The present study provided for the first time comprehensive data on the changes of Th17/Treg cells and cytokines IL-1β,IL-6,IL-17A,IL-21,IL-23 and TGF-β in the development and progression of CRC. It demonstrated in both CRA and CRC patients an increase of circulating Th17 cells in early stages and an increase of circulating Treg cells in advanced stages, and an increase of tumor infiltrating Th17 cells in advanced CRC tissues. The changes of Th17 cells along disease progression were accompanied by alterations of IL-1β, IL-6, IL-17A, IL-23 in serum and IL-1β, IL-6 in tumor tissues. In vitro studies further supported that the expansion of the Th17 cells were regulated by IL-1β, IL-6 and TGF-β in different combinations and/or concentrations.
Previously, the elevated percentage of Th17 cells were detected in blood, bone marrow, and spleen in mouse tumor models, and in peripheral blood, malignant ascites and tumor tissues in patients of advanced ovarian, pancreatic, renal cell carcinomas, melanoma, and breast and colon cancers [29, 30]. In gastric cancer, there were elevations of Th17 cells in the peripheral blood as well as tumor-draining lymph nodes, both of which were associated with clinical stages of cancer development . However, in ovarian cancer patients, the percentage of Th17 cells appeared higher in tumor but lower in peripheral blood or PBMCs and tumor-draining lymph nodes [32, 33]. Our data showed that the frequency of Th17 cells was markedly increased in the circulation of both CRA and CRC patients, but became significantly lower as the diseases progressed to the advanced stages. Moreover, our observations were consistent with an animal study, in which the Th17 cells were increased in tumor tissues as disease progressed and reached to a maximal level in advanced tumors .
A previous study showed that IL-1β, IL-6 and TGF-β were involved in differentiation and expansion of the Th17 cells in ovarian cancers; IL-1β and IL-6 promoted, whereas TGF-β inhibited, Th17 cell expansion . Our results showed that the changes in Th17 cell number along disease progression were accompanied by variations of IL-1β, IL-6 and IL-23 levels, suggesting the association of these cytokines with Th17 cell expansion. Given that the variation of cytokine levels, as seen in TGF-β, did not consistently follow the change of Th17 cells, we speculated the existence of an optimal level of these cytokines in regulating Th17 cell expansion. Indeed, we found that TGF-βlo, IL-6 lo or IL-1βhi increased Th17 cell number in PBMCs. However,IL-6hi plus TGF-βhi unexpectedly promoted Th17 cell expansion, suggesting that a complex cytokine context, rather than the change of individual cytokines, is more likely involved in regulating Th17 cells. In the early stages, accumulation of Th17 cells in tumor tissues may be supported by high concentrations of TGF-β and IL-6. However, following tumor progression, high level of IL-1β, and possibly IL-23 as well, with the reduced levels of IL-6 and TGF-β may become supporting cytokine milieu for the expansion of Th17 cells in tumor tissues.
In this study, we detected the reduced level of IL-1β and increased frequency of Th17 cells in the circulation of CRCs as compared with that of CRAs. Since the IL-23 level is commonly elevated in the circulation of CRC patients, we speculated that IL-23 or other unknown factors, rather than IL-1β, may be more responsible for the expansion of Th17 cells in the circulation of CRC patients. Nevertheless, new studies are needed to test this hypothesis.
Previous reports showed that IL-21 promoted differentiation of human naïve CD4+T cells into Th17 cells . IL-21 was found essential in patients of inflammatory bowel diseases (IBD) in promoting IL-17 production in anti-CD3/CD28-stimulated LPMCs, whereas IL-1β, IL-6, IL-23 and TGF-β exerted no effects . However, our observation showed no significant difference in IL-21 level between normal tissues and tumor tissues or between early and advanced CRC tissues. This discrepancy may be interpreted by the difference in immunological mechanisms for regulating Th17 cells between IBD and cancer.
Previous studies showed that Th17 cells can be recruited into tumor microenvironment from the circulation . These findings are supportive at least in part to our observations that, in advanced CRCs, the Th17 cells became reduced in the circulation but increased in tumor tissues. We cannot of course exclude another possibility that the Th17 cells were converted into Treg cells during tumor progression. The emerging evidence suggested that Th17 cells are of functional plasticity and can be converted into Treg cells in vitro and these cells cannot change back to Th17 cells even under the highly favorable conditions [35, 36]. Nevertheless, new studies to test these two possibilities are warranted.