Glucocorticoids and medroxyprogesterone acetate synergize with inflammatory stimuli to selectively upregulate CCL20 transcription

CCL20, or macrophage inflammatory protein (MIP)3α, is a pro-inflammatory chemokine that regulates the trafficking of interleukin (IL)17-producing T helper (Th17) cells (Lee and Korner, 2017; Maddur et al., 2012). CCL20 is constitutively expressed by epithelial and immune cells, while inflammatory stimuli such as tumor necrosis factor-alpha (TNFα) can upregulate CCL20 expression via nuclear factor (NF)-κB (Zhao et al., 2014). CCL20 has been implicated in inflammation and viral pathogenesis. In the female genital tract (FGT), blocking local CCL20 expression prevents simian immunodeficiency virus (SIV) infection in macaque monkeys following vaginal challenge (Haase et al., 2015; Li et al., 2009; Shang et al., 2017). Systemically, elevated CCL20 levels contribute to human immunodeficiency virus (HIV)-1 disease progression and severity (Fontaine et al., 2011; Aziz et al., 2016). CCL20 also directly promotes the growth and metastasis of some cancers (Lee et al., 2017) or mediates tumor suppression in others such as squamous cervical cancer, thus making CCL20 an important target for immune-based therapies (Yu et al., 2015; Walch-Ruckheim et al., 2015). Despite these findings, very little is known regarding which factors, in particular which steroids, regulate CCL20 gene expression systemically and in the FGT, and the mechanisms thereof.

Depot-medroxyprogesterone acetate (DMPA) and norethisterone enanthate (NET-EN) are synthetic steroids used in progestin-only injectable contraceptives, with DMPA being the most widely used hormonal contraceptive in sub-Saharan Africa (United Nations. Department of Economic and Social Affairs Population Division, 2020). Of concern, meta-analyses of higher quality observational studies suggest that DMPA, but not NET-EN use, is associated with a 40–50% increased risk of HIV-1 acquisition compared to no contraception or infrequent use of condoms (Brind et al., 2015; Ralph et al., 2015; Morrison et al., 2015; Polis et al., 2016). Clinical and in vitro data support multiple possible mechanisms whereby MPA may increase HIV-1 acquisition (Hapgood et al., 2018). Whether these mechanisms include upregulation by DMPA of specific chemokines such as CCL20 to affect the recruitment of HIV-1 target cells is unclear. This hypothesis is supported by in vitro, animal, and some clinical studies which suggest that DMPA increases the frequency of CD4+ T subtypes that are particularly vulnerable to HIV-1 infection, in the FGT and/or blood (Bradley et al., 2022; Bunjun et al., 2022; Edfeldt et al., 2022; Lajoie et al., 2019; Li et al., 2019; Tasker et al., 2017, 2020). Consistent with a role for CCL20 in this process, one study has shown that MPA upregulates CCL20 mRNA levels in vitro in primary genital epithelial cells (Woods et al., 2018), but its effects on protein levels are unknown. MPA may also cooperate with factors associated with other infections, to increase HIV-1 infection. Bacterial vaginosis (BV) is prevalent in regions in sub-Saharan Africa (United Nations Department of Economic and Social Affairs Population Division, 2015, Butler et al., 2013) and high CCL20 levels have been reported in the FGT of women with BV(Thurman et al., 2015). HIV-1 acquisition risk may be elevated in women with BV using DMPA compared to BV alone (Haddad et al., 2018; Dabee et al., 2019). MPA may thus cooperate with microbial factors associated with BV, such as lipopolysaccharide (LPS), to further enhance CCL20 expression in the FGT. Such cooperation may also occur systemically in HIV-1 patients. However, clinical data on the effects of DMPA and NET-EN on systemic and FGT frequency of CD4+ T subtypes and CCL20 levels, with or without other infections, are limited or controversial (Fichorova et al., 2015; Konstantinus et al., 2019; Lajoie et al., 2019; Morrison et al., 2014; Omollo et al., 2020; Radzey et al., 2022). Nevertheless, a large body of in vitro evidence suggests that MPA and NET potentially exert differential effects on select chemokine gene expression via the ubiquitous glucocorticoid receptor (GR) (Hapgood et al., 2018).

Although it was designed to act via the progesterone receptor (PR), MPA, unlike progesterone and NET, binds with relatively high affinity and activates the GR (Stanczyk et al., 2013; Ronacher et al., 2009). We have previously reported that MPA, like the synthetic GC dexamethasone (DEX), but unlike NET, acts via the GR to regulate the expression of select immune function genes in FGT epithelial cell lines and peripheral blood mononuclear cells (PBMCs) (Maritz et al., 2018; Hapgood et al., 2014b; Govender et al., 2014; Tomasicchio et al., 2019; Ray et al., 2019). The GR mediates the enhancement of TNFα-induced CCL20 expression by the synthetic GC budesonide in human primary bronchial epithelial cells (Zijlstra et al., 2014), suggesting its involvement in MPA responses on CCL20.

However, whether MPA or NET can act alone or cooperate synergistically with bacterial factors and other inflammatory stimuli to co-regulate CCL20 mRNA and protein expression is unknown, as are the possible ligand- and cell-specific effects, the role of the GR and whether the responses occur at the level of transcription. Given the potentially important clinical implications for women on DMPA or NET-EN, and the limitations of clinical studies with multiple confounding factors, we have investigated these questions ex vivo using human PBMCs and in vitro using cell line models.

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