Treg and Th 17 cells in inflammatory periapical disease : a systematic review

The process involved in periapical lesions, which occur as an outcome of pulpal necrosis, is regulated by the immune system including regulatory T cells (Treg) and T helper 17 cell (Th17) responses. The objective of this study was to conduct a frequency systematic review to determine the presence of Treg/Th17 responses and the influence of these cells in the progression of chronic inflammatory periapical lesions in humans. A systematic computerized search was carried out in Pubmed, Medline, Web of Science and Scopus electronic databases from their date of inception through the first week of May 2017. In addition, the reference lists of the included articles and the grey literature were hand-searched. Articles that evaluated the presence and influence of Treg/Th17 in the progression of human periapical lesions were included. Study selection and the quality assessment of the included articles (using the Newcastle-Ottawa scale) were carried out by two authors. Fifty-seven titles/abstracts were screened and eight studies met the eligibility criteria and were included in this systematic review. The included studies showed large variation in the type of periapical lesion assessed, mean age, age range, type of experiment and findings regarding the participation of Th17 and Treg in the status of inflammatory periapical lesions. The studies showed the involvement of Treg in the modulation of the inflammatory response in radicular cysts and periapical granulomas. This systematic review highlights the relationship between Treg and Th17 acting in a subtle balance inhibiting or promoting the progression of human periapical lesions.


Introduction
Apical chronic periodontitis is a common inflammatory osteolytic disease of the jaws.The process is regulated by several inflammatory mediators including cytokines released by leukocytes. 1 Leukocytes are recruited from the periapical tissues in response to intracanal bacterial infection, and the persistence of the antigenic stimulus can result in chronic periapical lesions such as periapical granuloma and radicular cyst. 2 The role of immunological mechanisms in the pathogenesis of periapical lesions has been widely documented. 3The interplay between immune regulatory mechanisms and effector T cell responses is a crucial Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.
determinant of innate and adaptive immunity. 4CD4 + T cells play a regulatory role and help to constrain the effector function of other cell types. 5More recently, 6 the identification of CD4+ Foxp3+ regulatory T cells (Treg) and T helper 17 cells (Th17) modified the paradigm Th1-Th2.The Treg-Th17 pair has explained the immunity/inflammation process in an increasing number of diseases, including periodontal disease and others involving progressive bone resorption. 7regs, defined by the expression of the lineagespecific transcription factor FoxP3 (Forkhead box P3), are cells required for immune induction. 8Th17 play critical roles in the progression of autoimmunity and inflammation by the production of IL-17 and require specific cytokines for their differentiation, such as the transforming growth factor-β (TGF-β), which may be combined with interleukin-6 (IL-6) or IL-21 and the transcription factor RORγt. 9 The participation of Treg and Th17 in the inflammatory response may help explain many unresolved issues in the immunobiology of periapical lesions, such as bone resorption occurring in different manners as a reflection of intracanal infection in similar cases of pulpal necrosis. 10However, the up or down regulation of both Foxp3 and Th17 in periapical inflammatory disease has been explored mostly in animal models, 1,9,11 with few studies conducted in humans, in different types of chronic lesions, such as radicular cysts or periapical granuloma.Thus, defining the role of Treg/Th17 is crucial for better understanding the immune system regulation of these lesions.
This frequency systematic review aimed to demonstrate the presence of Treg and Th17 in human inflammatory chronic disease and to describe the effects of these subsets of cells in the immuneinflammatory status of periapical lesions in humans.

Protocol and registration
This systematic review was designed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses checklist (PRISMA) as a template, 12 and was registered in the International Prospective Register of Systematic Reviews (PROSPERO) under the number CRD42017077714.

Eligibility criteria
Studies evaluating the role of Th17 and Treg in the progression of human periapical lesions were included.The pathway of the Treg/Th17 and the involved biomarkers is shown in Figure 1.The following PECOS question has been applied:  O (outcome) = Immune inflammatory response S (study design) = Observational studies Reviews, case reports, letters to editors, studies involving animals and meeting abstracts were excluded.No restriction regarding language, date and status of publication was applied.

