The Role of Retinoic Acid in Tolerance and Immunity

Introduction

In the early 20^th century E.V. McCollum and Thomas Osborne independently embarked on studies to identify dietary constituents that were essential for mammalian health and survival. Using different dietary supplements they arrived at the seminal conclusion that a single factor present in lipids was essential for growth and survival, which they coined “fat soluble factor A” (Wolf, 1996). Subsequently designated vitamin A, studies over the years have demonstrated the pleiotropic influence of this nutrient, ranging from eyesight and organogenesis to metabolism and immunological fitness (Acin-Perez et al.; Duester, 2008; Underwood, 2004; Ziouzenkova et al., 2007). Exposing its critical contribution to immunological health, vitamin A supplementation was shown to dramatically curb young-childhood mortality in endemic regions of malnutrition (Rahmathullah et al., 1990; Sommer, 2008; Sommer et al., 1986). The vitamin A metabolite, retinoic acid (RA), first received attention as an interventional therapy upon discovery that it could substitute for more toxic chemotherapeutic regimens to dramatically improve the prognosis of acute promyelocytic leukemia, a malignancy caused by genetic translocations with the retinoic acid receptor (RAR), RARα(de The and Chen, 2010). While numerous investigations of APL have highlighted the ability of RA to promote myeloid cell differentiation (Kastner et al., 2001), over the last 20 years it has become clear that this metabolite influences multiple immune cell lineages and an array immunological functions (Cantorna et al., 1995; Chun et al., 1992). In this review, we discuss recent advances that have established RA as central to both immunological tolerance and the elicitation of adaptive immune responses. Further, we provide a comprehensive overview of the cell types and factors that control the production of RA and discuss how host perturbations may affect the ability of this metabolite to control tolerance and immunity, or instigate pathology.

Acquisition, Storage, and Metabolism of Vitamin A

Vitamin A is a fat-soluble essential nutrient obtained through foods containing vitamin A precursors, i.e. carotenoids, or vitamin A itself in the form of retinyl esters (Harrison, 2005; Yeum and Russell, 2002) (Figure 1). Following absorption and arrival into circulation, retinyl esters enter the liver, where most of the vitamin A in the body is stored (Blomhoff and Blomhoff, 2006). Liver retinyl esters are continually hydrolyzed into retinol and deployed into circulation (Wolf, 2007). Bile, which drains from the liver into the small intestinal duodenum, is also enriched in retinol (Jaensson-Gyllenback et al., 2011). Once inside a cell, widely expressed alcohol dehydrogenases (ADH) oxidize retinol into retinal, which can then bind to more selectively expressed Retinal dehydrogenases (RALDH) for oxidation into retinoic acid (RA). RA can be generated in multiple isoforms; however, the all-trans isoform predominates in most tissues and, therefore, the immunological effects of this compound will be the focus in this review (Mic et al., 2003). Although RA is constitutively present in serum at low levels (Kane et al., 2008), RALDH induction is tightly controlled process and subject to change during perturbations to homeostasis (Figure 2).

Figure 1. Vitamin A metabolism and major cellular sources of retinoic acid during homeostasis.

(A) Dietary vitamin A is absorbed in the intestine and transported through the lymphatics into blood circulation where it enters the liver for storage. Retinol chaperoned by retinol binding protein (RBP) is constitutively deployed from the liver into circulation. It is also secreted in bile that drains into the small intestine. Upon entry into cells, retinol is reversibly oxidized into retinal via the alcohol dehydrogenase (ADH) family. Depending on the cell type (see table in B), retinal can undergo irreversible metabolism into retinoic acid (RA) via retinal dehydrogenases (RALDH). (B) Overview of major cellular sources of RA during steady-state conditions. Table includes cellular location, the isoform(s) of RALDH that are expressed, and the factors know to induce RALDH expression in each cell type.

Figure 2. The role of retinoic acid in the regulation of CD4⁺ T cell homeostasis and immunity in the GI tract.

The GALT is a retinoic acid (RA) rich environment, containing a vast presence of RA synthesizing cells, including: resident epithelia (IEC) and stroma (SC), as well as migratory CD103⁺ DCs. RA produced by IEC may confer characteristic features to Lp DCs, while RA produced by SC in the draining mesenteric lymph nodes may reinforce lymphocyte acquisition of mucosal homing markers and potentially other effector functions. (A) During steady-state conditions RA sustains oral tolerance and helps maintain barrier integrity. These processes are mediated in large part by the ability of RA to support the induction of Foxp3⁺ iTreg and Th17 cells. Lamina propria CD103⁺ DCs are important for the induction and recruitment of heterogeneous CD4⁺ T cell populations during steady-state, as result of their ability to respond to commensal microbial signals and produce both RA and TGF-β. This dual capacity likely involves autocrine, or paracrine, RA signaling through retinoic acid receptors, such as RARα, which drives RA synthesis. An RA rich intestinal milieu also potentially limits the pathogenic potential and turnover of Th17 cells through down regulation of IL-6R and IL-23R. (B) During inflammation or infection, the inflammatory milieu triggers altered cytokine production by CD103⁺ DCs, leading to RARα-dependent effector CD4⁺ T cell activation and differentiation. Innate cell populations, including antigen presenting cells, are recruited during inflammation and may support RA production. Factors in the inflammatory milieu including TLR-ligands and the cytokine, GM-CSF, promote RALDH activity in CD103⁺ DCs and potentially in recruited innate cells.

