Species and number of bacterium may alternate IL-1β levels in the odontogenic cyst fluid

Introduction

Odontogenic cysts (OCs) originating from odontogenic epithelium are commonly seen pathological jaw lesions in the oral and maxillofacial surgery practice. They are divided into two groups, one being developmental and the other being inflammatory. Inflammatory cysts are thought to arise from inflammation-induced epithelial proliferation with subsequent central liquefaction [1]. Inflammatory radicular cysts (RCs) and developmental odontogenic keratocysts (OKCs) are the two most commonly encountered types in our practice. Infection of necrotic teeth are responsible for the formation of RCs and progress as apical lesions whereas OKCs react with an inflammatory response to chronic irritation caused by necrotic teeth [2]. Although studies have shown that periodontal inflammations’ initation is often caused by oral bacteria including periapical lesions, there’s still limited research to define the responsible bacteria [3]. The proinflammatory cytokines and inflammation-associated growth factors are playing a role in the development of OKCs [4], [5]. Interleukin-1 (IL-1), IL-12, tumor necrosis factor-alpha (TNF-α) and IL-15 are secreted from macrophages during bacterial infections and regulate the immune response [6], [7]. Cytokines (IL-1, IL-12, TNF-α) are also responsible for systemic response to infections. IL-1α and TNF-α are important mediators contributing to the development of cysts. IL-1α and TNF-α were shown to stimulate the production of osteoclast-like multinucleated cells and enhance the activity of osteoclasts’ bone resorption [5]. IL-12 (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40) causes local release of interferon-gamma (IFN-γ) from natural killer cells (NK) and T lymphocytes, stimulates the cytolytic activity of NK cells and also alerts the adaptive immune response by forwarding type-1 helper T cells (Th1) [7]. IL-15 is a pleiotropic cytokine that plays an important role in both innate and adaptive immune systems [7], [8], [9]. There are limited studies investigating the levels of cytokines in OCs and these have usually examined pro-inflammatory cytokines [5], [10], [11] or growth factors [4]. The aim of this study is to detect the levels of macrophage-originated cytokines (IL-1β, IL-12, IL-15 and TNF-α) as response to bacterial infection and the bacterial content in OC fluids.

Materials and methods

Results

Cytokines levels: IL-1β, IL-12, IL-15 and TNF-α

Macrophage-derived cytokines IL-1β, IL-12, IL-15 and TNF-α levels in response to CFs content bacterial infections were determined by luminex (Table 3). The correlation (R) of curves obtained from the standards of all cytokines was 0.900–0.999. High and low concentrations of control serum of the kit were within the expected range. There was no significant difference compared with respect to gender, age, cyst size and type. After the division of CFs in four groups according to the number of bacteria content (0, 1, 2 and ≥3 bacteria, Table 4), a significant difference was detected in terms of IL-1β levels (KWp=0.009). IL1-β level of CF group containing three or more species of bacteria increased compared with CF groups containing two types of bacteria (Figure 1).

Table 3:

Comparison of cytokine levels between RC and OKC [median (min-max) pg mL].

Cytokine	Assay sensitivitiesa	OKC	RC	pb
IL-1β	0.19	5.33 (0–335.21)	11.5 (0–2043)	NS
IL-12	0.23	0 (0–9.42)	0 (0–10.38)	NS
IL-15	0.63	4.06 (0–16.95)	3.34 (0–16.31)	NS
TNFα	0.22	0 (0–61.24)	2.495 (0–111.01)	NS

1. IL-1β, Interleukine-1 beta; IL-12, interleukine-12; IL-15, interleukine-15; NS, non significant; OKC, odontogenic keratocyst; RC, radicular cysts; TNF-α, tumor necrosis factor-alpha. ^aMinimum detectable concentrations, pg/mL; ^bMann-Whitney U test.

Table 4:

Cyst fluid cytokine (IL-1β, IL-12, IL-15 and TNF-α) levels [median (min-max) pg/mL] grouped according to the number of bacteria species.

Number of bacteria species	n	IL-1β	IL-12	IL-15	TNF-α
Bacterium-free	5	10.58 (0–123.26)	0 (0–9.42)	0.19 (0–8.19)	0 (0–0.84)
1 bacterium	11	6.44 (0–335.21)	0 (0–10.38)	4.06 (0–16.95)	0.84 (0–61.24)
2 bacteria	13	0 (0–14.44)	0 (0–0.52)	4.06 (0–16.31)	1.17 (0–16.34)
≥3 bacteria	10	23.33 (0–2043.11)	0 (0–3.24)	2.12 (0–15)	15.42 (0–111.01)
pa		0.009	NS	NS	NS

1. IL-1β, Interleukine-1 beta; IL-12, interleukine-12; IL-15, interleukine-15; NS, non significant; OKC, odontogenic keratocyst; RC, radicular cysts: TNF-α, tumor necrosis factor-alpha. ^aKruskall-Wallis test.

