key: cord-351276-eatlugrm authors: Pinna, Graziano title: Sex and COVID-19: A protective role for reproductive steroids date: 2020-11-09 journal: Trends Endocrinol Metab DOI: 10.1016/j.tem.2020.11.004 sha: doc_id: 351276 cord_uid: eatlugrm Evidence shows COVID-19-induced symptom severity and mortality are more frequent in men than in women suggesting sex steroids may play a protective role. Female reproductive steroids, estrogen and progesterone and its metabolite, allopregnanolone are anti-inflammatory, reshape competence of immune cells, stimulate antibody production and promote proliferation and respiratory epithelial cell repair suggesting they may protect against COVID-19 symptoms. SARS-CoV-2 is a rapidly spreading pandemic characterized by a strong sex-bias with male patients showing double the odds of requiring intensive care unit (ICU) admission and higher mortality when compared to female patients [1] . This evidence points to female sex hormones as a possible explanation for the sexual dimorphism in COVID-19 symptom severity and mortality. Estrogen, progesterone and allopregnanolone are endogenous reproductive steroids, which are abundantly produced in the periphery by the adrenal glands and ovaries and de novo by the brain [2] . These steroids play important physiological roles by modulating inflammatory processes and behavior. Estradiol and progesterone exerts peripheral and neuronal functions mediated by genomic influencing nuclear hormone receptors, are anti-inflammatory, reshape competence of immune cells and stimulate antibody production. Allopregnanolone, instead, rapidly modulates GABA A receptors in neurons, which is associated with mood and cognition improvement, and affects immune-competent cells and induces potent anti-inflammatory functions. Allopregnanolone was recently approved by the Food and Drug Administration (FDA) as Zulresso™ for the treatment of postpartum depression [3] . In addition to the well-known role of progesterone in reproduction, this steroid regulates important immunomodulatory functions, including reshaping the competence of immune cells and inducing potent anti-inflammatory actions. Progesterone plays a significant role in the maternal reproductive apparatus, for example, induces immune adaptations and immune tolerance that promote and sustain pregnancy. There are several ways by which progesterone induces immunomodulatory effects. It stimulates T cellactivation and plays a direct role on their differentiation. Progesterone can also modulate T cell receptor signaling, suppress cellular cytotoxicity, and may also block degranulation by influencing progesteroneinduced blocking factor [4] . Intriguingly, progesterone binds to progesterone receptors in immune cells, including natural killer cells, T cells, macrophages, and dendritic cells but can also bind non-immune cells, including epithelial and endothelial cells in the respiratory tracts where it alters cellular signaling/activity improving infections. In influenza A virus, progesterone administration decreased inflammation and promoted pulmonary repair after clearance of the viruses by increasing regulatory CD39+ Th17 cells and stimulating cytokine (TGF-β, IL-6, and IL-22) levels ( Figure 1 ). IL-22 similarly to TGF-β can stimulate the proliferation of epithelial cells and promote repair of the damaged alveolar epithelium. Progesterone signaling through progesterone receptors stimulates the epidermal growth factor amphiregulin thereby promoting proliferation and respiratory epithelial cell repair [5] . A faster recovery of the lung tissue may reduce in females the susceptibility to secondary bacterial infection, which is the primary cause of mortality after influenza virus infection. This finding is substantiated by studies showing that following influenza A virus infection, the histological density of pulmonary inflammation was decreased during pregnancy. Thus, pregnancy-associated pulmonary physiology may protect females during severe influenza. Combination of estradiol with progesterone showed the strongest protective effects in the lungs after the stimulation of the inflammatory cascade involving toll-like receptor 4 (TLR4) function in cultured alveolar macrophages [6] . Like progesterone, estrogens are strong immune regulatory agents. Estrogens regulate immune cell responses and promote anti-inflammatory and neuroprotective effects. Increase in circulating estrogen concentration affects progenitor and mature cells of both the innate and adaptive immune systems. In the innate immune system, estrogens regulate the number of cells and their functions. In neutrophils, estrogens regulate chemotaxis, infiltration as well as the induction of cytokine-induced chemoattractants and cytokines (e.g., TNF-α, IL-6, IL-1β). In dendritic cells, they stimulate the differentiation and directly regulate expression of chemokines (e.g., IL-8) and cytokines (e.g., IL-6, IL-10). In macrophages, estrogens regulate chemotaxis, phagocytic activity, and the production of cytokines (e.g., IL-6, TNF-α). Estrogens influence the phenotype of T helper cells and have