Transgender women (TW) are an underserved population in medicine. Treatment with gender-affirming, feminizing hormone therapy (FHT) using estrogen-based supplementation can significantly improve quality of life (1, 2). Despite the success of FHT, long-term estrogen use may contribute to increased prevalence of chronic co-morbidities including metabolic, pulmonary, cardiovascular, and immunologic complications (3–5).

Prevalence of human immunodeficiency virus (HIV) and cardiovascular disease (CVD) is higher in TW compared to the general population (6–11). TW have an approximately 3-fold greater risk of myocardial infarction (MI) compared to their cisgender counterparts (11). Additionally, MI prevalence is greater in TW receiving FHT compared to TW not on FHT (9). Studies have identified a 3-fold increased risk of cardiovascular mortality with estrogen use in TW (4). Despite these findings, mechanisms underlying FHT-associated CVD risk have been incompletely explored.

Increased CVD risk as a consequence of FHT may be particularly concerning in TW with HIV as HIV is linked to heightened CV morbidity and mortality (12). Globally, the prevalence of HIV in TW is 19% compared to < 1% in the general population; the CDC reports TW are 49 times more likely to live with HIV than cisgender women (CW) (6, 7). Current antiretroviral therapy (ART) regimens successfully manage HIV progression, however, persistent immune activation is a strong predictor of CVD in people with HIV (PWH) (13–16). Chronic immune activation may be driven by multiple factors, including the activation of Toll-like receptors (TLRs) by microbial products that translocate through damaged gut lumen or by products of low-level HIV replication (14–30). Increased monocyte and macrophage activation has been implicated in the pathogenesis of HIV-associated CVD (31–33). Exposure to bacterial (e.g., lipopolysaccharide (LPS) or flagellin) or viral products (e.g., single-stranded polyuridine (ssPolyU), imiquimod, or HIV-1) triggers cellular activation and production of pro-inflammatory mediators. Compared to those without HIV, exposure of myeloid cells from PWH to TLR ligands can result in elevated inflammatory cytokines levels including IL-6, a molecule linked to increased morbidity and mortality in PWH (34–36).

Several studies demonstrate that estrogen may enhance inflammatory responses to TLR ligands (37–41). We have measured cardiometabolic profiles and associated inflammatory biomarkers in cisgender men (CM) and TW and reported increased immune activation in the latter, regardless of HIV status. We also observed slight elevations in the CVD-associated molecules TNF-α receptor I (TNFRI), oxidized low-density lipoprotein (oxLDL), and human extracellular newly-identified receptor for advanced glycation end products binding protein (EN-RAGE) in TW on FHT compared to those off FHT (42). Further work is needed to elucidate the mechanisms that contribute to cardiometabolic risk in TW with HIV. Here, we explored the in vitro consequences of estrogen (17-β estradiol or 17-α ethinylestradiol) exposure to TLR-stimulated peripheral blood mononuclear cells (cryoPBMCs) from CM who were HIV negative (HIV-), HIV-positive with ART suppression (HIV+SP), or HIV-positive and ART-naïve/unsuppressed (HIV+USP) to test our hypothesis that estrogen can enhance TLR responsiveness in monocytes from PWH compared to those without HIV. While this work will be limited in its generalizability to TW, as these individuals may experience greater health disparities associated with CVD due to minority stress and marginalization when compared to CM (43), this work serves as a model to elucidate the immune modulatory effects of exogenous estrogen in those assigned male at birth with HIV.

Transgender women are a clinically unique population who are especially vulnerable to both HIV and CVD. As HIV-induced chronic immune activation and inflammation can contribute to CVD risk, and estrogen exposure may exacerbate inflammatory responses, research is needed to elucidate the effects of FHT in TW with HIV.

ART reduces AIDS morbidity and mortality in PWH. Despite its ability to suppress viral replication to undetectable levels and allow restoration of peripheral CD4⁺ T cell counts in most individuals, gut permeability is often persistently altered in PWH due to incomplete restoration of CD4⁺ T cells in the gut mucosa. LPS, a bacterial product which can translocate through the gut lining, may drive HIV-induced inflammatory states through activation of myeloid cells by TLR4 ligation (50). As expected, levels of LPS-binding protein, monocyte activation markers, and inflammatory molecules tended to be increased in PWH compared to levels in people without HIV (51–53). In this study, significant positive correlations among markers of monocyte activation, inflammation, and our marker of microbial translocation were identified within PWH, relationships that were not seen among the HIV- group, signaling that microbial translocation may play an influential role in altering immune activation in this cohort.

