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Autoimmunity Reviews 11 (2012) A502–A514

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Autoimmunity Reviews

journal homepage: www.elsevier.com/locate/autrev

Review

Progesterone and autoimmune disease

Grant C. Hughes ⁎ University of Washington, 1959 NE Pacific St., Box 356428, Seattle, WA 98195–6428, USA

⁎ Tel.: +1 206 685 9557. E-mail address: [email protected].

1568-9972/$ – see front matter © 2011 Elsevier B.V. All doi:10.1016/j.autrev.2011.12.003

a b s t r a c t

a r t i c l e i n f o

Available online 13 December 2011

Keywords: Progesterone Autoimmunity Sex hormones Sex steroids Systemic lupus erythematosus SLE

Sexual dimorphism in human immune systems is most apparent in the female predominance of certain au- toimmune diseases (ADs) like systemic lupus erythematosus (SLE). Epidemiologic, observational and exper- imental evidence strongly suggest sex steroids are important modulators of genetic risk in human AD. In this regard, the roles of progesterone (Pg), an immunomodulatory female sex steroid, are poorly understood. Sev- eral lines of investigation indicate Pg and synthetic progestins impact risk of AD and immune-mediated inju- ry in different ways depending on their concentrations and their engagement of various Pg receptors expressed in immune organs, immune cells or tissues targeted by immune attack. At low physiologic levels, Pg may enhance interferon-alpha (IFN-α) pathways important in SLE pathogenesis. Commonly used synthet- ic progestins may have the opposite effect. At pregnancy levels, Pg may suppress disease activity in rheuma- toid arthritis (RA) and multiple sclerosis (MS) via inhibition of T helper type 1 (Th1) and Th17 pathways and induction of anti-inflammatory molecules. Importantly, Pg's immunomodulatory effects differ from those of estrogens and androgens. An additional layer of complexity arises from apparent interdependence of sex hor- mone signaling pathways. Identifying mechanisms by which Pg and other sex steroids modulate risk of AD and immune-mediated injury will require clarification of their cellular and molecular targets in vivo. These future studies should be informed by recent genetic discoveries in human AD, particularly those revealing their sex-specific genetic associations.

© 2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A503 2. Progesterone and mammalian biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A503 3. Sex steroids and the risk of AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A504

3.1. Sexual dimorphism in human AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A504 3.2. Sex hormones and risk of human AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A504 3.3. Sex hormones and genetic risk of AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A504 3.4. Effects of sex steroids on risk of autoimmunity vs. immune-mediated injury . . . . . . . . . . . . . . . . . . . . . . . . . . . A505

4. Progesterone — mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A505 5. Effects of progesterone on immune functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A506

5.1. Pregnancy-associated changes in the maternal immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A506 5.2. Progesterone birth control and risk of sexually acquired HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A506 5.3. Differential immunomodulation by progesterone and estrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A506 5.4. Progesterone effects on immune system components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A506

5.4.1. Complement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A506 5.4.2. Neutrophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A507 5.4.3. Eosinophils, mast cells and basophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A507 5.4.4. Natural killer (NK) cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A507 5.4.5. Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A507 5.4.6. Myeloid dendritic cells (mDCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A507 5.4.7. Plasmacytoid DCs (pDCs) and IFN-α . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A507 5.4.8. CD4+ T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A508 5.4.9. CD8+ T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A508

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A503G.C. Hughes / Autoimmunity Reviews 11 (2012) A502–A514

5.4.10. Thymic involution during pregnancy and after puberty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A509 5.4.11. B cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A509

6. Progesterone and autoimmune disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A509 6.1. Systemic lupus erythematosus (SLE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A509 6.2. Rheumatoid arthritis (RA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A510 6.3. Multiple sclerosis (MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A510 6.4. Autoimmune thyroid disease (AITD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A511 6.5. Other female-predominant autoimmune diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A511

7. Summary and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A511 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A511 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A511

1. Introduction

At roughly the same time 500 million years ago, two major adap- tations arose in primitive vertebrates: the adaptive immune system and highly specific steroid hormone receptors [1,2]. The subsequent development of placental reproduction posed immunological “prob- lems” for the adaptive immune system, as Sir Peter Medawar, pioneer in transplantation immunology, put it in 1953 [3]. He posited that the fetus might avoid attack from its mother's immune system by means of physical barriers, immunological inertness and suppression of ma- ternal immune responses. As Medawar himself suspected [3], physi- cal separation of maternal and fetal circulations is critical, but so too are certain adaptations of the mother's immune system. Accordingly, the human immune system bears evidence of sexual selection. This is most obvious in the female predominance of certain autoimmune dis- eases (ADs) (Fig. 1), and to a lesser extent, sexual dimorphism in re- sponses to infections. Several theories attempt to explain the female predominance of ADs: immunomodulatory effects of sex hormones; sex-dependent genetic factors; microchimerism; and gender-related factors [4]. The importance of sex hormones, particularly the sex ste- roids, in human AD is supported by considerable observational and experimental evidence. In this regard, much attention has been given to estrogen (Es) and androgens, important determinants of sex- ual dimorphism, but far less has been given to progesterone (Pg), the hormone of pregnancy. Long known for its immunoregulatory prop- erties, Pg's impacts on human AD are not well understood. This re- view article will summarize what is known about Pg and AD.

Fig. 1. Female:male prevalence ratios for selected autoimmune and inflammatory disorders. a non-weighted manner from Beeson et al. [174], Whitacre [184], Cooper and Stroehla [175], data, reported risk associations with IRF5 or STAT4 genes are shown as either detected (+) o Institute GWAS Catalog [33]. SLE, systemic lupus erythematosus; ITP, immune (idiopathic) t

First, I will outline observations supporting a role for female sex steroids in AD, correlating them with recent genetic discoveries. Next, I will briefly discuss the molecular actions of Pg, followed by a more detailed description of how Pg impacts functions of select com- ponents of the immune system. With this framework in place, I will discuss what is known about Pg, individual ADs and their animal models.

2. Progesterone and mammalian biology

Pg is a cholesterol-derived hormone critical to pregnancy [5]. In- deed, the name ultimately derives from the prefix pro and the Latin gestāre to carry. Through a variety of mechanisms, Pg is required for normal function at multiple stages of mammalian reproduction: oo- cyte maturation, differentiation of the endometrium, implantation of the embryo, growth of the placenta, quiescence of uterine muscle during fetal development and differentiation of mammary gland tis- sues. Pg also contributes to some of the changes in maternal physiol- ogy and metabolism during normal pregnancy. [5] More recently it has been appreciated that Pg actions in the brain also regulate repro- ductive behaviors and reparative responses to central nervous system (CNS) injury [6,7]. As will be discussed in Section 5, fluctuations in en- dogenous Pg during the ovulatory cycle and pregnancy are associated with reversible changes in a woman's immune system. How these changes contribute to successful pregnancy or risk of AD is not well understood.

Shown are the means and ranges of female:male ratios (log10 transformed) compiled in McCombe et al. [4] and Jørgensen et al. [157] For those disorders with published GWAS r not detected (−). GWAS data extracted from the National Human Genome Research

hrombocytopenic purpura; CIDP, chronic inflammatory demyelinating polyneuropathy.

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3. Sex steroids and the risk of AD

3.1. Sexual dimorphism in human AD

The female predominance of AD remains unexplained, but several non-competing hypotheses have been proposed. The first idea is that female sex hormones modulate the immune system to increase risk of AD in genetically susceptible individuals; the converse is that male sex steroids are protective. While this may be the most intuitive explana- tion, it is not the only one. X-linked immune genes, particularly if they escape inactivation or are duplicated [8], could play a major role in female predominance of AD, though convincing evidence that this is a common factor in female-predominant forms of AD is currently lacking. Microchimerism, the persistence of allogeneic cells acquired by the mother or the fetus during pregnancy, may contribute to loss of tolerance in certain female-predominant ADs, like systemic sclerosis (scleroderma) (SSc) [9]. Gender – those behavioral, cultural or psychological factors tightly linked to one's sex – likely play a role in some ADs, especially those where exposure to exogenous triggers or protective factors are determined by gender-related behavior. For example, female predominance of multiple sclerosis (MS) may in part reflect gender differences in protective exposure to sunlight [10]. Finally, given an increasing appreciation that microbial environ- ments influence adaptive immunity and autoimmunity [11], it may be that factors unique to vaginal microbial environments contribute to female-predominance of certain ADs — a possibility that remains largely unexplored. These various hypotheses are well summarized by McCombe, Greer and Mackay [4]. The following section will high- light evidence supporting a major role for female sex hormones in AD, particularly that for Pg and Es.

