Dietary and commensal derived nutrients: Shaping mucosal and systemic immunity

Introduction

The gastrointestinal (GI) tract is charged with a difficult task: it must absorb nutrients while simultaneously establishing détente with commensal microflora and defending against invading pathogens. Critical to achieving this task is the immune network of the GI tract, which is home to one of the largest reservoirs of immune cells in the body [1]. Contained within this complex immunologic network of the GI tract are highly specialized antigen presenting populations, a large population of IgA secreting plasma cells, and an abundance of CD4+ T regulatory (Treg) and Th17 polarized T cells [2]. All of these factors work in concert to promote barrier function of the GI tract and to maintain tolerance to food and commensal bacteria. Our understanding of the intestinal immune system has grown immensely over the past decade. With this understanding has come a greater appreciation of the dominant signals immune cells are exposed to in the GI tract. In addition to tonic commensal microflora derived signals, the intestinal immune system appears to be uniquely well equipped to respond to dietary derived factors and micronutrients and, in some cases, relies on these nutritional cues for its function [3].

Nutrient deficiencies underscore the importance of micronutrients in regulation of mucosal immunity. One dietary micronutrient that has emerged as a critical mediator of mucosal immune responses is Vitamin A and, particularly, its metabolite retinoic acid (RA) [4]. Vitamin A deficiency, one of the most common micronutrient deficiencies [5], is associated with higher rates of mortality [6], while supplementation of deficient children with Vitamin A is able to reduce mortality [7]. Although the importance of Vitamin A in supporting mucosal immunity has long been appreciated, recent findings have enhanced our understanding of the diverse mechanisms by which Vitamin A accomplishes this task and its contextual role in promoting immunity. As well, additional signals controlling immune function in the GI tract have come to light consisting of dietary derived factors and commensal metabolites.

Vitamin A: A keystone dietary micronutrient for intestinal immunity

Vitamin A is a fat-soluble essential micronutrient obtained from foods containing either vitamin A precursors in the form of carotenoids or from vitamin A itself in the form of retinyl esters [8]. After absorption, retinyl esters are hydrolyzed into retinol in the liver for release into the circulation or are recycled into the gut via bile drainage. Retinol taken up by cells is converted to retinoic acid (RA) by sequential oxidation by alcohol dehydrogenases (ADH) and retinaldehyde dehydrogenases (RALDH). RA is mainly generated in the all-trans isoform [8] and, therefore, the immunological effects of this compound are the focus of current research efforts. Recent advances in understanding the role of RA have uncovered some of the relevant sources of RA and the signals necessary for RA production by these cells. As well, the complex and contextual role of RA continues to be clarified.

Although RA is constitutively present in serum at low levels [9], RALDH induction is subject to tight regulation and is controlled by environmental cues. Many cell types in the intestine are capable of synthesizing RA, including epithelial, stromal and dendritic cells. An important source of RA in the lamina propria of the intestine is thought to arise from a specialized subset of CD103+ migratory DCs with high basal expression of Aldh1a2, encoding the enzyme responsible for RA generation, RALDH2 [10–12]. Initial reports indicated that RA expression could be driven by the cytokines GM-CSF and IL-4 [13]. Subsequently, TLR signaling was shown to promote RALDH2 expression in both mucosal and peripheral DCs [14]. Another factor that has been shown to promote RALDH expression in intestinal DCs is beta-catenin [15]. Interestingly, RA itself has been shown to re-enforce RALDH2 expression in DCs [16,17]. These data suggest a model in which gut specific factors (cytokines, TLR ligands, or RA) prime DCs to generate RA, which is then sensed by DCs in order to re-enforce RA production.