Figure 1:

After being grouped according to the number of detected bacteria species [0 (n=5): bacterium-free, 1 (n=11) and 2 (n=13) are positive for 1 or 2 bacteria species, ≥3 (n=10): equal or more than three bacteria species] IL-1β levels of cyst fluids were significantly different (Kruskal Wallis p=0.009, Mann-Whitney U test p=0.005, α=0.008).

IL-1β levels of the groups, formed depending on whether the identified bacterial species in CF are positive or negative, were compared (Figure 2). A significant difference was found in C. rectus positive n=6; 1006.27 (6.44–2043.11) pg/mL and T. denticola positive n=6; 13.49 (6.44–1706.19) pg/mL, with higher levels of IL-1β observed than relative bacterium negative cases. Different from these, the presence of higher levels of IL-1β in E. faecalis negative CFs n=31; 6.44 (0–2043) pg/mL than E. faecalis positive fluids was observed.

Figure 2:

Levels of IL-1β are higher in Campylobacter rectus positive (Cr pos, n=6), Treponema denticola positive (Td pos, n=6) and Enterecoccus faecalis negative (Ef neg, n=31) cyst fluids when compared with Campylobacter rectus negative (Cr neg, n=33), Treponema denticola negative (Td neg, n=33) and Enterecoccus faecalis positive (Ef pos, n=8, Mann-Whitney U test).

Monocyte derived TNF-α, IL-12 and IL-15 cytokine levels were not significantly different.

Discussion

The OKCs are usually asymptomatic and are more commonly found in the posterior mandible of males [15]. RCs represent an inflammatory response to a chronic irritation, whereas OKCs are mostly non-inflammatory lesions [2], [16]. Studies report that the major part of the bacterial content in the OCs are consisted of anaerobes [3], [17]. Scalas et al. [18] observed the number of bacterial species per sample as 2.06±0.93, but they identified bacterial species Propionibacterium acnes (P. acnes) (7/16; 43.75%) and Prevotella spp. (2/16; 12.50%) by microbiological culture techniques. According to our results, the number of bacterial species per sample was similar and we have observed more bacteria species in the RC fluids. On the contrary, common bacteria in both types of CF were P. gingivalis, F. nucleatum and Prevotella spp. (46.15%). P. gingivalis was associated generally with E. faecalis. The discrimination depends on using species-specific PCR primers for identification of bacteria in cysts. We were able to identify bacteria in a more sensitive way however the spectrum was limited to these primers. We can suggest that these anaerobic bacteria may trigger the pathogenesis of cyst formation.

The cytokine IL-1β is a key mediator of host immune response and plays an important role in the development of inflammatory disease and tissue destruction [19], and its precursor is induced in the cytosol of monocytes and macrophages in response to proinflammatory stimuli including pathogenic bacteria [6], [20]. Similarly to IL-6 and TNF-α, it is thought to play a role in bone remodeling, bone resorption, and new bone deposition.

In our study, we have screened IL-1β. IL-12, IL-15 and TNF-α. Although no significant difference for any cytokine level was determined between cysts groups, IL-1β and TNF-α were higher in RC fluids similar to report of Hayashi et al. [21]. Our results, where we identified the protein levels on CF, using Luminex, are in correlation with the results of the quantitative multiplex ELISA kit and Human Cytokine Antibody Array [16], [21]. High IL-1β and TNF-α levels in RC fluid, in addition to higher number of bacteria species in RC, might suggest that a proinflammatory response created by the macrophages might be induced in response to bacteria. Local IL-1 and IL-6 are known to affect epithelial cell proliferation, and as such, IL-1β and TNF-α could affect cyst formation too [22].

Hayashi et al. [21] considered that the expansion mechanisms of RC and OKC involve similar biologic mechanisms other than infection. Alhashimi et al. [23] showed that after the application of orthodontic force on the maxillary first molars of rats, messenger RNA (mRNA) expression IL-1β and IL-6 were inducted but the mRNA of TNF-α was not detected. It can be proposed that the trigger of cytokine production could be also due to the application of orthodontic force. IL-1β level in cysts with no bacteria isolation were not statistically significant yet higher compared to one type of bacteria containing CF group and two types of bacteria containing CF group; this can be explained by the orthodontic forces applied on these subjects.