3.2. Sex hormones and risk of human AD

AD can strike both children and adults. Most often, pediatric AD is defined as having onset before age 16–18. This is not necessarily a bio- logical cutoff, because puberty onset is around age 10, at least in the U.S. [12], and for diseases like rheumatoid arthritis (RA), SLE and SSc, the distribution of age of onset is uni-modal, not bimodal [13,14]. This arti- ficial segregation of pediatric and adult AD has tended to obscure an im- portant relationship between sex hormones and the incidence of AD: i.e., that extreme female predominance occurs mainly after puberty. In SLE, for example, the overall female:male ratio is close to 9:1 (Fig. 1), but in pre-pubertal SLE, the ratio is around 3–4:1 [15,16]; and in SLE pa- tients presenting under the age of 5, it is essentially 1:1 [17]. A strikingly similar pattern is seen in autoimmune thyroid disease (AITD) [18], an- other female predominant group of ADs (Fig. 1, Hashimoto's thyroiditis and Grave's disease). Pediatric Sjögren syndrome (SS) appears to be quite rare [19], so analyses of sex ratios in pre-pubertal vs. pubertal/ post-pubertal onset cases are not readily available. Pediatric onset pri- mary biliary cirrhosis (PBC) is exceedingly rare [20], and pre-pubertal cases have apparently not been reported. Interestingly, in atopic disease and asthma, the male predominance in pre-pubertal disease is reversed after puberty [21], suggesting that sex hormones can modulate inci- dence other forms of immune-mediated disease. Finally, the sole AD in Fig. 1 with mean age of onset before puberty, type 1 diabetes mellitus (T1D), shows no female predominance. Overall, these observations in- dicate female reproductive factors such as sex steroids are important determinants of risk for female-predominant ADs. This hypothesis is supported by considerable evidence in human and animal studies, dis- cussed in Sections 5 and 6. This body of literature was recently reviewed by González et al. [22] and Martocchia et al. [23]

3.3. Sex hormones and genetic risk of AD

In general, human ADs demonstrate polygenic patterns of inheri- tance. A prevailing model holds that a person's overall risk for any

given AD reflects the sum of genetic, environmental and stochastic factors. Environmental factors could include both endogenous trig- gers (e.g., endogenous sex hormones, endogenous retroviruses) and external exposures (e.g., toxins, UV light, exogenous hormones, infec- tions). Over the last several years, genome wide association studies (GWAS) have identified numerous genetic variants associated with risk of individual ADs. These variants are common (>10% prevalence) and tend to confer low risk (e.g., odds ratios [ORs] 1.1–2.0). Candidate gene studies have identified rarer (b1% prevalence) genetic variants, particularly loss-of-function mutations and gene copy number vari- ants, associated with much higher ORs. [24–26] An individual's genet- ic risk burden for any given AD varies widely. Theoretically, someone with extremely high genetic risk burden might develop AD early in life, because fewer environmental or stochastic triggers are needed to reach critical risk burden, above which development of AD is es- sentially guaranteed. In this setting, sex steroids and other environ- mental triggers are unnecessary. Conversely, a person with relatively low genetic risk burden may present later in life, only after accrual of sufficient triggers (here, hormones) and stochastic events, if at all. Again, SLE provides a good example. The strongest known genetic risk factor for SLE is complete deficiency in the com- plement factor C1q [24]. Over 90% of individuals with this rare homo- zygous mutation will develop SLE [27]. The age of onset in C1q- deficient children is frequently before age 5 [27], and the female: male ratio is close to 1 [28] — consistent with the idea that extreme genetic risk obviates the need for additional stochastic or hormonal factors. These and other observations have prompted the hypothesis that some very early onset ADs are largely genetic diseases [29]. What about adult-onset SLE in males? If female sex hormones are im- portant environmental risk factors, and male sex hormones like tes- tosterone (T) are protective (see Section 6), this model would predict that in order to develop SLE, men would require a higher ge- netic burden than women. An initial report [30] from a large adult- onset SLE cohort indicates this is likely the case — men had signifi- cantly higher frequency of SLE risk loci than women. It is reasonable to hypothesize that similar studies comparing pediatric-onset SLE to adult-onset SLE would reveal higher genetic risk burden in pediatric-onset disease.

Can recent genetic advances inform our understanding of the fe- male bias of certain ADs and how sex steroids might be involved? We already know that the most female-predominant ADs share cer- tain phenotypes. At least 5 of the top 6 ADs shown in Fig. 1 are strong- ly associated with anti-nuclear Abs (ANA): SS, SLE, Hashimoto's thyroiditis (HT), PBC, Grave's disease (GD), and SSc [31]. This suggests that sex steroids impact tolerance to ubiquitous antigens (Ags) such as those contained in dead and dying cells. Another aspect that links several of them (SS, HT, PBC, SSc) is evidence for the pathogenic in- volvement of the anti-viral cytokine interferon-alpha (IFN-α), whose production and signaling appears to be under hormonal regu- lation (Section 5.4.7). Consistent with this idea, genetic variation of the IRF5 gene, whose protein product is integral to IFN-α production [32], is reported in several GWAS studies to be associated with risk of SLE, PBC, SSc and RA, but not any of other ADs for which there are published GWAS data [33], except ulcerative colitis (UC) [32,33] (Fig. 1). A similar if not more compelling association can be made be- tween the STAT4 gene and the most female-predominant ADs. STAT4 is an IFN-α-inducible intracellular signaling molecule important for Th1- and Th17-related responses, and cell-mediated immunity [34]. In published GWAS studies, STAT4 is reported to be associated with risk of SLE, PBC, SSc and RA, but none of the other ADs (Fig. 1) [33]. That a genetic association of both IRF5 and STAT4 is reported only among the most female predominant diseases suggests IFN-α and Th1/Th17 pathways could be important nodes where hormonal ac- tion and genetic risk interact. If true, then IRF5 and STAT4 may even- tually prove to be risk genes for AITD and SS, but not other ADs lacking strong female predominance, like pemphigoid or ITP. Indeed,

Table 1 Reported effects of progesterone and synthetic progestins on functions of individual im- mune cell types. Results from human cells underlined. All effects are direct and involve Pg unless otherwise noted in parentheses. When Pg receptor usage for given effect is known, that receptor is shown in parentheses. Evidence supporting iPR or mPR protein ex- pression in immune cell types indicated by letter: H, human; m, mouse; r, rat. Blank spaces indicate lack of data. Receptor expression data based on Gilliver et al. [52] See Section 5 for specific citations.

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candidate gene studies link variations in both IRF5 and STAT4 to risk of SS [8]. Another prediction is that risk variants should result in changes in gene function concordant or synergistic with effects of fe- male sex hormones, or antagonistic with those of androgens. Finally, differences in any gene's risk association in male vs. female adult- onset AD could reflect pathways where male vs. female sex hormones enhance or counteract genetic risk. For example, in genetic risk stud- ies comparing male and female SLE, risk alleles in three IFN-α path- ways genes, TLR7 [35], IRF5 [30] and SPP1 [36], show significantly higher ORs in men vs. women. One explanation for the enrichment of these variants in male SLE is that in order for a man to develop SLE, more dysregulation of the IFN-α pathway is needed to overcome the protective effects of androgens, or to compensate for lack aug- mentation conferred by female sex steroids. Clearly this is an over- simplification, but it illustrates one way sex-specific genetic analysis might be used to inform future investigation into the hormonal basis of sex bias in human AD. More sex-specific genetic risk analyses need to be performed in human ADs.

