POTENCIJALNI UTICAJ IL-33/ST2 SIGNALNOG PUTA NA GUBITAK ALVEOLARNE KOSTI POTENTIAL EFFECT OF IL-33/ST2 SIGNAL PATHWAY ON ALVEOLAR BONE LOSS

Uvod: ST2 je član IL-1R familije receptora, dok je interleukin-33 (IL-33) njegov prirodni ligand. Kako se u ekstracelularni prostor oslobađa iz nekrotičnih ćelija, IL-33 može da ima ulogu „alarmina“ koji obaveštava imunski sistem o postojanju destrukcije tkiva. S obzirom da je ST2 receptor eksprimiran na gotovo svim ćelijama imunskog sistema, IL-33/ST2 signalni put ima važnu ulogu u patogenezi brojnih bolesti u kojima uglavnom aktivacija ovog puta podstiče razvoj Th2 imunskog odgovora. Protektivna ili proinflamatorna uloga IL-33/ST2 signalne osovine direktno je zavisna od dominantnog imunskog odgovora koji je u osnovi ovih oboljenja. Podaci iz literature o uticaju IL-33/ST2 signalnog puta na resorpciju alveolarne kosti su veoma oskudni. Mi smo ispitali uticaj delecije ST2 gena i administracije IL-33 na periapeksnu inflamatornu destrukciju kosti kod BALB/c miševa. Periapeksne lezije ST2-/miševa sadržale su veći procenat CD4+ T limfocita, CD3+CCR6+ T limfocita, IFN-γ-, IL-17-, TNF-αi IL-6produkujućih ćelija u okviru gejtovanih CD4+ T limfocita u poređenju sa lezijama WT miševa. Nasuprot tome, administracija IL-33 kod WT miševa prouzrokovala je smanjenje procenta CD4+ T limfocita koji produkuju proinflamatorne citokine i povećanje procenta IL-4-produkujućih ćelija. Zaključak: IL-33/ST2 signalni put negativno reguliše intenzitet periapeksne destrukcije alveolarne kosti prevencijom razvoja Th1/Th17 imunskog odgovora i ukazuju na moguću protektivnu ulogu IL-33 u terapiji gubitka alveolarne kosti. Buduća istraživanja implementiraće novu strategiju lečenja u stomatološku praksu.


Introduction Structure and function of interleukin-33 (IL-33)
Interleukin-33 (IL-33) is a new member of IL-1 cytokine family.It was identified in 2005 by Schmitz et al. as an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces Th2-associated cytokines 1 .Unlike IL-33, the other IL-1 family members, IL-1, IL-1Rа and IL-18, primarily induce Th1 immune response 2 .IL-33 is chromatinassociated nuclear factor in high endothelial venules with transcriptional properties.IL-33 acts intracellularly as a nuclear factor and extracellularly as a cytokine 3 .This cytokine is constitutively expressed in many tissues (lung, brain, skin, spinal cord).Fibroblasts, endothelial and epithelial cells are the main cellular source of IL-33 4,5 .In mice, IL-33 was also found in macrophages, dendritic and muscle cells 1,6 .In the absence of proinflammatory cytokines and inflammation, IL-33 is chromatin-associated factor in the nucleus of endothelial cells.IL-33 chromatin-binding peptide shares striking similarities with a motif found in herpesvirus LANA (latency associated nuclear antigen) 3,7 .IL-33 function as an "alarmin" released following cell necrosis to alert the immune system to tissue damage or stress 8,9 .Processing of released IL-33 by caspase-7 and caspase-3 dramatically attenuate IL-33 bioactivity 10,11 .It was previously believed that IL-33 requires maturation by caspase-1 for optimal biological activity.Recently, it was reported that processing by caspase-1 results in IL-33 inactivation, rather than activation 12 .
Soluble ST2 molecule regulates the activation of IL-33/ST2 signal pathway by the negative feedback mechanism.Cells produce large amounts of ST2 in the presence of cytokines and chemokines generated by IL-33/ST2 pathway activation.Soluble ST2 molecule may actually be a decoy to bind circulating IL-33 and prevent its binding to ST2L 22 .IL-33 may modulate the function of ST2 positive cells.As previously described, ST2 molecule is preferentially expressed on Th2 cells and is important for Th2 effector function.In the presence of IL-33 naïve T cells differentiate into the Th2 lineage, even in the absence of IL-4, the key driver of Th2 immune response 23 .IL-33-dependent stimulation of Th2 cytokine producing cells induce the IL-33 deluje na naivne T limfocite, oni diferenciraju u Th2 limfocite, čak i u odsustvu IL-4, ključnog citokina koji usmerava imunski odgovor u Th2 smeru 23 .Stimulacija efektorskih Th2 limfocita IL-33 indukuje produkciju IL-4, IL-5 i IL-13 i hemotaksu Th2 limfocita 24,25 .
More recent evidence suggests that NK and NKT cells express ST2 receptor.Although it was expected that IL-33 application induces the Th2 immune response, both Th1 and Th2 cytokines were produced in these cells 24 .
IL-33 activates antigen-presenting cells.The administration of IL-33 amplifies the polarization of alternatively activated macrophages and stimulate the production of CCL17 and CCL24 chemokines 29 .Mayuzumi et al. have shown that IL-33 promotes dendritic cell development 30 .IL-33-activated dendritic cells are functionally and phenotipically immature with low capacity to activate naïve T cells, but possibility to drive polarization of the immune response toward Th2 subset 31 .
The data on the role of IL-33/ST2 signaling in the pathogenesis of periapical inflammation and alveolar bone loss are lacking.We hypothesized that IL-33/ST2 signaling pathway is involved in the modulation of immune responses during the development and progression of periapical inflammatory bone destruction.To test this hypothesis, we investigated the effects of ST2 gene deletion and IL-33 administration on the formation of experimentally-induced periapical lesions in BALB/c mice.

