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

Abstract

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.

Figure 1

Expression of individual mRNAs, with normalization to housekeeping genes, in periapical granulomas. Total RNA was extracted from periapical granulomas (experimental groups, N=110) and periodontal ligament (control group, N=26), and levels of RANKL and OPG mRNA were measured quantitatively by RealTimePCR using TaqMan chemistry. Based in profile of RANKL/OPG expression29 the lesions were then categorized into active (RANKL>OPG) or inactive (RANKL»OPG and RANKL

Figure 2

Patterns of cytokine expression in active and inactive periapical granulomas. Total RNA was extracted from periapical granulomas (N=110) and periodontal ligament control samples (N=26), and levels of TNF-α(classic pro-inflammmatory cytokine), IL-10 (Treg and Tr1 marker), IFN-γ (Th1 marker), IL-4 (Th2 marker), FOXp3 (Treg marker), CTLA4 (Treg marker), TGF-β (Treg and Th3 marker), IL-9 (Th9 marker), IL-17A (Th17 marker), IL-17F (Th17 marker), IL-21 (Th17 or Tfh marker), IL-23 (Th17 marker) and IL-22 (Th22 marker) were measured quantitatively by RealTimePCR using TaqMan chemistry. Based on the profile of RANKL/OPG expression29, the lesions were then categorized into active (RANKL>OPG) or inactive (RANKL≈OPG and RANKL

Figure 3

Patterns of cytokine expression in the clusters associated with active and inactive periapical granulomas nature. Hierarchical analysis demonstrated that the inactivity pole was characterized by the highest OPG levels, sequentially followed in a downward way by FOXp3, IL-10, IL-9, IL-4 and IL-22. OPG and IL-22 expression profiles were relatively stable within inactive clusters; FOXP3 and IL-10 levels prevail in the clusters located in the inactivity pole edge, while a significant variation in expression of IL-4 and IL-9 was verified in specific clusters. On the other hand, the lesions activity pole was characterized by the highest expression of TNF-α, downward followed by RANKL, IL-21, IL-17 and IFN-γ. High levels of TNF-α expression were a hallmark of all active clusters, and RANKL expression prevail in the clusters located in the activity pole edge; also, a fairly specificity in the IFN-γ, IL-17 and IL-21 expression peaks was verified in definite clusters within active lesions. Interestingly, one cluster from inactive lesions subset, presented a relatively high expression of IL-17. The cytokines IL-23 and IL-33 were not significantly associated with lesions’ status