3.4. Effects of sex steroids on risk of autoimmunity vs. immune-mediated injury

It is essential to appreciate that the biological processes leading to autoimmunity, i.e., generation of auto-reactive lymphocytes, are not necessarily the same as those mediating immune injury and organ damage. Autoimmunity does not necessarily mean autoimmune dis- ease. For example, disease-specific anti-thyroid Abs, e.g., anti- thyroperoxidase Abs [37], are frequently found in people without clinically evident thyroid dysfunction. Strikingly, RA- and SLE- specific autoAbs appear in the blood years before diagnosis [38,39]. Moreover, results from GWAS studies in SLE [40] and genetic analysis of lupus-prone mice [41] together demonstrate that loci associated with development of autoimmunity are different from those associat- ed with kidney damage. Therefore, the effects of sex steroids on risk of AD may be different than effects on immune-mediated injury (i.e., disease activity). RA is a useful example in this regard. The strong female predominance of RA (Fig. 1) suggests female sex steroids may enhance risk of disease in genetically predisposed individuals. Yet RA often remits during pregnancy [42], when Es and Pg are extremely elevated systemically [5]. The same is not true, however, for all female-predominant ADs, as will be discussed in Section 6. Thus, when talking about the impact of female sex steroids on AD, it is essen- tial to distinguish effects on risk from those on immune-mediated injury.

4. Progesterone — mechanisms of action

Steroid hormones exert their influence on physiological processes via direct effects on cellular functions. Sex steroids are synthesized primarily in the gonads, adrenal glands and, during pregnancy, the placenta. They are delivered via the bloodstream, largely protein- bound, to target tissues. Steroid hormones signal via highly specific receptors found in the plasma membrane, cytosol and nucleus of cells. [5]

Pg utilizes both cell surface and intracellular receptors. Best char- acterized are intracellular Pg receptors (iPRs). iPRs are ligand- sensitive transcription factors of the nuclear receptor superfamily most closely related to glucocorticoid receptors (GRs) and mineralo- corticoid receptors [1]. The expression, functions and regulation of iPRs in female reproductive tissues (and their related cancers) are well characterized and underlie the use of synthetic progestins and anti-progestins in modern gynecology and obstetrics. Nevertheless, it remains controversial whether steroid hormones enter target cells via passive diffusion or active transport. Within the last decade, a group of membrane-associated molecules was found to act as highly specific Pg receptors [43]. Structurally unrelated to iPRs, these

membrane Pg receptors (mPRs: mPR-α, mPR-β, mPR-γ) are linked to inhibitory G-coupled proteins, potentially explaining some of Pg's rapid non-transcriptional actions [44]. The functions of mPRs in re- productive and non-reproductive tissues are just now being uncov- ered [45]. A putative membrane-associated Pg receptor, Pg receptor membrane component 1 (PGRMC1), binds Pg but also to many other molecules. PGRMC1's functions are largely unknown. [44] Pg has also been shown to alter cellular functions through specific inter- actions with plasma membrane ion channel proteins.

Pg binds iPRs and mPRs with half-maximal inhibitory concentra- tions around 7 nM and 90 nM, respectively [43,44]. In addition, Pg at high-physiologic and pharmacologic concentrations (>200 nM) binds GRs [46], which are highly expressed in most immune cell types [47]. Synthetic progestins show unique patterns of Pg receptor usage. For ex- ample, medroxyprogesterone acetate (MPA), one of the commonest forms of hormonal birth control world-wide [48,49], is both an iPR and GR agonist [50]; its binding to mPRs has not yet been characterized. Mifepristone (RU-486), a widely used abortifacient and emergency con- traceptive, is an iPR and GR antagonist [51] but does not bind mPRs with significant affinity [44]. Conversely, several synthetic molecules can bind with high affinity to mPRs but not to iPRs [43,44]. For iPR signaling, an additional layer of complexity arises from the fact that iPR's tran- scriptional activity varies depending on the ligand activating it. Thus, the immunomodulatory effects of various progestins depend on their concentration and their receptor usage.

What are the patterns of Pg receptor expression in tissues of the im- mune system? This question has not been addressed in a systematic fashion, and thus we must rely on a collection of studies employing dis- parate methods. Still, some important patterns emerge. The expression of various Pg receptors in some immune cell subsets (Table 1) strongly supports the idea that progestins can act directly on immune cells to

A506 G.C. Hughes / Autoimmunity Reviews 11 (2012) A502–A514

modulate their functions. iPR expression in human immune cells ap- pears to be restricted to certain subsets like peripheral natural killer (NK) cells and tissue macrophages [52]. We and others have failed to detect either iPR protein or PGR mRNA in human total peripheral blood mononuclear cells (PBMCs), CD4+ T cells, CD8+ T cells, or plas- macytoid dendritic cells (pDCs) [52,53] (and unpublished results). The expression and functions of mPRs in immune cells remain largely unex- plored. Nor is it known, as it is for reproductive tissues, if expression of these receptors in immune tissues is regulated by sex steroids. Answers to these basic questions are currently lacking but are essential for full understanding of Pg's immunomodulatory actions during physiologic states when both Pg and Es are elevated, such as pregnancy, ovulatory cycle and treatment with combined oral contraceptive pills (OCPs) or hormone replacement therapy (HRT). Finally, it is important to note that Pg may impact human AD by acting directly on tissues of immune attack, e.g. the brain, or indirectly on immune cells via paracrine or en- docrine actions of non-hematopoietic tissues bearing Pg receptors, e.g., thymic stromal cells [54].

5. Effects of progesterone on immune functions

Four general lines of evidence together indicate that Pg modulates immune functions in vivo: 1) post-pubertal female predominance of ADs; 2) systemic immunomodulation during times of high circulating Pg (pregnancy, luteal phase of the ovulatory cycle); 3) immunomo- dulation after Pg treatment; and 4) in vitro experiments showing di- rect effects on immune cells. In this section, I will discuss these last three lines of evidence, reserving the first for Section 6, “Progesterone and Autoimmune Disease.”

5.1. Pregnancy-associated changes in the maternal immune system

Successful human pregnancy is associated with marked and re- versible changes in maternal immune functions. For comprehensive reviews on this subject, please see Poole and Claman [55] and Munoz-Suano et al. [56]. In the decades since Medawar's famous pos- tulate, it has become clear that the fetus is not immunologically inert; nor is the mother systemically immunosuppressed. Rather, pregnan- cy is a state of immunomodulation: intense at the maternal-fetal in- terface and more subtle systemically in the mother. Many of these changes are believed to be induced by Pg, in part because maternal serum Pg and estradiol (E2) levels rise 5- to 10-fold throughout ges- tation then normalize quickly post-partum. In mouse pregnancy, Pg is produced by the ovaries, but in human pregnancy, most of the Pg is synthesized in the placenta; tissue levels can be 10–100 times higher than those in the maternal circulation, high enough to engage GRs. [5] In the uterine lining where the (fetal) placenta invades, called the de- cidua, there is marked suppression of cytotoxicity [56], infiltration by specialized uterine natural killer (NK) cells [57], blockade of fetal an- tigen (Ag) delivery to maternal lymph nodes (LNs) [58], and accumu- lation of fetus-specific regulatory T cells (Tregs) [59]. Some of these effects likely involve induction by Pg at the maternal–fetal interface of soluble immunomodulatory molecules like Pg-induced blocking factor (PIBF) [60] and glycodelin A (GdA) [61]. It is important to re- member that the fetal and maternal circulations do not intermingle but are kept separate by a highly specialized interface that allows se- lective exchange of circulating molecules, and in some cases, cells. Nevertheless, several important pathogens like Toxoplasma, cytomeg- alovirus (CMV), Plasmodium (malaria), and HIV are able to breach if not exploit this interface to cause serious congenital infection in the fetus [62].

