In this study, we investigated the effects of Po exposure on pulmonary inflammatory responses and evaluated the role of sex in this process in female and male mice. Most importantly, based on the above findings, we elucidated the role of ERβ in this process. To our knowledge, this is the first investigation about the role of sex in Po-induced adverse effects.
Existing epidemiological and experimental studies have shown that PAHs in PM2.5 can promote the release of inflammatory cytokines, such as IL-1β, IL-8, TNF-α, and IL-6 (Falcon-Rodriguez et al. 2016, Kuang et al. 2020, Manzano-Leon et al. 2016). Our previous study has proved that Po contained more than half (53.75%) of the total polycyclic aromatic hydrocarbons (PAHs) in PM2.5 (Wei et al. 2016). Therefore, we further evaluated whether Po could induce pulmonary inflammation in mice. We found that Po could induced inflammatory cell infiltration in lung tissues of mice, increased the inflammatory cell numbers and upregulated the levels of several inflammatory cytokines (IFN-γ, TNF-α, IL-1β, IL-5, IL-6, GRO/KC and IL-12p70) in the BALF of the mice (Fig. 2; Table 1 and 2). Thus, Po could cause pulmonary inflammation in mice, and might have an influence on the immune microenvironment through regulating the inflammatory cytokines, which might lead to lung injury. Park et al. reported that Po can induce inflammatory responses of lung cells, which is consistent with this study (Park et al. 2021).
Existing evidences have suggested that pulmonary inflammation may arise from a combination of genetic susceptibilities and environmental influences (Racanelli et al. 2018), and there are differences in the susceptibility to tobacco between women and men (Dransfield et al. 2006). In addition, there are sex differences in some inflammatory diseases (Carey et al. 2007, Klein &Flanagan 2016, Laws et al. 2018, Ngo et al. 2014, Rainville et al. 2018, Samet et al. 2009, Zore et al. 2018). Among nonsmoking patients with COPD, there were more women than men (Birring et al. 2002). These studies indicated that sex might plays an important role in the pulmonary inflammation. In this study, we found that the increase of inflammatory cell numbers, and the fluctuation of TNF-α, IL-5, and GRO/KC levels in BALF induced by Po were different between female and male mice (Table 2). TNFα, GRO/KCC and IL-5 are pro-inflammatory factors that have been reported to participate in inflammatory and immune responses. The function of GRO/KC is similar to IL-8 in rat (Dong et al. 2012). IL-5 is important to eosinophils which is related to the fibrosis and long-term tissue injury. Some asthma patients will suffer from diseases characterized by eosinophilic airway inflammation (Dougan et al. 2019). Studies reported that inflammatory cytokines may contribute to some inflammatory diseases, such as COPD, pulmonary fibrosis, asthma (Hou et al. 2018, Narendra &Hanania 2019, Russell &Brightling 2016, Zhang et al. 2019). Therefore, Po might cause differences in these diseases between men and women through regulating the levels of inflammatory cytokines.
As we all know, hormonal difference is an important reason why sex differences appear in many diseases. Our previous study revealed that Po contained more than half (53.75%) of the total PAHs with DBA, PHE, BPE, IPY, BaP, BbF, BkF and CHR as the main compositions (Wei et al. 2016), and these substances, as environmental endocrine disruptors, have been confirmed to have estrogenic activity (Zhang et al. 2016). An existing study shown that PAHs could selectively induce ERβ transcriptional activity, while did not activating ERα (Sievers et al. 2013); Additionally, PAHs were able to competitively bind ERβ, induce ERβ homodimers, and regulate ERβ target genes (Sievers et al. 2013). ERα, ERβ and GPER belong to the estrogen receptor family, and they are major mediators of estrogenic signals (Barzi et al. 2013, Prossnitz &Barton 2011). The present study was the first to explore the association of Po with ERs (ERα, ERβ and GPER). The results shown that Po exposure could regulate the protein levels of ERβ, but not ERα and GPER, in lung tissues of both female and male mice, which indicating that Po exposure could activate ERβ signaling in lung tissues of mice.
ERβ not only can acts on the breast and uterus whose function were closely related to estrogen, but also can acts on the lung (Chen et al. 2011, Niikawa et al. 2008). Abnormal ERβ signaling can lead to inflammation in different organs, such as the lung (Jia et al. 2015, Watanabe et al. 2019), cardiovascular (Novella et al. 2019) and uterus (Spence &Voskuhl 2012). Considering that Po exposure might activate ERβ signaling in the process of Po-induced pulmonary inflammation in mice, we further evaluated whether ERβ played a key role in this process by using PHTPP, an ERβ antagonist. The results shown that PHTPP treatment can interfere with Po-induced inflammatory cell infiltration in lung tissues and Po-induced increases of the inflammatory cell numbers and the levels of IFN-γ, TNF-α IL-5, IL-6, GRO/KC and IL-12p70 in the BALF of the mice (Fig. 4; Table 1 and 2). Therefore, we speculated that ERβ was one of the contributors for Po-induced pulmonary inflammation in mice. Moreover, ERs have been reported to contribute to sex differences in pulmonary fibrosis, asthma and allergic inflammation (Elliot et al. 2019, Keselman &Heller 2015). According to the above results, we focused on the ERβ signaling pathway and explore whether ERβ participate in the process of Po-induced inflammatory response differences by sex. Intriguingly, we found that ERβ did play a role in the Po-induced inflammatory response differences (including the increase of inflammatory cell numbers and the fluctuation of the TNF-α, IL-5 and GRO/KC levels in BALF) between female and male mice and the differences were diminished by PHTPP (Table 2).
In summary, we explore the effects of sex in Po-induced inflammatory responses and evaluate the role of ERβ in this process, which provided a theoretical basis for understanding the sex difference in the adverse effects of environmental pollutants.