RISING STARS: Androgens and immune cell function

Androgens can modulate immune cell function and may contribute to differences in the prevalence and severity of common inflammatory conditions. Although most immune cells are androgen targets, our understanding of how changes in androgen bioavailability can affect immune responses is incomplete. Androgens alter immune cell composition, phenotype, and activation by modulating the expression and secretion of inflammatory mediators or by altering the development and maturation of immune cell precursors. Androgens are generally associated with having suppressive effects on the immune system, but their impacts are cell and tissue context-dependent and can be highly nuanced even within immune cell subsets. In response to androgens, innate immune cells such as neutrophils, monocytes, and macrophages increase the production of the anti-inflammatory cytokine IL-10 and decrease nitric oxide production. Androgens promote the differentiation of T cell subsets and reduce the production of inflammatory mediators, such as IFNG, IL-4 and IL-5. Additionally, androgens/androgen receptor can promote the maturation of B cells. Thus, androgens can be considered as immunomodulatory agents, but further work is required to understand the precise molecular pathways that are regulated at the intersection between endocrine and inflammatory signals. This narrative review focusses on summarising our current understanding of how androgens can alter immune cell function and how this might affect inflammatory responses in health and disease.


Invited Author's profile
Dr Douglas Gibson is a principal investigator and Sir Henry Dale Fellow in the Institute for Regeneration and Repair at the University of Edinburgh.His research focusses on understanding how hormones, particularly androgens, control immune cell function in the womb and how this might affect women's reproductive health.His lab uses fate-mapping techniques, transcriptomics analysis, multiparameter flow cytometry and immunohistochemistry to characterise the phenotype and function of immune cell populations that are required for endometrial repair and remodelling.

Introduction
Androgens play important roles in human health, including the development and maintenance of the reproductive system, regulating cardiovascular function and metabolism (Davey & Grossmann 2016).Androgen signalling has broad physiological effects, but how it regulates the immune system is poorly understood.
Evidence for the impact of androgens on immune cells comes from in vitro assessment of isolated populations or deduced from cohort differences due to sex or suppression of endogenous hormones.It is well known that sexual dimorphism in immune responses affects the prevalence and severity of a range of inflammatory conditions, but our understanding of the specific contribution of androgens to immune responses and the impact androgens may have on different immune cell subsets is incomplete.Interpretation is limited by the attribution of male-predominant responses to androgens and female-predominant responses to estrogens.Moreover, the potential for androgens to be aromatised to estrogens is often not controlled for in experimental studies.
Immune cell populations from both the innate and adaptive immune systems express the androgen receptor (AR) and may therefore be sensitive to fluctuations in androgen bioavailability, which may change with age, sex and as a result of endocrine disorders.This narrative review focusses on how androgens regulate immune cell phenotype and function, and how this affects inflammatory responses in health and disease.

Search method
A review of published literature was performed using PubMed to search for articles and reviews containing the following main keywords: androgens, AR, testosterone, immune system, immune cells, sexual dimorphism, and other key terms related to these subjects.

Androgens and androgen signalling
Androgens are sex steroid hormones that are classically considered 'male' due to their important roles in the development and maintenance of the male reproductive system (Barsoum & Yao 2006).However, androgens also play important roles in regulating the function of various tissues and organs in both sexes, including muscle, heart, kidney and bone (Davey & Grossmann 2016), as well as the female reproductive tract (Simitsidellis et al. 2018).They are predominantly produced by the gonads (testes and ovaries) and the adrenal glands (Bienenfeld et al. 2019) but are also produced through the conversion of precursors in peripheral tissue sites, such as adipose tissue.The bioavailability of androgens can vary with age and different developmental and endocrine disorders, resulting in high (hyperandrogenism) or low (androgen deficiency) systemic androgen concentrations (Jordan & DonCarlos 2008).Androgen-related disorders can be caused by different mechanisms, including defects in androgen synthesis, metabolism or receptor signalling (Khera & Lipshultz 2006).
Androgens consist of both inactive precursors, dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA) and androstenedione (A4), and active androgens, namely, testosterone and dihydrotestosterone (DHT).In addition, recent evidence supports an emerging role for the importance of 11-oxygenated androgens, a group of androgen metabolites that are present in the circulation at concentrations that can be equivalent to those of 'classical' androgens and can bind the androgen receptor in target tissues (Alemany 2022, Storbeck & O'Reilly 2023).
The de novo synthesis of androgens requires sequential enzymatic modification of cholesterol to generate active androgens.This is mediated by cytochrome P450 enzymes, hydroxysteroid dehydrogenases and aldo/keto reductases (Manenda et al. 2016), and their differential expression throughout the body regulates local androgen action.Testosterone is the main circulating androgen in men, and it can be converted within target tissues to the potent androgen DHT by 5α-reductase or aromatised into 17β-estradiol by aromatase, therefore contributing to both androgenic and estrogenic effects in tissues.
Upon reaching the target tissue, testosterone can passively diffuse across the cell membrane or exert its actions at the cell surface (Oren et al. 2004).Additionally, androgens can act in an intracrine manner where activation and action occur within target tissues via activation of inactive precursors such as DHEAS, DHEA and A4.Intracrine androgen action has been documented in a range of tissues, including the prostate, adipose tissue and the endometrium (Schiffer et al. 2018, Simitsidellis et al. 2018).Notably, intracrine androgen action has been proposed to affect immune cell function and has been described in human alveolar macrophages (Milewich et al. 1983) and synovial macrophages (Cutolo et al. 1996), as well as murine T lymphocytes (Samy et al. 2001).

Mechanisms of androgen signalling
Classically, androgen signalling is mediated through specific binding and activation of the androgen receptor (AR) protein (reviewed in Davey & Grossmann (2016)).AR is primarily located in the cytoplasm where, in the absence of androgens, it is found in a complex with heat shock and chaperone proteins, such as HSP70 (Bennett et al. 2010).