Although pregnancy is not thought to cause general immunosup- pression in the mother, certain pathways may be targeted for modu- lation. Complement (C) protein levels and activation are increased (though C activation at the maternal–fetal interface is strongly sup- pressed), and there are increased levels of circulating levels of anti-

inflammatory molecules IL-1 receptor antagonist (IL-RA) and soluble TNF-α receptor (TNF-R), along with decreased IL-1β and TNF-α. Cir- culating maternal NK cell functions are diminished as is the capacity for T cell proliferation. This is accompanied by decreased numbers of IFN-γ-producing CD4+ T cells and alterations in numbers of FoxP3-expression regulatory T cells (Tregs). [55,56,63,64] Small changes have also been reported in serum IgM, IgG, IgE and IgA levels, but more important may be pregnancy-associated functional changes in circulating immunoglobulin (Ig) resulting from altered glycosyla- tion [65]. Strikingly, mammalian pregnancy is associated with tran- sient reduction in cellularity of both the thymus [66] (Section 5.4.10) and the bone marrow [67], although an impact on immune functions has yet to be demonstrated. That some of these phenomena can be induced by maternal serum/plasma factors, Pg or E2 (Section 5.4) strongly supports the idea that circulating sex ste- roids modulate systemic maternal immune functions during pregnancy.

5.2. Progesterone birth control and risk of sexually acquired HIV

The use of a particular injection Pg birth control, depot medroxy- progesterone acetate (DMPA), appears to significantly increase a woman's risk of sexually acquired HIV-1 infection [48]. This is of major concern because over 50 million women use injection Pg birth control, mainly DMPA [49], increasingly in Sub-Saharan Africa where HIV is pandemic [48]. DMPA may increase risk of HIV infec- tions through altered mucosal defenses, suppression of anti-viral IFN-α, and suppression of CD8+ T cell functions [48,68]. Because of selective Pg receptor and GR usage, Pg and MPA may have very differ- ent effects on anti-viral immune pathways and AD (see Sections 5.4.7, 5.4.9 and 6.1).

5.3. Differential immunomodulation by progesterone and estrogen

It is important to understand that Pg and Es, the chief female sex steroids, do not necessarily modulate immune functions in the same way. In fact, there are many examples where the effects of Pg and Es oppose one another: Uterine inflammation [69], Th1-related auto- immune processes [70], dendritic cell (DC) activation [71] and B cell differentiation [72] are just a few examples. This fact should be borne in mind when interpreting immunomodulation during states when both hormones are increased simultaneously, such as the ovu- latory cycle, pregnancy and treatment with combined OCPs or HRT. Moreover, the effects of Pg and Es in immune organs and cells may be interdependent, as is the case in female reproductive tissues.

5.4. Progesterone effects on immune system components

This section summarizes what is known about the effects of Pg on select components of innate and adaptive immunity, highlighting the most recent discoveries and those pertinent to AD. An overview is provided in Table 1. For a more comprehensive discussion on the sub- ject of female sex steroids and immune functions, see the recent re- view article by Muñoz-Cruz et al. [73].

5.4.1. Complement Little is known about the effects of Pg on C functions. Maternal cir-

culating C protein levels [74] and C system activity [75] rise signifi- cantly with serum Es and Pg levels during normal human pregnancy, and this effect is mirrored after use of combined HRT in women [76], but the relationship of these phenomena with autoim- mune processes of C activation, e.g. SLE and antiphospholipid syn- drome (APS), is not clear. Given the likely involvement of C activation in APS [77], however, it is reasonable to speculate that hor- monal effects on C activation could synergize with anti-phospholipid antibodies (Abs) to increase the risk of APS-associated thrombotic

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disease in pregnancy (preeclampsia, fetal loss, premature delivery) or after OCP/HRT use. The relationships between pregnancy-related C activation and SLE disease activity remain to be studied.

5.4.2. Neutrophils Very little is known about the effects of Pg on neutrophil func-

tions. In human neutrophils, Pg can suppress both superoxide release and apoptosis and antagonize enhancement of chemotaxis by Es [73,78]. These effects could relate to Pg's ability to suppress end- organ damage in models of SLE, MS and RA (Section 6). An unex- plored area of potential importance is how Pg affects release by neu- trophils of extracellular chromatin traps, which, in addition to bactericidal actions, may induce IFN-α in SLE [79] and tissue damage in anti-neutrophil cytoplasmic Ab (ANCA)-associated vasculitis syn- dromes [80].

5.4.3. Eosinophils, mast cells and basophils Sex steroid regulation of atopy and asthma is suggested by re-

versed sex ratio after puberty, exacerbation (and sometimes im- provement) of these conditions during pregnancy or use of exogenous Pg [81], and expression of iPRs in mast cells (Table 1). Pg, in combination with Es, enhances human eosinophil degranula- tion [82], an effect inducible by Pg alone in mouse eosinophils [83]. Pg also appears to induce uterine infiltration by mast cells during pregnancy, at least in mice [84], but whether this is true of mucosal tissues involved in atopy and asthma is not clear. Pg also can enhance Th2-related Ab responses that are associated with generation of IgE in atopy (see Section 5.4.8). Accordingly, it will be important to study how Pg impacts functions of basophils, which can present Ag and drive Th2 responses in mice [85].

5.4.4. Natural killer (NK) cells NK cells are lymphocytes involved in innate anti-viral defense,

tumor surveillance [86] and differentiation of the endometrial decid- ua [87]. Pregnancy and hormonal changes of the ovulatory cycle are associated with altered endometrial and peripheral NK cell numbers and functions [56,73,78,87]. In vitro studies indicate Pg may both di- rectly and indirectly inhibit NK cell functions. High levels of Pg pro- duced by the human placenta likely induce two factors at the maternal–fetal interface that inhibit NK cytolytic activity and IFN-γ secretion: major histocompatibility complex, class I, G (HLA-G) [88] and PIBF, a soluble factor released by uterine lymphocytes [89]. Re- cently, it was shown that Pg can directly induce apoptosis and sup- press IFN-γ production in human peripheral blood NK cells in vitro, possibly via iPRs [90]. Substantiating a requirement for iPR in this re- gard would identify a possible immunologic mechanism linking the finding that certain iPR gene polymorphisms are associated with re- current spontaneous abortion and premature delivery and elevated serum IFN-γ levels in these conditions [91,92]. Regulation of NK func- tions by Pg in AD is essentially unexplored but deserves more atten- tion given increasing appreciation of a role for NK cells in AD.

5.4.5. Macrophages Macrophages (MPs) are tissue-resident phagocytes that have im-

portant roles in inflammation, tissue repair and innate and adaptive immune responses [86]. Sex-dependent differences in MP functions may contribute to sexual dimorphism in atherosclerosis, wound heal- ing and bone metabolism [52]. MP numbers and functions in the human endometrium appear to be under hormonal control during pregnancy [93] and the ovulatory cycle [94]. In vitro, Pg has largely inhibitory effects on MP functions, including suppression of lipopoly- saccharide (LPS)-induced nitric oxide synthase, TNF-α and release of pro-thrombotic microparticles [73,95,96]. Treatment of rodents with MPA resulted in in down-regulation of FcγR expression on spleen MPs and decreased clearance of IgG-coated sheep red blood cells in vivo, the opposite effect of Es treatment [97,98]. Differential

regulation of FcγR functions by MPA (or Pg) and Es could be one way these two hormones exert opposite effects on autoimmunity and kidney damage in a model of SLE (Section 6.1) or other immune complex (IC)-mediated ADs like RA or cryoglobulinemia. That iPRs mediated these Pg effects is at least suggested by evidence for the ex- pression of this receptor in MPs (Table 1). Direct Pg effects on synovi- al (joint) MPs in RA, particularly suppression of TNF-α or FcγR expression, could be an important mechanism of pregnancy- induced remission (Section 6.2). Finally, because dysregulated clear- ance of dead and dying cells by MPs likely contributes to loss of toler- ance against nuclear Ags [99], common among the most female- predominant ADs (Section 3.3), hormonal regulation of this pathway should be investigated.

