This study was designed to elucidate the contributions of sex and sex hormones to the progression of lung fibrosis and associated lung function decline in a murine model. To this end, the established and well-characterized bleomycin model was used to induce pulmonary fibrosis in reproductive-age male and female C57BL/6 mice. Fibrosis was assessed with common biochemical and histological endpoints, and invasive lung function analysis was employed to measure respiratory mechanics. Our data suggest that lung function analysis is a unique indicator of lung damage not captured by other more commonly used endpoints. Lung function decline also displays a sex specificity not observed in the other endpoints, a finding that may allow researchers to more accurately model human diseases in which sex differences exist. Furthermore, decreased lung function may not necessarily mirror immune cell influx, collagen deposition, or total lung fibrosis in the murine model. This is consistent with our previous observations in cyclooxygenase-2–deficient mice (10
). Furthermore, we demonstrated an exacerbatory role for androgen in mediating the decline in lung function associated with fibrosis, but not the progression of fibrosis itself. By comparison, estrogen appeared to play no role in modulating lung function after bleomycin administration, as αERKO, βERKO, and ovariectomized female mice responded identically to intact wild-type females. Female mice responded to bleomycin with decreased lung function if and only if they had been treated with DHT, further illustrating the exacerbatory role of androgen in decreasing lung function after bleomycin administration.
The possibility of sexual disparity in the response to bleomycin in the murine model has been raised before (25
) and is consistent with recently published findings in humans that describe sex differences in the severity of emphysema (29
). However, our data are in contrast to recent findings suggesting that estrogen plays an important role in pulmonary fibrosis in a different species (30
). We currently have no explanation for the differences in our study compared with that of Gharaee-Kermani and colleagues (30
); however, it is important to note that our study was performed with mice while theirs was performed with rats, and that we have included invasive lung function analysis as an endpoint. Further work is required before we can say definitively why the dichotomy exists between the two rodent models.
Bleomycin hydrolase is a neutral cysteine protease of the papain superfamily (31
) named for its ability to catalytically deaminate and inactive bleomycin (23
). In humans, bleomycin hydrolase is widely expressed (32
), although evidence exists to suggest that activity in lungs is heterogeneous (24
) and may be decreased as a result of bleomycin administration (33
). Bleomycin hydrolase–null mice display a profoundly increased sensitivity to bleomycin (34
), suggesting that bleomycin hydrolase is the predominant, if not sole, bleomycin-detoxifying enzyme. Moreover, bleomycin hydrolase is a candidate gene for a recently identified sex-specific genetic locus conferring susceptibility to bleomycin-induced pulmonary fibrosis (25
). In our studies, we observed no difference in bleomycin hydrolase levels between untreated wild-type male and female mice as assessed by real-time quantitative PCR. Furthermore, while exposure to bleomycin increased bleomycin hydrolase expression, there was no observed difference in expression between male and female mice. Based on these data, we think it unlikely that differential bleomycin hydrolase expression accounts for the observed differences in lung function. This conclusion is supported by the observed similarities in total lung collagen deposition and fibrotic response independent of the difference in lung function.
Lung function as an endpoint in the murine model of bleomycin-induced lung fibrosis is a relatively new and important development and, in our view, allows researchers to more accurately reflect the human pathological condition that the model was designed to emulate. Declining lung function more directly correlates with poor prognosis in humans with pulmonary fibrosis than do fibrotic endpoints (35
) and may have more predictive value than the commonly reported collagen content, immune cell influx, and Ashcroft score (36
). The importance of including lung function analysis as part of the murine model is underscored by our findings that there is a quantifiable and significant difference between males and females in their static and dynamic compliance after bleomycin despite having similar cellular and histological responses, and that androgen appears to impact lung function while not altering other commonly measured endpoints associated with bleomycin. Other studies that have included lung function in the bleomycin model lead to disparate conclusions regarding the correlation between compliance and fibrotic endpoints. We have previously reported discordance between the fibrotic endpoints and lung function (10
), while others have reported an agreement between the compliance and fibrotic endpoints (11
). It is worth noting that Lovgren and colleagues (11
) used multiple strains of mice and administered a dose of bleomycin that was not adjusted for body weight. Indeed, the resulting doses of bleomycin administered by Lovgren and coworkers were higher than what we used for most of our experiments. It is possible that higher doses may obscure the point at which lung function and other endpoints diverge. We specifically selected a dose of bleomycin that was in the middle to low range of reported doses to maximize our ability to detect subtle changes or changes that may be masked by larger doses.