The expression and function of TLRs are thought to be altered in PWH due to chronic exposure to bacterial and viral products from microbial translocation, HIV replication, or co-infections. In this cohort, exposure of PBMCs to either TLR4 or TLR8 agonists, LPS and ssPolyU, increased inflammatory cytokine production and monocyte activation marker expression regardless of HIV serostatus. TLR-induced activation was notably higher in PWH despite reduced baseline levels. Altered immune responses to microbial products may have arisen in these individuals after long-term exposure to chronic inflammatory stimuli prompting epigenetic or metabolic reprogramming of innate immune cell responses, a phenomenon known as trained immunity. In PWH, trained immunity may optimize inflammatory responses to successive pathogens; in the setting of chronic TLR activation, however, a persistently elevated inflammatory response may promote pathways associated with cardiometabolic risk (54, 55). Additional studies are needed to further assess the role of trained immunity in chronic HIV infection.

Several in vivo markers of monocyte activation and inflammation were inversely related to levels of cytokine production following in vitro LPS exposure among ART-suppressed PWH in our cohort. LPS desensitization, or reduced responsiveness to repeated exposure, in myeloid cells is an adaptive response to limit inflammation during chronic LPS exposure (56). Similar trends existed in HIV- individuals with elevated immune activation and LBP compared to those with reduced profiles of immune activation. While similar relationships were not consistently seen in HIV+USP individuals, there may be a unique interplay between high levels of HIV replication and products of microbial translocation that should be considered. Viral and bacterial products may trigger independent immune pathways and may differentially alter functional responses to TLR stimuli.

Chronic exposure to inflammatory stimuli in vivo may play a role in producing altered functional responses to TLR ligation. Studies of endogenous estrogen in human and animal models suggest estrogen modulates inflammation and TLR responsiveness (37). We hypothesized that estrogen exposure to HIV- cells would result in downregulation of immune activation, as estrogen may play an atheroprotective role, but these effects could be reversed in PWH due to functional reprogramming of cells after exposure to chronic inflammatory environments in vivo. While estrogen had mild effects on cellular activation in all donor groups, the directionality of the response to estrogen differed by HIV serostatus, with increases in inflammatory cytokines in PWH. Interestingly, estrogen’s atheroprotective role in HIV- individuals may be reflected by the reduced cytokine production in those with greater in vivo activation profiles. In contrast, PWH tended to increase inflammatory cytokines in response to estrogen in individuals with greater in vivo immune activation, indicating a potential shift in the cellular response to estrogen with concurrent HIV infection.

Studies of HIV in CW have revealed faster progression to AIDS compared to men with HIV and similar viral loads, as well as altered levels of plasma markers of microbial and immune activation after treatment among CW versus CM with HIV (57, 58). These differences may be explained by estrogen’s ability to modulate TLR-mediated pro-inflammatory pathways, which contribute to chronic immune activation associated with CV morbidity and mortality (57–61). The addition of 17-β estradiol tended to enhance LPS-induced expression of the activation marker HLA-DR on monocytes, and production of cytokines IL-6 and TNF-α from the PBMCs of PWH regardless of ART status; enhancement of these molecules by the addition of estrogen was reduced in those without HIV. Among HIV- individuals, increased immune activation was inversely associated with LPS-induced cytokine production following estrogen exposure, while cytokine production was directly related to in vivo immune activation in PWH. In the latter group, this effect differs from the indirect relationship seen with LPS alone indicating the presence of estrogen may alter functional desensitization to LPS in suppressed individuals with greater activation profiles. Previous cross-sectional biomarker data identified increased levels of inflammatory molecules associated with CVD risk, such as EN-RAGE, oxLDL, and TNFRI, in TW both on and off FHT compared to CM (42). In the presence of estrogen, elevated immune activation profiles in TW with HIV may indicate risk for enhanced inflammatory responses associated with CVD.

Estrogen can modulate the phosphorylation, and activation, of important intracellular signaling molecules in the TLR pathway including JNK and MAPK p38 (48, 49). Both of these kinases can induce upregulation of transcription factors associated with immune activation. While phosphorylation of JNK seemed to play a minor role in either HIV- or suppressed donors, phosphorylation of MAPK p38 in the presence of LPS stimulation was induced by 17-β estradiol in only ART-suppressed individuals. This may suggest that estrogen can upregulate activation of important TLR4 signaling molecules, and subsequent inflammation, during suppressive ART, but not among HIV- individuals. Inhibition of MAPK p38 normalized LPS and estrogen-induced cytokine production to levels seen with LPS alone in HIV+ SP, suggesting that MAPK p38 may play a partial role in additive effects of estrogen and LPS on cytokine production. While MAPK p38 phosphorylation in ART-suppressed individuals may be one mechanism by which estrogen alters HIV-induced inflammatory pathways, estrogen’s effects are likely complex and multifactorial, as many factors are hypothesized to influence the effects of endogenous estrogen in humans. Multiple cellular receptors have been identified for estrogen, but their functions are incompletely understood. Further research is needed to unravel the intersections among estrogen, TLRs, and drivers of inflammation in PWH.

