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George Whipple Laboratory for Cancer Research, University of Rochester Medical Center, Rochester, New YorkDepartment of Pathology, University of Rochester Medical Center, Rochester, New YorkDepartment of Urology, University of Rochester Medical Center, Rochester, New YorkDepartment of Radiation Oncology, University of Rochester Medical Center, Rochester, New YorkWilmot Cancer Center, University of Rochester Medical Center, Rochester, New York
George Whipple Laboratory for Cancer Research, University of Rochester Medical Center, Rochester, New YorkDepartment of Pathology, University of Rochester Medical Center, Rochester, New YorkDepartment of Urology, University of Rochester Medical Center, Rochester, New YorkDepartment of Radiation Oncology, University of Rochester Medical Center, Rochester, New YorkWilmot Cancer Center, University of Rochester Medical Center, Rochester, New York
George Whipple Laboratory for Cancer Research, University of Rochester Medical Center, Rochester, New YorkDepartment of Pathology, University of Rochester Medical Center, Rochester, New YorkDepartment of Urology, University of Rochester Medical Center, Rochester, New YorkDepartment of Radiation Oncology, University of Rochester Medical Center, Rochester, New YorkWilmot Cancer Center, University of Rochester Medical Center, Rochester, New York
George Whipple Laboratory for Cancer Research, University of Rochester Medical Center, Rochester, New YorkDepartment of Pathology, University of Rochester Medical Center, Rochester, New YorkDepartment of Urology, University of Rochester Medical Center, Rochester, New YorkDepartment of Radiation Oncology, University of Rochester Medical Center, Rochester, New YorkWilmot Cancer Center, University of Rochester Medical Center, Rochester, New York
George Whipple Laboratory for Cancer Research, University of Rochester Medical Center, Rochester, New YorkDepartment of Pathology, University of Rochester Medical Center, Rochester, New YorkDepartment of Urology, University of Rochester Medical Center, Rochester, New YorkDepartment of Radiation Oncology, University of Rochester Medical Center, Rochester, New YorkWilmot Cancer Center, University of Rochester Medical Center, Rochester, New York
George Whipple Laboratory for Cancer Research, University of Rochester Medical Center, Rochester, New YorkDepartment of Pathology, University of Rochester Medical Center, Rochester, New YorkDepartment of Urology, University of Rochester Medical Center, Rochester, New YorkDepartment of Radiation Oncology, University of Rochester Medical Center, Rochester, New YorkWilmot Cancer Center, University of Rochester Medical Center, Rochester, New YorkSex Hormone Research Center, China Medical University/Hospital, Taichung, Taiwan
Upon insult, such as infection or tissue injury, the innate and adaptive immune systems initiate a series of responses to defend the body. Recent studies from immune cell-specific androgen receptor (AR) knockout mice demonstrated that androgen and its receptor (androgen/AR) play significant roles in both immune regulations. In the innate immunity, androgen/AR is required for generation and proper function of neutrophils; androgen/AR also regulates wound healing processes through macrophage recruitment and proinflammatory cytokine production. In adaptive immunity, androgen/AR exerts suppressive effects on development and activation of T and B cells. Removal of such suppression causes thymic enlargement and excessive export of immature B cells. Altogether, androgen/AR plays distinct roles in individual immune cells, and targeting androgen/AR may help in treatment and management of immune-related diseases.
The immune system includes both innate and adaptive immune responses. Once insult to the body (eg, infection) is initially encountered, the innate system acts within minutes, followed some hours later by early induced responses conducted by recruited effector cells, such as neutrophils and macrophages, in a nonspecific manner.
In most cases, infections are eliminated by the innate immune system. When pathogens breach the first line of immune defense, however, the adaptive immune system is activated with antigen-specific effectors (eg, T and B cells), and the generation of memory cells ensures a long-lasting and prompt response to recurrent infection with the same pathogens. On the other hand, the context of the activated innate immunity also shapes the outcome of adaptive immune responses.