Direct AR signalling
Following androgen binding, the androgen/AR complex alters its conformation, dissociates from chaperone proteins, and translocates to the nucleus where it binds as a dimer to consensus androgen response element regions in DNA.Transcriptional regulation of target gene expression is modulated by co-regulatory proteins that form complexes with the activated AR to enhance or repress transcription (Fig. 1(i), (Davey & Grossmann 2016)).Specifically, regulation of gene expression by AR can be mediated via interaction with general transcription factors (such as SRC-1 and TF II H), components of the chromatin remodelling complex (such as SWI/SNF), histone-modifying enzymes and proteins involved in RNA splicing and metabolism (for a comprehensive review on the subject see Heemers & Tindall (2007)).AR-dependent signalling can also occur independently of androgens via activation of growth factor receptors and downstream signalling pathways that promote direct phosphorylation and activation of cytoplasmic AR (Fig. 1(ii)) (Davey & Grossmann 2016).This activation promotes nuclear translocation of the AR and transcriptional regulation Direct and indirect mechanisms of androgen signalling.(i) Ligand-dependent direct androgen receptor signalling: Upon androgen binding to the cytoplasmic androgen receptor (AR), AR begins to dissociate from the chaperone proteins and translocates to the nucleus.Once inside the nucleus AR binds to androgen target genes through androgen response elements to alter the transcription of target genes (Davey & Grossmann 2016).(ii) Ligandindependent androgen receptor signalling: Cytoplasmic AR can be activated through phosphorylation due to activation of growth factor receptor mediated signalling pathways.The phosphorylated AR is then able to translocate to the nucleus and induce the expression of androgen target genes (Bennett et al. 2010, Davey & Grossmann 2016, Kadel & Kovats 2018).(iii) Indirect membrane-bound androgen receptor signalling: Androgens can bind to a membrane bound form of the AR and induce signalling transduction which results in the binding of transcription factors to target genes altering gene expression.This induction of intracellular signalling pathways can also result in the phosphorylation of the cytoplasmic form of the AR which can then translocate to the nucleus and induce AR-dependent direct signalling pathway (Kousteni et al. 2001, Estrada et al. 2003, Kang et al. 2004, Davey & Grossmann 2016, Thomas 2019).(iv) Indirect membrane associated androgen receptor signalling: Androgens can also exert their effects via the cytoplasmic AR that is localised at the cell membrane.Binding of androgens to this receptor leads to activation of intracellular signalling pathways that result in the transcription of target genes (Kousteni et al. 2001, Estrada et al. 2003, Kang et al. 2004, Davey & Grossmann 2016, Thomas 2019).AKT, protein kinase B; AR, androgen receptor; DHT, dihydrotestosterone; ERK1/2, extracellular signal-regulated kinase 1/2; MAPK, mitogen-activated protein kinases; PI3K, phosphoinositide 3-kinases; Ras, rat sarcoma; Src, proto-oncogene tyrosine-protein kinase; TF, transcription factor.Created with BioRender.com.(Kadel & Kovats 2018).This mechanism of ligandindependent AR signalling has been described in prostate cancer where upregulation of androgen target genes occurs through pathways such as the protein kinase A (PKA), proteins kinase C and mitogen-activated protein kinases (MAPK) signalling pathway in the presence of non-androgen ligands, such as IL-6 (Lyons et al. 2008, Davey & Grossmann 2016).

Indirect AR signalling
Several studies have identified a further mechanism of androgen action that occurs too rapidly (seconds to minutes rather than hours to days) to be via a direct mechanism (Davey & Grossmann 2016).This led to the discovery of membrane-bound AR that can be found in a range of cell types including Sertoli cells (Gorczynska & Handelsman 1995), prostate cancer cells (Hatzoglou et al. 2005, Sun et al. 2006), satellite cells from human skeletal muscles (Sinha-Hikim et al. 2004) and some immune cell types such as T lymphocytes (Benten et al. 1999a) and macrophages (Benten et al. 1999b).Indirect androgen signalling can occur through direct binding of androgens to membrane-bound ARs or via cytoplasmic ARs that are tethered to the cell membrane (Fig. 1(iii) and (iv)), (Thomas 2019)).The binding of androgens to these receptors leads to the release of intracellular second messengers such as calcium and/or activation of a range of signalling pathways including extracellular signalregulated kinase (ERK), protein kinase B (PKB or Akt) and MAPK (Migliaccio et al. 2000, Kousteni et al. 2001, Estrada et al. 2003, Kang et al. 2004).The activation of these signalling pathways results in androgen-dependent changes in the expression of target genes without direct binding of the AR to DNA (Thomas 2019).
Due to variations in ligand bioavailability, as well as different cell-and tissue-specific expressions of coregulatory proteins, the impact of androgens must be investigated in a context-specific way.Thus, the different cell types, AR expression/localisation and concomitant inflammatory signals must all be accounted for when seeking to understand how androgens affect immune cell function.

The immune system
The immune system is a complex network of cells, proteins and organs that function to protect the body from infection and disease, as well as aid in the repair of damaged tissues.The immune system is broadly divided into two parts: the innate immune system and the adaptive immune system (Marshall et al. 2018).

Innate immune system
The innate immune system is the body's first line of defence against invading pathogens and is therefore highly responsive and fast-acting (within minutes of exposure).The innate immune system is comprised of a range of immune cells including monocytes, macrophages, dendritic cells (DCs), mast cells, neutrophils, eosinophils, basophils, natural killer cells and innate lymphoid cells (Turvey & Broide 2010).During infection, resident phagocytic cells, such as macrophages and DCs, identify pathogens through the recognition of foreign cell-surface markers, which leads to encapsulation of the pathogen through a process known as phagocytosis.This internalisation event stimulates the release of a cascade of pro-inflammatory mediators known as cytokines and chemokines.These proteins initiate a state of inflammation in the local area which attracts other immune cells, such as neutrophils and monocytes, that aid in the clearance of an infection through direct killing and orchestration of the immune response.Another important component of innate immunity is the propagation of a prolonged immune response through the activation of the adaptive immune system by antigen-presenting cells (APCs), such as macrophages and DCs (Murphy & Weaver 2016).

