In this study, we have compared the role of IL-23R in the EAE and OTII and DTH models and found concordance of the results. During the initial 5 to 7 days of the response, OTII cells were unaffected by IL-23R deficiency and their frequencies in the blood were similar to wild-type cells. Likewise, at the early time point in the chimera EAE system the ratio of wild-type to Il23ra−/− CD4+ cells in the CNS was unaffected, although numbers were very low. At day 10, there were obvious functional defects apparent in the phenotype of the Il23ra−/− OTII cells, and their frequency in the blood remained low compared to the large increase of wild-type cells. Similarly, at onset of EAE (day 11), it was clear that the number of Il23ra−/− CD4+ cells had not greatly increased in the CNS compared to wild-type cells, and the Il23ra−/− CD4+ cells that were present were not producing IL-17, consistent with a low frequency of Il23ra−/− antigen-specific effector cells available to enter theCNS from the circulation at this time point. Therefore, we can combine results from these two systems to understand the requirements for IL-23 signaling during TH-17 development in the lymph node and function in target tissues.
Our results have identified a far more central function for IL-23 in TH-17 differentiation than previously thought. As expected, early TH-17 development appeared normal in the absence of IL- 23R, with expansion of antigen-specific T cells and induction of IL-17. However, these cells did not progress in their differentiation and did not down-regulate CD27 or re-upregulate IL7Rα. Il23ra−/− cells also failed to generate large numbers of IL-17-producing progeny that could enter the bloodstream and therefore did not infiltrate tissues in large numbers or successfully initiate inflammation. Importantly, neutralizing antibody experiments demonstrated that these effects relied on IL-23 signals received between days 4 to 6, before the appearance of large numbers of effector TH-17 cells. Furthermore, the timing of this requirement for IL-23 signaling also directly preceded the effects on proliferation of IL-17+ cells. Taken together, these results show that rather to increase the number or to maintain already-differentiated TH-17 cells, IL-23 is in fact required for the differentiation process to generate large numbers of effector TH-17 cells.
Studies have demonstrated that IL-23p19-deficient mice have a normal response to both toxoplasma36
infections. However, while IL-23 does not appear to play a central role in protective responses to these infections, the presence of IL-23 can partially compensate for the absence of IL-1236
. Our data further confirms that IL-23R is not required for a fulminant TH1 response, and also that IL-23 does not repress an outgrowth of TH1 cells since there was no increase in IFN-γ+ cells in its absence. However we did observe a reduction in IFN-γ+IL-17+ cells. These double-positive T cells are found in many inflammatory sites, but their role in disease pathogenesis is still unclear. Our results suggest that the double IFN-γ IL-17 producers are more TH-17-like than TH1-like, as they were reduced IL-23R-deficient mice, just as are IL-17+ cells. Finally, the normal TH1 response in absence of IL-23R suggests that the defects in activation seen in Il23ra
−/− T cells are specific to TH-17-inducing conditions rather than to global activation of T cells.
The number of activated T cells in the lymph node at any given time is a function of the rates of proliferation, death and migration. The results presented here suggest the reduced proliferation rate of Il23ra
−/− OTII cells, particularly IL-17+ cells, later in the response results in the failure to generate large numbers of cells that egress into the blood, and together these two factors balance so that the overall number of OTII cells in the lymph node is not greatly altered compared to wild type. Given that the Il23ra
−/− population never attains a high frequency of single IL-17+ or CD27lo cells, and that there is not a great increase in apoptotic cells, it seems likely that it is the actual generation of effector cells that is affected, rather than the survival of these effector cells. This would be consistent with the decreased BrdU uptake and reduced proliferation rate in the absence of IL-23, as cell division is known to be linked with cellular differentiation32
. We therefore conclude that the major function of IL-23 in TH-17 biology is to drive terminal differentiation; in its absence, TH-17 cells experience ‘arrested development’ leading to impaired function.