The AR gene is located on the X chromosome in both the human and the murine genome. AR is a prototypical nuclear receptor, containing an N-terminal domain, a ligand-binding domain, a DNA-binding domain, and a C-terminal domain.
After binding of its ligands, testosterone or 5α-dihydrotestosterone, AR translocates into the nucleus and binds to androgen responsive elements on the promoters or enhancers of target genes, thereby turning on their expression.
Human mast cells express androgen receptors but treatment with testosterone exerts no influence on IgE-independent mast cell degranulation elicited by neuromuscular blocking agents.
implying the involvement of androgen and its receptor (androgen/AR) in regulating the development and function of the immune system.
It has long been suspected that, in both animals and humans, the male sex hormones may modulate the development and function of the immune system. Males are at higher risk of developing sepsis, acute respiratory distress, and multiorgan failure after soft-tissue traumatic hemorrhagic shock and thermal injury, in part because of immune suppression and abnormal activation of neutrophils.
On the other hand, males are less prone to autoimmune diseases. Of more than 70 chronic disorders categorized as autoimmune diseases, many affect predominantly women.
Various studies have uncovered important immune regulatory functions of androgen/AR (as discussed below), and such nonclassical roles of androgen/AR outside the reproductive system are important for understanding the pathogenesis of these immunological conditions.
Recent studies using conditional AR knockout (ARKO) mice, with knockout of AR in selective immune cells, opened a new era for investigating the nonclassical roles of androgen/AR involved in immune regulation; these studies also suggest potential for the development of new therapeutic strategies for treating immune-related diseases.
In this review, we summarize the roles of androgen/AR in both the innate and adaptive components of the immune system (specifically neutrophils, macrophages, T cells, and B cells), as well as new evidence from studies using conditional ARKO mice. We also address the potential influences of androgen/AR on immune-related diseases.
Androgen/AR in the Innate Immune System
Roles of Neutrophil AR in Defending the Body
Granulopoiesis is a dynamic process leading to the production of 120 billion granulocytes daily in humans; its synthesis capacity can be increased at least 10-fold in response to certain stress conditions, including infection. Neutrophils are the most abundant type of granulocyte; eosinophils and basophils are much rarer. Upon insult, neutrophils arrive on site within minutes and usually peak at 12 to 24 hours, establishing the first line of immune defense. In addition to their major function for the phagocytosis of invading infections/pathogens or damaged tissues, neutrophils are able to secrete several chemokines and cytokines to attract and activate monocytes/macrophages.
Originating from hematopoietic stem cells and progenitors, neutrophils in the bone marrow are composed of a precursor pool and a storage pool. Peripheral blood neutrophils, which are postmitotic, consist of a free circulating pool and a marginal pool. AR is universally expressed in neutrophil lineages from the proliferative precursors (myeloblasts, promyelocytes, and myelocytes) to mature neutrophils (metamyelocytes, band forms, and neutrophils), with no difference in AR expression patterns between males and females.
In myelocytes, AR is stained positively in the cytoplasm and shows perinuclear dot staining. Similarly, AR is localized in the cytoplasm of myeloblasts, but without perinuclear dot staining.
Young women who suffer from polycystic ovarian syndrome with hyperandrogenism have higher neutrophil counts, which can be normalized by treatment with the antiandrogen flutamide, suggesting that androgen/AR signals can promote neutrophil production.
High neutrophil count in girls and women with hyperinsulinaemic hyperandrogenism: normalization with metformin and flutamide overcomes the aggravation by oral contraception.
Consistent with this notion, we found almost 90% reduction of neutrophil counts in general ARKO (G-ARKO) mice, compared with wild-type mice, and a similar reduction was also found in male mice carrying the testicular feminization (Tfm) mutation in the AR gene.
Castration of male mice also resulted in neutrophil reduction; however, the effect of castration was less dramatic than in ARKO or Tfm mice. These results suggest that AR confers a direct and more profound effect than androgen to control neutrophil production. Although neutropenia was observed in some prostate cancer patients treated with antiandrogens,
castration in humans does not always result in neutropenia but may instead lead to only a mild reduction of neutrophil counts, suggesting that AR, but not androgen, is more critical for neutrophil homeostasis.
