Age and sex dimorphisms contribute to the severity of bleomycin-induced lung injury and fibrosis

Abstract

Fibrotic interstitial pneumonias are more prevalent in males of advancing age, although little is known about the underlying mechanisms. To evaluate the contributions of age and sex to the development of pulmonary fibrosis, we intratracheally instilled young (8-12 wk) and aged (52-54 wk) male and female mice with bleomycin and assessed the development and severity of fibrotic lung disease by measurements of lung collagen levels, static compliance, leukocyte infiltration, and stereological quantification of fibrotic areas in histological sections. We also quantified proinflammatory and profibrotic chemokine and cytokine levels in the bronchoalveolar lavage fluid. Aged male mice developed more severe lung disease, indicated by increased mortality, increased collagen deposition, and neutrophilic alveolitis compared with aged female mice or young mice of either sex. Aged male mice also exhibited increased levels of transforming growth factor-β, IL-17A, and CXCL1 in their bronchoalveolar lavage fluid. Young male mice developed a more fibrotic disease after bleomycin instillation compared with female mice, regardless of age. There was no difference in fibrosis between young and aged female mice. Taken together, these findings suggest that the variables of advanced age and male sex contribute to the severity of pulmonary fibrosis in this model. Our findings also emphasize the importance of stratifying experimental groups on the basis of age and sex in experimental and epidemiological studies of this nature.

Fig. 1.

Aged male (AM) mice have increased lung tissue volume and mortality after bleomycin (Bleo) instillation. A: lung tissue volume does not change with age, but is significantly increased in young female (YF) and male (YM) mice and AM mice after Bleo (P < 0.001). AM mice have a significant increase in lung tissue volume compared with YF and aged female (AF) (***P < 0.001) mice, and a significantly lower lung tissue volume compared with YM mice (***P < 0.001). Values are means ± SE. B: AM mice exhibit increased mortality after intratracheal administration of Bleo compared with female mice and YM mice. Sal, saline.

Fig. 2.

AM mice have increased pulmonary fibrosis following Bleo administration. A: hydroxyproline levels were measured in upper right lobe from Sal- and Bleo-treated mice. There was no significant difference between groups of Sal-instilled mice by ANOVA (P > 0.05). All Bleo groups were significantly increased vs. Sal controls. YM mice have significantly greater hydroxyproline levels compared with females (*P < 0.05), and AMs have increased levels compared with all other groups (***P < 0.001). B: Bleo-instilled male mice have decreased static compliance measurements compared with female mice and Sal controls (***P < 0.001). There was no significant difference between groups of Sal-instilled mice by ANOVA (P > 0.05). Values are means ± SE.

Fig. 3.

Bleo induces greater collagen deposition in the lungs of male mice. A: mouse lung sections stained with Masson's trichrome. Collagen deposition (blue) is observed adjacent to airways and large vessels in Sal control mice, as well as in fibrotic areas in response to Bleo. B: quantitative assessment of collagen deposition indicates male mice have a significantly greater percentage of lung collagen compared with female mice (*P < 0.05). There was no significant difference between groups of Sal-instilled mice by ANOVA (P > 0.05). AM mice have a higher percentage of collagen vs. YM mice (**P < 0.01). YM and YF mice and AM Bleo-instilled mice have significantly more collagen them Sal-treated mice (at least P < 0.05). Values are means ± SE.

Fig. 4.

Male mice have increased epithelial cell injury and inflammatory cell infiltration after Bleo instillation. A: AM mice have a significant increase in lavaged albumin compared with YF (**P < 0.01) and AF (*P < 0.05) mice, but no significant difference (nsd) compared with YM mice (P > 0.1). Male mice have a significant increase in albumin after Bleo administration compared with Sal controls (***P < 0.001). B: total bronchoalveolar lavage (BAL) cell numbers. All Bleo-treated groups are significantly elevated compared with Sal controls (at least **P < 0.01). AMs treated with Bleo were significantly higher than all groups (***P < 0.001). C: differential BAL counts identifying macrophages, lymphocytes, and neutrophils. All mice have a significant increase in cells after Bleo instillation (**P < 0.01, ***P < 0.001) compared with Sal controls. AM mice have significantly increased neutrophil infiltration after Bleo administration compared with AF mice and young mice of either sex (#P < 0.001). Values are means ± SE.

Fig. 5.

AM mice have increased IL-17A, CXCL1, and CXCL2 in the BAL fluid (BALF) following Bleo instillation. A: AM mice have a significant increase in lavaged IL-17A compared with YM (**P < 0.01) and YF and AF mice (***P < 0.001). Male mice have a significant increase in IL-17A after Bleo administration vs. Sal control (***P < 0.001). B: AM mice have a significant increase in CXCL1 measured in BALF compared with YM (***P < 0.001) and YF (***P < 0.001) and AF mice (**P < 0.01). C: YM and AM mice and AF mice have a significant increase in CXCL2 after treatment with Bleo (**P < 0.01 and ***P < 0.001). Values are means ± SE.

Fig. 6.

Male mice exhibit increased phospho-Smad2/3 lung tissue staining and total transforming growth factor (TGF)-β in their BALF following Bleo instillation. A: Sal-instilled mice have positive nuclear staining of phospho-Smad2/3 in the airway and alveolar epithelium. YM and AM mice have increased staining in the airway epithelium, macrophages, and fibrotic areas following Bleo instillation compared with female mice. Final magnification of images is ×400. B: quantitative analysis of positive phospho-Smad2/3 nuclei indicates that male mice have a significant increase in staining after Bleo instillation compared with female mice (*P < 0.05). Sal instilled mice from all groups have a baseline level of staining that is not significantly different by ANOVA (P > 0.05). C: AM mice have a significant increase in total TGF-β in the BALF compared with all other groups treated with Sal and Bleo (**P < 0.01). YM mice have a significant increase in total TGF-β compared with Sal control (***P < 0.001). Values are means ± SE.