5.4.6. Myeloid dendritic cells (mDCs) The prominent role of mDCs in innate defense against infection,

adaptive immunity and tolerance [86] suggests mDCs could be impor- tant targets of Pg in modulating AD. In 2007, we published a review of this topic [71], but several points deserve reiteration and refinement. In general, Pg inhibits toll-like receptor (TLR) activation of human and mouse mDCs (Table 1). Published in vitro experiments reveal that pregnancy-level Pg can significantly inhibit induction of TNF-α, IL-1β, IL-6, IL-12p40, CD80, CD86 and MHC II in TLR-activated mDCs of rat or mouse origin [71,73], possibly via iPRs [100]. In contrast, Pg can enhance IL-10 release by rodent [100] and human [101] mDCs. This finding is consistent with the idea that elevated Pg in pregnancy programs DCs to present Ag to CD4+ T cells in a manner that favors Treg and/or Th2 development but hinders that of Th1 cells [71] (Section 5.4.8).

Pg and synthetic progestins appear to regulate mDC functions in both signal-specific and gene-specific manners, indicating Pg controls select immune programs in mDCs. For example, we have observed that high physiologic doses of Pg (likely via GRs) significantly inhibit in human monocyte-derived mDCs release of TNF-α, IL-6, IL-12p70, and IL-10 after ligation of CD40 (by CD40L) but not after TLR4 (LPS) stimulus (unpublished results), suggesting Pg targets for suppression mDC functions related to adaptive immunity. Jones et al. [102] recent- ly showed that in mouse bone marrow-derived mDCs, a form of Pg highly selective for iPRs (norgestrel) could inhibit TLR3/Mda5- but not TLR4-activated up-regulation of CD40, indicating Pg may also se- lectively suppress pathways involved in CD4+ T cell responses after virus infection. Moreover, we showed that treatment of mice with MPA, an iPR/GR agonist, impaired in vivo up-regulation of a costimu- latory molecule CD86 on spleen mDCs after virus infection [68]. To- gether, these observations suggest endogenous Pg modulates select immune programs in mDCs related to anti-viral defense, Ag presenta- tion, and CD4+ T cell differentiation. Through differential engage- ment of steroid hormone receptors, synthetic progestins may vary widely in terms of these effects. Future studies should focus on how signals via iPR, mPR and GR differentially regulate TLR and non-TLR activation in mDCs, and how this relates to immunologic outcomes in pregnancy, T cell dependent immunity and AD.

5.4.7. Plasmacytoid DCs (pDCs) and IFN-α pDCs are circulating and lymphoid tissue-resident cells important

for anti-viral defense and in some forms of AD, most notably SLE. pDCs are poised, through accumulation of intracellular signaling mol- ecules, to release large amounts of IFN-α upon sensing viruses through TLRs 7, 8 and 9. [86,103] In SLE, autoAb-containing ICs acti- vate pDCs through these same pathways to induce IFN-α, which is believed to act on multiple immune cell types to break tolerance and amplify autoimmunity [103]. Despite their prominence in one of the most female ADs, SLE, very little is known about how female sex steroids regulate pDC functions. Two studies [104,105] indicate that blood pDCs from women produce more IFN-α than do those from men, but only after stimulation through TLR7 or TLR8, and not

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via TLR9. This difference does not appear to reflect either increased transcription of the X-linked TLR7 or TLR8 genes in female pDCs or Es effects [104]. Subsequently, Meier et al. [105] observed in blood of HIV-infected individuals a similar sex-dependent difference in per- centages of blood pDCs making IFN-α after TLR7/8 but not TLR9 stim- ulation. In the female subjects, serum Pg was significantly and positively correlated with IFN-α+ pDCs upon TLR7/8 stimulation. To- gether, these studies suggest Pg augments TLR7/8-induced IFN-α re- lease by pDCs in women, a potentially important mechanism by which Pg increases a woman's risk of SLE. Similarly, we have observed that physiologic levels of Pg (10−7 M) significantly enhanced IFN-α- induced expression of several immune genes in PBMCs from healthy premenopausal female donors, but not in those from male donors. In these experiments, Pg enhancement of IFN-α-induced gene ex- pression correlated positively with mPR-α gene (PAQR7) mRNA levels (unpublished results). Indeed, mPR-α expression in blood CD8+ T cells is increased in the luteal phase (high Pg+E2) compared with follicular phase (high E2) [53], together suggesting hormonal milieu in females may enhance downstream effects, and thus, patho- genicity, of SLE-related IFN-α. This may be one reason female pDCs make more IFN-α than do male pDCs, because exposure of pDCs to IFN-α potentiates their production of IFN-α. Overall, these data sug- gest endogenous Pg may increase risk of SLE through enhanced IFN- α production and signaling. How androgens like testosterone (T) im- pact these pathways remains to be determined, but enrichment of TLR7, IRF5 and SPP1 risk alleles in male SLE suggest important interac- tions between male sex factors and the IFN-α pathway (Section 3.3).

Modulatory effects on the IFN-α pathway seem to depend on the dose and form of progestin involved. We showed that high- physiologic -pharmacologic doses of Pg could inhibit TLR9-induced IFN-α production by human and mouse pDCs in vitro [68]. In vivo, these levels of Pg occur at the maternal–fetal interface, potentially explaining the fetus's susceptibility to infection by select viruses, e.g., HIV and HSV [62], against which pDCs may be innate sentinels. Treatment of mice with the synthetic progestin birth control, DMPA, resulted in selective impairment of TLR9-induced IFN-α production by pDCs ex vivo and serum IFN-α responses after virus infection [68]. This likely reflects MPA's ability to signal through GRs, since Pg inhibition of TLR9-induced IFN-α production by mouse spleen pDCs does not require iPRs (our unpublished results), and in human pDCs, GR agonists are potent inhibitors of TLR-induced IFN-α induc- tion [47]. These distinctions are clinically important. We and others have postulated that DMPA's ability to suppress this particular path- way helps explain why women using DMPA birth control are at in- creased risk of sexually acquired HIV infection (Section 5.2). By the same token, in women with SLE, DMPA may have dual benefit as an effective contraceptive and suppressor of pathogenic IFN-α and Th1-related autoimmunity.

5.4.8. CD4+ T cells CD4+ T cells are critical to development of long-lasting Ab re-

sponses, cell-mediated immunity (CMI) and tolerance [86]. They are also involved in chronic inflammation found in several ADs. A useful scheme classifies CD4+ T helper cells based on their effector func- tions: 1) Th1 cells produce IFN-γ and IL-2, important inducers of CMI and induction cytotoxic and inflammatory IgG subclasses be- lieved to be pathogenic in SLE and AITD; 2) Th2 cells produce IL-4 and IL-5 and are important inducers of neutralizing IgG subclasses and IgE; 3) Tregs suppress functions of other CD4+ T cells in part through release of IL-10 and TGF-β; 4) Th17 cells release IL-17 and IL-21 and are important in chronic inflammation during chronic in- fections and ADs like RA and MS. [86,106] Overall, in vivo and in vitro evidence indicates Pg has some capacity to suppress CD4+ T cell proliferation and Th1/Th17 differentiation and effector functions. In contrast, Pg can enhance Th2 and Treg differentiation (Table 1) [59,73,107]. These effects are prominent at the maternal–fetal

interface, where Pg levels are extremely elevated, but appear much weaker or are absent in the mother's circulating CD4+ T cells (reviewed in [59]). In vitro, high-physiologic concentrations of Pg, but not Es, are able to enhance IL-4 secretion by human CD4+ T cell clones [108,109], suggesting one way Pg might favor Th2 memory re- sponses in pregnancy. One report [110] indicates pregnancy-level Pg can inhibit differentiation of human Th1 cells, driving them toward an IL-10-secreting Th2-like phenotype. In contrast, E2 treatment in mice primes naïve CD4+ T cells towards Th1 differentiation through transcriptional promotion of the T-bet gene [111]. Interestingly, PIBF can suppress activation of STAT4 protein in human lymphocytes [112], while E2 has the opposite effect in mouse splenocytes [113]. Together, these results suggest that Th1 or Th17 differentiation may be an important pathway on which Es and Pg differentially influence risk of certain ADs associated with STAT4 (Fig. 1) (see Section 6).