Further analysis of neutrophil lineages showed that myelocytes/metamyelocytes and mature neutrophils, but not myeloblasts and promyelocytes, were significantly reduced in ARKO mice. Additionally, restoring the AR expression in the ARKO granulocyte-macrophage progenitors rescued neutrophil production, implying that AR is essential in regulating neutrophil differentiation.
Further studies also suggested that AR regulates primarily the transition between proliferation of precursors (myeloblasts, promyelocytes, and myelocytes) and maturation of neutrophils (metamyelocytes, band forms, and neutrophils).
Mechanistically, AR appears to stimulate neutrophil production by enhancing granulocyte colony-stimulating factor (G-CSF) signaling. This is achieved by activating ERK1/2 and also by sustaining Stat3 activity via diminishing the inhibitory binding of protein inhibitor of activated STAT protein 3 (PIAS3).
Such AR-PIAS3 interaction does not rely on ligand binding, and could be considered an androgen-independent regulation. Notably, the major induction of Stat3 reporter activity was caused by AR expression and was only slightly enhanced by adding androgens, suggesting that androgens are not essential for the function of AR in promoting Stat3 activity.
Other lines of evidence using in vitro and transgenic mouse studies have demonstrated that Stat3 plays an essential role in G-CSF-dependent granulopoiesis.
Conditional overexpression of Stat3alpha in differentiating myeloid cells results in neutrophil expansion and induces a distinct, antiapoptotic and pro-oncogenic gene expression pattern.
Sustained receptor activation and hyperproliferation in response to granulocyte colony-stimulating factor (G-CSF) in mice with a severe congenital neutropenia/acute myeloid leukemia-derived mutation in the G-CSF receptor gene.
Moreover, several reports have shown that mice with knockout of suppressor of cytokine signaling 3 (SOCS-3), in which Stat3 activity is persistent, developed severe neutrophilia.
Neutrophils are a key component of the innate immune system in clearing bacterial pathogen infection. As a result of neutrophil reduction, the ability of ARKO mice to survive pathogenic bacteria challenge is severely compromised.
In addition to reduced neutrophil counts, functional defects of neutrophils also contributed to the susceptibility to septic challenges in the ARKO mice. Although the residual neutrophils in ARKO mice retained the normal capacity for phagocytosis and respiratory burst, they produced less proinflammatory cytokines IL-1β, IL-6, and TNF-α and of chemokines CCL2, CCL3, CCL4, CXCL1, CXCL4, and CXCL7.
It has long been suspected that chronic inflammation can promote prostate tumorigenesis and progression. Several lines of evidence show that mice lacking proinflammatory cytokines are resistant to carcinogenesis, tumor invasion, and angiogenesis.
AR may play a pivotal role in remodeling the tumor-promoting microenvironment via release of proinflammatory cytokines from neutrophils.
Neutrophils can be attracted by IL-8 released from castration-resistant prostate cancer cells; they then migrate toward the tumor lesion and release enzymes (eg, MMP-9, collagenase) to remodel the extracellular matrix of nearby tissues.
Thus, neutrophils could serve as enhancers of cancer cell migration through remodeling of the extracellular matrix. Moreover, the reactive oxygen species produced by neutrophilic oxidases to kill invading organisms have the potential to interact with tumor cells, attenuating their apoptotic cascade and increasing their mutation rate and malignancy.
Roles of Macrophage AR in Regulating Inflammatory Responses
Immediately after neutrophil activation, monocytes are recruited from the circulation into inflamed tissues in response to chemokines secreted from neutrophils and damaged tissues.
The infiltrated monocytes subsequently differentiate into macrophages, which in turn become the master regulators of the inflammatory response. The hallmark functions of macrophages include not only phagocytosis of the infecting microbes, dead neutrophils, and tissue debris, but also the production of proinflammatory cytokines and growth factors to regulate the succeeding immune responses and tissue regeneration.