Prior to its function, RA binds to nuclear receptors, including: retinoic acid receptors (RAR), retinoid X receptors (RXR) and, under certain circumstances, PPARβ (Chambon, 1996; Schug et al., 2007). Notably, all-trans RA exclusively binds RXR via heterodimers with the RAR family, which consists of three receptors RAR alpha (RARα), beta (RARβ), and gamma (RAR γ) (Chambon, 1996).

Retinoic Acid Sythesis and Migration into Mucosal Sites

Appropriate immune responses depend on the ability of effector and regulatory lymphocytes to home to the site of infection or injury. In this regard, DCs have been shown to foster lymphocyte migration into tissues where antigen was initially encountered (Campbell and Butcher, 2002). For instance, DCs from gastrointestinal tract and associated lymphoid tissue (GALT), but not the periphery were shown to induce the mucosal homing markers – integrin heterodimer α₄β₇ and chemokine receptor CCR9 – on stimulated effector T cells (Johansson-Lindbom et al., 2005; Johansson-Lindbom et al., 2003; Mora et al., 2003). The insight that mucosal DCs triggered α₄β₇ and CCR9 through the capacity to synthesize RA was based on the seminal observation that adding RA to T cells during activation selectively induced these gut homing markers (Iwata et al., 2004). In a reciprocal fashion, blockade of retinoic acid receptor mediated signaling and transcription in cultures containing GALT DCs reversed induction of α₄β₇ and CCR9 (Iwata et al., 2004; Svensson et al., 2008). Recently, RA was revealed to predominantly affect the α₄ subunit of α₄β₇ via binding of RARα to a retinoic acid receptor response element within the regulatory region of the α₄ gene (DeNucci et al., 2010; Kang et al., 2011). A retinoic acid response element half-site was also recently discovered in the promoter region of CCR9, which RAR α/RXR heterodimers were able to bind to (Ohoka et al., 2011). These data coupled with the prominent baseline expression of Rara, the gene encoding RAR α, in CD4⁺ T cells, pinpoint RARα as an important mediator of lymphocyte trafficking. Nevertheless, they do not exclude a role for other RARs in mediating the regulation of these markers on other defined subsets.

An assessment of vitamin A synthesizing enzymes demonstrated that GALT DCs express mRNA for Aldh1a2, the gene encoding RALDH2 (Iwata et al., 2004; Schulz et al., 2009; Yokota et al., 2009). Subsequent studies have shown that basal Aldh1a2 expression in GALT DCs is enriched in CD103⁺ DC subsets, which induce α₄β₇ and especially CCR9 far more potently than the CD103^neg compartment (Coombes et al., 2007; Johansson-Lindbom et al., 2005; Yokota et al., 2009) (Figure 1). In the small intestinal Lp, this population is uniquely equipped with migratory capacity and therefore accumulates in the mesenteric lymph nodes (mln) (Bogunovic et al., 2009; Jaensson et al., 2008; Jang et al., 2006; Schulz et al., 2009). CD103⁺ DCs within the mln and Peyer’s patches (Pp) are comprised of two subsets based on expression of integrin CD11b. The CD103⁺CD11b^neg subset is related to the CD103⁺CD11b^neg and CD8 α⁺ DC subsets outside of the GALT (Edelson et al., 2010; Ginhoux et al., 2009; Hildner et al., 2008). However, CD103⁺CD11b^neg DCs residing at other sites fail to produce appreciable RALDH, suggesting that factors within the GI tract induce RA synthesizing capacity in this subset (Guilliams et al., 2010). In this regard, vitamin A itself was indispensible for DC production of Aldh1a2 during homeostasis (Jaensson-Gyllenback et al., 2011; Molenaar et al., 2011; Yokota et al., 2009). This may involve Wnt/β-catenin driven signals, as ablation of β-catenin in CD11c cells was shown to attenuate their expression of RALDH (Manicassamy et al., 2010). Microbial stimuli also appear to have an additional influence on RALDH expression, as moderate decreases were observed in GALT DCs isolated from mice reared in germ-free conditions or genetically deficient in the microbial signaling adaptor, MYD88 (Guilliams et al., 2010). TLR2 stimulating ligands, in particular, were found to most potently induce RALDH expression {Manicassamy, 2009 #1398; Wang, 2011 #1687}.

