Simultaneous analysis of T helper subsets (Th1, Th2, Th9, Th17, Th22, Tfh, Tr1 and Tregs) markers expression in periapical lesions reveals multiple cytokine clusters accountable for lesions activity and inactivity status

Previous studies demonstrate that the balance between pro- and anti-inflammatory mediators determines the stable or progressive nature of periapical granulomas by modulating the balance of the osteoclastogenic factor RANKL and its antagonist OPG. However, the cytokine networks operating in the development of periapical lesions are quite more complex than what the simple pro- versus anti-inflammatory mediators' paradigm suggests. Here we simultaneously investigated the patterns of Th1, Th2, Th9, Th17, Th22, Thf, Tr1 and Tregs cytokines/markers expression in human periapical granulomas. METHODS: The expression of TNF-α, IFN-γ, IL-17A, IL23, IL21, IL-33, IL-10, IL-4, IL-9, IL-22, FOXp3 markers (via RealTimePCR array) was accessed in active/progressive (N=40) versus inactive/stable (N=70) periapical granulomas (as determined by RANKL/OPG expression ratio), and also to compare these samples with a panel of control specimens (N=26). A cluster analysis of 13 cytokine levels was performed to examine possible clustering between the cytokines in a total of 110 granulomas. RESULTS: The expression of all target cytokines was higher in the granulomas than in control samples. TNF-α, IFN-γ, IL-17A and IL-21 mRNA levels were significantly higher in active granulomas, while in inactive lesions the expression levels of IL-4, IL-9, IL-10, IL-22 and FOXp3 were higher than in active granulomas. Five clusters were identified in inactive lesion groups, being the variance in the expression levels of IL-17, IL-10, FOXp3, IFN-γ, IL-9, IL-33 and IL-4 statistically significant (KW p<0.05). Three clusters were identified in active lesions, being the variance in the expression levels of IL-22, IL-10, IFN-γ, IL-17, IL-33, FOXp3, IL-21 and RANKL statistically significant (KW p<0.05). CONCLUSION: There is a clear dichotomy in the profile of cytokine expression in inactive and active periapical lesions. While the widespread cytokine expression seems to be a feature of chronic lesions, hierarchical cluster analysis demonstrates the association of TNF-α, IL-21, IL-17 and IFN-γ with lesions activity, and the association of FOXP3, IL-10, IL-9, IL-4 and IL-22 with lesions inactivity.