In addition, macrophages can present antigens to T cells, and thus macrophage activity shapes the successive T cell activation and bridges innate immunity and the adaptive immune response. Increasing evidence shows that macrophages play an important role in regulating tissue regeneration and inflammatory responses during wound healing and traumatic-hemorrhagic shock.
suggesting that AR might modulate macrophage functions involved in these inflammatory conditions.
Macrophage AR Promotes Inflammation in Cutaneous Wound Healing
In the early phase of cutaneous wound healing, macrophages are among the major inflammatory cells recruited to the injured tissues. Although inflammation is necessary for removing tissue debris, an excess of it hinders wound healing.
Castration of animals or blockade of androgen action by antiandrogen (flutamide) accelerated wound healing and suppressed macrophage recruitment to the wounds.
In a mouse model of wound healing, the proinflammatory cytokine TNF-α, but not IL-6, at the site of injury was down-regulated by castration or flutamide treatment. Conversely, in vitro studies found that lipopolysaccharide-induced TNF-α production in macrophages was enhanced by testosterone treatment.
and therefore using castration or antiandrogens to decrease androgen levels cannot clarify whether the androgen effects are through AR or non-AR pathways to inhibit wound healing.
Interestingly, a recent report using the Cre-loxP system to generate conditional ARKO mice found that mice lacking AR had accelerated wound healing, and such acceleration could not be reversed by 5α-dihydrotestosterone restoration, suggesting that AR signals, rather than androgen itself, are critical to suppression of wound healing.
Using reciprocal bone marrow transplantation, it was found that AR deficiency in the infiltrating cells, and not in mesenchymal cells, was responsible for the accelerated wound healing in ARKO mice. AR in the wild-type donor bone marrow cells, even in the low-testosterone environment of ARKO recipients, could still suppress wound healing, suggesting that AR can go through androgen-independent pathways to suppress wound healing.
Although the detailed mechanisms of these androgen-independent AR effects in wound healing remain unclear, these results are in accord with earlier studies showing that AR could also be activated by other factors in addition to androgens, such as estrogens,
Further studies are warranted, to determine whether any of these mechanisms are involved in suppression of wound healing. Taken together, these studies reveal the novel concept that AR, but not androgen, is critical for wound healing suppression.
The essential roles of monocyte/macrophage AR in wound healing suppression were further corroborated in a study using myeloid-specific ARKO mice (M-ARKO), which found that AR in the infiltrating monocytes/macrophages executed its suppressive roles in cutaneous wound healing by enhancing local TNF-α expression via the following mechanisms: i) increasing circulating inflammatory monocyte population, ii) increasing recruitment of monocytes by promoting their chemotaxis through up-regulation of CCR2 expression, and iii) increasing TNF-α expression at the transcriptional level in macrophages.
Although the results from bone marrow transplantation imply androgen-independent AR functions in the suppression of wound healing, it is noteworthy that the AR regulation of TNF-α and CCR2 expression is androgen-dependent and directly regulates the promoter activities of these two proteins.
These findings demonstrate the essential roles of AR in the macrophage-associated inflammatory regulation toward the pathological conditions (such as wound healing) and raise an important question, whether monocyte/macrophage AR may also play essential roles in other inflammatory diseases that demonstrate sex differences, such as atherosclerosis
A positive answer may then point toward development of a potential therapeutic approach via targeting AR in treatment and management of these sexually dimorphic inflammatory diseases.
Role of Androgen/AR Signals in the Adaptive Immune System
Unlike macrophages and neutrophils, which play a central role in the innate immune system,
T and B cells represent the central players in adaptive immune response. The activation of T and B cells requires proper antigen presentation, cell-cell interaction (engagement of T cell receptor TCR, or B cell receptor BCR, and costimulation), and specific environmental cytokine profiles. Studies using castration and Tfm mouse models suggest that androgen/AR signals are involved in both differentiation and activation of T cells and B cells, and might contribute to the sexual dimorphism of autoimmune diseases.
