We report the presence of Th17 cell sub-populations with distinct in vivo tissue tropism which is regulated by the RA signal. The tissue tropism of these Th17 cells is determined by differential expression of gut homing receptors. Strikingly, the Th17 cell sub-populations with different tissue tropism induced inflammation in different parts of the intestine. We believe that this is the first demonstration of the presence of Th17 cell subsets with inflammatory activities in distinct compartments of the intestine.
What concentration of RA is really available for T cells undergoing antigen activation in vivo? The RA concentration in normal human blood plasma is ~4.9 ng/ml (28
). Comparably, ~2 ng/ml of RA was detected in rat serum. It is estimated that 50–75% of the RA in serum or plasma is At-RA. Therefore, the concentration of At-RA in normal blood plasma is estimated to be ~5 nM. The balance between RA-synthesizing retinaldehyde dehydrogenases (RALDHs) and RA-inactivating cytochrome P450RAI (CYP26) determines the availability of RA in a given tissue microenvironment. The expression patterns of CYP26 and RALDH2 are largely complementary in developing embryos (29
), and it is expected that a similar pattern of complementary expression of the two enzymes would occur in adults. Based on the sensitivity of the expression of CCR9 and α4β7 in response to RA, it is estimated that the intestinal antigen priming environment would have at least 2–3 nM and potentially higher levels of active RA (up to 30 nM). The concentration of RA in peripheral lymph nodes, where CCR9 is not induced, is expected to be < 1 nM. We estimate that RA levels between 0.1 and 30 nM are within the physiological range. RA concentrations higher than this range could be found in vivo but the need for that high RA signal is unclear.
A most characteristic feature of the Th17 cells induced at physiological levels (10–20 nM) of RA is the expression of both CCR9 and α4β7. These two trafficking receptors are somewhat different from each other in requirement for RA for persistent expression. α4β7 initially induced by RA persists even after subsequent antigen priming in the absence of exogenous RA. In contrast, expression of CCR9 on Th17 cells is transient requiring continued presence of RA. Moreover, CCR9 is not induced well in response to RA on the Th17 cells that were previously activated in the presence of suboptimal concentrations of RA. We found that α4β7 is a general homing receptor of Th17 cells for the intestine and associated lymphoid tissues, while CCR9 is a receptor more specific for the small intestine. This suggests that at least several populations of Th17 cells (CCR9+ α4β7+, CCR9− α4β7+, and CCR9− α4β7−) with different homing behaviors can be generated in different sites of the intestine depending on the RA availability during repetitive priming processes.
The potent activity of retinoid-induced Th17 cells in inducing small intestinal inflammation is notable and is associated with their expression ofα4β7 and CCR9. α4β7 is widely expressed by intestinal lymphocytes and serves as the major homing receptor (32
).α4β7 is an adhesion molecule for MAdCAM-1 (32
) and can guide the Th17 cells to the entire gut system. It appears that even basal levels of α4β7, induced in the presence of low (serum) levels of RA, is required for migration of Th17 cells into the intestine and associated lymphoid tissues. Thus, higher expression of α4β7, induced at optimal levels of RA over the physiological range, confers Th17 cells with enhanced ability to stay within the intestine and to induce inflammation. CCR9 is expressed more specifically by the T cells in the small intestine and acts as a homing receptor specific for this tissue (35
). CCR9 can guide the Th17 cells to the small intestinal lamina propria compartment close to epithelial cells where CCL25 is expressed (35
), and therefore would cause the more focused migration and inflammation in the small intestine. Consistently, in our study, Itgβ7 deficiency had a broader impact on Th17 cell migration to the intestine, while CCR9 deficiency had a specific effect on their homing to the small intestine. In line with the differential roles of these trafficking receptors in determining the tissue tropism of Th17 cells, Itgβ7 deficiency had greater impact than CCR9 deficiency on the inflammatory activity of the retinoid-induced Th17 cells. This is supported by the fact that without proper expression of Itgβ7 (or α4β7), Th17 cells fail to migrate to any site of the entire intestinal system including the small intestine despite their normal expression of CCR9. This finding is in line with the established roles of these trafficking receptors for other T cells (32
), and suggests that Th17 cells do not deviate from the general trafficking behavior of gut T cells. Some reported that IL-22 has a protective role in intestinal inflammation (12
) while others reported pro-inflammatory roles of IL-22 in intestinal inflammation (42
). We would like to point out that there was no significant difference in expression of IL-22 by control and RA Th17 cells (Figure S5
Because of the previously reported in vitro
phenomenon that induction of Th17 cells is suppressed by RA (19
), it has been assumed that vitamin A antagonized the population of Th17 cells in the intestine. Our finding with vitamin A-deficient mice indicates that the RA signal is actually important for presence of Th17 cells in the small intestine. A similar decrease of small intestinal Th17 cells in vitamin A deficiency was observed in a mouse model of chronic intestinal inflammation (44
), suggesting that it occurs broadly in both normal and inflammatory conditions. We don’t rule out the possibility that induction of Th17 cells could be somewhat suppressed by the RA in the small intestine in vivo
. However, this small decrease due to the presence of RA would be greatly compensated by the increase of gut homing receptor-expressing Th17 cells. Thus, there would be an overall increase in numbers of gut Th17 cells in the intestine in response to RA.
Our results provide useful targets of intervention in regulation of the inflammatory activities of Th17 cells in the intestine. It is possible to regulate the inflammatory activity of Th17 cells in the small versus other compartments of the intestine by selective blocking of CCR9 or Itgβ7. This projection is well supported by the U.S. Food and Drug Administration (FDA)-approved and other proposed therapies targetingα4 or α4β7 integrin. For examples, blocking of the Itgα4 using monoclonal antibodies to integrin α4 is a FDA-approved treatment for Crohn’s disease (45
). It was shown effective in amelioration of colitis in animal models too (45
). While this therapy targets all α4 integrins including α4β7 and α4β1, monoclonal antibodies more specifically blocking Itgβ7 or MAdCAM-1 were also effective in inhibiting colitis in animal models (48
). We propose that these reagents can act on the α4 integrins of Th17 cells and, thus, can block their migration and inflammatory activities in the intestine. If the inflammatory activity of Th17 cells is localized to the small intestine, blocking of CCR9 would be more specific in treating the localized inflammation without the potential side effects of broad immune-suppression in the body.