Although insufficient stores of vitamin A have long been linked to impaired immunity to pathogens, the role of vitamin A metabolism in the regulation of CD4+ T cell responses remains poorly understood. In our current study, we reveal that the retinoic acid/RARα signaling axis is essential for adaptive CD4+ T cell immunity. Specifically, we demonstrate that mucosal TH-1 and TH-17 responses to oral infection and vaccination are compromised upon loss of vitamin A. These impairments are unlikely to be manifestations of a developmental defect propagated upon loss of vitamin A, as RA rapidly restores mucosal TH-1 and TH-17 responses. This finding, in particular, indicates that this metabolite is the cardinal mediator of vitamin A dependent immunity in vivo. Strikingly, genetic ablation of Rara is sufficient to recapitulate the phenotype of VAI mice, both at steady-state and during infection. Furthermore, T cells lacking Rara or subjected to RA receptor antagonism display early TCR activation defects and proliferate less efficiently in response to T cell stimulation. Thus, the RA/RARα axis controls the fate of adaptive immunity, at least in part, via cell autonomous effects on CD4+ T cells and reveals one potential explanation for the broad control of this pathway over various T cell fates.
RA has been proposed to serve as a switch factor in the induction of regulatory versus inflammatory T cells. However, our data demonstrate that RA is a physiological mediator of not just GALT Treg induction, but also inflammatory TH
-1 and TH
-17 responses. Although we attribute these defects to a role of RA/RARα in activation, as discussed below, we do not discount the possibility that indirect effects of RA in the milieu can also affect adaptive T cell responses. Importantly, the majority of CD4+
T cells that displayed an RA signature (based on α4β7), co-expressed T-bet, the transcription factor required for TH
-1 commitment (Szabo et al., 2000
). Further, in vivo
add-back experiments demonstrated that RA was capable of restoring TH
-1 responses in VAI mice. These data are somewhat in conflict with previous reports that have suggested that RA is a negative regulator of TH
-1 inflammation (Cui et al., 2000
). For instance, VAI mice produced abnormally high levels of IFN-γ during infection with the nematode, Trichinella Spiralis
and failed to elicit a proper and robust TH
-2 response (Carman et al., 1992
). In this system, RA was shown to decrease IFN-γ production when added to in vitro
restimulated cell suspensions and was, hence, characterized as a suppressor of TH
-1 responses (Cantorna et al., 1994
). However, this type of “add-back” experiment is difficult to interpret, especially when considering that the cells treated in culture were likely of a heterogeneous activation status. In this regard, RA was shown to be able to inhibit effector/memory T cell cytokine production (Hill et al., 2008
). Integrating these data into a working model suggests that RA signaling is potentially biphasic - driving T cell activation/differentiation during the early stages of an immune response, but regulating the amplitude of effector responses at later stages. This could be a particularly effective strategy to allow proper initiation of immune responses while minimizing tissue damage.
-17 cells are elicited via the actions of multiple cytokines, including TGF-β, and any combination of IL-6, IL-21, IL-23 and/or IL-1 (Korn et al., 2009
). We found that diminished vitamin A prevented the acquisition of a robust TH
-17 response in vivo
. The ability of RA to restore TH
-17 responses in VAI mice initially appears discordant with other studies that have reported negative effects of RA on IL-17 production in vitro
(Elias et al., 2008
; Mucida et al., 2007
) and in certain animal models of autoimmune disease (Xiao et al., 2008
). However, in systems that have scrutinized the effects of RA at low doses (Wang et al.), and in conjunction with microbial stimuli, such as TLR5 ligands (Uematsu et al., 2008
-17 generation was shown to be unaffected or enhanced, respectively. Thus, in physiological settings and microbial rich environments, RA can potentially amplify the inflammatory tone of the mucosal environment. In this regard RA may act in a comparable fashion to TGF-β on T cell differentiation, leading to tolerance or immunopathology depending on the presence of inflammatory stimuli (Zhou et al., 2008
). Lending further support for a role of RA in the generation of TH
-17, several recent studies showed that TH
-17 cells were virtually ablated in the Pp and Lp of VAI mice during steady-state (Cha et al.; Wang et al.). In one of these studies, the authors attributed this finding to impeded migration of these cells into the gut; yet, TH
-17 cells were not increased elsewhere (Wang et al.). In another study, the deficit in TH
-17 was noted in young mice on a vitamin A deficient diet, before the defect in T cell homing capacity to mucosal sites should have set in (Cha et al.). Taken together, physiological concentrations of retinoids appear to sustain TH
-17 development and maintenance.
