Though gender-based differences in the development of protective or pathological adaptive host responses have been widely noted, it is becoming apparent that sex may also influence the early perception of microbial challenges and the generation of inflammatory immune responses. These differences may be due to the actions of reproductive hormones, and such a hypothesis is supported by the presence of receptors for these hormones in a variety of immune cell types. Androgens such as testosterone have been shown to decrease immune functions, including cytokine production. However, the mechanisms by which testosterone limits such responses remain undefined. In this study, we have investigated the acute effects of testosterone on the level of expression of a key trigger for inflammation and innate immunity, Toll-like receptor 4 (TLR4), on isolated mouse macrophages. We show that in vitro testosterone treatment of macrophages, generated in the absence of androgen, elicits a modest but significant decrease in TLR4 expression and sensitivity to a TLR4-specific ligand. In addition, we have studied the effect of in vivo removal of endogenous testosterone on TLR4 expression and endotoxin susceptibility. We report that orchidectomized mice were significantly more susceptible to endotoxic shock and show that macrophages isolated from these animals have significantly higher TLR4 cell surface expression than those derived from sham gonadectomized mice. Importantly, these effects were not apparent in orchidectomized animals that received exogenous testosterone treatment. As such, these data may represent an important mechanism underlying the immunosuppressive effects of testosterone.

Gender-based differences exist in infectious disease susceptibility. In general, females generate more robust and potentially protective humoral and cell-mediated immune responses after antigenic challenge than their male counterparts. Furthermore, evidence is accumulating that sex may also influence the early perception of microbial challenges and the generation of inflammatory immune responses such as sepsis. These differences have previously been attributed to the actions of reproductive hormones. Whereas androgens have been shown to suppress acute host immune responses to bacterial endotoxin challenge, estrogens have been found to promote increased resistance to bacterial infections. However, the mechanisms by which estrogens exert immunoprotective effects have not been established. In this study, we investigated the in vivo effects of 17beta-estradiol on endotoxin susceptibility in mice. Importantly, we have examined the actions of this female reproductive hormone on the expression of pattern recognition receptors that recognize bacterial endotoxin by key innate immune sentinel cells. We show that removal of endogenous estrogens decreases both pro- and antiinflammatory cytokine production, with a concomitant reduction in circulating levels of lipopolysaccharide-binding protein and cell surface expression of Toll-like receptor 4 on murine macrophages. Exogenous in vivo replacement of 17beta-estradiol, but not progesterone, significantly elevates sera lipopolysaccharide-binding protein levels and cell surface expression of Toll-like receptor 4 and CD14 on macrophages. Furthermore, this effect corresponds with significantly higher inflammatory cytokine levels after in vivo lipopolysaccharide challenge and a marked increase in endotoxin-associated morbidity. Taken together, these data provide a potential mechanism underlying the immunoenhancing effects of estrogens.

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Numerous studies have shown that estrogens augment immune responses and have been demonstrated to increase resistance to bacterial infections. In addition, it has been shown that production of inflammatory cytokines following administration of LPS is attenuated in ovariectomized animals, which is reversed following exogenous estrogen administration. We have recently demonstrated that chronic in vivo exposure to estrogen, but not acute in vitro exposure, augments Toll-like receptor 4 (TLR4) expression on peritoneal macrophages although the mechanisms that underlie this disparity are unclear. In this study we have investigated the effect of in vivo removal of endogenous estrogen and high dose estrogen replacement on TLR4 expression on bone-marrow derived macrophages and/or their progenitor cells. We report that these cells isolated from ovariectomized animals that received high dose estrogen treatment have significantly less (~20%) cell surface TLR4 expression than those derived from sham treated mice or untreated gonadectomized animals following 7 days in culture. Importantly, this effect was not reflected by changes in total cell TLR4 content suggesting that estrogen does not affect TLR4 production. However, longer-term (14-16 day) culture of these cells resulted in an apparent reversal of this pattern with restoration of TLR4 expression on bone-marrow derived macrophages isolated from estrogen treated animals and reduction in the expression of this pattern recognition receptor on cells from untreated gonadectomized mice in a manner that is consistent with our previous observations in acutely isolated peritoneal macrophages. Interestingly, in vitro treatment of bone-marrow derived cells with varying doses of estrogen failed to mimic these effects suggesting that this reproductive hormone exerts its effects prior to macrophage maturation.

Gender has long been known to be a contributory factor in immune responses following infection, with females generally exhibiting greater immune responses than males. These differences have generally been assumed to be a consequence of the actions of reproductive hormones and such a hypothesis is supported by the presence of receptors for these hormones in a variety of immune cell types. Androgens such as testosterone have been shown to decrease immune functions including cytokine production. However, the mechanisms by which testosterone limits such responses remain undefined. In this study we have investigated the acute effects of testosterone on the level of expression of a key microbial pattern recognition receptor, Toll-like receptor 4 (TLR4), on isolated murine macrophages. We show that in vitro testosterone treatment of macrophages, generated in the absence of androgen, elicits a modest but significant decrease in cell surface TLR4 expression within 24 hours. In addition, we have studied the effect of in vivo removal of endogenous testosterone on TLR4 expression. We report that macrophages isolated from orchidectomized animals have significantly less (~30%) TLR4 cell surface expression than those derived from sham gonadectomized mice. Importantly, this effect was not apparent in orchidectomized animals that received exogenous testosterone treatment. As such, these data represent a novel means by which reproductive hormones can modulate immune responsiveness to infectious organisms.