Role of Androgen/AR in the Development and Activation of T Cells
Androgen/AR Signals in T Cell Development
The thymus is the major organ in which naïve T cells develop. Several studies have shown that T cell generation from thymus is significantly reduced after puberty,
suggesting that sex hormones may play critical roles in maintaining the homeostatic T cell populations and repertoires. It was more than a century ago that castration of male animals was found to result in thymic enlargement.
The effects of castration on the reversal of thymic involution have since been analyzed in great detail. The role of androgen on thymic involution was confirmed by the reversal of thymic enlargement by androgen replacement in castrated animals.
and Lai et al (unpublished data) in ARKO and Tfm mice, as well as castrated mice, found thymic enlargement and increase of thymic cellularity in these mice, which could be due to more proliferating and less apoptotic thymocytes.
The net outcome of castration-induced reversal of thymic involution is increased output of naïve CD4 and CD8 single-positive T cells.
comparison of thymic and peripheral TCR repertoires showed no noticeable differences between castrated and normal mice (Lai et al, unpublished data). The latter observation appears to argue against a role of androgen/AR signals in the negative selection of thymocytes. The aforementioned reduction of thymocyte apoptosis in both ARKO and castrated mice may indicate that there is no role for androgen/AR signals in enhancing negative selection.
Importance of AR Expression in the Thymic Epithelium for Thymocyte Development
To determine the target cells involved in androgen/AR effects on thymopoiesis, the expression of AR in thymocytes and thymic stromal cells have been extensively analyzed. Although some earlier ligand-binding studies indicated that thymocytes do not express AR,
AR expression in thymocytes can also be detected by RT-PCR (Lai et al, unpublished data). Flow cytometry analysis showed that AR is expressed in all thymocyte subpopulations defined by the expression of CD3, CD4, and CD8, with the highest expression in CD3lowCD8+ immature thymocytes.
Reciprocal bone marrow transplantation between normal male mice and Tfm male mice was used to determine whether AR expression in thymocytes or thymic epithelial cells is important for androgen/AR effects on thymic development. Bone-marrow chimeric mice with bone marrow grafts from Tfm mice to normal mice showed normal response to androgen and thymic involution. In contrast, chimeric mice receiving bone marrow from normal mice to Tfm mice developed enlarged thymus and were insensitive to androgen.
These findings, together with the evidence for AR expression in thymic stroma, suggest that AR expression in thymic stroma, rather than thymocytes, is responsible for inhibition of T cell development by androgen/AR signals. Using G-ARKO mice, we have performed similar bone marrow chimeric studies and confirmed the importance of AR expression in thymic stroma (Lai et al, unpublished data).
Based on the residing compartment, thymic epithelial cells can be further divided into cortical thymic epithelial cells (cTECs) and medullar thymic epithelial cells (mTECs). These two populations have distinct functions in thymocyte development.
After positive selection, the double-positive thymocytes encounter other stromal cells (including macrophages, dendritic cells, and mTECs) in the medullar compartment for negative selection. The role of mTECs in central tolerance has been of great interest since the discovery of promiscuous expression of tissue-restricted self-antigens in these cells.
Thus, mTECs can directly induce or, through cross-presentation of self-antigens, promote negative selection of self-reactive T cells. We have established TEC-ARKO mice by using bovine keratin-5 promoter Cre to delete AR in thymic epithelial cells and found that AR was ablated in both cTEC and mTEC populations (Lai et al, unpublished data). Upon further breeding of ARKO with TCR transgenic mice, results suggested that AR plays an essential role in regulating positive selection. In contrast to TEC-ARKO mice, T cell–specific ARKO (T-ARKO) mice with AR deletion in thymocytes revealed normal size and cellularity of thymus (Lai et al, unpublished data). These results are in accord with findings in bone marrow transplantations showing that AR in thymic epithelial cells plays a more crucial role than thymocytes in thymic development.