In addition to DCs, several non-hematopoietic lineages within the gastrointestinal tract and associated lymphoid tissues (GALT), such as epithelia (Aldh1a1/RALDH1) and stromal cells (Aldh1a1, Aldh1a2 and Aldh1a3), share the capacity to synthesize RA (Edele et al., 2008; Hammerschmidt et al., 2008; Iliev et al., 2009; Iwata et al., 2004; Molenaar et al., 2011) (Figure 1). Intestinal epithelial cells have been shown to imprint bone marrow DCs with signature characteristics of Lp DCs, via provision of soluble factors, including RA (Edele et al., 2008; Iliev et al., 2009). They may also provide RA in trans and reinforce the expression of mucosal homing markers on lymphocytes. Stromal cells within the mln were also found to support mucosal homing, likely through indirect effects on APCs (Hammerschmidt et al., 2008; Stock et al., 2011) (Figure 2). Altogether, these findings support the observation of a prominent reduction in the number of effector T lymphocytes within the intestinal effector sites in adult mice reared on a vitamin A deficient diet, as well as in Rara deficient (Rara^−/−) mice (Hall et al., 2011; Iwata et al., 2004).

A recent study found that other DC populations, particularly in the draining lymph nodes of the skin and lung expressed Aldh1a2 (Guilliams et al., 2010) (Figure 1). The finding that RA signaling occurs in sites not typically associated with mucosal homing raises several interesting points of discussion. First, crosstalk with other cells in these tissues may modulate the capacity of DCs to induce mucosal homing. For example, prostaglandin E2, from the stroma of peripheral tissues, was recently shown to antagonize RALDH expression (Stock et al., 2011). Second, RA signaling may also influence migration to peripheral sites. In this regard, integrin α₄ can also form heterodimers with integrin subunit, β₁, which is rapidly upregulated in response to TCR stimulation and impedes formation of the α₄β₇ heterodimer (DeNucci et al., 2010). As α₄β₁ binds to VCAM-1, which is present on endothelial cells and upreguated during inflammation, RA may additionally influence migration to peripheral sites during inflammation (Henninger et al., 1997; Muller, 2011) (Figure 3). Further studies are essential to understand how crosstalk between DCs and other cells can modulate RALDH activity, which may produce a better understanding of how accessory cells contribute to the regulation of immunity during both homeostasis and inflammation. Collectively these findings indicate that RA signaling occurs in tissues throughout the host and suggest that apart from mucosal homing it plays a general role in both tolerance and inflammation.

Figure 3. RA synergizes with an inflammatory milieu to promote pathology.

During inflammation, RA signaling can induce the production of inflammatory cytokines by DCs, and in turn promote effector T cell differentiation. Direct interactions of RA with T cells may further contribute to the activation status of the cells and promote localization to inflamed tissues. RA may also engender the formation of tertiary lymphoid structures and facilitate chronic inflammation.

Retinoic acid in plasma cell differentiation and mucosal IgA

The discovery that RA was critical for the generation of immunoglobulin A (IgA) secreting B cells offered further evidence of a multifactorial role for RA in mucosal immunity (Mora et al., 2006). A number of studies have demonstrated the potent capacity of DCs from the intestinal Lp, mln and Pp to drive naïve B cell differentiation into IgA⁺ B cells (Macpherson and Uhr, 2004; Mora et al., 2006; Uematsu et al., 2008), and the ability of stromal derived cells to support IgA⁺ class switching in activated B cells (Fagarasan et al., 2001; Suzuki et al., 2010). Synthesis of RA by GALT DCs was crucial for the generation of IgA⁺ B cells, as antagonism of RA signaling significantly reduced IgA⁺ production (Mora et al., 2006; Uematsu et al., 2008). Complementing this finding, addition of RA to DC cocultures in which DCs lacked the capacity to synthesize RA restored IgA⁺ production. Notably, microbial induced cytokines, such as IL-6, were also integral cofactors in this process (Mora et al., 2006; Uematsu et al., 2008). Although inhibitory at high concentrations (Mora et al., 2006), TGF-β mediated signals have also been observed to play a decisive role in IgA⁺ production (Cazac and Roes, 2000). This has been verified in systems analyzing the capacity of mucosal stromal cells to foster IgA⁺ B cell generation (Fagarasan et al., 2001; Suzuki et al., 2010). In a manner analogous to peripheral DCs, peripheral follicular dendritic cells were able to efficiently support IgA⁺ production only when treated with RA and a Myd88 dependent microbial stimulus (Suzuki et al., 2010). This gain of function was dependent on the ability of RA signaling to induce secretion of TGF-β in peripheral follicular dendritic cells. RA signaling was shown to promote a similar effect (i.e. induction of TGF-β production) in bone marrow derived DCs via inhibition of suppressor of cytokine signaling 3 activity (Feng et al., 2010). These findings suggest interdependency between TGF-β and RA propagated signals in several cell lineages.

