The Role of Estrogen Receptor β in PM2.5 Organic Extract-Induced Pulmonary In ammation in Female and Male Mice

Huaqi Guo Shanghai Jiao Tong University School of Medicine Hengyi Yu Shanghai Jiao Tong University School of Medicine Yan Feng Shanghai Jiao Tong University School of Medicine Wei Cheng Shanghai Jiao Tong University School of Medicine Yan Li Shanghai Jiao Tong University School of Medicine Yan Wang (  wangyan@shsmu.edu.cn ) The ninth people's hospital of Shanghai Jiao Tong University School of Medicine https://orcid.org/0000-0003-4008-6200


Introduction
With rapid urbanization and economic growth, the use of motorized transport in China is increasing Several epidemiological studies have reported that there were sex differences in many in ammatory diseases. For example, there are gender biases in disease incidences for many autoimmune diseases, such as Addison's disease, scleroderma, systemic lupus erythematosus, Sjogren's syndrome, and thyroiditis (Cooper &Stroehla 2003). The incidence rates of some types of cancer were signi cantly different between men and women, such as kidney cancer (Lucca et al. 2015), lung cancer (Mederos et al. 2020) and colorectal cancer (Nguyen et al. 2009). The pulmonary in ammatory disease -chronic obstructive pulmonary disease (COPD) was originally recognized as a male disease in history, but now COPD is more and more common in women (Aryal et al. 2013). The prevalence of COPD in women was higher than that in men among the non-smoking people (Birring et al. 2002). Chronic in ammation in the lung may arise from a combination of environmental in uences and genetic susceptibilities (Racanelli et al. 2018). For example, the susceptibility to tobacco smoke among patients with COPD was different between men and women (Drans eld et al. 2006).
Clougherty reviewed 209 epidemiological studies to discuss the role of sex in respiratory effects of air pollution. The author reported that more studies observed stronger adverse effects in women, although the sources of effect modi cation by sex remained unclear and some studies reported controversial results (Clougherty 2010). However, few experimental articles reported sex differences in the in uences of environmental pollutants. And to our knowledge, there is no experimental evidence of sex differences in the Po-induced adverse effects. Considering that PAHs in PM 2.5 could promote in ammatory responses and there were sex differences in many in ammatory diseases, we supposed that there might be differences in the process of Po-induced pulmonary in ammation between men and women.
Estrogen receptors (ER) family, including estrogen receptor α (ERα), estrogen receptor β (ERβ) and GPER, not only can act as signal transducers or transcription factors to regulate the expression of target genes . Therefore, we supposed that ER might be involved in the sex differences of Po-induced pulmonary in ammatory responses.
In this study, we performed an animal experiment in female and male mice. We investigated the effects of Po on pulmonary in ammation in mice, evaluated the differences of Po-induced in ammatory responses between female and male mice and further explored whether ERs took part in this process.

Materials & Methods
2.1 Collection of PM 2.5 and preparation of PM 2.5 organic extract PM 2.5 and its organic extract were prepared according to our previous study (Wei et al. 2016). In order to obtain comparable components of the samples to the most extent, we followed strictly the same procedure, including the sampler, sampling season, sampling place, and the extraction of organic components. Brie y, the PM 2.5 samples were collected in winter (November to December) by a largevolume PM 2.5 sampler (Intelligent 2031, Qingdao Laoying Inc., China), which was set on the top of a building (approximately 30 meters high) near Chongqing South Road (a two-way two-lane road with very dense tra c). The sampling lter membranes were taken out from the sampler after collecting PM 2.5 for 5 to 7 days, balanced in the dryer for 24 hours, wrapped in tin foil and stored at -20℃. The extraction method of Po has been demonstrated in our previous articles (Guo et al. 2020, Wei et al. 2016). Finally, the Po extract was dissolved in dimethyl sulfoxide in 400 µg/ml and stored at -20°C.
2.2 Animals and experimental design 6-7 weeks old C57BL/6 mice (24 female and 24 male mice) were obtained from Sino-British SIPPR/BK Lab Animal Ltd. (Shanghai, China) and raised in the Animal Experimental Center of the Ninth People's Hospital of Shanghai, and their care was in accordance with institution guidelines which was approved by the Committee on Animal Use and Care of Shanghai Jiao Tong University School of Medicine, China. All mice were fed in cages of the same size with a maximum of three mice per cage and placed in the same room with fresh air in the SPF level animal experimental center. An inhalation exposure system (Shanghai Raymain Information Technology Co., Ltd.) was used for the animal experiment, which can real-time monitor dynamic parameters in the exposure chamber, including the concentration of Po aerosols, temperature, humidity, O 2 and CO 2 concentration. The experimental procedure has been described in our previous article (Luo et al. 2020), with additional male mice in the present study. Brie y, the female and male mice were respectively divided randomly into three groups (8 mice/group): a, Control groups; b, Po groups; c, Po + PHTPP groups. The mice in the control groups were left untreated; the mice in other two groups were exposed to 100 µg/m 3 Po for 12 weeks (four hours a day, ve days a week) by the inhalation exposure system in a polycarbonate chamber. Meantime, the mice of Po + PHTPP groups were injected with PHTPP (Sigma-Aldrich) once per week (1 mg/kg, i.p.) from the fth week. The weight of mice was measured and recorded on the rst day of each week during these experiments. The mice were sacri ced at the day after the last exposure. The lung tissues and bronchoalveolar lavage uid (BALF) were collected.