Retinoic acid (RA) came into focus as an important factor for promoting intestinal immunity when it was found to directly induce the gut homing markers α4β7 and CCR9 on both T cells and B cells [12,18]. These studies highlighted the importance of RA in specifically supporting mucosal immunity by controlling the trafficking of responding cells. Indeed, the generation of key components of the mucosal immune system: IgA+ plasma cells, CD4+ Tregs and Th17 cells are all regulated by RA derived signals. In addition to controlling the tissue tropism of these cells, RA also influences the differentiation of cells towards lineages important for mucosal defense (Figure 1). For instance, RA signaling on B cells results in the selective promotion of intestinal IgA via enhancement of class switching to IgA [18,19]. Notably, vitamin A metabolism plays a central role in the acquisition of oral tolerance, a process that requires peripheral generation of Foxp3+ Treg cells [20,21**]. Mice in which Foxp3+ Treg cells cannot be induced, failed to establish tolerance at mucosal surfaces and develop aberrant Th2 responses in both the lung and GI tract [20]. The extrathymic differentiation of Foxp3+ Tregs in the GI tract is tightly controlled at steady state by the capacity of a specialized population of gut tropic DCs expressing CD103 that have a potent capacity to activate latent TGF-β and produce RA [22–25**]. The effect of RA is not limited to regulatory responses as RA, in a context dependent manner, can promote effector T cell responses. Indeed, vitamin A deficient mice have greatly diminished Th17 composition in their small intestines at steady state [26,27] and lower, physiologic, concentrations of RA can promote the differentiation and trafficking of Th17 cells to the small intestine [26–28]. As well, antigen presenting cells from vitamin A deficient mice are impaired in their ability to secrete IL-6, indicating that RA is important for the functional capacity of DC’s to promote Th17 differentiation [29]. Thus, RA can promote both Treg and Th17 lineages in a context specific manner [4].

Recent evidence suggests that retinoic acid may have a more general role in promoting both mucosal and peripheral immunity [29,30**]. Indeed, impaired RARα signaling revealed a cell intrinsic role for RA sensing by naïve CD4+ T cells in order to achieve a proper threshold of TCR signaling and to become fully activated in the context of infection and skin allograft [29,30]. Both of these studies demonstrate that RA synthesis increases during systemic inflammation, likely in order to support various aspects of immune responses including T cell activation. All together, recent work on the multifaceted role of retinoic acid begins to offer a mechanistic explanation for the profound immune deficiencies associated with increased susceptibility to infections and poor vaccine responses observed in vitamin A deficiency.

Notably, several recent studies have begun to apply these findings to clinical settings. Recent work demonstrated that the addition of RA at the time of peripheral vaccination has a profound capacity to increase the accumulation of memory cells in the gut mucosa [31*]. Given the key role of RA in supporting effector responses, the peripheral administration of vaccines with RA provides an opportunity to not only target vaccines to mucosal surfaces, but to utilize RA as a vaccine adjuvant. The role of RA in supporting effector responses during inflammatory settings has also been found to have deleterious effects. In the context of Celiac disease, RA in combination with IL-15 can drive dendritic cell activation and production of IL12/23 p40 [32*]. Such activation results in impaired Treg priming and augmented effector responses to the dietary gluten antigen, gliadin [32]. Given the expanded knowledge of RA in regulating immunity, it will be important to further understand the contextual roles of RA, particularly in clinical settings.

Other dietary micronutrients

Micronutrients are important for systemic immunity and deficiencies lead to immunosuppression and/or immune dysregulation. These include, but are not limited to: iron, folate, zinc, vitamin A, vitamin C, vitamin E [33,34]. Of these, vitamin D has recently been shown to have profound and direct effects on T cell activation [35*]. Notably, vitamin D can control various aspects of intestinal immunity (Figure 1). Intraepithelial lymphocytes of the CD8αα TCRβ+ subset are reduced in Vitamin D receptor (VDR) deficient mice and appear to be developmentally dependent on VDR signaling [36]. Several studies have linked vitamin D deficiency to inflammatory bowel disease (IBD) susceptibility both in mouse studies of vitamin D receptor deficient mice and in correlative studies of IBD patients [37]. Of interest, activated Tregs and effector T cells express high levels of folate receptor 4 (FR4), although a role for folate in influencing Treg function remains unclear [38]. Because of the constant exposure of the GI mucosa to dietary components, animal models of micronutrient deficiencies warrant future exploration. Future studies will need to mechanistically dissect the effects of different micronutrients on the immune system and specifically their impact on mucosal immunity.

