TGFβ and Retinoic Acid Intersect in Immune-Regulation

Abstract

Transforming growth factor (TGFβ) prevents T_H1 and T_H2 differentiation and converts naïve CD4 cells into Foxp3-expressing T regulatory (Treg) cell.^1,2 In sharp contrast, in the presence of pro-inflammatory cytokines, including IL-6, TGFβ not only inhibits Foxp3 expression but also promotes the differentiation of pro-inflammatory IL17-producing CD4 effector T (T_H17) cells.^3–5

This reciprocal TGFβ-dependent differentiation imposes a critical dilemma between pro- and anti-inflammatory immunity and suggests that a sensitive regulatory mechanism must exist to control TGFβ-driven T_H17 effector and Treg differentiation. A vitamin A metabolite, retinoic acid (RA), was recently identified as a key modulator of TGFβ-driven-immune deviation capable of suppressing T_H17 differentiation while promoting Foxp3⁺Treg generation.^6–10

RA and TGFβ are both abundantly produced in the gut and TGFβ is crucial for both systemic and mucosal immune-regulation.^11,12 Although the so-called thymus-derived naturally occurring Treg cells (nTreg) are important for the control of a variety of autoimmune processes, it has been shown using monoclonal TCR transgenic mice devoid of nTreg that peripheral neoconverted Foxp3⁺ Treg cells are efficient and sufficient for oral tolerance induction.² Furthermore, Mucida et al. also showed that blocking of TGFβ during feeding of the antigen (OVA) inhibited both; the establishment of oral tolerance and the peripheral conversion of OVA-induced Treg cells.² The abundant production of TGFβ and RA in the mucosa and the ability of RA to promote TGFβ-dependent Treg differentiation may thus be directly related to the increased frequency of Foxp3-expressing Treg cells in the lamina propria in normal mice.^6–10,13

TGFβ and RA each are known to play significant roles in a variety of developmental processes, including the differentiation of lymphocyte lineages. Whereas TGFβ mediates the direct inhibition of T_H1 and T_H2 cytokine polarization concomitant with the generation of Tregs.¹⁴ RA, in contrast, is a potent stimulator of T_H2 differentiation but a profound inhibitor of IFNγ synthesis.¹⁵ In addition to their separate actions, the functions of TGFβ and RA are also known to merge in a variety of biological processes, including embryogenesis, organ development and carcinogenesis.¹⁶ For example, TG-interacting factor (TGIF) is a transcriptional repressor common to the TGFβ and retinoic acid signaling pathways.¹⁷ Moreover, mice with deficiency in the enzyme required for RA production, retinaldehyde dehydrogenase-2 (Raldh2), die before birth with several developmental defects and reduced TGFβ1.^18–20 On the other hand, RA can also inhibit TGFβ mediated effects, such as lung fibrosis.²¹

The finding that RA plays a central role in directing the immunological function of TGFβ expands the consequences of their interrelationship to the adaptive immune system.^6–10,13,22 Recent evidence shows that the signaling through RA-receptors may play an important role in the control of inflammation in the gut.^6–10,13 Exogenous RA was shown to inhibit induction of T_H17 cells in vivo using an infection model whereas injection of RAR antagonists resulted in decrease of Foxp3⁺Treg cells in the lamina propria.⁶ Iwata and co-workers have previously shown that RA production by mucosal DCs is crucial for the homing of T cells to the intestinal lamina propria,²³ and Mora et al. extended this finding to B cells migration to the gut and IgA class switching.²⁴ Whether the production of RA by mucosal DCs is crucial for the development of oral tolerance and for the conversion of naïve T cells into Foxp3⁺Treg in the gut, as well as for the mucosal in situ control of T_H17 cells, is not yet known.

Our study and those of others show that RA signaling through RAR receptors in the T cell blocks the inhibitory effects of inflammatory cytokines, such as IL-6, on the TGFβ mediated Foxp3 induction. Similarly to the RA and TGFβ pathways interaction, several studies have shown that RA may synergize or antagonize with IL-6 signaling or production.^21,24 Moreover, RA has been shown to improve clinical symptoms and reduce the levels of inflammatory cytokines, including IL-6, TNFα and IFNγ in a model of arthritis,²⁵ an autoimmune disease shown to correlate with increased production of IL-17.²⁶ Finally, it was shown that RA directly inhibits retinoic acid orphan receptor γ T (RORγt) that is involved in T_H17 differentiation and which requires IL-6 for its expression.²⁷ It is not known, however, whether RA antagonistic effects on IL-6 signaling extend to the recently described IL-21 pathway of T_H17 differentiation.^28–30

Transcription factors STAT5 and STAT3 have been shown to be important for the transcription of Foxp3 and IL-17 respectively.^22,27,31 The enhanced expression of Foxp3 in the presence of RA suggests a potential relationship between STAT5 and RARs in a similar fashion as the cooperation between STAT3 and RORγt. It is therefore perhaps not a coincidence that RORγt shows strong homology with the RARs and also appears to function in the context of transcriptional activators and repressors.³² STAT5 and RARs have also been shown to physically interact in vivo to promote RAR-mediated transcription.³³ In addition, it was shown that the STAT5 consensus binding site overlaps with a RAR-response element which leads to promoting coordinated transcription activity rather than competition for the same site.³³ The cooperation between STAT5 and RARs resulted in STAT5-enhanced responsiveness of the RARs to RA induced transcription of target genes.³³ It was further demonstrated that RAR and STAT5 can bind the same repressor of transcription, SMRT, which can be released by RA.³⁴ RA mediated effects may thus reflect the intense communication between the STAT and RAR families of transcription factors, which has not been explored for the differentiation of T lymphocytes. It is also possible that RA might synergize with Smads that act downstream of TGFβ receptor signaling, and/or with the transcription factor Runx3, which is involved in the induction of CD103 expression and which physically interacts with Smads to cooperate in TGFβ mediated signaling.³⁵

The immune regulatory mechanism we have delineated has particular relevance for the mucosal immune system. The intestinal mucosa forms the largest surface that is exposed to microbes, innocuous and pathogenic, and diet proteins; and also houses the largest proportion of lymphocytes that in physiological conditions have an immune quiescent state.³⁶ Therefore, an improper balance between inflammatory and suppressive immunity can jeopardize mucosal homeostasis. The abundant production of RA by the intestinal epithelium and dendritic cells, and the dominance of RA over IL-6 in balancing the effects of TGFβ, may account for the predominance of Foxp3⁺ T cells in the intestine, allowing tolerance to prevail in the face of the extensive microbial load.