In vitro studies have been demonstrated IL-1β and TNF-α secretion inducted by live bacteria or by their lipopolysaccharide (LPS) and plays a stimulatory role in maxillofacial abscess formation [6], [24]. Additionally, in adult periodontitis, significant elevation of LPS-stimulated IL-1β and TNF-α was indicated by the gingival crevicular fluid analysis [25]. IL-1β level increased when the CF’s number of species of bacterial content was three or more. Especially in the RC fluids of our study, more bacteria species and elevated IL-1β levels were detected. Increasing bacteria species could trigger IL-1β production. The cytokine response being lower in the few bacteria species containing CFs (one or two bacteria species containing cysts) can be associated with the proteolytic effect of the bacteria. P. gingivalis’s proteolytic effect, down regulating the IL-8, is previously shown in oral epithelial cell cultures in vitro [26]. Other bacteria, including the P. gingivalis need to be investigated in regards to their skipping the innate immunity mechanisms by down regulating the cytokines. Our results suggest that E. faecalis might have a similar effect on IL-1β. However there was no statistically difference in between P. gingivalis positive and P. gingivalis negative proteolytic effect, down regulating the IL-8, is previously shown in oral epithelial cell cultures samples’ IL-1β levels, yet we found high IL-1β level in C. rectus positive and T. denticola positive CFs; this can stimulate cyst formation. According to our results, C. rectus, T. denticola bacteria exacerbated the pathogenesis by increasing the production of IL-1β. However, it was observed that E. faecalis suppresses the secretion of IL-1β. The reason of less IL-1β in CFs with two species bacteria in comparison with one type could be the antagonistic interaction of bacteria or prevention of host inflammatory response by some bacteria such as E. faecalis. In this study, bacterial DNAs were defined as the agent of infection in CF and their relationship was shown with IL-1β.

Although our study reflects local response to cyst, macrophage derived cytokines (IL-1, IL-12, TNF-α) are also responsible of systemic manifestations of infections. TNF-α, as a pleiotropic cytokine, is considered to play a primary role in modifying inflammatory and immune responses to various inflammatory diseases and tumor. It mostly triggers apoptosis and necrosis in sensitive tissues. It has been linked to osteomyelitis and periodontitis, however it can also participate in osteoclast formation and thus help in haemostasis, and the bone resorption in the maxillofacial region [27]. Investigations showed that TNF-α is significantly higher in the RC fluids [5], [27]. In our study, although there was no significant difference between the OKC and RC, the TNF-α levels in RC were higher than OKC that were consistent with aforementioned studies. The idea of anti-inflammatory response, being more dominant in the development of RCs, is also supported by the aforementioned finding.

It has been suggested by previous literature that the gradual diminishing of IFN-γ as the T helper cytokine type 1 response or the transformation of chronic inflammation into cyst formation due to absence of the adaptive immune response [28] however getting chronic innate immune response was supported with these findings. This study focuses on determination of the types of bacteria causing infections in OKCs and RCs and it reveals levels of some cytokine secreted by macrophages as local immune response to infection. Previous microbiological studies on OCs evalueted the bacteriological spectrum or cytokines individually, while in our investigation, however these factors have been investigated together in our study [5], [17], [18], [29], [30], [31]. One of the advantages of Luminex method was that it is supposed to detect the concentration of many different types of protein in OC fluid, so it can be obtained in a small sample volume. The presence of immune-competent cells in OC fluid samples was not investigated and, the relatively small number of the samples in bacteria groups can be considered as limitations of the present study. There’s a need for further in vitro studies are required to clarify the role of each bacterium and other cytokines in the pathogenesis of the OCs.

Conclusion remarks

This study shows the revelation of the relationship between cytokines and bacterial content of OC fluids. It was detected that the increased number of type of bacteria in the CF may cause increased secretion of IL-1β as a sign of activated macrophages. The most common type of bacteria observed in OC fluids were P. gingivalis and Prevotella spp. While some bacteria (C. rectus and T. denticola) cause the secretion of IL-1β at high levels, others (such as E. faecalis) suppress it. IL-1β is thought to be involved more than IL-12, IL-15 and TNF-α in the cyst formation, but not identified as a specific cyst type. However, the IL-1β level was slightly elevated in the RC fluid specimens with increased number of type of bacteria. These findings supported the thought that anti-inflammatory response is responsible for the development of this type of cyst. Further studies are required to clarify the role of these bacteria in the pathogenesis of the OCs. The relatively small number of the samples for bacterial types groups of OC can be considered as a limitation of this study.