Another significant source of IgA⁺ production is B1-B cells, which contribute to mucosal integrity during homeostasis and early responses to pathogens. RA was recently shown to enforce the homeostatic maintenance of this compartment through direct regulation of NFATc1 (Maruya et al., 2011). Combined with the capacity of RA to generate IgA⁺ B cells and facilitate their mucosal localization, vitamin A deficiency leads to a severe decrease in intestinal IgA and a significant decrease in the serum (Maruya et al., 2011; Mora et al., 2006). Altogether, these findings underscore the importance of RA in IgA responses and humoral immunity.

Retinoic Acid in Extrathymic Treg (iTreg) Induction and Oral Tolerance

Foxp3 regulatory T (Treg) cells maintain both peripheral and mucosal homeostasis throughout the lifespan of the host (Josefowicz and Rudensky, 2009; Kim et al., 2007). Treg cells typically develop during thymic selection processes; however, they also develop extra-thymically in response to chronic antigen stimulation or exposure to environmental and food antigen at mucosal sites (Curotto de Lafaille and Lafaille, 2009). Development of inducible Treg (iTreg) cells, but not thymic derived Treg, requires transcription factor binding to the intronic enhancer element (enhancer-1) of the foxp3 locus, also known as conserved non-coding sequence 1 (CNS1). iTreg development is also dependent on several soluble mediators, including: TGF-β, IL-2 and, as recent data demonstrate, RA (Hall et al., 2011; Knoechel et al., 2005; Kretschmer et al., 2005; Mucida et al., 2005; Tone et al., 2008; Zheng et al., 2010).

The insight that RA served as a cofactor in the generation of iTreg cells stemmed from in vitro findings that relative to splenic DCs, mln and Lp DCs potently induced iTreg cell differentiation in the presence of TGF-β (Coombes et al., 2007; Mucida et al., 2007; Sun et al., 2007). Separation of mln DCs and Lp DCs based on CD103 expression revealed that the CD103⁺ subsets were specifically able to yield iTreg cells in the absence of exogenous factors, implying that RA signaling potentially contributed to this process (Coombes et al., 2007; Sun et al., 2007). Indeed, a pan RAR antagonist strongly inhibited iTreg cell generation (Coombes et al., 2007; Mucida et al., 2007; Sun et al., 2007).

Blockade of TGF-β in cocultures with CD103⁺ GALT DCs also diminished iTreg cell generation. The coordinate ability of GALT DCs to produce both RA and TGF-β may involve conditioning signals from RA during developmental maturation (Feng et al., 2010) (Figure 2). Assessment of DCs in a retinoic acid response reporter mouse revealed abundant RAR binding activity in GALT CD103⁺ DCs (Jaensson-Gyllenback et al., 2011). The level of RAR binding activity in DCs from several tissues correlated with both the concentration of retinol and the percentage of CD103⁺ DCs expressing RALDH2 in those tissues, which suggests that DC conditioning by RA may occur in situ. Furthermore, GALT CD103⁺ DCs highly expressed mRNA for the gene encoding RAR α, implicating this receptor as a dominant mediator of such conditioning signals (Jaensson-Gyllenback et al., 2011).

The addition of RA to cocultures with splenic DCs and TGF-β also dramatically enhanced iTreg induction (Benson et al., 2007; Mucida et al., 2007; Mucida et al., 2009; Sun et al., 2007). This effect was dependent on T cell expression of RARα (Hill et al., 2008), which was upregulated upon stimulation in the presence of TGF-β (Schambach et al., 2007). Remarkably, exogenous RA could sustain iTreg generation in conditions that typically opposed it, such as the presence of certain inflammatory cytokines (IL-6, IL-21) and high costimulatory environments (Benson et al., 2007; Mucida et al., 2007; Xiao et al., 2008). Stimulation of CD4⁺ T cells in the presence of TGF-β was revealed to induce IL-6R expression, which addition of RA reversed (Hill et al., 2008; Xiao et al., 2008) (Figure 2). Since RA did not appear to enhance the inherently unstable phenotype of iTreg (Floess et al., 2007; Hill et al., 2008), RA mediated repression of IL-6R could potentially explain why iTreg cells generated in the presence of RA were reportedly more stable in vivo following adoptive transfer (Benson et al., 2007). Corroborating in vitro findings, the generation of iTreg cells in response to antigen feeding was abrogated in animals deficient in vitamin A and, therefore, lacking RA (Hall et al., 2011).