To investigate the potential targets of androgen/AR in thymocyte development, we demonstrated that ARKO thymic stromal cells expressed less mRNA of TGFβ1, IL-6, and CD80/CD86, but more of IL-7 and CCL21, compared with wild-type thymic stromal cells on 5α-dihydrotestosterone treatment (Lai et al, unpublished data). Interestingly, androgen/AR can up-regulate CD80 promoter activity through direct promoter binding (Lai et al, unpublished data). Notably, it has been shown that in CD28 or B7 (CD80/CD86) knockout mice, the selection of mature CD4 and CD8 single-positive thymocytes was increased,
suggesting that CD28-B7 engagement serves as a negative regulator for positive selection. This may explain, at least in part, how androgen/AR signaling inhibits positive selection of thymocytes.
Effect of Androgen/AR on Peripheral T Cell Function
In addition to effects in development, the effect of androgen/AR on T cell–mediated immune responses has been recognized in many models of cellular immunity. Administration of androgens to female mice alleviated rejection of skin grafts and alleviated CD4 T cell–mediated autoimmune diseases such as experimental autoimmune encephalomyelitis, whereas castration in male mice exacerbated this autoimmune disease.
Testosterone therapy ameliorates experimental autoimmune encephalomyelitis and induces a T helper 2 bias in the autoantigen-specific T lymphocyte response.
The effects of androgen/AR on peripheral T cell response are twofold: first, androgen/AR suppresses T cell proliferation; second, androgen/AR modulates the balance of Th1 and Th2 responses. In terms of T cell proliferation, T cells from castrated mice proliferated more vigorously in vitro than T cells from normal mice regardless of the modes of T cell activation, although the effect seemed to be transient.
Because the enhancement of T cell activation could occur in the absence of antigen-presenting cells or any other accessory cells, androgen/AR appeared to exert a direct effect on T cells. This is an issue of debate, however, given that some studies have found no AR expression in peripheral T cells,
In models of burn injury and hemorrhagic shock in which delayed-type hyper-responsiveness and T cell proliferation were suppressed, blockade of androgen/AR restored T cell response, which was accompanied by increased IL-2 production and IL-2R expression in T cells.
Thus, androgen/AR appears to suppress T cell proliferation by diminishing IL-2 signaling in T cells.
Regulation of Th1 and Th2 Responses by Androgen/AR
The inhibitory action of androgen/AR on T cell proliferation may be a contributing factor for the lower incidence of autoimmune diseases in males than in females. In addition, evidence has accumulated to support a role of androgen/AR in regulating the functional differentiation of peripheral CD4 T cells, which may have even greater implications for the sexual dimorphism of autoimmunity. Organ-specific autoimmune diseases such as experimental autoimmune encephalomyelitis are typically associated with biased Th1 immune responses to autoantigens. In a study using experimental autoimmune encephalomyelitis, autoantigen-specific T cells from immunized male animals were less capable of transferring disease, compared with T cells from female animals.
Analyses of cytokine profiles of the autoreactive T cells showed higher IFN-γ production in female than in male mice. In in vitro cultures, the presence of androgen diminished both the differentiation of Th1 cells from naïve precursors and the production of IFN-γ by T-helper effector cells induced by TCR stimulation.
Although the study was performed on resting CD4 T cells that were previously activated by concanavalin A, one may extrapolate a role of androgen-mediated inhibition of Stat4 activation in dampening Th1 differentiation from naïve CD4 T cells as well, because IL-12 is a potent inducer of Th1 cell differentiation.
Finally, it should be pointed out that the Th1 and Th2 divergence of immune response between males and females is not restricted to autoimmunity. It was also observed in hemorrhagic shock that androgen/AR suppressed IFN-γ and IL-2 expression by splenocytes in male mice, whereas female mice maintained strong production of these two cytokines.