Additional dietary and commensal derived substances

Dietary intake, either directly or via its processing by commensal communities, provides a multitude of metabolites with the potential to influence immune function (Figure 1). Additionally, dietary intake of food provides non-nutritive ligands for the immune system as well. Two recent studies indicate that dietary derived Ayrl Hrydrocarbon Receptor (AHR) ligands from cruciferous vegetables, such as broccoli, may be important signals for intestinal immune development and immune responses [39,40**]. Indeed, AHR deficient mice have a defect in intraepithelial lymphocyte (IEL) development in both skin and intestine [39]. Using diets with low amounts of AHR ligands or enriched in AHR ligands, this study demonstrates that dietary derived AHR ligands control intestinal but not skin IEL development [39]. Complementing these findings, intestinal lymphoid tissue inducer (Lti) populations are developmentally regulated by dietary AHR Ligands and AHR deficient mice are highly susceptible to infection with the attaching effacing intestinal pathogen Citrobacter rodentium [40]. These findings further highlight the central role of dietary components in controlling mucosal immunity.

Another node of interaction that is important to consider in the GI tract is the sensing of commensal metabolites by the immune system. An example of such communication is the metabolism of dietary fiber by commensal bacteria of the Bacteroidetes phylum into immunomodulatory short chain fatty acids (SCFA) such as butyrate and acetate [41]. SCFA binding to GPR43 receptor on neutrophils acts to restrain their activation [41]. Consequently, mice lacking GPR43 have augmented inflammatory responses, while exogenous administration of SCFA to wild type mice was clinically beneficial [41].

Interdependence of diet, commensals and immune interactions

Understanding the role of micronutrients in intestinal immune responses requires integration of the complex and intimately linked relationship between diet, commensals and the immune system (Figure 2). A high fat diet is an example of dietary effects on both commensals and immunity. Mice fed a high fat diet have altered commensal communities that not only affect energy harvest, but that can also alter immunity and increase the severity of disease states such as colitis [42–44*]. Additionally, dietary deficiency can alter commensal communities as demonstrated by a study showing that Vitamin A deficient mice have altered commensal communities as well as immune deficits [26].

Immunodeficiency can also have profound effects on nutrient absorption and metabolism. For instance, mice lacking B cells have a dietary malabsorption [45]. Conversely, mice deficient in the receptor for bacterial flagellin, TLR5, have a dysbiotic commensal microflora that leads to increased weight gain and the development of metabolic syndrome [46**]. Further, these metabolic alterations can be transmitted to wild type germ-free mice by colonizing them with gut flora from TLR5 deficient mice [46]. This implies that immunodeficiencies can alter the gut flora’s composition and the metabolic capacity of both the flora and the host.

Children with severe malnutrition represent an example of both immunodeficiency and dietary deficiency coinciding in a clinically relevant and important way. Malnourished children have dramatically altered commensal communities likely with diminished metabolic capacity and impaired ability to promote host energy absorption; a problem that could persist even after refeeding [44,47,48]. Thus, through the measurement of commensal metabolites in healthy states, as in the case of SCFA, the mucosal immune system may be able to monitor the diversity and metabolic function of intestinal commensal communities. Perhaps, appropriate intestinal immunity requires a diversity of signals that are lacking in a malnourished state. Thus, considering the interdependency of these three factors, an important challenge over the next few years will be to develop experimental strategies aimed at defining critical regulators of this complex and interdependent network.

Conclusion and Future directions

Dietary derived substances and commensal metabolites are critical regulators of intestinal immunity. Both homeostasis and response to infection in the GI tract requires the integration of environmental signals derived from commensal bacteria as well as dietary sources. Of these, RA has emerged as a dominant regulator of immune responses in the GI tract as well as systemically. Recent advances have also brought to light the importance of other dietary substances, such as AHR ligands and commensal derived SCFA, in controlling mucosal immune responses. Are these unique examples or does the mucosal immune system respond to the multitude of available dietary cues? It will be important to test the generalizability of these two sets of findings. We could speculate that because the primary role of the gut is food absorption, the immune system of the GI tract is more finely tuned to respond to dietary status than we currently appreciate [49].