Oral tolerance - the active suppression of inflammatory responses to food and other orally ingested antigens - is critically dependent on the generation of iTreg cells (Curotto de Lafaille et al., 2008; Weiner et al., 2011). Recent evidence indicates that in addition to supporting iTreg differentiation, RA mediated trafficking is required for a sustained expansion of iTreg cells in the gut (Hadis et al., 2011). This expansion is propagated through IL-10 mediated interactions with resident CD103^neg APCs. In previous studies similar interactions were shown to contribute to both the induction and maintenance of Treg cells (Denning et al., 2007; Murai et al., 2009). Altogether, these findings indicate that RA signaling is a keystone in the development of oral tolerance.

In the absence of RA, other mechanisms may compensate for the loss of these tolerogenic processes. For instance, elevated frequencies of Foxp3^neg IL-10⁺ Tr1 cells have been reported in vitamin A deficient mice and could be generated in vitro in the presence of RAR antagonists and DC induced microbial production of IL-6 and IL-21 (Maynard et al., 2009). Despite the importance of RA for generation of iTreg cells, neither the frequency nor absolute numbers of Treg cells were reduced during vitamin A deficiency (Hall et al., 2011). Thymic Treg cell differentiation was also intact in Rara^−/− animals (Hill et al., 2008). Although these findings suggest that there are distinct requirements for inducible versus thymic derived Treg, it still remains unclear whether intrathymic Treg differentiation and regulatory function is impacted in the absence of RA.

In vivo requirements notwithstanding, RA was insufficient to induce Foxp3 in the absence of TGF-β (Benson et al., 2007; Mucida et al., 2007; Sun et al., 2007). These findings imply that responsiveness to TGF-β is prerequisite for RA to access Foxp3 differentiation programs. In this regard, blockade of TGF-β RI kinase activity, which inhibits TGF-β-induced Smad2/3 phosphorylation (Sorrentino et al., 2008), diminished TGF-β mediated iTreg generation and abrogated the additive effect of RA (Xu et al., 2010). Several preceding studies noted that RA enhanced the total expression of Smad3 in activated CD4⁺ T cells; however, TGF-β was required to trigger Smad3 activation (i.e. phosphorylation) (Nolting et al., 2009; Xiao et al., 2008). Furthermore, TGF-β was observed to prevent the intracellular degradation of RA (Takeuchi et al., 2011), prompting the possibility that RA and TGF-β cooperatively promote enhanced Smad3 activity. Based on the importance of Smad3 activation in CNS1-mediated Foxp3 expression (Tone et al., 2008), the role of RA in the regulation of CNS-1 was examined, which led to the identification of a potential RAR binding site (Xu et al., 2010). Accordingly, RA was shown to dramatically enhance TGF-β induced chromatin accessibility and Smad3 binding in CNS1. The strong association between RA and the regulation of CNS1 not only provides a tentative molecular mechanism for the enhancement of iTreg cell generation, but further suggests that RA synthesis pathways may be manipulated during infection and inflammation to shift the balance between Treg and effector T cells, and in turn influence immunopathology.

Influence of Retinoic Acid on effector CD4⁺ T cell differentiation and function

The role of RA in TGF-β dependent responses has been further evaluated in Th17 cell differentiation. Th17 cells, which produce IL-17-A (IL-17), IL-17-F, IL-21 and IL- 22 promote control of bacteria and fungal infections at mucosal sites (Littman and Rudensky, 2010). They are induced in response to TGF-β, combinations of the Stat3 signaling cytokines - IL-6, IL-21 and IL-23 – and IL-1 (Korn et al., 2009). Although linked to the pathogenesis of autoimmune responses (Iwakura et al., 2011), Th17 cells provide an important layer of protection at mucosal interfaces and are typically detected during steady state in these regions (Ivanov et al., 2008; Ivanov et al., 2006). Several recent studies revealed that Th17 cells were virtually ablated in the GALT of mice reared on a vitamin A deficient diet during steady-state (Cha et al.; Wang et al.). In concert with the loss of Th17 cells, the ability of APCs to produce IL-6, which promotes Th17 polarization, was reduced in vitamin A deficient mice (Hall et al., 2011). Despite the likelihood that additional factors were complicit in the diminished number of Th17 cells during vitamin A deficiency, these data suggest that RA is critical for the in vivo differentiation and/or survival of Th17 cells. In support of this, in conjunction with microbial stimulation, a low dose of RA (~1nM) in splenic DC cocultures was found to potentiate Th17 cell generation (Uematsu et al., 2008). These findings suggest that a synergy between RA and microbial driven signals promote Th17 differentiation in vivo (Figure 2). Importantly, the ability of RA to support Th17 differentiation likely results from combined actions on DCs and T cells, as addition of RA to Th17 polarizing conditions in APC-less cultures did not enhance Th17 differentiation (Wang et al., 2010).