Adaptive immune system
The adaptive immune system is a secondary line of defence that is initiated by cells of the innate immune system when an innate immune response fails to clear a new infection.The adaptive immune system is composed of two main cell types (T lymphocytes and B lymphocytes) that have the ability to mount an antigenspecific response against pathogens (Murphy & Weaver 2016).Adaptive immunity also has the capacity to remember exposure to previous pathogens, enabling a rapid and more efficient response upon future exposure to the same or similar pathogens.T lymphocytes have important roles in the direct killing of infected cells, activation of other immune cells and regulation of the immune response.B lymphocytes produce antibodies against foreign antigens, and unlike T lymphocytes, they can recognise antigens directly through unique antibodies expressed on their surface without the assistance of APCs (Marshall et al. 2018).

Sexual dimorphism in immunity
It is well established that sexual dimorphism (differences between males and females) exists within the immune system (Shepherd et al. 2021), which impacts responses to infection as well as the risk of autoimmune diseases (Cook 2008, McCombe et al. 2009, Zuk 2009).In general, women can generate greater innate and adaptive immune responses than men (Klein & Flanagan 2016).This is associated with reduced rates of bacterial, viral and parasite infections in women and a higher rate of survival for several cancer types compared to men (Jung et al. 2012).In contrast, there is a greater prevalence of autoimmune disease in women; approximately 80% of all patients with autoimmune diseases are women (Angum et al. 2020).This is consistent with heightened immune reactogenicity towards molecular patterns that originate from both self and non-self in females (Shepherd et al. 2021).The key drivers of sexual dimorphism are genetics (chromosomal sex) and sex hormones.We know that autoimmune diseases have a complex genetic basis and that associations between genetics and autoimmune diseases include the presence of certain major histocompatibility complex genes (Taneja 2021) and the expression of toll-like receptors (TLRs), which are encoded by the X chromosome (Rubtsova et al. 2015).This review focusses on the association between androgens and the immune system; details of the effects of genetics and other sex hormones (estrogens) on sexual dimorphism in the immune system are reviewed elsewhere (Rubtsova et al. 2015, Moulton 2018, Taneja 2021, Harding & Heaton 2022, Sciarra et al. 2023).

Androgen receptor expression in the immune system
Immune cells from both the innate and adaptive immune system express AR (summarised in Fig. 2), where receptor protein expression is commonly localised to the cytoplasm and, in some cases, the cell membrane.Flow cytometry, immunofluorescence and gene expression analyses have been predominantly used to determine AR expression in macrophage subsets of humans and mice (Cutolo et al. 1996, Benten et al. 1999b, Mantalaris et al. 2001, Wunderlich et al. 2002, Liu et al. 2004, 2005, Ahmadi & McCruden 2006, Bizzaro et al. 2018, Rubinow et al. 2018, Lee et al. 2019, McCrohon et al. 2000, Campesi et al. 2012).These studies revealed the presence of either cytoplasmic or membrane-bound AR in subsets of murine macrophages and confirmed the expression of cytoplasmic AR in bone marrow-derived and synovial macrophages in humans (Cutolo et al. 1996, Benten et al. 1999b, Mantalaris et al. 2001, Wunderlich et al. 2002, Liu et al. 2005).RT-PCR analysis identified AR mRNA expression in human peripheral blood monocytes and the monocyte THP-1 cell line (Liu et al. 2004).AR expression has not been comprehensively described in neutrophils, but cytoplasmic AR protein has been detected by immunohistochemistry throughout stages of neutrophil development in humans (Mantalaris et al. 2001, Markman et al. 2020).
Investigations into expression of AR in the adaptive immune system have involved the use of binding assays, flow cytometry and western blot analysis.Benten et al. assessed the binding of the conjugated ligand testosterone-BSA-FITC, which cannot cross the cell membrane, combined with confocal microscopy and flow cytometry to demonstrate ligand binding to membrane AR in murine CD4 + and CD8 + T cells (Benten et al. 1999a).Further in vitro studies also confirmed the expression of cytoplasmic AR in human and murine T cells (Kovacs & Olsen 1987, Benten et al. 1999a).A combination of techniques, including western blot, RT-PCR, flow cytometry and confocal microscopy, have been used to investigate the expression of AR in murine B cells and their progenitors, leading to the identification Androgen receptor expression in immune cells.Innate immune cells such as neutrophils, monocytes and macrophages express AR.AR is predominantly expressed in the cytoplasm with some evidence for membrane expression in macrophage subsets.T and B lymphocytes of the adaptive immune system are also AR positive with AR detected in the cytoplasm and membrane of T cells and cytoplasm of B cell progenitors (Milewich et al. 1983, Kovacs & Olsen 1987, Viselli et al. 1997, Benten et al. 1999a, Mantalaris et al. 2001, Wunderlich et al. 2002, Liu et al. 2004, Sinha-Hikim et al. 2004, Liu et al. 2005, Ahmadi & McCruden 2006, Bolego et al. 2013, Bizzaro et al. 2018, Rubinow et al. 2018, Lee et al. 2019, Markman et al. 2020, McCrohon et al. 2000, Campesi et al. 2012).•, mouse; °, human; +, mouse and human.Created with BioRender.com.
of cytoplasmic AR expression in B cell progenitors.Notably, AR was absent in peripheral or mature B cells (Viselli et al. 1997, Benten et al. 2002b) consistent with a potential role for AR in B cell development.
In addition to the expression of AR in mature immune cells, haematopoietic stem cells, common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) in bone marrow also express AR (Mierzejewska et al. 2015).Due to the expression of AR across these cell types, androgen signalling may have an important role in regulating the immune system, affecting both immune cell development and/or the function of mature immune cells.