The following question then arises: is IL-23 merely driving proliferation of developing TH-17 cells and thus facilitating their differentiation? There are several observations that suggest this is not the case. In the initial description of the predominant role of IL-23 rather than IL-12 in EAE induction, we demonstrated that forced expression of IL-23 in the brain of IL-23-deficient mice was able to restore their susceptibility to inflammation2
. Using the OTII model, we have shown here that injection of IL-23 in the site of antigen challenge was able to partially restore the DTH response in IL-23-deficient mice. These two findings suggest that although TH-17 effector generation in the lymph node is greatly impaired in the absence of IL-23, for the few cells that do migrate into the blood, exposure to IL-23 in the periphery may be sufficient to activate effector functions (i.e. drive terminal differentiation) to a sufficient level to induce inflammation. In the DTH model, one injection of soluble cytokine was used and the response was temporary. In the EAE experiments, adenoviral expression resulted in more sustained IL-23 production most likely explaining the greater efficacy in disease induction. In addition, IL-23 enhanced IL-17 production by CNS-derived T cells in a short time period in vitro, and IL-23 has previously been shown to be required for production of other TH-17-associated pro-inflammatory cytokines such as IL-2215,16
. Taken together, these studies support the hypothesis that IL-23 can act both in the lymph node and peripheral tissues to drive terminal differentiation of effector cells, and that proliferation of cells developing in the lymph node is only one facet of IL-23 functions in promoting TH-17-mediated inflammation.
The failure of IL-23R-deficient T cells to re-express IL-7Rα after activation may have long-term consequences for these cells. Although it is down-regulated during early activation, IL-7Ra re-expression is required for survival of effector and memory T cells29,30,34
. There is currently little data available whether effective TH-17 memory responses can be generated in autoimmune models, but two vaccination studies using the pathogens Mycobacterium tuberculosis
and Bordetella pertussis
suggested this is possible43,44
. Elson et al
have reported increased numbers of apoptotic CD4+ T cells following long-term in vivo
neutralization of IL-23 in a colitis model, although the mechanisms were not clear19
. Indeed, we also observed a slight increase in caspase3+ IL17+ Il23ra
−/− cells on day 10, although the low frequency of IL-17+ cells makes it difficult to assess the significance of this observation. Overall then, it seems likely that survival of effector TH-17 cells and/or TH-17 memory formation would be impaired in the absence of signals from IL-23.
TH-17 development is known to depend on STAT3 signaling17,24,25
and expression of the transcriptional regulator RORγt10
. It has also been suggested that STAT4 may contribute to TH-17 development, since cells from STAT4 ‘knockout’ mice have partially reduced IL-17 production25
, although others report no role for STAT446,47
and we were unable to detect high levels of STAT4 phosphorylation in T cells following stimulation with IL-23 (data not shown). IL-6, IL-21 and IL-23 are all strong activators of STAT3 phosphorylation, and since IL-6 and possibly IL-21 are required for early differentiation of TH-17 cells, it is very difficult to investigate the requirement for STAT3 phosphorylation by IL-23 during later differentiation. However, we were able to confirm that STAT3 is phosphorylated by IL-23 ex vivo
in developing TH-17 cells, and also that STAT3 is required for TH-17 production of IL-17 in vitro
. Hence we believe that IL-23 acts through STAT3, although there may also be additional pathways activated.
In conclusion, we have shown that IL-23R is required for terminal differentiation and therefore function of TH-17 cells in vivo
. These findings also have important implications for targeting IL-23 and its receptor therapeutically in TH-17-mediated diseases in humans. Our results suggest that the beneficial effects of neutralizing IL-23 may not be instantaneous since IL-23 promotes but does not appear to be required for effector functions of already generated TH-17 cells. However, the effects should be long-lasting since ongoing TH-17 generation should be blocked, and potentially TH-17 memory responses will be impaired thereby impacting rates of disease relapse, as we have observed in the EAE model. In addition, the ‘upstream’ position of IL-23 in inflammatory cascade compared to downstream mediators such as TNF allows hope for greater efficacy. In some diseases such as psoriasis, there are strong indications that IL-23 acts not only on TH-17 cells but also innate immune cells, and in these circumstances targeting IL-23 may prove even more effective. The success of anti-IL-12 p40 clinical trials in psoriasis48
suggest this may be the case, but clinical testing of neutralizing IL-23 will specifically address this question.