Limitations exist in this study, including the small number of participants. As an exploratory study, these numbers were satisfactory in revealing trends that require further elucidation. Another limitation of this study was the use of cryopreserved PBMCs, as baseline activation of cells may be elevated, although cell viability was not a concern. To mitigate this issue, cells were rested overnight with supplemented media and human serum. A dose-response study was utilized to select a concentration for estrogen. While the dose chosen was greater than physiological estrogen levels, higher levels of estrogen are associated with more anti-inflammatory responses (37) furthering our hypothesis that estrogen use in biological males may have altered effects on inflammation. Due to limitations in human subject enrollment from COVID-19 restrictions, we utilized cells from CM to best replicate the biological environment present in TW pre-FHT. While a convenient solution, studies would better represent this population with cells from TW, as these individuals have increased risk for co-morbidities such as obesity, diabetes, lipid dysregulation, and CVD regardless of HIV. Further study is also needed to understand the full effects of FHT, as FHT can utilize both estrogens and anti-androgens that block the effects of testosterone. We report here that testosterone may play a role in lowering LPS-induced inflammation in PWH. Despite these factors, our findings demonstrate the need for clinical studies aimed at understanding the mechanistic role of estrogen to reduce CV mortality in TW with HIV.

Overall, this work suggests complex interactions may exist among HIV, CVD, and FHT in TW. As PWH are living longer, both increased frequency and severity of multiple end-organ diseases, such as CVDs, have been uncovered. While estrogen is a known modulator of TLR expression and function, the intensified responses to TLR activation with estrogen treatment in PWH cannot be fully explained by this phenomenon when compared to responses in HIV- individuals. This work demonstrates a unique synergistic effect of microbial-and-estrogen-induced immune activation in PWH independent of the increased activation seen with TLR ligation or estrogen treatment alone. Our findings may reflect alterations in cellular response due to chronic inflammatory stimuli that result in enhanced inflammatory pathways associated with CV morbidity.

To appropriately address clinical disparities in TW, current research assessing treatments aimed to reduce pivotal drivers of inflammation and downstream immune signaling underlying HIV-induced co-morbidities should be considered. As previously reviewed, studies range from altering the microbiota in the gut to reduce translocation of microbial products, to blocking the actions of important inflammatory molecules associated with HIV morbidity and mortality such as IL-6 (62). A multi-pronged approach with ART may be a beneficial route to provide equitable care. By understanding the mechanism behind estrogen-induced modulation of TLR pathways, we may identify important clinical indications for additional therapies in TW on FHT or identify alternative estrogen-like drugs designed to reduce deleterious immune effects in TW with HIV.

Estrogen has been recognized as an important modulator of immune activation including alterations in inflammation and Toll-like receptor function in innate immune cells. Mechanisms by which estrogen exerts these effects are incompletely understood and may depend on estrogen concentration, cell type, and cellular environments. While the use of estrogen in feminizing hormone therapy can improve quality of life in transgender women, research is needed to understand the side effects of long-term high dose usage. This is particularly concerning in the setting of HIV-associated chronic inflammation as Toll-like receptor 4 activation is linked to elevated systemic lipopolysaccharide and inflammation. Based on current literature, we propose that estrogen may upregulate immune activation of myeloid cells in people with HIV as a result of Toll-like receptor functional changes. The elevated immune activation seen here has been associated with increased risk for co-morbidities such as cardiovascular disease and higher mortality rates in people with HIV. As estrogen use in transgender women has been linked to three-fold increased risk of cardiovascular mortality, these results may provide a potential mechanism by which estrogen exerts deleterious effects and potential biomarkers that may indicate a need for additional immunotherapies or alternative estrogen-based therapies in people with HIV.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Institutional Review Board at Ohio State Wexner Medical Center. The patients/participants provided their written informed consent to participate in this study.

All authors contributed to experimental design, data analysis, and writing of the manuscript. AK and EB: performed experiments. BH: provided analytical expertise.

Research reported in our publication was supported by the National Institutes of Health supported by the National Institute of Allergic and Infectious Disease of the National Institutes of Health under grant numbers P30AI161943, 5R21AI143452 to JEL. This work was also supported, in part, by the National Center for Advancing Translational Sciences of the National Institutes of Health under Grant Numbers TL1TR002735 & UL1TR001450.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NF has served as a consultant for Gilead.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.