Experimental animals
Male BALB/c (WT) and ST2 knockout (ST2-/-) mice on BALB/c background, 6 to 8 weeks old, were used for the induction of periapical lesions.The experiments were approved by the Ethics Board of the Faculty of Medical Sciences, University of Kragujevac, Serbia.

Induction of periapical lesions in mice
WT and ST2-/-mice were anesthetized with i.p. injection of ketaminehydrochloride and xylazinen, and mandibular molar pulps were exposed and left open to the oral environment.Mice that did not undergo pulp exposure were used as controls.

Statistical Analysis
Data were analyzed using statistical package SPSS, version 13.The results were considered significantly different when p<0.05.

ST2 deletion enhances the influx of mononuclear cells and proinflammatory cytokines production in periapical lesions
The number of MNCs in the periapical lesions was significantly higher in ST2-/mice compared with WT mice (p<0.05).Periapical lesions of ST2-/-mice had increased percentages of CD4 + T cells and CCR6 + cells among gated CD3 + T cells (p < 0.05), while the frequencies of other cell populations studied were similar between the two genotypes (data not shown).

FIGURE LEGEND Figure 1. Flow cytometric analysis of periapical lesion mononuclear cells (MNCs) was done at day 14 after periapical lesion induction in mice.
The total MNCs number was significantly higher in periapical lesions of ST2 -/-mice compared to WT mice.Percentages of CD4 + T cells and CCR6 + cells in gated CD3 + T cells, as well as TNF-α + , IL-6 + , IFN-γ + and IL-17 + cells among gated CD4 + T cells were significantly higher in ST2-/-mice compared to WT mice.The number of CD4 + cells, CXCR3 + and CCR6 + cells in gated CD3 + cells in periapical lesions were significantly lower in IL-33 treated mice compared to untreated WT mice.IL-33 treated mice had significantly lower percentages of TNFα + , IL-6 + , IFN-γ + and IL-17 + cells and significantly higher percentages of IL-4 + cells in gated CD4 + T cells (*p<0.05).

Significance of the obtained results in relation to the available data
We were first to provide the evidence that IL-33/ST2 signal pathway plays an important role in the pathogenesis of alveolar bone loss in the periapical region.Targeted disruption of ST2 gene in BALB/c mice led to massive infiltration of effector cells and production of proinflammatory cytokines, whereas the treatment of WT mice with IL-33 had the opposite effects (Fig. 1).

Napomene
Autori nemaju nikakvu finansijsku korist ili sukob interesa.lomas and radicular cysts, as well as in healthy periapical tissues.Increased numbers of IL-33 and ST2-positive cells in periapical lesions, when compared to healthy periapical tissues, suggested that IL-33/ST2 signaling may be involved in periapical inflammatory bone destruction 41 .Further, we investigated the effects of ST2 gene deletion or IL-33 administration on the formation of experimentally-induced periapical lesions in BALB/c mice.Periapical lesions of ST2-/mice had increased percentages of CD4+ T cells and CCR6+ cells among gated CD3+ T cells.The percentages TNF-α+, IL-6+, IFN-γ+ and IL-17+ cells in gated CD4+ T cells in periapical lesions were significantly higher in ST2-/-mice compared with WT mice.The exogenous IL-33 significantly downregulated the influx of CD4+ T, CXCR3+ Th1 and CCR6+ Th17 lymphocytes.The percentage of TNF-α-, IL-6-, IFN-γ-and IL-17-producing CD4+ T lymphocytes was significantly lower in the periapical lesions of IL-33-treated mice compared with untreated mice.In contrast, exogenous IL-33 significantly increased the percentage of IL-4-positive CD4+ T lymphocytes (Fig. 1).The obtained findings suggest that IL-33/ST2 signaling negatively regulates the severity of periapical alveolar bone destruction by preventing Th1/Th17 cell-mediated immune responses.

Conclusion
IL-33 presents a new target for therapeutic intervention across a range of diseases.This cytokine mediates signal transduction through the ST2 receptor and potently enhances Th2 immune response.Manipulation of IL-33/ST2 system by using monoclonal anti-ST2 antibodies, ST2 fusion proteins or monoclonal anti-IL-33 antibodies may present new approach in the therapy of Th1/Th17-mediated alveolar bone loss.Future research will implement new therapeuthic strategy in dental practice.

Slika 1 .Figure 1 .
Figure 1.Flow cytometric analysis of periapical lesion mononuclear cells (MNCs) was done at day 14 after the induction of periapical lesion in mice