Maternal Tregs are required for maintenance of allogeneic preg- nancy in mice [114], but it remains to be determined how fluctuations in maternal circulating Treg numbers during the ovulatory cycle and human pregnancy relate to fertility, and to what extent Pg is involved [56,59]. However, a recent study [115] showed that high-physiologic doses of Pg could directly enhance differentiation of neonatal (but not adult) naive CD4+ T cells into functional Foxp3-expressing Tregs while suppressing their differentiation into Th17 cells. Together, these observations indicate that high levels of Pg can alter CD4+ T cell differentiation and effector functions, generally enhancing those of Th2 and suppressing those of Th1/Th17 cells. These processes could play important roles in the remission of RA and MS during pregnancy (Section 6).

Finally, we have observed that iPRs selectively suppress CD4+ T cell-dependent immune responses in vivo. Mice lacking functional iPRs (PRKO mice) [116] show exaggerated Ag-specific IgM and IgG re- sponses after immunization with T-dependent (TD) but not T- independent (TI) Ags (unpublished results), indicating that iPRs (and probably endogenous Pg) are selective suppressors of CD4+ T cell functions and adaptive immunity. Further experiments defining the cellular and molecular mechanisms by which Pg modulates CD4+ T cell responses are critical to understanding modulation of Th1, Th2, and Th17 responses during pregnancy and the potential therapeutic effects of natural and synthetic progestins in ADs involv- ing dysregulation of these pathways.

5.4.9. CD8+ T cells CD8+ T cells are cytotoxic T cells important for recognizing and

killing virally infected or neoplastic cells [86]. CD8+ T cells also have regulatory functions and are aberrantly activated in several ADs [117]. Some reports indicate CD8+ T cell functions are under the control of Pg and/or Es. White et al. [118] found that the cytolytic activity of CD3+CD8+ T cells in the uterine lining of women in the se- cretory phase of the menstrual cycle (Pg+E2 effects) was greatly di- minished compared to that in the proliferative phase (E2 dominant), suggesting Pg and E2 together suppress cytotoxicity. Pg effects in this setting might be direct, via mPR-α, because this receptor's expression is selectively upregulated on blood CD8+ T cells during the luteal phase [53]. However, Pg may also regulate uterine CD8+ T cell indi- rectly, via induction in the E2-primed endometrium of GdA, a potent inhibitor of CD8+ T cell cytotoxicity [119]. Pg effects on CD8+ T cells (and/or CD8+ DCs) appear to be important in preventing stress- induced abortions in mice [120]. One idea is that uterine CD8+ T cells, when exposed to Pg, elaborate immunoregulatory molecules like indolamine 2,3-dioxegenase (IDO) or PIBF, involved in Treg in- duction and NK cell suppression, respectively [120]. These Pg- induced molecules, or Pg receptors expressed in CD8+ T cells, may prove to be therapeutic targets for the prevention of recurrent mis- carriage and premature delivery, a major health issue [121]. The abil- ity of MPA to suppress virus-specific IFN-γ and lytic granule release

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by CD8+ T cells [122] may be another mechanism by which DMPA in- creases a woman's risk of HIV infection (Section 5.2).

5.4.10. Thymic involution during pregnancy and after puberty The thymus is the organ where T cells develop. Beginning with pu-

berty, the thymus undergoes a gradual decline in cellularity (thymic involution) that likely involves testosterone effects in males and com- bined estrogen and progesterone effects in women. Marked and tran- sient thymic involution also occurs during mammalian pregnancy. [66] While thymic involution during pregnancy does not result in re- duced numbers of circulating T cells, it appears to be required for nor- mal fertility, at least in mice [54]. The mechanisms linking thymic involution and fertility are unknown, but one hypothesis is that the processes associated with thymic involution are important for induc- tion of maternal T cell tolerance to fetal alloAgs, perhaps via induction of natural Tregs. Importantly, thymic involution of pregnancy in mice can be mimicked by administration of pregnancy-level Pg and/or Es; and this process requires iPR expression in thymic stromal cells [54]. Thus, Pg, through paracrine actions of iPR-bearing cells, is a neg- ative regulator of T cell development. The interplay between male and female sex steroids, thymic functions and risk of AD, particularly after puberty, remains to be explored.

5.4.11. B cells B cells are Ab-secreting and Ag-presenting cells critical for humor-

al immunity and autoimmunity [86]. Hormone effects on B cell differ- entiation and function are at least suggested by the fact that women have modestly higher serum levels of IgM and IgG than do men [73,78]. In mice, pregnancy reversibly suppresses B cell development in the bone marrow, a phenomenon likely involving direct E2 action on B cell precursors [67], and its potentiation by Pg [123]. How these phenomena contribute to the immune requirements of preg- nancy remains to be determined.

Surprisingly few studies have examined the effects of Pg on hu- moral immune responses. Vermeulen et al. [124] showed that after treatment with pharmacologic doses of MPA, mice exhibited in- creased primary and secondary Ab responses against sheep red blood cells (TD Ab responses); iPRs were detected in B cells, but GR agonism by MPA was not excluded as a mechanism. Indeed, our re- sults with iPRKO mice indicate iPRs are suppressors of TD Ab re- sponses, suggesting MPA modulates TD Ab responses via other receptors, i.e., GRs or mPRs. Consistent with this idea, two recent re- ports [72,125] indicate Pg, via iPRs, and Es, via Es receptors, exert op- posing effects on mouse B cell differentiation via competing transcriptional regulation of a gene whose product, activation- induced deaminase (AID), is required for B cell class switch recombi- nation (CSR) and somatic hypermutation (SHM). Es enhances CSR and SHM [72], while Pg suppresses them [125]. These observations further suggest that the relative effects of Pg vs. Es may be important determinants of B cell differentiation in immunity and autoimmunity. Finally, Pg may induce important post-translational modifications of secreted Ig molecules that alter their avidity and effector functions [126]. In RA, serum IgG shows abnormally low levels of terminal ga- lactose residues [127], increases in which are associated with success- ful pregnancy and RA disease remission during pregnancy [128]. In vitro, Pg can induce some of these pregnancy-related Ig glycosylation patterns [65]. Thus, pregnancy-induced remission of RA may rely in part on Pg-mediated changes in Ig glycosylation. Interestingly, a re- cent study [129] showed that terminal galactose residues on glyco- proteins are natural suppressors of pDC IFN-α production via ligation of the human pDC-specific molecule BDCA-2. Together, these observations suggest that Pg, via iPRs, may suppress pathways of B cell differentiation involved in many ADs, and that Pg may mod- ulate immune-mediated injury in AD through post-translational modification of Ig. MPA may have different effects on these processes. Clearly, much more research is needed to clarify the impact of Pg and

synthetic progestins on B cell differentiation and functions in human AD.