Western blotting
The protein levels of lung tissues were quanti ed by western blotting. The standard protocol for western blotting has been described previously (Luo et al. 2017). Brie y, the proteins were separated and transferred onto membranes. The membranes were incubated with primary antibodies against ERα (1:1000, #8644, CST), ERβ (1:1000, ab3576, abcam), GPER (1:1000, ab137479, abcam), overnight with agitation at 4°C. Then, the appropriate secondary antibodies were used to incubate the bands for one hour at room temperature. The protein band density was detected using Quantity One Software (Bio-Rad, USA), and quanti ed using Image J software.

Preparation and Cell Counts of Bronchoalveolar Lavage Fluid (BALF)
The detailed method of obtaining BALF has been described in a previous article (Luo et al. 2020). Brie y, BALF were centrifuged at 1000×g for 5 min at 4°C. The supernatant was stored at -80°C to measure the levels of in ammatory cytokines, and the cells at the bottom were resuspended with 1 ml PBS (contains 0.1% BSA). The total cell numbers for each sample were counted by the hemocytometer.

Hematoxylin and Eosin (HE) staining
The left lung tissues were xed in 4% paraformaldehyde and then were embedded in para n after dehydration. After embedding with para n, these tissues were cut into 5-µm-thick sections and stained with hematoxylin and eosin. The sections were examined, photos were taken with a Pannoramic SCAN (3DHISTECH, Hungary).

Statistical analysis
All statistical analyses were performed using SPSS 19.0 software. The experiment data are shown as the mean ± SD. The signi cance level was de ned as 0.05. Interaction between the factors was analyzed by factorial analysis of variance (P<0.05, there is an interaction between the factors; P>0.05, there is no interaction between the factors). In addition, when there was a signi cant difference in the test of homogeneity of variance for experimental data, the data were log10-transformed before statistical modeling. If there was still a signi cant difference in the homogeneity test of variance for log10transformed data, the data were analyzed by Scheirer-Ray-Hare test (Hughes et al. 2020). The graphs were made by GraphPad Prism 7.0 software.

Changes in body weight
We evaluated the body weights of female and male mice once a week to see if the mice were healthy. As shown in Fig. 1 and our previous report(Luo et al. 2020), during the entire period of the animal experiment, there was no obvious difference in the changes of body weight between groups of female or male mice.

The effects of Po on pulmonary in ammation in female and male mice
Our previous study has proved that Po can induce pulmonary in ammation in female mice (Luo et al. 2020). We further included male mice in the present experiment. Histological analysis shown that obvious in ammatory cell in ltration was observed in the lung tissue of female and male mice after Po exposure (Fig. 2a). Po could also increase total cell numbers in the BALF of the mice ( Fig. 2b; Table 1 and 2). Meanwhile, Po could upregulate the levels of several in ammatory cytokines (IFN-γ, TNF-α, IL-1β, IL-5, IL-6, GRO/KC and IL-12p70) in the BALF of the mice (Fig. 2c-2i; Table 1 and 2).
We further investigated if there were signi cant differences in in ammatory cell numbers and cytokine levels in BALF between female and male mice. We found that the in ammatory cell numbers and IFN-γ level in the BALF of the female mice were signi cantly higher than those of the male mice ( Fig. 2a and 2b; Table 1 and 2), and the IL-1β level in the BALF of the female mice was signi cantly lower than that of the male mice ( Fig. 2e; Table 1 and 2). There were no signi cant differences in the levels of TNF-α, IL-5, IL-6, GRO/KC and IL-12p70 in BALF between female and male mice ( Fig. 2d and 2f-2i; Table 1 and 2). The results indicated that there were differences in the numbers of in ammatory cell and the levels of IFN-γ and IL-1β between female and male mice.
To explore the cause of in ammatory response differences induced by Po between female and male mice, factorial analysis of variance was used to analyze the interaction of sex and Po exposure in experimental mice. The results shown that there was an interaction between sex and Po exposure in the in ammatory cell numbers and the levels of TNF-α, IL-5, and GRO/KC (Table 1 and 2). The results implied that the increase of in ammatory cell numbers and the uctuation of TNF-α, IL-5, and GRO/KC levels induced by Po were different between female and male mice.