profound effects on B cell maturation, differentiation, activity, and survival. Estradiol also modulates cytokine secretion by CD4+ T cells (7) . Intriguingly, in a mild SARS-CoV-infected mouse model, mortality accounted for 90% of males and only 20% of females. In a more severe infection, all males died within 5 days, whereas 50% of females survived. Gonadectomy increased mortality rate in females but failed to change it in males, supporting a protective role of female reproductive steroids against SARS-CoV. Furthermore, sex bias to SARS-CoV outcomes increased through age reflecting SARS-CoV observations in humans [8] . Estradiol decline during menopause reduces the number of B and T cells while increasing production of pro-inflammatory cytokines. Intriguingly, during menopause women are at higher risk for developing diseases. Thus, men and older females are generally less protected by estrogens. SARS-CoV-2 replicates in the respiratory epithelium after gaining access through the angiotensinconverting enzyme 2 (ACE2) receptor (Figure 2 ). High estradiol concentrations decrease lung ACE2 expression while lower levels increase it [9] , which may explain the increased vulnerability to COVID-19 through sex and age biases. Furthermore, estradiol potently stimulates higher concentrations of antibodies as well as cells involved in antibody production in response to viral infection. Immune cells express receptors for several neurotransmitters, neuropeptides, and hormones. Allopregnanolone is a physiologically-active progesterone derivative and a neurosteroid abundantly produced by the brain where it acts by potently and positively modulating the inhibitory neurotransmission mediated by GABA A receptor [2] . GABA A receptor regulates emotional behavior and is a target of psychotropic drugs, including benzodiazepines. Allopregnanolone is an "endogenous tranquillizer" that shows protective functions in neuropsychiatric disorders, including postpartum depression, posttraumatic stress disorder, alcohol use disorder, epilepsy, and Alzheimer's disease [10] . These pathophysiological conditions are characterized by enhanced proinflammatory signaling mediated by TLR4-activation in peripheral organs and brain. In recent studies conducted in macrophages and monocytes, allopregnanolone inhibited the binding of TLR4 with its specific ligand, lipopolysaccharide (LPS), which promoted pro-inflammatory cytokines and chemokines [11] . This effect was also demonstrated in brain, where TLR4 signal cascade has been demonstrated in neurons and glia. Allopregnanolone acted independently of GABA A receptors. Indeed, both the steroid precursor, pregnenolone, which is devoid of GABAergic actions, and allopregnanolone, but not the GABAergic tetrahydrodeoxycorticosterone (THDOC) blocked the entire TLR4 signaling pathway. Pregnenolone and allopregnanolone inhibited the activation step which involves TLR4 binding to the adaptor proteins MD2 J o u r n a l P r e -p r o o f Journal Pre-proof in macrophages and MyD88 in brain. While TLR4 also binds a GABA A receptor subunit (alpha-2) in brain, this binding was not observed in macrophages [11] . Other studies have shown that pregnenolone and allopregnanolone inhibit TLR4 signals through enhancement of TLR4 degradation. Furthermore, progesterone and allopregnanolone at equivalent efficacy inhibited TLR4 signaling pathway activated by traumatic brain injury in rats. All of these effects were independent of GABA A receptors and suggest a direct action of allopregnanolone to inhibit TLR4 mediated pro-inflammatory signaling in the innate immune system and the brain. Since COVID-19 is characterized by excessive TLR4 signals in the lungs, marked by the overexpression of pro-inflammatory cytokines, including IL-6 and TNF-α and culminating with the cytokine storm, allopregnanolone may protect against COVID-19 -induced inflammation. Inhibition of pro-inflammatory processes following allopregnanolone blockade of TLR4 underlies a novel function in the regulation of periphery and brain immune response [11] . The summary above suggests that reproductive steroids may play a role in COVID-19 sex bias by explaining why more severe symptoms and higher mortality following SARS-CoV-2 infection are observed in men and older subjects. Generally, men and women who are exposed to other viral infections also show differences in prevalence and outcomes with women being less susceptible than men owing a general more efficient immune response. Indeed, women may be more protected than men during physiological conditions, including pregnancy or across the menstrual cycle when fluctuation of reproductive steroids warrants a stronger immune protection. Although, data are mixed and currently the Center for Disease Control and Prevention (CDC) reports that pregnant women may have a worse course [12] , only a few studies evaluated COVID-19 symptom severity in late pregnancy (high hormone levels) and after delivery (low hormone levels). Observational studies have noted that some SARS-CoV-2-positive pregnant women with mild or absence of COVID-19 symptoms on admission