In parallel with the initial discovery that RA enhanced iTreg cell differentiation, RA was observed to suppress Th17 cell generation (Elias et al., 2008; Kang et al., 2007; Mucida et al., 2007). RA was shown to inhibit IL-6R and IL-23R upregulation induced by TGF-β and IL-6, respectively (Xiao et al., 2008; Zhou et al., 2007). Accordingly, RA supplementation in vitamin A-replete settings could suppress Th17 responses and IL-23 driven immunopathology during Listeria monocytogenes infection and autoimmune experimental encephalitis (Mucida et al., 2007; Xiao et al., 2008). Thus, while RA is required for the in vivo promotion of Th17 differentiation, it also may directly contribute to Th17 cell regulation.

In addition to Th17 cells, RA can exert direct regulatory effects on other effector T cell populations (Hill et al., 2008). For instance, RA was shown to inhibit IFN- γ production from CD8⁺ T cells and Th1 cells (Cantorna et al., 1996; Cantorna et al., 1995; Stephensen et al., 2002). A previous study argued that such regulation might have contributed to impaired Th2 responses to the parasitic infection, Trichinella spiralis during vitamin A deficiency (Carman et al., 1992). RA was also shown to relieve the inhibitory influence of effector T cells in the generation iTreg cells by directly suppressing their production of IL-4 and IL-21, which were previously demonstrated to potently antagonize iTreg generation (Hill et al., 2008; Korn et al., 2007; Nurieva et al., 2007; Wei et al., 2007). Further investigation into the temporal regulation of retinoid receptor expression during helper T cell polarization should clarify the molecular mechanisms by which RA controls cytokine production in various effector T cell subsets. Although the direct effects of RA on effector T cells should continue to be explored in more pathological contexts, these findings support a model in which RA-rich microenvironments can limit cytokine production by terminally differentiated effector T cells and, thus, T cell mediated tissue pathology.

Retinoic Acid/Retinoic Acid Receptor Signaling in CD4⁺ T cell activation

The recent descriptions of RA in immunoregulation have in some ways overshadowed the importance of this metabolite in generating functional immunity. Indeed, several studies have noted potent adjuvant effects of RA during infection (Dawson et al., 2009; Yamada et al., 2007). One aspect in the amplification of these responses may involve the vital role of RA in T cell activation. For instance, in serum free cultures, RA was observed to dramatically enhance TCR mediated CD4⁺ T cell proliferation in an IL-2 dependent manner (Engedal et al., 2006). The NFAT family of transcription factors regulates an array of functions in multiple cell types; in T cells these include production of IL-2 and the full acquisition of effector properties (Macian, 2005; Peng et al., 2001). mRNA levels of multiple NFAT isoforms, which were reduced in B1-B cells during vitamin A deficiency, rebounded to normal levels following treatment with RA. NFAT proteins were also significantly reduced in T cells during vitamin A deficiency and it is possible that RA regulates NFAT transcription and/or stability in these cells, as well (Maruya et al., 2011). Interestingly, NFATc2 was recently shown to cooperate with RARα/RXR heterodimers to induce CCR9, while NFATc1 inhibited CCR9 induction (Ohoka et al., 2011). These findings suggest that RA establishes an intimate link between T cell activation, effector function and homing properties. It will be important to determine whether T cell expression and localization of NFAT proteins are differentially regulated in distinct anatomical sites and how their modulation converges with RA availability in these sites to influence expression of homing markers.

Naïve CD4⁺ T cells basally express the genes encoding RARα (Rara) and RARγ (Rarg) (Hall et al., 2011; Ohoka et al., 2011). While, previous data suggested that RARγ was dispensable for CD4⁺ T cell activation (Dzhagalov et al., 2007), recent data revealed that RARα was important for this process (Hall et al., 2011). Specifically, Ca²⁺mobilization in response to TCR/CD3 engagement was impaired in CD4⁺ T cells from Rara^−/− mice or normal cells exposed to a pan-RAR antagonist (Hall et al., 2011). As Ca²⁺ mobilization results in NFAT translocation into the nucleus, these data suggest that both early and sustained T cell activation are impaired when RA signaling is deficient (Feske, 2007). In this regard, activation of the Mammalian TOR (mTOR) pathways, which play important roles in directing helper T cell responses, were also reduced in Rara^−/− T cells (Delgoffe et al., 2009; Delgoffe et al., 2011; Zhang et al., 2011). The reductions in these signaling pathways may, in part, contribute to the immunodeficient state observed in animals devoid of RA/RARα signaling, which will be discussed below.