Role of Androgen/AR Signals in the Development and Activation of B Cells
The major function of B cells is to produce antibodies on stimulation. Most of the B cells respond to foreign antigens, such as virus, bacteria, and toxins; however, some autoreactive B cells escape from negative selection during B-cell development and are released into the periphery. These autoreactive B cells are able to produce antibodies against self antigens and subsequently result in autoimmunity and in autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, and therefore B cells have been one of the primary targets for autoimmune therapy.
Moreover, profound sexual dimorphism in autoimmune diseases implies that sex hormones might involve the regulation of B-cell responses and/or their development.
B-cell development is negatively regulated by androgen/AR. Castration of male mice caused splenic enlargement, due primarily to the increase of B cells.
Further studies found that B cells were increased not only in the spleen but also in the bone marrow of castrated mice. The B cell increase could occur in thymectomized mice, and therefore it was independent of the effect of castration on T cells.
Closer examination of the B-cell lineage in the bone marrow of castrated mice revealed the most dramatic increase in the late pro-B population, and to lesser degrees in the pre-B- and immature B-cell populations.
The increase of B-cell progenitors in the bone marrow led to the increase of the peripheral B-cell pool, with altered composition. Specifically, the proportion of newly emigrated immature B cells was found to be up to 45% of total circulating B cells in castrated and Tfm mice, although these cells represent only 10% to 15% in normal mice.
and both cell types could be the targets of androgen/AR in regulating B-cell lymphopoiesis. In bone marrow chimeric studies between Tfm and normal mice, the expression of intact AR on bone marrow stroma was critical for the inhibition of B-cell lymphopoiesis by androgen/AR.
On the other hand, mice with either general or B cell–specific knockout of the AR gene showed enhanced B-cell lymphopoiesis, demonstrating that B cells are direct targets of androgen.
However, the B cell–specific ARKO mice, showed a milder effect on B-cell lymphopoiesis, compared with the general ARKO mice. The full effect of androgen on B-cell lymphopoiesis therefore depends on its action on both B cells and the stroma. In both the general and the B cell–specific ARKO mice, the pre-B- and immature B-cell populations were increased in the bone marrow, apparently due to both increased proliferation and reduced apoptosis.
These findings came mainly from primary culture of immature B cells cultured in a medium (MesenCult) containing very low levels of androgen. The above-mentioned changes in gene expression could therefore be due primarily to androgen-independent pathways, although androgen-dependent regulation could not be completely ruled out in this study.
The enlarged B-cell pool could potentially increase the frequency of autoreactive B cells in castrated or AR deficient mice. In fact, in both the general and the B cell–specific ARKO mice, levels of anti–double-stranded DNA antibodies were significantly elevated.
Thus, removal of the brake on B-cell lymphopoiesis imposed by androgen/AR could lead to autoimmune diseases in which autoantibodies play pathogenic roles. Examples of such consequences could be found in hypogonadal men with rheumatoid arthritis and/or systemic lupus erythematosus
Conversely, androgen replacement therapy of hypogonadal men with rheumatoid arthritis ameliorated clinical symptoms and reduced the concentrations of IgM rheumatoid factors.
Concluding Remarks
The study of immune regulation by androgen/AR has attracted long-lasting interest. The recent availability of cell lineage-specific ARKO mice has opened new avenues of research.
Up-to-date studies using these animal models and other approaches have shown that androgen/AR exerts different regulatory effects on the immune system, which may also be influenced by the pathophysiological conditions of the hosts (Figure 1; Table 1). For neutrophils, androgen/AR plays a positive role for both development and function. These effects appear to be beneficial for hosts, to survive bacterial challenge.
it inhibits cytokine production after traumatic-hemorrhagic shock and burns. For the adaptive arm of the immune system, androgen/AR has generally inhibitory effects on T- and B-cell development. In the periphery, androgen/AR dampens T cell activation and inhibits Th1 differentiation. These inhibitory effects of androgen/AR on B and T cells may help lower the risk of autoimmunity in males.