Monocytes and macrophages
Monocytes and macrophages are innate immune cells with important roles in the initiation and maintenance of a non-specific immune response, as well as the activation of the adaptive immune system (Murphy & Weaver 2016).Monocytes and macrophages are mononuclear phagocytes that play distinct roles in immunity, with monocytes having important roles in orchestrating inflammation as well as protecting the body from pathogens, while tissue-resident macrophages are key players in tissue homeostasis and the resolution of inflammation.Monocytes are continuously replenished from the bone marrow, where they are derived from haematopoietic stem cells.These cells are released from the bone marrow and travel around the body to target tissues where they can then differentiate into macrophages (Ginhoux & Jung 2014).As seen in Fig. 2, peripheral blood monocytes in both humans and mice express the cytoplasmic form of the AR.AR expression and localisation (cytoplasmic or membrane) in macrophages varies depending on tissue context in both humans and mice.
Several animal studies have investigated the impact of androgens on monocyte and macrophage function.These studies, summarised in Table 1, have utilised in vitro experiments where different macrophage cell lines are exposed to exogenous androgens in the presence or absence of lipopolysaccharide (LPS) stimulation to investigate the impact of androgens on macrophage activation.
One study compared unstimulated macrophages with LPS-stimulated macrophages from the murine cell line J774 to investigate the effects of testosterone on macrophage cytokine production.Testosterone treatment induced the production of interleukin 10 (IL-10) in unstimulated macrophages, and this production was significantly increased in LPSstimulated macrophages.Treatment with testosterone in stimulated macrophages also led to reduced production of nitric oxide (NO) and tumour necrosis factor alpha (TNF-α) (D'Agostino et al. 1999).Similarly, when the macrophage cell line RAW 264.7 was exposed to increasing concentrations of testosterone (0.1-40 µM) and stimulated with LPS, testosterone had a dosedependent inhibitory effect on the protein expression of NO and both the protein and mRNA expression of NO synthase (NOS) (Friedl et al. 2000).Further studies in which peritoneal macrophages were cultured with testosterone or DHT identified that androgen treatment led to increased gene expression of IL-6 (IL6) but had no effect on the expression of TNF and transforming growth factor beta 1 (TGFB1) (Gilliver et al. 2006).Furthermore, testosterone-induced apoptosis of bone marrow-derived macrophages increased both the mRNA and protein expression of caspase 3, caspase 8 and poly (ADPribose) polymerase (PARP) as determined by RT-PCR and western blot (Jin et al. 2006).Treatment of RAW 264.7 cells with testosterone led to a decrease in TLR4 protein expression consistent with androgens priming a reduced response to inflammatory stimuli that act through TLR4 (Rettew et al. 2008).
In summary, these studies (Table 1) demonstrate that androgens, in general, promote an anti-inflammatory macrophage phenotype through the upregulation of key anti-inflammatory mediators, such as IL-10, reduced production of pro-inflammatory mediators, such as NO and TNF, and decreased expression of TLRs (D'Agostino et al. 1999, Friedl et al. 2000, Gilliver et al. 2006, Jin et al. 2006, Rettew et al. 2008).

Dendritic cells
DCs are an innate immune cell type that bridges the gap between innate and adaptive immune responses.The main function of DCs is to digest phagocytosed pathogens to process and present foreign antigens to adaptive immune cells (Martin-Gayo & Yu 2019).For the most part, studies investigating the expression of AR on DCs are either lacking or have utilised western blot analysis to demonstrate that myeloid-derived DCs do not express AR (Paharkova-Vatchkova et al. 2004).However, more recent chromatin immunoprecipitation sequencing analysis of tumour-associated DCs as well as splenic DCs identified AR expression in these cell types (Thompson et al. 2017).Further studies are required to definitively confirm the expression of AR on DC cell subsets.
Regardless, functional studies suggest that DC activity can be androgen-regulated.One in vitro study utilised murine bone marrow-derived DCs (BMDCs) and exposed them to LPS and varying concentrations of DHT (1 -500 nM) to investigate the effect of DHT on BMDC function.After 24 h, it was demonstrated that there was a dose-dependent downregulation of IL-6 as well as an upregulation in the expression of TNF, IL-10 and IL-4 in BMDCs exposed to 50-500 nM of DHT (Thompson et al. 2017).A study investigating DCs from hypogonadal men found that CD16+ DCs stimulated with CpG oligodeoxynucleotides had significantly higher expression levels of the activation marker CD107b compared to healthy controls consistent with androgens inhibiting DC activation (Corrales et al. 2012).Collectively, these limited studies suggest androgens promote an anti-inflammatory DC phenotype.

Wound healing
Macrophages play an important role in the regulation of tissue regeneration and wound healing (Martin & Leibovich 2005).Biological sex contributes to age-related differences in wound healing, which has been attributed to differences in circulating androgens; elderly males experience delayed healing of acute wounds compared to females of the same age (Guo & DiPietro 2010).
To expression and enhanced TNF production by macrophages (Lai et al. 2009).Reciprocal bone marrow transplantation experiments demonstrated that the suppressive effects of androgens on wound healing can be mediated by both androgen-dependent and -independent pathways, but the effects are exclusively mediated by AR.This was further investigated through the topical application of the anti-AR compound, ASC-19, which degrades the AR protein.This treatment accelerated cutaneous wound healing and dampened TNF production (Lai et al. 2009).Taken together, these data illustrate that androgens have a suppressive effect on wound healing and that targeting the AR directly may present a better therapeutic option for blocking this suppression than altering systemic androgen concentrations.

Asthma
Asthma is a chronic heterogeneous disease of the lungs that results in restricted airflow and respiratory problems (Papi et al. 2020).There are sex differences in both disease prevalence and the phenotype of asthma (Laffont et al. 2017), such that adult women have an increased prevalence and severity of asthma compared to adult men (Chowdhury et al. 2021).This sex difference could be partly attributed to a protective effect of greater circulating androgen concentrations in males, which can act as bronchodilators and immunosuppressors (Montaño et al. 2020).In a mouse model of allergic lung inflammation, castrated male mice administered with a DHT pellet had reduced inflammation compared to castrated mice that received a placebo pellet due to reduced immune cellularity in bronchoalveolar lavage fluid (Becerra-Díaz et al. 2018).Alveolar macrophages have important roles in suppressing inflammation in the lung and maintaining homeostasis (Draijer & Peters-Golden 2017).Studies using the same myeloid-specific ARKO mouse model (LyzM Cre AR flox ) described previously revealed that loss of myeloid AR in males, but not females, led to a reduction in eosinophil recruitment as well as lung inflammation due to impaired polarisation of antiinflammatory alveolar macrophages (Becerra-Díaz et al. 2018).Interestingly, testosterone was investigated as an experimental asthma treatment in the 1960s which showed improved asthma symptoms in 88% of women with asthma, although the mechanisms were not elucidated (Wulfsohn et al. 1964).The current literature is consistent with androgens and the AR modulating myeloid cell function by inducing an anti-inflammatory phenotype and contributing to tissue repair during lung inflammation.