The effects of Po on the levels of estrogen receptors in lung tissues of mice
To determine whether Po exposure affect the expression of estrogen receptors, the protein levels of ERα, ERβ and GPER in the lung tissues of experimental mice were investigated. As shown in Fig. 3, while the mice were exposed to Po, the protein levels of ERβ in the lung tissues of female and male mice were upregulated, and the protein levels of ERα and GPER were no obvious change in the lung tissues of female and male mice ( Fig. 3a and 3b).

The effects of ERβ on Po-induced pulmonary in ammation in mice
We further explored whether ERβ could play an important role in Po-induced pulmonary in ammation in mice by using the ERβ antagonist, PHTPP. As shown in Fig. 4, PHTPP could signi cantly reduce the in ltration degree of in ammatory cell in lung tissues as well as the in ammatory cell numbers in BALF induced by Po (Fig. 4a and 4b; Table 1 and 2). Meanwhile, the Po-induced increases of IFN-γ, TNF-α IL-5, IL-6, GRO/KC and IL-12p70 levels in BALF were diminished by PHTPP (Fig. 4d and 4h; Table 1 and 2). The results suggested that ERβ contributed to Po-induced in ammatory cell in ltration in lung tissues as well as Po-induced increases of some proin ammatory cytokines levels in the BALF of the mice. Therefore, we summarized that ERβ was one of the contributors for Po-induced pulmonary in ammation.
As shown in Table 1 and 2, we speculated that the Po-induced increase of in ammatory cell numbers and the uctuation of TNF-α, IL-5, and GRO/KC levels in BALF were different between female and male mice by factorial analysis of variance. Then, we further evaluated whether ERβ participated in the Po-induced in ammatory response differences. We investigated the interaction between sex and Po+PHTPP treatment in experimental mice. The results indicated that there was no interaction in in ammatory cell numbers and the levels of TNF-α, IL-5, and GRO/KC between sex and Po+PHTPP treatment (Table 1 and 2). It means that PHTPP might diminish the interaction between sex and Po exposure which have been reported in 3.2. The results implied that PHTPP could eliminate the differences in the Po-induced increase of in ammatory cell numbers and the uctuation of TNF-α, IL-5 and GRO/KC levels in BALF between female and male mice.

Discussion
In this study, we investigated the effects of Po exposure on pulmonary in ammatory responses and evaluated the role of sex in this process in female and male mice. Most importantly, based on the above ndings, we elucidated the role of ERβ in this process. To our knowledge, this is the rst investigation about the role of sex in Po-induced adverse effects. Therefore, we further evaluated whether Po could induce pulmonary in ammation in mice. We found that Po could induced in ammatory cell in ltration in lung tissues of mice, increased the in ammatory cell numbers and upregulated the levels of several in ammatory 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  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 in ammation. In this study, we found that the increase of in ammatory cell numbers, and the uctuation 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-in ammatory factors that have been reported to participate in in ammatory 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 brosis and long-term tissue injury. Some asthma patients will suffer from diseases characterized by eosinophilic airway in ammation (Dougan et al. 2019). Studies reported that in ammatory cytokines may contribute to some in ammatory diseases, such as COPD, pulmonary brosis, 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 in ammatory 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 con rmed 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 rst 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 ( Table 1 and 2). Therefore, we speculated that ERβ was one of the contributors for Po-induced pulmonary in ammation in mice. Moreover, ERs have been reported to contribute to sex differences in pulmonary brosis, asthma and allergic in ammation (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 in ammatory response differences by sex.
Intriguingly, we found that ERβ did play a role in the Po-induced in ammatory response differences (including the increase of in ammatory cell numbers and the uctuation 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 in ammatory 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.

Conclusion
Po could induce pulmonary in ammation in both female and male mice. While exposed to Po, the increase of in ammatory cell numbers induced by Po was different between female and male mice. And the uctuation of several in ammatory cytokine levels in BALF shown different pattern between female and male mice. Further analysis suggested that ERβ was involved in these processes.

Declarations
Availability of data and materials The data that support the ndings of this study are available on request from the corresponding author.  Tables   Table 1, 2 is available in the Supplemental Files section. Figure 1 The body weight changes of experimental mice. C57BL/6 mice (24 female and 24 male mice) divided randomly into three groups (8 mice/group), and the mice were weighed once a week. Control groups:

Figures
untreated, Po groups: Po exposure, Po+PHTPP groups: Po exposure and PHTPP treatment.   The effects of ERβ on pulmonary in ammation induced by Po in female and male mice. C57BL/6 female mice and male mice exposed to Po with or without PHTPP treatment (8 mice/group).

Supplementary Files
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