Rara−/− mice complemented VAI mice on multiple levels. First, the number of Lp effector T cells was reduced in both animals compared to their control counterparts (~3 fold for VAI; ~2 fold for Rara−/−). These findings suggest that during homeostatic activation, RA/RARα signaling is critical for the upregulation of homing receptors. Remarkably, transient treatment with RA restored both T cell equilibrium and the CD4+ T cell response within the Lp of VAI mice upon challenge. Since RA restored both of these parameters, it is difficult to dissect the contribution of homing versus activation to the rescue of immune responses in this tissue. However, despite defects in gastrointestinal homing, VAI mice that were infected systemically with T. gondii also mounted a markedly impaired TH-1 response. This outcome could be the product of both direct and indirect actions of RA/RARα signaling on T lymphocytes.
As evidence that this pathway can function directly through T cells, we demonstrate that RARα mediates signal transduction events downstream of the TCR that govern T cell activation. Recent findings indicate a role for nutrient metabolism in T cell activation. For instance, vitamin D/vitamin D receptor (VDR) signaling was shown to promote the proliferation of human T cells in response to TCR stimulation via the induction of PLCγ (von Essen et al.). The DNA binding capacity of VDR presumably mediates this induction via transcription. RARα is also recognized to regulate gene expression in the same fashion (Chambon, 1996
). As RA, as well as other retinoids, are present in the serum and tissues of mice (Kane et al., 2008a
; Kane et al., 2008b
), it is intriguing to speculate that these compounds exert constitutive effects on the phosphorylation status, localization and/or conformation of RARα in T cells, which may in turn regulate proteins involved in T cell signal transduction pathways. Short-term incubation (< 1 hr) with a pan-RAR antagonist impaired T cell Ca2+
mobilization in a manner similar to that observed in Rara−/−
T cells. One explanation for this finding is that transcriptional modification via RARα regulates the expression or activity of a mediator of T cell activation. Alternatively, RARα may potentially facilitate TCR dependent signal transduction through extranuclear activity. For example, in a neuroblastoma cell line, RARα was described to interact with the p85 subunit of phosphoinositide 3-kinsase in an RA dependent manner (Masia et al., 2007
Although we identify a novel function for RA/RARα signaling in T cells, RARα may also affect the function of APC, including DC. For instance, RARα ligands were shown to synergize with inflammatory mediators to enhance the activation of human Langerhans cell-type dendritic cells (Geissmann et al., 2003
). Thus, it is possible that altered APC function also contributes to impaired adaptive immune responses in VAI and Rara−/−
mice. In this regard, we noted a specific defect in the capacity of LpDC from VAI mice to produce IL-6, while other proinflammatory mediators, such as TNF-α and IL-12/23p40 remained intact (Figure S4
). This finding is consistent with the reduction of TH
-17 cells in VAI mice during steady-state conditions, as previously reported (Cha et al.; Wang et al.), and in response to oral vaccination, as reported in our study. RA signaling may also influence other cell types that can shape T cell responses. Recently, RA was revealed to promote TGF-β activation in non-hematopoietic follicular DC (Suzuki et al.). This effect may extend to other cell types of stromal/mesenchymal origin as well as DC and potentially contributes further to the concomitant loss of both Foxp3+
-17 induction in VAI mice. Thus, RA/RARα signaling may converge on both innate and adaptive arms of immunity.
In summary, the GI tract must be able to tolerate constant exposure to food antigen and commensals, while maintaining the capacity to rapidly respond to encounters with pathogen. These conflicting pressures confront the host immune system defending the GI tract with a unique challenge. One would predict that the most judicious strategy to respond to this spectrum of recurring challenges would involve a conserved pathway that can readily adjust to environmental cues. Here we identify the RA/RARα signaling pathway as fitting this mode of host control, promoting Treg generation and likely tolerance during steady-state conditions, while adaptive T cell responses in the face of pathogen. As such, we propose that RA regulates adaptive immunity in a manner that is symmetrical to TGF-β where accompanying signals dictate whether a response ultimately becomes regulatory in nature or inflammatory. An important consideration is that adaptive immune responses often involve multiple waves of antigen presenting cell recruitment. Based on the systemic RA mediated signals that we observe during infection, it will be interesting to examine how newly recruited APC contribute to the RA/RARα signaling axis during inflammation. Finally, the requirement of RARα for T cell activation suggests that this pathway may have evolved early with the development of adaptive CD4+ T cell responses to coordinate host protection.