Precisely how RA/RARα signals mediate early T cell activation events is unclear. RARα is potent transcriptional regulator of gene networks and known to constitutively bind to DNA. Such binding may exert a tonic influence on the DNA binding capacity of other proteins involved in the regulation of T cell activation. For instance, RA/RARα was previously shown to regulate DNA binding of the AP-1 transcription factor, c-Jun, which is involved in responses to stress and TCR mediated signals (Schule et al., 1991). In addition to its well-appreciated nuclear activity, extra-nuclear functions of RARα have also been described (Rochette-Egly and Germain, 2009). In this regard, RARα was observed to regulate expression of the phosphatase MKP-1, which is an important mediator of effector T cell differentiation (Lee et al., 1999; Zhang et al., 2009). Furthermore, RARα may associate with a membrane and/or cytosolic signaling scaffold important in T cell activation (Rochette-Egly and Germain, 2009). Recent findings indicate that RXR expression is barely detectable in naïve T cells, suggesting the effects of RARα on T cell activation may proceed independently of RXR heterodimerization. Conversely, RAR/RXR heterodimerization is required for the acquisition of mucosal homing markers (Ohoka et al., 2011). While further research into the mechanism by which RA/RARα regulates T cell signaling is needed, one can construct a model in which RA regulates adaptive T cell responses through dichotomous roles: on the one hand promoting the initiation of effector T cell differentiation and, on the other hand, restraining inflammatory T cell responses in tissues.

Retinoic acid in infection and immunity

Recent human data highlight the correlation between vitamin A status and T cell function (Ahmad et al., 2009). Although vitamin A has gained widespread acceptance as a clinical health intervention, skepticism of its efficacy has lingered due to inconsistency in outcomes of various vitamin A supplementation programs and a subpar understanding of the mechanisms it employs to combat infectious disease (Sommer, 2008; Wintergerst et al., 2007). The latter, in and of itself, poses a significant challenge to developing efficacious supplementation programs. Recent studies utilizing various animal models of vitamin A or retinoid receptor deficiency have begun to close this knowledge gap, revealing an integral role for RA in vitamin A dependent immunity. Impaired and/or dysregulated T cell responses have been observed in various models of infection and vaccination strategies during vitamin A and/or retinoid receptor deficiency (Carman et al., 1992; Dzhagalov et al., 2007; Hall et al., 2011; Stephensen et al., 2004). During infection with T. gondii, an intracellular replicating pathogen controlled by IFN-γ (Suzuki et al., 1988), the acute Th1 response and parasite clearance were significantly impaired in vitamin A deficient mice (Hall et al., 2011). Similarly, vaccination with an E. coli derived heat-labile enterotoxin mucosal adjuvant, LT(R129G), which simultaneously elicits Th1 and Th17 cells (Hall et al., 2008), yielded diminished Th1 and Th17 responses in these animals. Short-term treatment with RA immediately prior to and during challenge completely rescued CD4⁺ T cell responses both to acute T. gondii infection and vaccination in vitamin A deficient mice (Hall et al., 2011). These findings demonstrated an essential role for RA in the development of Th1 and Th17 cell responses.

RA signaling appears to control the fate of T cell immunity largely through RARα and RAR γ. The CD4⁺ T cell response to vaccination with LT(R129G) was strongly diminished in Rara^−/− mice (Hall et al., 2011). While CD4⁺ T cell activation was reduced in the absence of RARα, additional functional impairments may have also factored into this outcome. Preliminary evidence suggests that RARα regulates not just the proper maturation of DCs in the GALT (Jaensson-Gyllenback et al., 2011), but also their ability to drive inflammatory responses in certain pathologic settings, which will be discussed below (Depaolo et al., 2011). Future studies employing lineage-targeting strategies will be integral for discerning the contributions of RARα in individual cell types to immunological outcomes. Although the role of RARα in CD8⁺ T cell function remains to be explored, RAR γ, which was dispensable for CD4⁺ T cell and humoral responses, was shown to be required for full effector differentiation of CD8⁺ T cells (Tc) in response to infection with Listeria monocytogenes (Dzhagalov et al., 2007). A direct role for RARγ in CD8⁺ T cell activation has not yet been addressed; however, this receptor was observed to control optimal macrophage production of inflammatory cytokines in response to microbial stimuli (Dzhagalov et al., 2007). A role for RA signaling in macrophages is consistent with another study, which showed that RA enhanced macrophage activation in response to in vitro infection with Mycobacterium tuberculosis (Yamada et al., 2007). Altogether, these data illustrate the ability of RA to regulate a network of innate and adaptive immune cell functions, which through non-redundant receptor signaling pathways power functional immune responses. More research is required to elucidate how RARβ signals fit into the control of vitamin A dependent immunity. In contrast to the other RARs, RARβ expression appears to largely depend on RA itself and, therefore, may play a prominent role in the regulation of mucosal immune responses (Molenaar et al., 2011; Suzuki et al., 2010). However, RARβ was also found to regulate the recruitment of lymphoid tissue inducer cells during embryogenesis via stromal cell induction of CXCL13 (van de Pavert et al., 2009). Thus, it will be worthwhile to examine the role of RA in the formation of tertiary lymphoid structures during chronic inflammation.

