In the present study, we successfully established a mouse model of ARDS by intratracheal instillation of LPS in vivo and investigated sex differences in the effects of GPER activation on acute inflammatory responses in lung. In parallel, we used a cell line (MH-S) derived from a murine alveolar macrophage cell to explore related molecular mechanisms in vitro. Our results indicate that pretreatment with the GPER-specific agonist G1 protected mouse lung injury in a sex-specific manner. G1 administration significantly ameliorated LPS-induced pathological lung damage and alveolar capillary barrier destruction in male and OVX female mice. Administration of G1 also inhibited the recruitment of inflammatory cells into alveoli and decreased concentrations of pro-inflammatory factors TNF-α and IL-6 in BALF of male and OVX female mice. However, such salutary effects of GPER activation were not observed in intact female mice. In vitro experiments demonstrated that G1 pretreatment significantly inhibited LPS-mediated MH-S changes in expression of TNF-α, IL-6, and MIP2.
Overactivated inflammation is believed to be the major pathophysiological driver of ARDS, and sex differences in the inflammatory responsiveness of lung have been reported[10, 21]. In addition, the anti-inflammatory effect of E2 was reported in an animal model of ARDS. Use of knockout mice and receptor blockers revealed that ER-α, but not ER-β, plays a key role in mediating the anti-inflammatory effects of estrogen in lung[10, 22]. The current study, for the first time, found that pretreatment with the GPER-selective agonist G1 alleviated histopathological lung injury, inhibited inflammatory cell infiltration, and decreased IL-6 and TNF-α levels in BALF of male and OVX female mice following LPS injection. However, no such anti-inflammatory effects were observed in intact female mice. These sex-specific effects of G1 administration are consistent with a study of traumatic brain injury in which G1 treatment significantly attenuated IL-1β, IL-6, and TNF-α production in males and OVX females, but induced pro-inflammatory effects in intact females.
In addition, we observed sex-dependent reductions of total protein and albumin concentrations in BALF, which reflect alveolar capillary barrier function during ARDS. Previous reports showed sex differences in alveolar capillary barrier disruption within the lung. In these studies, males and OVX females exhibited higher albumin contents in BALF compared with intact females, and estrogen administration reversed LPS-induced leakage of albumin into alveoli in OVX female mice. In the present study, albumin levels in BALF did not differ between sham and OVX females administered LPS, although a slight trend was noticed. We considered the possibility that these differences resulted from differential dosages and routes of LPS administration, as well as differences in sample numbers. In addition, we observed a similar trend of albumin decrease following G1 administration, but no significant difference was detected between G1-treated and vehicle-treated OVX females after LPS stimulation. In a previous study, estrogen exerted a lung-protective effect on the alveolar capillary barrier in male rats exposed to paraquat or the glutamate agonist N-methyl-d-aspartate, indicating that infusion of estrogen into the pulmonary circulation significantly attenuated albumin leakage. Consistent with this finding, our results also demonstrated attenuation of albumin after G1 administration in males. Few studies have attempted to identify the specific receptor mediating the beneficial effects of E2 on alveolar capillary barrier function. Here, we provide the first evidence that GPER mediates such effects.
As a crucial mediator of pulmonary inflammation during ARDS, alveolar macrophages are a promising cell target to regulate processes involved in the inflammatory response. Upon injury by pulmonary insult (e.g., intratracheal LPS injection), activated macrophages recruit neutrophils into the lung by secreting chemokines and cytokines. Evidence indicate that inhibiting activation of alveolar macrophages can alleviate lung injury by inhibiting inflammatory cytokine expression and neutrophil recruitment. We selected the MH-S cell line to simulate alveolar macrophage responses to G1 in vitro. Our results show that pretreatment with G1 inhibited LPS-mediated production of IL-6, TNF-α, and CXCL2/MIP2 in a dose-dependent manner. These results are consistent with previous studies in which inhibition of TNF-α and IL-6 secretion was observed in primary human macrophages and RAW 264.7 (mouse macrophage) cells via GPER signaling[26, 27].
The current study had several limitations. First, we did not explore the mechanism by which G1 is engaged. A previous study indicated a role for Akt in mediating G1-inhibited inflammation of males and OVX females with brain injury. Moreover, in vitro studies demonstrated that G1 administration downregulated expression of Toll-like receptor 4 and nuclear factor κB p65 in macrophages following LPS exposure[15, 27]. Therefore, additional in vivo study is necessary to uncover the signaling pathway involved in attenuation of lung inflammation by G1. Furthermore, a binding study revealed that the anti-inflammatory activity of estrogen on human monocytes is mediated by ERa36 (a 36-kDa splice variant of ER-a) together with GPER, demonstrating crosstalk between ERa36 and GPER. Thus, investigating the role of ERa36 on G1-mediated anti-inflammatory processed during ARDS is necessary. Another limitation is that we did not monitor circulating estradiol levels in female mice at the time of injury, although previous studies showed that hormonal cycling plays a crucial role in pulmonary inflammation. In this study, we monitored the estradiol levels in female 24h after lung injury, which may partly represent the estradiol states in females. Nonetheless, it is still necessary to further investigate the effects of G1 in females during various estrous cycles by using cycling female mice, whose estrus cycle can be determined by vaginal smear.
Perspectives and significance
Although numerous studies have attempted to identify efficient drugs to treat ARDS owing to its high mortality, no major breakthroughs have been achieved so far. Previous studies demonstrated a protective role of estrogen in ARDS, but it remains unclear which receptor mediates such salutary effects. This study demonstrated a lung-protective role of the GPER-selective antagonist G1 against LPS-induced ARDS in male and OVX female mice, but not in gonadally intact females. To the best of our knowledge, this is the first report showing a sex-specific role of GPER in ARDS. Although further efforts are required to fully evaluate the efficacy and mechanisms of GPER activation following pulmonary inflammation, our findings may provide novel perspectives for gender influence in ARDS.