6. Progesterone and autoimmune disease

As summarized in the previous section, Pg can impact many im- mune pathways involved in AD. This section will examine what is known about Pg and the female-predominant ADs, drawing on the previous sections to highlight possible mechanisms and future lines of investigation. For general review of the topic of female sex steroids and AD, see McCombe et al. [4] and Matocchia et al. [23]

6.1. Systemic lupus erythematosus (SLE)

While the relationships between Es and SLE autoimmunity have been relatively well studied, much less is known about Pg in this re- gard. Several observations indicate female sex steroids increase risk of SLE. As mentioned in Section 3, incidence and female predomi- nance of SLE rise after puberty. Moreover, risk of SLE is associated with early menarche (i.e., increased exposure to endogenous sex ste- roids) [130]. Two large observational studies [130,131] indicate use of exogenous Es in the form of OCPs or HRT is associated with an ap- proximately 1.5- to 3.0-fold increased risk of SLE. In contrast, one unpublished study [132] suggests use of Pg-only contraception pro- tects against incident SLE, but this has not been replicated. Thus, Pg and Es appear to modulate risk of SLE in different ways. Interestingly, women with SLE show luteal phase deficiency in serum Pg [133], al- though it is unknown whether this precedes illness or is a conse- quence of it. However, a causal link between abnormal sex steroid signaling and risk of SLE is suggested by the discovery of the SLE risk gene UBE2L3 [33], whose protein product is a co-activator of iPRs and other sex steroid receptors [134]. Accordingly, it will be in- formative to assess if UBE2L3 risk variants result in altered Pg sensi- tivity in terms of its effects on IFN-α production or sensitivity (Section 5.4.7).

The model that Pg and Es have different effects on the develop- ment of SLE autoimmunity is further supported by animal studies. In the NZB×NZW F1 (NZB/W) model of SLE, multiple risk loci from both parental strains combine to result in spontaneous lupus-like dis- ease, earlier and more severe in female mice compared to male mice. In the NZB/W strain, early mortality is tightly linked to emergence of class-switched Th1-related IgG2a anti-DNA Abs and immune complex (IC) glomerulonephritis. [135,136] A series of incisive experiments by Talal et al. (reviewed in [136]) clearly showed that autoimmune dis- ease in NZB/W mice was under gonadal and thymic control. In these experiments, pre-pubertal castration of male mice led to anti-DNA IgG and mortality similar to those of intact females, and this could be prevented with continuous T exposure. Thus, T has a dominant protective effect in this model. Interestingly, increased autoAb levels after castration of male NZB/W mice could be prevented by neonatal thymectomy, suggesting that protective effects of T might involve modulation of thymic functions (see 5.4.10). In contrast to male mice, pre-pubertal ovariectomy in female NZB/W mice had little ef- fect on survival, indicating ovarian factors (e.g., Pg and E2) were not required for full expression of disease. This is consistent with the idea, outlined in Section 3.3, that when genetic risk burden is high, as it is in NZB/W mice, female sex steroids become a subordinate risk factor. In the NZB/W model, E2 treatment invariably increases mortality, a process that likely involves a combination of enhanced Th1-related autoimmune responses [71] and induction of lymphoid neoplasia [137], possibly through oncogenic activation of AID in B cells [72].

Studies assessing Pg treatment in the NZB/W model have led to different results. Keisler et al. [137] showed that treatment of pre- morbid female NZB/W mice with any of three synthetic progestins (including MPA) had little to no effect on either survival or serum

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anti-DNA reactivity. However, Roubinian et al. [138] showed that continuous Pg treatment of pre-morbid female NZB/W mice whose ovaries had been removed modestly worsened survival, but markedly increased levels of serum anti-DNA IgG, far more than did E2 treat- ment. In castrated male NZB/W mice, Pg also markedly increased anti-DNA IgG but decreased mortality, suggesting an uncoupling of autoimmunity from end-organ damage, perhaps through suppression of FcγR expression in the kidneys or diversion of pathogenic Th1- related anti-DNA IgG2a towards protective IgG1 [106]. Consistent with the latter of these mechanisms, we recently showed that treat- ment of pre-morbid female NZB/W mice with continuous MPA resulted in markedly decreased death, kidney damage and selective suppression of pathogenic Th1-related anti-dsDNA IgG2a in the serum and kidneys [70]. How DMPA suppresses Th1 autoimmune re- sponses is not clear but could involve direct actions, likely via GRs, on the differentiation of autoreactive Th1 cells, suppression of Th1- related IL-12, IFN-α or CSR in autoreactive B cells. Together, these re- sults indicate Pg and MPA have different effects on the development of autoimmunity in this model, likely due to differential engagement of iPRs, mPRs, and GRs. The importance of these pathways could be sorted out by treating lupus mice with selective Pg receptor agonists and by introducing Pg receptor knockout mutations into models of SLE.

Compared with their impact on disease development, female sex steroids appear to have weaker effects on SLE disease activity. Nu- merous studies together indicate that pregnancy can increase the risk of disease “flare” [31,139]. However, interpretation of these data is confounded by inconsistent disease classification, inadequate measures of disease activity, and a tendency by patients and physi- cians to alter medical therapy during pregnancy. The most important predictor of SLE flare during pregnancy, however, is high disease ac- tivity in the 6 months before conception [31,140], indicating that the effects of pregnancy are subordinate. Do exogenous hormones af- fect SLE disease activity? Results of a recent meta-analysis [141] indi- cate that for women with mild or inactive SLE, use of combined (Es +progestin) OCPs poses no increased risk of flare. The same is true for post-menopausal women with SLE taking combined HRT [142]. Two recent studies examined the risk of flare of in women with SLE taking (synthetic) progestin-only HRT [143] or contraception [144] and found either no or reduced rates of flare, respectively. Indeed, DMPA may be able to suppress certain pathways of SLE immune- mediated injury, like IFN-α induction in pDCs and FcγR-signaling in MPs and Th1 differentiation (Table 1). These results together suggest that in women with SLE, synthetic forms of Pg-only contraception, particularly DMPA, are safer than those containing Es and may even have therapeutic effects.

Overall, observations in humans and animal models indicate that individually, Es and Pg have very different effects on SLE disease de- velopment: Es can promote it, and progestins in some situations can inhibit it. Future studies should compare how natural and synthetic progestins modulate SLE risk and immune-injury pathways. In terms of risk pathways, special attention might be given to those as- sociated with TLR7, IRF5, SPP1 and STAT4.

6.2. Rheumatoid arthritis (RA)

RA is a chronic inflammatory polyarthritis syndrome associated with serum rheumatoid factor (RF) and Abs recognizing post- translationally modified (citrullinated) peptides [145]. Little is known about the effects of Pg on risk of disease. A protective effect of parity (the number of pregnancies a woman has experienced) has been observed in some studies but not others [146]. In the largest retrospective studies, OCP use was not associated with risk of RA [147]. In animal models of RA, such as collagen-induced arthritis (CIA), pregnancy-level E2 treatment before immunization with colla- gen retards disease development in association with reduced anti-

collagen Abs, TNF-α, IFN-γ and prostaglandin E2 (PGE2) responses [148–150]. In rat CIA, pre-treatment with pregnancy-level Pg had lit- tle effect on joint swelling or serum TNF-α and prostaglandin E2 (PGE2), but appeared to reduce the beneficial effects of pregnancy- level Es treatment in terms of these parameters [148]. Interestingly, neither Pg nor Es treatment appeared to alter anti-collagen Ab re- sponses in these mice, suggesting hormones were not modulating au- toimmunity but rather acting primarily at the level of joint inflammation. Indeed, recent evidence [151] suggests that Es exerts most of its protective effects in CIA on the effector phase (immune in- jury) rather than induction of autoimmunity.

While use of low-dose exogenous HRT (usually Es+progestin) does not appear to impact disease activity in post-menopausal women with RA [142], pregnancy-level sex steroids appear to have pronounced effects. RA disease usually remits during pregnancy, pro- vided disease was active beforehand, then flares post-partum [42]. A similar pattern is seen in mouse models of arthritis [152,153]. Several Pg-inducible changes in systemic immune functions could be in- volved in this form of RA remission, including increased IL-1RA, in- duction of Tregs, suppression of Th1 or Th17 responses, increased Th2-related responses, and increased levels of terminal galactose res- idues on circulating Ig. A role for Th1-suppression/Th2-enhancement is supported by experiments [152] in a model pregnancy-induced re- mission of immune-mediated arthritis after infection with Borrelia burgdorferi (causative agent in human Lyme disease). Infection dur- ing pregnancy results in less severe arthritis, decreased spirochete- specific Th1-related responses (IFN-γ, IgG2a), and increased spirochete-specific IL-4; and these effects could be mimicked by Pg pre-treatment of non-pregnant mice. Interestingly, in the study [148] of Pg in CIA mentioned above, the combination of pregnancy level Pg+Es did not alleviate arthritis, suggesting that pregnancy- induced remission of certain forms of inflammatory arthritis requires additional factors, such as those released at the maternal–fetal inter- face. Dissecting the mechanisms of pregnancy-induced remission in RA and its animal models is important because it will likely reveal new pathways, or new combinations of known pathways, that could be targeted to treat people with RA.

