2 and JAK2 phosphorylated (Y1007+Y1008) expression and effect of ozone exposure. Left panels: a Representative Western blot images of JAK2 and JAK2 phosphorylated (Y1007+Y1008) expression in males and females with filter air and O3 exposure; univariate analysis of JAK2 (b) and JAK2 (e) phosphorylated (Y1007+Y1008), expression in males and females, with FA and O3 exposure; two-way ANOVA interaction effect of sex (c) and exposure (d), for JAK2 expression and sex (f) and exposure (g), for JAK2 phosphorylated (Y1007+Y1008) expression. Right panels: h Representative Western blot images of JAK2 and JAK2 phosphorylated (Y1007+Y1008) expression in estrous cycle stages of females, with filter air and O3 exposure; univariate analysis of i JAK2 and l JAK2 phosphorylated (Y1007+Y1008), expression in estrous cycle stages of females, with FA and O3 exposure. Two-way ANOVA interaction effect of exposure (j) and estrous cycle stages (k), for JAK2 expression and exposure (m) and estrous cycle stages (n) and for JAK2 phosphorylated (Y1007+Y1008) expression. Univariate analysis data expressed as Ranks-Kruskal-Wallis test of densitometric analysis; the values are depicted as mean with SD, where *p ≤ 0.05 and **p ≤ 0.01 are the levels of statistical significance compared to controls (n = 6–8 per group). Two-way ANOVA for JAK2 and JAK2 phosphorylated (Y1007+Y1008) analysis is given in Tables 7 and 8, respectively

Fig. 4

Fig. 4

NF-κB (p105/p50) expression and effect of ozone exposure. Left panel: a Representative Western blot images of NF-κB (p105/p50) expression in males and females with filter air and O3 exposure; b univariate analysis of NF-κB (p105/p50) expression in males and females with FA and O3 exposure; two-way ANOVA interaction effect of sex (c) and exposure (d) for NF-κB (p105/p50) expression. Right panel: e Representative Western blot images of NF-κB (p105/p50) expression in estrous cycle stages of females, with filter air and O3 exposure; f univariate analysis of NF-κB (p105/p50) expression in estrous cycle stages of females with FA and O3 exposure. Two-way ANOVA interaction effect of exposure (g) and estrous cycle stages (h) for NF-κB (p105/p50) expression. Univariate analysis data expressed as Ranks-Kruskal-Wallis test of densitometric analysis; the values are depicted as mean with SD, where **p ≤ 0.01 is the level of statistical significance compared to controls (n = 6–8 per group). Two-way ANOVA for NF-κB (p105/p50) expression analysis is given in Table 9

Fig. 5

Fig. 5

AKT1 expression and effect of ozone exposure. Left panel: a Representative Western blot images of AKT1 expression in males and females with filter air and O3 exposure; b univariate analysis of AKT1 expression in males and females with FA and O3 exposure; two-way ANOVA interaction effect of sex (c) and exposure (d) for AKT1 expression. Right panels: e Representative Western blot images of AKT1 expression in estrous cycle stages of females, with filter air and O3 exposure; f univariate analysis of AKT1 expression in estrous cycle stages of females with FA and O3 exposure. Two-way ANOVA interaction effect of exposure (g) and estrous cycle stages (h) for AKT1 expression. Univariate analysis data expressed as Ranks-Kruskal-Wallis test of densitometric analysis; the values are depicted as mean with SD where **p ≤ 0.01 and ***p ≤ 0.001 are the levels of statistical significance compared to controls (n = 6–8 per group). Two-way ANOVA for AKT1 expression analysis is given in Table 10

Fig. 6

Fig. 6

Schematic of ozone-induced lung inflammation and role of IL-6 signaling pathway: ozone-associated lung inflammation is primarily driven by lipid ozonation products (LOP) and generation of free radicals primarily in lung epithelial cells. LOP and free radicals can further mediate activation of downstream biochemical events and cascades of secondary cellular responses leading to the inflammatory damage. The production of free radicals and LOP can trigger (i) increased expression of IL-6 and STAT3 and (ii) activation of NF-κB and (iii) activation of PI3K/AKT pathway

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