Figure 1Roles of AR in the innate and adaptive immune compartments, including macrophages, neutrophils, thymic epithelial cells, T cells, and B cells. The model incorporates phenotypic observation and associated diseases from conditional ARKO mice with AR deletion in macrophages (cutaneous wound healing, organ failure, and immune suppression), neutrophils (organ failure, immune suppression, and sepsis), thymic epithelial cells (thymic involution and bone marrow transplantation grafting efficacy), T cells (autoimmune diseases), and B cells (autoimmune diseases).
Table 1Immune-Related Phenotypes in Tissue-Specific ARKO Mice
Characteristic
Castration
Tfm
G-ARKO
T-ARKO
B-ARKO
M-ARKO
TEC-ARKO (K-ARKO)
F-ARKO
Mutation/Cre promoter
Natural AR mutant with AR mRNA instability
β-actin promoter
proximal region of lck promoter
CD19 promoter
lysozyme M promoter
bovine keratin-5 promoter
Fsp1 promoter
Specific tissue/cell
Systemic androgen level decreased
general
general
thymocyte and T cell
B cell
myeloid cell
epithelial cell
fibroblast
Phenotype
T-cell development
thymus enlargement
thymus enlargement
thymus enlargement
normal thymus size
NA
NA
thymus enlargement
normal thymus size
B-cell development/function
increased immature B-cell population
immature B-cell population increased
immature B-cell population increased; autoantibodies increased
NA
immature B-cell population increased; autoantibodies increased
NA
NA
NA
Neutrophil development/function
Moderate reduction of neutrophil counts
Neutropenia
Neutropenia and susceptible to bacterial infections
NA
NA
normal granulopoiesis in bone marrow; moderate reduction of neutrophil counts in circulation
NA
NA
Wound healing
accelerated cutaneous wound healing
NA
accelerated cutaneous wound healing
NA
NA
accelerated cutaneous wound healing
wound healing rate similar to wild-type but re-epithelialization rate decreased
wound healing rate similar to wild-type but re-epithelialization rate decreased
Related human disease
Autoimmune disease; wound healing
Autoimmune disease; septicemia; wound healing
autoimmune disease; septicemia; wound healing
autoimmune disease
autoimmune disease
wound healing
autoimmune disease; wound healing
wound healing
The mice from castration treatment, Tfm mutation, G-ARKO, T-ARKO, B-ARKO, M-ARKO, TEC-ARKO (K-ARKO), and F-ARKO are included for comparison. Also included is usage of specific Cre promoter, deficiency of AR in selective cells/tissue types, phenotypes, and related human diseases.
Further studies applying these valuable mouse models could not only advance our knowledge and understanding of the communication between androgen/AR and the immune system, but could also suggest novel strategies to modulate the immune system under clinical conditions in which regulation of immune responses by androgen/AR plays critical roles.
Acknowledgment
We thank Karen Wolf for manuscript preparation and critical review.
Human mast cells express androgen receptors but treatment with testosterone exerts no influence on IgE-independent mast cell degranulation elicited by neuromuscular blocking agents.
High neutrophil count in girls and women with hyperinsulinaemic hyperandrogenism: normalization with metformin and flutamide overcomes the aggravation by oral contraception.
Conditional overexpression of Stat3alpha in differentiating myeloid cells results in neutrophil expansion and induces a distinct, antiapoptotic and pro-oncogenic gene expression pattern.
Sustained receptor activation and hyperproliferation in response to granulocyte colony-stimulating factor (G-CSF) in mice with a severe congenital neutropenia/acute myeloid leukemia-derived mutation in the G-CSF receptor gene.
Testosterone therapy ameliorates experimental autoimmune encephalomyelitis and induces a T helper 2 bias in the autoantigen-specific T lymphocyte response.
Supported by the NIH (NIH-NIDDK R01-DK73414), the George Whipple Professorship Endowment, and the Taiwan Department of Health Clinical Trial and Research Center of Excellence grant DOH99-TD-B-111-004 (China Medical University, Taichung, Taiwan, to C.C.).
J.J.L. and K.P.L. contributed equally to this work.
CME Disclosure: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interest to disclose.
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