Although not the focus of this study, total respiratory system resistance (R) was observed to increase in bleomycin-treated male but not female mice (data not shown), and we believe this may have been at least partially due to the increased collagen deposition seen around the airways in males. Other studies did not demonstrate increased R after bleomycin, but they differed from the current study in that they used either female mice only (10
) or a mix of sexes (11
). The implications of this observation are currently unclear, but will be investigated in future studies. Furthermore, our findings are consistent with those of Lovgren and coworkers and others who have reported that administration of bleomycin results in a decrease in lung compliance. This is consistent over a number of mammalian models including mice, rats (37
), and rabbits (38
), and is consistent with findings in humans demonstrating decreased lung function in patients with fibrosis (36
The mechanism by which androgen exacerbates the decrease in static and dynamic compliance in our model remains undetermined. Our data suggests that multiple mechanisms may be involved, as castration changes both baseline quasi-static compliance and the response of the animal to bleomycin (). Indeed, both of these changes are necessary to observe statistically significant differences after castration. Moreover, addition of exogenous DHT concomitant with castration increases baseline Cst and normalizes the response to bleomycin. Furthermore, as illustrated in , addition of exogenous DHT similarly alters the response of both saline- and bleomycin-treated mice. It should be noted that the only statistically significant difference that was observed in quasi-static compliance in females was in the presence of DHT, and both the baseline and the bleomycin responses changed. A number of potential explanations exist and are the subject of ongoing experimentation. Injury to specific cell types within the lung and/or the immune system, changes in noncollagen components of the extracellular matrix, differences in collagen maturity, and micro-environmental differences in extracellular matrix changes are some of the potential mechanisms being investigated. Among these, we have examined regional differences in collagen deposition in this study. While our observations are consistent with fibrosis being a predominantly parenchymal phenomenon, there were differences between the sexes in the amount of collagen associated with small and intermediate-sized airways that tended to be greater in males than in females. These differences underscore the challenges involved in describing and quantifying the morphological changes associated with a condition as heterogeneous as pulmonary fibrosis while highlighting the utility and importance of including invasive lung function in studies of pulmonary fibrosis.
A number of recent studies have highlighted the myriad of ways in which sex hormones can modulate lung development, physiology, and pathology (reviewed in Refs. 26
). Sex hormones have been demonstrated to play important roles in models of allergic airway disease, immune response, and lung injury, and the influence of sex hormones is often dependent upon the model studied. In this article we have described a model in which male mice suffered a more severe decrease in lung compliance than female mice after bleomycin administration. Conversely, in a Pseudomonas aeruginosa
infection model in the same C57BL/6 strain of mice, females are demonstrably more susceptible to infection and display a more robust immune response than males (39
). Our study further showed that castration attenuated the observed differences while ovariectomy did nothing to modulate the phenotype, and that addition of exogenous androgen resulted in a return of castrated males to their pre-surgery response level as well as masculinizing the response of female mice. These data suggest that estrogen plays an insignificant role in protecting females from lung function decline after bleomycin exposure despite the mounting of a fibrotic response similar to that of males. Furthermore, these data indicate that androgen can exacerbate the lung function decline in males and females after bleomycin administration, a finding consistent with previously published data demonstrating an exacerbatory role for androgens in modulating airway responsiveness to methacholine during LPS-induced inflammation (40
In closing, the findings described herein reveal an androgen-mediated sex difference in lung function decline in a murine model of pulmonary fibrosis that is not reflected in more common biochemical and histological measures of fibrosis. Our data indicates that more specialized histopathologic measurements of airways in the lung may be helpful to capture regional fibrotic responses. Furthermore, our data highlights the utility of invasive lung function analysis in experimental studies of pulmonary fibrosis and indicate the need to stratify experimental design and data analysis based on sex. Future investigations will focus on elucidating the mechanism(s) by which androgens promote lung function decline in pulmonary fibrosis with the expectation that such insights might lead to the development of novel approaches to combat this condition.