6.3. Multiple sclerosis (MS)

MS is a female-predominant demyelinating disease of the CNS as- sociated with activation of Th1 and Th17 pathways [154]. Like with RA, female sex steroids appear to have stronger effects on MS disease activity compared to risk of disease. For example, even though MS is a female-predominant AD (Fig. 1), multiple observational studies have failed to find an association between either OCP use [155,156] or par- ity [156,157] and disease risk. However, in the experimental autoim- mune encephalomyelitis (EAE) model of MS, pre-treatment with pregnancy-level Es invariably reduces subsequent neurologic disease [156], perhaps through direct suppression of Th1 and Th17 cell func- tions [158]. In this model, Pg pre-treatment protects against neuro- logical damage [159], but its effects on immunological parameters were not addressed. Interestingly, in rats whose ovaries were re- moved, Pg treatment before EAE induction worsened brain inflamma- tion and neurological symptoms [160], indicating that the protective effects of Pg require gonadal factors. Overall, however, these observa- tions do not support a significant role for either Es or Pg in explaining the female predominance of MS.

In contrast, female sex steroids appear to have major impacts on MS disease activity. Pregnancy is associated with remission of MS and reduced rates of flare, which rise markedly post-partum [161]. How this occurs is not known but is believed to involve the ability of Pg to prevent neurological damage and foster regeneration [162,163]. Consistent with this idea, Pg treatment after onset of EAE halts disease progression and fosters CNS myelin regeneration [162,164]. Moreover, Yates et al. [164] recently showed that the

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protective effects of Pg treatment after induction of EAE autoimmuni- ty involves suppression of IL-2 and IL-17, consistent with Pg's abil- ity to suppress Th17 effector functions and T cell proliferation (Section 5.4.8). Another potential target in this regard is release by DCs of IL-23, a critical inducer of Th17 differentiation in EAE and probably MS [154]. Thus, it is likely that pregnancy-induced improvement in MS reflects Pg's ability to 1) suppress Th17 differ- entiation or effector functions, 2) protect against CNS injury, and 3) foster repair of prior neurological damage. A clinical trial [165] is underway testing the efficacy of progestin treatment in the prevention of post-partum flares in women with MS. If this therapy proves effec- tive, it will be important to determine which Pg receptors expressed in the CNS mediate protective effects, because these receptors could be selectively targeted in MS and other diseases of inflammatory injury to the CNS.

6.4. Autoimmune thyroid disease (AITD)

Autoimmune thyroid diseases are characterized by immune- mediated attack of the thyroid gland. Two important forms of AITD are Hashimoto's thyroiditis (HT) and Grave's disease (GD). [166] HT and GD are among the ADs with the highest female:male ratios (Fig. 1). Epidemiological data strongly implicate female reproductive factors in the pathogenesis of AITD. First, like with SLE, the extreme female to male ratio arises after puberty [18]. Second, large retrospec- tive studies show that increasing parity is positively associated with risk of AITD or anti-thyroid autoimmunity [157,167], although this could reflect immune responses to increasing fetal alloantigen [168] rather than hormone effects. In one retrospective study [167], use of Es-containing OCPs appeared to protect against thyroid disease (here, presumed GD) independent of parity, consistent with Es's pro- tective effects in a mouse model of AITD [169]. In this same model, Pg treatment modestly increased anti-thyroid Abs and thyroiditis. Thus, Pg and Es appear to modulate risk of AITD differently than they do in SLE (Section 6.1). Why Es in particular would protect against GD but enhance risk of SLE is unclear, but one possibility relates to differ- ential IgG subclass usage by autoAbs in GD (IgG1 and IgG4) [170] and SLE (IgG2 and IgG3 in severe GN) [171]. Through modulation of CD4+

T cell differentiation and B cell CSR, Es could differentially impact ADs mediated by neutralizing IgG subclasses (e.g., IgG4) vs. complement- fixing and cytotoxic subclasses (e.g., IgG3) [172]. Less is known about the impact of Es or Pg on AITD activity, but GD apparently can remit during pregnancy and flare post-partum [173]. Much more research is needed to clarify hormonal risk factors for AITD.

6.5. Other female-predominant autoimmune diseases

Very little is known about the impact of Pg on Sjögren syndrome (SS), SSc or PBC. SS is a highly female-predominant AD characterized by ANA and immune-mediated dysfunction of salivary and other mu- cosal exocrine glands [145]. Though SS shares many features with SLE, including risk association with IRF5 and STAT4, its median age of onset is 40–60, 1–2 decades later than SLE [174]. Thus, risk modu- lation by female sex steroids in SS, if present, appears to be slower than in SLE, perhaps because SS comes to clinical attention only after sufficient damage to exocrine glands has accrued to cause symp- toms, as is the case with thyroid gland damage in HT, which also shows peak incidence between 40 and 60 [174,175]. It is unclear whether female sex steroids impact immune-mediated injury in SS, but there is evidence that androgens [176], Es, and to a lesser extent, Pg [177], modulate exocrine gland function and thereby disease symptoms.

SSc is characterized by ANA, progressive skin hardening (sclero- sis), vascular dysfunction and sometimes fibrosis of internal organs [145]. Studies examining the relationship of parity and risk of SSc have shown either negative, positive, or no correlation [157,178].

OCP use did not appear to be correlated with risk of SSc in one study [179]. In PBC, a female-predominant disease of progressive liver scarring, rheumatologic symptoms and ANA, a large retrospec- tive study [180] found no correlation with parity or use of HRT with risk of disease, but found a modest protective effect of past OCP use. Two trials [181,182] tested the safety of HRT for bone health in women with PBC and found no adverse effects on liver disease, while a therapeutic trial [183] of MPA in PBC found no benefit. Clearly, more research, particularly with animal models, is needed to explain the female predominance of these ADs.

7. Summary and future directions

The evolutionary requirements of placental reproduction have driven sexual dimorphism in mammalian immune systems. This di- morphism is most obvious in the extreme female predominance of certain human ADs. Sexual immune dimorphism almost certainly in- volves modulatory actions of sex steroids like T, Es and Pg. Pg exerts these effects via direct actions on immune cells and induction of im- munomodulatory molecules in non-hematopoietic tissues. The effects of Pg and synthetic progestins depend greatly on their concentrations and differential engagement of various intracellular and membrane- associated Pg receptors expressed in immune cell subsets, immune organs and tissues of autoimmune attack. Via these mechanisms, Pg has profound and complex modulatory actions, different than those of Es or T, on inflammation, adaptive immune responses and autoim- munity. Moreover, Pg modulates risk of AD and immune-mediated injury in different ways in different ADs. Since the effects of Pg and Es on immune functions appear to be interdependent, as they are in female reproductive tissues, relationships between hormonal status and risk of AD and immune-mediated injury will defy simple classifi- cation. Thus, basic research is needed to clarify the cellular and mo- lecular mechanisms by which Pg and Es modulate immune functions, both individually and together. Similar mechanistic dissec- tion of pregnancy-induced remission in RA and MS should reveal novel therapeutic approaches. Finally, emerging understanding of sex-specific genetic risk should be used to guide basic scientific inves- tigation into the hormonal causes of sexual dimorphism in human AD.

Acknowledgments

I would like to thank Edward A. Clark, Ph.D., Peter Thomas, Ph.D., and Parul Sharma, D.O., for their helpful comments during the writing of this review.

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