Polycystic ovarian syndrome
Polycystic ovarian syndrome (PCOS) is a complex disorder that affects 4-20% of women of reproductive age worldwide (Deswal et al. 2020).Seventy to eighty percent of women with PCOS have hyperandrogenism (Baptiste et al. 2010, Huang et al. 2010) (Kierdorf & Fritz 2013).Although this evidence is indirect, it suggests that chronically elevated androgens in PCOS patients may promote an inflammatory phenotype in circulating monocytes.However, further investigation is required to understand the relationship between elevated androgens and monocytes/macrophage function in women's health.

Neutrophils
Neutrophils are the most abundant leucocytes in human blood and are important effector cells in the innate immune system.In the presence of infection, they rapidly respond by trapping and killing pathogens through mechanisms such as phagocytosis, degranulation and the release of extracellular traps (Rosales 2018).
Immunohistochemistry analysis has revealed that all neutrophil lineages, from precursors to mature neutrophils, express the cytoplasmic form of the AR in humans (Mantalaris et al. 2001, Markman et al. 2020).
Various studies have shown that androgens have an important role in neutrophil differentiation and function (Chuang et al. 2009, Marin et al. 2010, Inamdar & Jayamma 2012, Scalerandi et al. 2018).For example, studies utilising GARKO or mice with a testicular feminisation mutation (Tfm) that have loss of AR function, have a 90% reduction in neutrophils isolated from the bone marrow (Chuang et al. 2009).In particular, the proportion of myelocytes, metamyelocytes and mature neutrophils was reduced in GARKO mice.In vitro experiments in which granulocyte-myeloid progenitors were isolated from GARKO mice and exposed to retroviruses to restore the expression of AR revealed that the defects in neutrophil maturation could be restored by re-expressing AR, consistent with an essential role for AR in neutrophil differentiation (Chuang et al. 2009).These findings were further supported by another study in which mice were treated with the anabolic-androgenic steroid stanozolol (testosterone analogue), which increased myelocytes, metamyelocytes and mature neutrophils (Inamdar & Jayamma 2012).These studies demonstrate the important role of AR-mediated signalling in neutrophil development and maturation.
AR signalling has also been found to play a role in the ability of neutrophils to mount an efficient immune response to infection.GARKO studies demonstrated a reduction in neutrophil number resulting in a severely diminished capacity to survive bacterial challenge (Chuang et al. 2009).In vitro studies using human neutrophils revealed that testosterone treatment leads to a significant decrease in superoxide production, alterations in calcium mobilisation and altered NO production (Marin et al. 2010).Notably, modulation of human neutrophil function by androgens may be dose dependent, since neutrophils treated with 10 nM of testosterone exhibited increased phagocytic capacity while neutrophils treated with 10 μM of testosterone exhibited decreased microbicidal activity (Marin et al. 2010).In a rat model of bacterial prostate inflammation, testosterone-treated rats experienced higher neutrophil recruitment which was associated with increased tissue damage (Scalerandi et al. 2018).Interestingly, neutrophils exposed to exogenous testosterone exhibited diminished bactericidal ability as well as reduced myeloperoxidase activity (Scalerandi et al. 2018).This altered neutrophil profile was characterised by increased expression of IL-10 and TGFB1, which resemble the 'N2-like' neutrophil phenotype that has previously been demonstrated in tumours (Scalerandi et al. 2018).Thus, androgens reduce the overall effector response of neutrophils in experimental settings.

Impact of androgens on neutrophils in disease
In the clinical setting, the effects of androgens on neutrophils can be inferred from scenarios that alter androgen bioavailability.For example, women with hyperandrogenism due to PCOS exhibit neutrophilia, and treatment with an anti-androgen attenuated neutrophilia (Ibáñez et al. 2005).Men with prostate cancer who received anti-androgen treatment, nilutamide or flutamide, suffered from severe neutropenia, which was reversed upon cessation of treatment, and neutrophil counts returned to normal (McDonnell & Livingston 1994, Eaton & Blackmore 2001).These studies demonstrate the impact androgens can have on the abundance of circulating neutrophils, but also that these effects can be context-dependent.
Altering neutrophil abundance using androgens may have utility in certain clinical scenarios, but further studies will be required to determine the potential kinetics of these effects.

Impact of androgens on the adaptive immune system T lymphocytes
T lymphocytes, also known as T cells, are an important adaptive immune cell type that is derived from CLPs in the bone marrow and is split into subtypes based on maturity and function.Naive T cells have yet to encounter their corresponding antigen and therefore are inactive (Kumar et al. 2018), but differentiate into an effector T cell following activation.CD8 + T cells, also known as cytotoxic T cells, kill target cells expressing a specific antigen by releasing perforin and granzymes into the immunologic synapse resulting in apoptosis of the target cell (LaRosa & Orange 2008).CD4 + T cells, also known as helper T cells, suppress immune responses to maintain homeostasis and prevent tissue damage.Further differentiation of CD4 + T cells takes place in the presence of certain cytokines and these subpopulations exhibit specific functions (Luckheeram et al. 2012).
As seen in Fig. 2, both murine and human T cells are reported to express both the classic cytoplasmic and membrane-bound forms of the AR.
The effects of androgens on T cell development and function have been studied both in vivo using rodent models and in vitro using cell lines as outlined in Table 2. Several studies have utilised models of castration in male mice to deplete androgens and investigate the downstream effects of this depletion.It has been shown that castration leads to thymic enlargement and a significant decrease in the proportion CD4 − CD8 + thymocytes, which was reversed by exogenous testosterone treatment (Olsen et al. 1991).Studies have also shown that treatment with testosterone or methyltestosterone led to a decrease in the percentage of CD4 + CD8 + thymocytes (Olsen et al. 1991, Dulos & Bacchus 2001).Production of IL-4, IL-5 and interferon gamma (IFNG) by murine splenic T cells was reduced by DHT after anti-CD3 activation (Araneo et al. 1991).The same study revealed that androgen supplementation using physiologically relevant doses of DHT and DHEA in aged (>60 weeks) murine splenic T cells restored the capacity of this cell type to produce IL-2, IL-4 and IFNG to the same extent as younger mice (Araneo et al. 1991).
Kissick et al. utilised various androgen modulation methods, including castration and administration of the androgen analogue R1881, to investigate the impact of androgens on murine CD4 + splenic T cells.These studies demonstrated that androgen depletion led to significant gene expression changes in pathways involved in IFNG signalling and T-helper cell differentiation.It was also demonstrated that androgen treatment significantly reduced IFNG production (Kissick et al. 2014).CD4 + T cells treated with androgens expressed FOXP3 and produced IL-10 in a dose-dependent manner, and this effect could be abolished by the addition of the antiandrogen flutamide (Fijak et al. 2015).
In summary, androgens largely reduce T cell number and dampen T cell responses through the reduced production of inflammatory mediators, such as IL-5 and IFNG, and the increased production of IL-10 (Araneo et al. 1991, Olsen et al. 1991, Olsen et al. 1998, Dulos & Bacchus 2001, Kissick et al. 2014, Fijak et al. 2015).