to obstetrical service escalated symptoms severity immediately postpartum in coincidence with the drastic hormonal decrease following childbirth. Some women required unplanned ICU admission [13, 14] . Progesterone, estradiol and allopregnanolone concentrations increase up to 100 times, mainly from the first to the second trimester, and then remain elevated until delivery. This progression is consistent with a hormonal protective role during pregnancy and postpartum pathophysiology, which in 1 each 9 women is associated with perinatal psychopathology and sustained inflammation [3] . The CDC reports that on October 22 nd , 2020, the mortality rate among SARC-CoV-2-positive pregnant women in the USA is 0.16% (44 total deaths for 27,566 cases) compared to 2.24% of the American female population [12] , pointing to immunological and hormonal protective factors in lowering the risk of COVID-19-related deaths in pregnant women [15] . This protection may be also guaranteed during the administration of oral combinations of hormonal contraceptive or by treatment with hormone replacement therapy against hypoestrogenism in postmenopausal women. Nutrition may also play a role when diets are enriched with phytoestrogens (e.g., soy beans, lentils, oats) with ability to bind directly to human estrogen receptors or that can be converted to estradiol by the microbiome. There is currently no specific treatment available for COVID-19, however, the US Food and Drug Administration (FDA) has approved the antiviral medicine remdesivir and the steroid dexamethasone following positive clinical trials showing faster recovery and higher survival rates. Thus, there is an J o u r n a l P r e -p r o o f Journal Pre-proof urgent need to develop novel efficient treatments and to unveil biological risk factors to protect vulnerable subjects. Estrogens, progesterone and its metabolite allopregnanolone are involved in multiple pharmacological effects ranging from improvement of mood disorders to analgesic properties and improving cognitive deficits [2, 3, 10] . The anti-inflammatory action, the role in reshaping immunocompetence and increasing number of immune cells, and the stimulation of higher antibodies concentrations against viral infections raise the hypothesis that these reproductive steroids may be beneficial to prevent or improve COVID-19 symptom severity and mortality. Clinical trials should test whether these hormones offer benefits in men and in post-menopausal women at risk of developing severe COVID-19 symptoms. J o u r n a l P r e -p r o o f Glossary Immune tolerance: the state of immune system unresponsiveness to substances or tissues that potentially may induce an immune response. Degranulation: a cellular process of secretory vesicles by releasing antimicrobial cytotoxic molecules, which are called granules and are contained inside cells, including granulocytes (neutrophils, basophils, and eosinophils) as well as mast cells. Chemotaxis: the movement of an organism in response to chemical stimuli. Phagocytic activity: the activity of cells that protect the body by ingesting exogenous and potentially harmful bacteria, viruses as well as dead cells. Respiratory epithelial cells produce pro-inflammatory cytokines following influenza virus infections and they can also activate immune cells, including macrophages and neutrophils, which initiate inflammatory processes. Together, these actions result in clearance of the virus. The damaged respiratory epithelium (brown epithelial cell) following viral infection is more vulnerable to secondary bacterial infection. However, physiological conditions (in women), or treatments that increase P4 levels stimulate the production of citokines (e.g., IL-6 and TGF-β), that together with recruitment of CD39/Th17 regulatory cells, promote anti-inflammatory processes. By binding at progesterone receptors (PR), P4 can also stimulate amphiregulin (AREG), which is a growth factor that signals via the epidermal growth factor receptor (EGFR). AREAG induces respiratory epithelial cell proliferation (light green cells). Likewise, Th17 cells by producing IL-22, promote regeneration of epithelial cells. A repaired alveolar epithelium impedes secondary bacterial infections that constitute the main cause of mortality after influenza virus infection [5] . Estrogens (E) stimulate the differentiation of chemokines and cytokines, regulate chemotaxis, phagocytic activity, influence the phenotype of T helper cells, and affect B cell maturation, differentiation, activity, and survival. Estrogens can also stimulate higher antibody concentrations and increase the number of cells devoted in antibody production in response to viral infections. SARS-CoV2 replicates in the respiratory epithelium after binding at the angiotensin-converting enzyme 2 (ACE2) receptor. Importantly, elevated estradiol levels decrease ACE2 expression (red arrow) in lung epithelium while lower concentrations stimulate ACE2 expression [9] , which may explain why women are generally more resistant to COVID-19 adverse outcomes. 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