Retinoic acid in inflammation

Chronic inflammatory syndromes arise as a consequence of genetic polymorphisms in concert with accumulating environmental exposure to toxins, pathogens, and diet (Yazdanbakhsh et al., 2002). Western diets are becoming increasingly associated with a higher prevalence of inflammatory disorders, including allergies and inflammatory bowel diseases (Garrett et al., 2010; Wang and Sampson, 2011). Several studies have suggested that in vivo iTreg generation can prevent and/or mitigate these manifestations (Curotto de Lafaille and Lafaille, 2009; Lacy-Hulbert et al., 2007; Travis et al., 2007). In this regard, treatments that promote RA metabolism may constitute an effective strategy to restore the regulatory balance during chronic inflammation. For example, treatment with the TLR2 agonist, zymosan, was shown to induce RALDH production by non-mucosal DCs and ameliorated pathology in a model of autoimmunity (Manicassamy et al., 2009) (Figure 2). Nevertheless, in previous studies, mice fed a diet high in vitamin A exhibited more vigorous responses against grafts and tumors, suggesting that elevated retinoid levels were potentially detrimental in certain inflammatory contexts (Malkovsky et al., 1983a; Malkovsky et al., 1983b) (Figure 3). In this regard, a recent study demonstrated that RA driven signals in an inflammatory environment fostered reactivity to dietary glutens in a mouse model of Celiac disease (Depaolo et al., 2011; Jabri and Sollid, 2009) (Figure 3). In particular, RA was demonstrated to synergize with an IL-15 rich milieu and potentiate production of IL-12 and IL-23 by mucosal DCs, diminishing their capacity to promote iTreg cells and leading to exacerbated responses to Gliadin (Depaolo et al., 2011). These data reflect several reports describing a possible association between pharmacological retinoid treatment and spontaneous development of inflammatory bowel disease and point to vitamin A metabolic pathways as potential instigators of chronic inflammation (Crockett et al., 2010; Reddy et al., 2006) (Figure3).

Concluding Remarks

Recent insights into the role of RA in the promotion and regulation of multiple immunological pathways draw new attention to the sweeping influence of vitamin A in immunity. Though long on the radar of health experts trying to combat immunodeficiencies in developing nations, Vitamin A is often overlooked in developed regions where access to this nutrient is plentiful. New data reveal that multiple factors influence the generation of RA, including: vitamin A itself, fatty acids, TLR ligands, and GM-CSF, which promote RA synthesis and prostaglandin E2, which inhibits RA synthesis (Manicassamy et al., 2009; Stock et al., 2011; Szatmari et al., 2006; Yokota et al., 2009). Greater understanding of how these factors play into RA synthesis during homeostasis and inflammation will be essential for assessing their efficacy as therapeutic modalities in the treatment of syndromes in which retinoid imbalances may be involved. In summary, the potential of RA to transform from an essential to pathological mediator of immune responses raises many questions on how vitamin A metabolism affects disease. Furthermore, as retinoids are prevalent in clinical settings (de Lera et al., 2007), an additional consideration may be the patient’s risk factors for inflammatory disease.

Abbreviations used

Th1

type 1

Th17

type 17

RA

retinoic acid

RAR

retinoic acid receptor

RXR

retinoic X receptor

DC

dendritic cell

GALT

gastrointestinal tract and associated lymphoid tissue

Lp

lamina propria

mln

mesenteric lymph nodes

Pp

Peyer’s patches

ADH

alcohol dehydrogenases

RALDH

retinal dehydrogenase enzyme

Treg

Foxp3⁺ regulatory T cells

CNS1

non coding sequence 1

TGF-β

transforming growth factor beta

IgA

immunoglobulin A

IFN-γ

interferon gamma

T. gondii

Toxoplasma gondii

mln

mesenteric lymph nodes

IL-17

interlukin-17A

Pp

Peyer’s patch

mTOR

mammalian target of rapamycin kinase

PLCγ1

phospholipase C gamma-1