Impact of androgens on T cells in disease Autoimmunity
It is well known that males have reduced risk of developing various autoimmune diseases, such as multiple sclerosis (MS), systemic lupus erythematosus and rheumatoid arthritis (RA), when compared to females (Angum et al. 2020).To prevent the development of autoimmune diseases, the immune system must be able to recognise self-produced antigens without eliciting an immune response.This is known as self-tolerance, and loss of tolerance is known to underpin several autoimmune disorders (Zhang & Lu 2018).In the thymus, T-cell self-tolerance is enforced by the expression of the autoimmune regulator (Aire) gene, which promotes the expression of tissue-specific antigens (TSAs) by thymic epithelial cells.Immature thymocytes that come into contact with and recognise these TSAs with high affinity will be selectively removed from the population, preventing the maturation of self-reactive T cells.One study investigating sexual dimorphism in autoimmune disorders utilised mouse models and demonstrated that DHT treatment may have a protective effect against autoimmunity due to AR-dependent upregulation of Aire mRNA expression in thymic stromal cells.This increase in Aire mRNA expression leads to an upregulation in TSA expression and therefore negative selection of selfreactive T cells, limiting their release into the periphery (Zhu et al. 2016).The results of this study suggest that androgen therapy could be utilised to enhance Aire mRNA expression and therefore protect against autoimmunity, and opens new therapeutic avenues for autoimmune disorders.

Multiple sclerosis and experimental autoimmune encephalomyelitis
MS is an autoimmune condition that affects the brain and spinal cord as the immune system attacks the myelin that covers and protects nerve fibres in the central nervous system (CNS).MS is more predominant in women with a ratio of approximately 3:1 (Voskuhl et al. 2020).Experimental autoimmune encephalomyelitis (EAE) is a widely used mouse model of MS.EAE is a helper T cell-mediated autoimmune disease that causes inflammation in the CNS characterised by infiltration of monocytes and T cells, demyelination and axonal loss (Robinson et al. 2014).
Insertion of a DHT pellet in female mice resulted in significantly less severe EAE when compared to mice that received a placebo pellet.Furthermore, T cells specific for myelin basic protein from females treated with DHT expressed significantly higher levels of IL-10 than controls (Dalal et al. 1997).Similar results were found in another study where female mice were treated with physiological doses of DHT (5 mg), which increased IL-10 production in AR + CD4 + splenic T cells (Liva & Voskuhl 2001).Furthermore, administration of DHT 45 days after the induction of EAE in male rats led to an improvement in clinical scores and decreased inflammation in the spinal cord (Giatti et al. 2015).Taken together, these studies indicate that androgens have a protective effect in a mouse model of EAE that is in part mediated by increased IL-10 production by autoantigen-specific CD4 + T cells.

Prostate cancer and benign prostatic hyperplasia
Androgen deprivation therapy (ADT) is the main treatment for patients with prostate cancer; however, prostate cancer often recurs and becomes hormone therapy resistant.Recent evidence suggests that shortly after androgen deprivation, there is an expansion of T cells in both mouse models and prostate cancer patients.Wang et al. set out to investigate the role that different T cell subtypes play in hormone therapy-resistant prostate cancer.This study demonstrated that there was a novel population of CD4 low HLA-G + T cells that expanded in prostate cancer patients post ADT.These cells were found to promote the growth of prostate cancer cells through androgen-independent mechanisms that modulate the migration and activity of CD11b low F4/80 hi macrophages.
It was found that after androgen deprivation, there was an elevation of PGE2-EP2 signalling which led to the polarisation of this novel population of CD4 low HLA-G + T cells.Inactivation of PGE2, via celecoxib, during the appearance of CD4 low HLA-G + T cells significantly suppressed the onset of hormone therapy-resistant prostate cancer, suggesting that combining ADT with PGE2 inhibition may be effective in prostate cancer (Wang et al. 2018).
ADT has also been shown to sensitise prostate cancer patients to effective checkpoint blockade due to the enhanced function of CD8+ T cells.Inhibition of AR signalling via ADT prevented the exhaustion of CD8+ T cells and increased IFNG expression, resulting in improved responsiveness to programmed cell death protein 1 (PD-1) targeted treatment.The AR has the capacity to bind Ifng directly, repressing its expression, and preventing this binding using enzalutamide significantly increased IFNG production in CD8+ T cells.These findings demonstrate a novel mechanism of immunotherapy resistance in prostate cancer (Guan et al. 2022).This knowledge can be expanded to incorporate different tumour and treatment types to investigate this mechanism in other areas of cancer research.
Another condition that is androgen sensitive is benign prostate hyperplasia (BPH), which is the non-malignant growth or hyperplasia of prostate tissue (Roehrborn 2005).Emerging evidence from clinical studies suggests that androgens may be linked to the infiltration of T cells in BPH, but T cell subset information and potential mechanisms of this action are unknown.To investigate if DHT increases the infiltration of T cells via BPH epithelial cells, prostate tissues were collected from 64 BPH patients after transurethral resection of the prostate, split into two groups: (1) no medication history and (2) treated with finasteride (5α-reductase inhibitor) 5 mg daily for at least 6 months prior to surgery.Tissues from BPH patients treated with finasteride had significantly higher infiltration of CD8 + T cells into the prostate with no changes in CD4 + T cells and increased epithelial CCL5 expression (Fan et al. 2014).This study illustrated that intraprostatic DHT may have an important role in the regulation of immune responses through the recruitment of CD8 + T cells by human prostatic epithelial cells via their secretion of CCL5 (Fan et al. 2014).Thus, androgen  availability may affect T cell composition and function in the prostate and impact disease outcomes in BPH and hormone therapy-resistant prostate cancer.

B lymphocytes
B lymphocytes, also known as B cells, are adaptive immune cells that provide humoral immunity through antibody production.B cells develop from CLPs in the bone marrow and undergo a series of differentiation steps that result in the maturation and formation of the B cell receptor (BCR) (Cano & Lopera 2013).B cells can express a range of immunoglobulin (Ig) receptors including IgM, IgG, IgA, IgE and IgD, and these antibody molecules exert different immune functions such as neutralisation, opsonisation and high-affinity receptor-mediated killing of pathogens (Alberts et al. 2002).B cell progenitors, but not mature or peripheral B cells express, the cytoplasmic form on AR.B cells are therefore sensitive to the actions of androgens primarily during their development in the bone marrow (Benten et al. 2002a).
Androgens play an important role in B cell maturation and homeostasis.Altuwaijri et al. investigated the effect of androgens on B cell function using a variety of male mouse models.These models included castration, GARKO (Actb Cre Ar flox ) mice, B-cell-specific ARKO (BARKO -Cd19 Cre Ar flox ) mice, and Tfm mice to demonstrate that AR deficiency led to higher B cell numbers in the blood and the bone marrow due to increased proliferation of B cell precursors in the bone marrow.It was also seen that B cells with deficient AR had higher resistance to apoptosis and that implantation of a DHT pellet restored B cell numbers in castrated mice, but not GARKO mice, supporting the hypothesis that B cell maturation is androgen mediated and AR dependent (Altuwaijri et al. 2009).In vitro studies investigating the effect of androgens on B cell function demonstrated that testosterone treatment inhibited the production of IgG and IgM by B cells.This reduced production could be partially restored by the addition of exogenous IL-6.This study demonstrated that androgens could alter B cell function through the inhibition of antibody production (Kanda et al. 1996).
Ellis et al. investigated the effect of castration-dependent androgen ablation on B cell lymphopoiesis and the phenotype of peripheral B cell populations.Castration led to a significant increase in spleen weight and the total number of peripheral blood B cells (Ellis et al. 2001).
Further experiments were carried out to determine whether the increase in peripheral blood B cells was due to an increase in the number of mature lymphocytes and/ or due to an increase in newly formed B cells that have emigrated from the bone marrow.Analysis of peripheral blood B cells from castrated mice revealed an increase in mature B cells as well as B cells that had low expression of B220 and high expression of CD24.These markers identify newly emigrated immature B cells which proportionally were increased to a greater extent than mature B cells.This demonstrates that the observed increase in circulating B cells after castration is in part due to increased bone marrow egress of immature cells which remained elevated up to 54 days after castration (Ellis et al. 2001).These results demonstrate that loss of androgens can increase B cell lymphopoiesis and emigration of immature B cells from the bone marrow; however, it is unclear if this is a direct or indirect effect.An increase in the number of splenic B cells was demonstrated in a study that utilised general ARKO male mice (ubiquitous Pgk1; Pgk1 Cre Ar flox ).These mice also had increased serum levels of BAFF, a cytokine that is an essential splenic B cell survival factor (Wilhelmson et al. 2018).Increased serum BAFF levels are detected in healthy men with low testosterone, which suggests an association between testosterone, BAFF production, and B cell number.Taken together, these studies demonstrate the importance of androgens and AR signalling in the maturation and homeostasis of B cells.

Rheumatoid arthritis
RA is a chronic inflammatory autoimmune disorder that primarily affects the joints.RA is caused by abnormalities in the cellular and humoral immune response resulting in the generation of autoantibodies against targets, such as rheumatoid factors (RF).RA also results in the migration of T and B cells into the synovium and activation of the innate immune system within the affected tissues (Scherer et al. 2020).Male RA patients are reported to have low androgen levels (Cutolo et al. 1984, Spector et al. 1988), and prostate cancer patients receiving ADT have an altered risk of developing RA.This is characterised by a higher 5-year incidence rate of RA diagnosis in patients who received ADT compared to those who did not (Yang et al. 2018).To investigate the role of B cell AR in the development of autoimmune diseases, B cell ARKO mice (Cd19 Cre Ar flox ) were generated, and serum antibody levels were analysed.B cell ARKO mice had increased levels of IgG3 in their serum compared to wildtype mice as well as significantly elevated levels of autoantibodies that bind DNA (double-stranded DNA-IgG), which is a hallmark of autoimmune susceptibility.GARKO and B cell ARKO mice also show increased susceptibility to collagen-induced arthritis (CIA) with increased levels of basal IgG-RF autoantibodies and significant CIA manifestations, including enlarged synovial membrane and lymphocyte infiltration, 10 weeks post-bovine collagen II immunisation (Altuwaijri et al. 2009).Taken together, the data from clinical studies as well as the results generated in these B cell-deficient mouse models suggest that AR may be a plausible target for new therapies to treat autoimmune diseases, such as RA.

Final summary
Androgens and AR play an important role in the modulation of immune responses.Several innate and adaptive immune cell subsets express the cytoplasmic and/or membrane-bound form of AR and therefore have the capacity to be modulated by androgens (Fig. 2).Androgens/AR can modulate the development of immune cell subsets in both primary (bone marrow) and secondary (spleen) lymphoid organs with important roles in neutrophil and B cell maturation.Androgens exert their effect on immune function through various mechanisms in a cell-specific manner (Fig. 3).This includes a reduction in immune cell numbers, such as T cells, polarisation of monocyte and macrophage towards an anti-inflammatory phenotype, or inhibiting immune cell activation in the case of DCs.Androgens are therefore broadly associated with having immunosuppressive effects.
Changing the bioavailability of androgens in both health and disease can therefore alter immune cell function and responses.This has been explored in the context of sexual dimorphism, where the simple dichotomy of males as a high androgen state and females as a (relatively) low androgen state has been used to account for androgens having a role in the presentation/progression of sexually dimorphic diseases.However, variation in androgen bioavailability is not solely accounted for by sex.Endocrinopathies in both males and females can result in elevated (e.g.PCOS, congenital adrenal hyperplasia) or lowered (e.g.hypogonadism, premature ovarian insufficiency) androgens.Furthermore, surgical or medical castration can ablate endogenous androgens, and androgen replacement therapies are utilised in prostate cancer treatment and as part of post-menopausal hormone replacement therapy.Thus, understanding how androgens directly and indirectly affect immune cell function is key to determining their potential role in immune disorders.
Studies investigating the effects of androgens on immune cells in disease have shown that they are contextdependent and can differ even in the same immune cell type depending on the disease state.For example, it has been shown that androgens have an inhibitory effect in cutaneous wound healing by supressing pro-repair macrophages and production of TNF, whereas, in the context of asthma, it has been demonstrated that DHT treatment in experimental models reduces inflammation and promotes polarisation of alveolar macrophages into a pro-repair phenotype.These studies demonstrate that the impact of androgens on immune cells is highly nuanced, which can impact both the efficacy and specificity of androgen-targeting treatments, whether as direct therapies or as an indirect consequence of altered endocrine states.

Future directions
Androgen therapy impacts immune cells but has largely not been used to target immune function directly.Beyond endogenous ligands and classic antagonists, selective androgen receptor modulators (SARMs) have been developed, which have both agonist and antagonist effects on AR.SARMs exploit context-dependent impacts of AR signalling and have been developed to facilitate tissue-specific benefits without off-target side effects.There is currently no SARM approved by the Food and The impact of androgens on immune cell function in health and disease.In general, androgens tend to exhibit an anti-inflammatory effect on immune cells.This is demonstrated by the upregulation of inflammatory mediators, such as IL-10, in various immune cell types including T lymphocytes (Fijak et al. 2015), macrophages (D'Agostino et al. 1999), dendritic cells (Thompson et al. 2017) and neutrophils (Scalerandi et al. 2018) in the presence of androgens.It has also been demonstrated that androgens can reduce production of several inflammatory mediators, such as IFNG, IL-4 and IL-5 in T lymphocytes (Araneo et al. 1991) and NO in macrophages (D'Agostino et al. 1999, Friedl et al. 2000).Androgen have also been found to promote immune cell maturation and differentiation in cells such as T lymphocytes (Olsen et al. 1991), B lymphocytes (Altuwaijri et al. 2009) and neutrophils (Chuang et al. 2009, Inamdar & Jayamma 2012).Green arrows represent upregulation or increase.Red arrows represent downregulation or decrease.For abbreviations see text.Created with BioRender.com.
Drug Administration; however, there are many ongoing phase I and II clinical trials examining these treatment strategies for a range of conditions, including breast cancer, prostate cancer, BPH, and chronic obstructive pulmonary disease.This is a promising treatment avenue that has the potential to treat a wide spectrum of disorders, including those listed above, and others such as osteoporosis, muscle wasting and hypogonadism (Christiansen et al. 2020).Detailed information regarding both approved and ongoing clinical trials utilising androgen modulation as a treatment strategy is discussed in detail elsewhere (Gamat & McNeel 2017, Ben-Batalla et al. 2020, Christiansen et al. 2020).Importantly, none have a primary indication for modulating immune cellmediated diseases.
One challenge of investigating the effects of androgens in androgen-responsive tissues, such as the prostate, is that while androgens might mediate specific effects on immune cells, there are other cell types in the tissue, such as epithelial and stromal cells, that will also express the AR.Thus, delineating the effects of androgens/AR activation in tissue homeostasis and disease contexts will need to take this into account to understand how the cells in the tissue are being directly and indirectly modulated by androgens.Androgens may induce an anti-inflammatory phenotype in one cell type, but if AR activation in another cell type causes the release of mediators that bring about the opposite effect, the net result may not necessarily be anti-inflammatory.Further studies utilising in vitro techniques and conditional knockout models in vivo would be required to explore the direct regulation of androgens on cell types of interest and to identify downstream mediators of androgen signalling.Future research should take into account the more complete context of hormone availability, receptor expression and sites of cell/ tissue action when exploring roles for androgens in modulating immune cell function.

Conclusion
Androgens can have a wide variety of effects on immune cells, and these can differ depending on disease context and anatomical location.There remain various unanswered questions surrounding the effect of androgens on the immune system, including effective ways to modulate androgen-regulated processes and understanding androgen balance in the context of endocrinopathies and disease states.These will need to be accounted for to progress drug development and clinical use of treatments that target androgen action in inflammatory contexts.Embracing androgens as immunomodulatory agents will help to improve understanding of endocrine aspects of inflammatory disorders and open new avenues for repurposing and developing androgen therapies to modulate immune cell function.

Table 1 A
summary of primary research studies investigating the effects of androgens on murine macrophages in vitro.
investigate the molecular mechanisms contributing to this difference,Lai et al.utilised two mouse models with genetic deletion of AR.Global AR knockout (GARKO) mice were generated by breeding transgenic mice with loxP-flanked AR allele (Ar flox ) with mice expressing the Cre transgene under the control of the Actb promoter to generate mice that lack the AR in all cell types (Actb Cre Ar flox ).The second model involved breeding Ar flox mice with mice expressing the Cre transgene under the control of the LyzM promoter to generate myeloidspecific AR knockout (LyzM Cre Ar flox ) mice.It was observed that cutaneous wound healing was accelerated in both AR knockout models.The mechanism of immunosuppression by AR in this context was due to the loss of three AR-dependent mechanisms; increased monocyte recruitment, upregulation of monocyte CCR2

Table 2 A
summary of primary research studies investigating the effects of androgens on T lymphocytes in vitro and in vivo.