Information sources
Systematic computerized searches were carried out using the electronic databases Pubmed, Medline (Ovid SP), Web of Science (Thomson Reuters) and Scopus (Elsevier) from their date of inception through the first week of October 2015 and later updated to the first week of May 2017.Hand searches of the references of the included articles were also carried out in order to identify relevant studies that might have been missed in the electronic search.Finally, a search of the grey literature was conducted using Google Scholar search engine, limited to the first 100 hits.References were managed using the EndNote software (EndNote™ Thomson Reuters, Philadelphia, PA, USA).Duplicate hits were removed upon identification.

Search
The searches were conducted with the assistance of a senior librarian.The keywords and appropriate truncations were specific to Pubmed and Medline.The same search strategy was used for Web of Science, Scopus and Google Scholar.The keywords and the number of hits found in each electronic search are presented in Table 1.

Study selection
Study selection was carried out in two phases.During the first phase, two authors (AONT, MCFA) independently reviewed the list of titles and abstracts for inclusion; the ones that did not meet the eligibility criteria were excluded.Once potentially adequate abstracts were selected, full-text articles were retrieved.
During the second selection phase, the same reviewers independently evaluated full-texts, and those studies that met all eligibility criteria were included in the systematic review.In both phases, disagreements between reviewers were resolved through discussion until achievement of a consensus.A third reviewer was involved when needed for a final decision (MFMM).

Data collection process
Data collection was independently performed by two authors (AONT, MCFA).Discrepancies were dealt with as above.In case of missing information, contact with authors were planned.

Data items
Data on the following items were collected: author and date of publication, country where the study was conducted, study design, sample size, type of periapical lesion assessed, mean age, age range, type of experiment used in the included study and findings regarding the participation of Th17 and Treg in the progression of periapical lesions.Data were organized in a standardized table.

Risk of bias
Risk of bias in individual studies was evaluated according the Newcastle-Ottawa scale. 13The scores range of the scale are dependent on the study design.For case-control studies, a quality score was calculated based on group selection (four items), comparability between groups (one item), and outcome and exposure assessment (three items) categories.A maximum of 1 point was awarded for each item in the group selection, and outcome and exposure assessment categories.A maximum of two points was awarded for comparability.Thus, the maximum score of nine points represented the highest methodological quality.A study was categorized as high quality if the total score was 7 or higher.For cross-sectionals studies, the score was calculated based on the same three categories.However, those categories had a different number of items: group selection (two items), comparability (one item), and outcome and exposure assessment (two items).Therefore, the maximum score was 6 for the highest quality.A study was categorized as having high quality if the total score was 4 or higher.

Summary measures
Any type of statistical method (e.g.: p-value) examining the participation of Th17 and Treg in the progression of periapical lesions was considered.

Synthesis of results
Heterogeneity was evaluated by means of variability assessment across included articles regarding study design, analytical assay used and the statistical analysis examining the role of Th17 and Treg in the progression of periapical lesions.If data were homogeneous, a meta-analysis would be performed.Instead, if data were heterogeneous, a qualitative summary of results would be carried out.

Risk of bias across studies
Bias regarding multiple publications was investigated in the present systematic review.The unit of analysis considered was the study, not the publication.

Study selection
A total of 121 records were initially identified through the electronic databases.The updated search retrieved 18 records.Following the removal of 82 duplicates, 57 titles/abstracts were screened.Of those, 39 did not meet the eligibility criteria and were excluded.In phase 2, the full-text of 18 studies were retrieved and evaluated.Of those, only eight 14,15,16,17,18,19,20,21 met the eligibility criteria.No additional study was identified in the reference lists of the included articles and in the grey literature.A flowchart of the study selection for this systematic review is depicted in Figure 2.

Study characteristics
Among the eight included articles, four were crosssectional studies 16,17,19,20 and four were case-control studies. 14,15,18,21All studies involved human periapical lesion fragments.Samples of six articles 14,15,18,19,20,21 were obtained directly through surgery and samples of the other two studies were obtained through institutional archives collected from previous surgical procedures. 16,17ou r a r t icles repor ted t he mea n age of participants 18,19,20,21 and five studies defined the range between 17 and 64 years. 14,15,19,20,21The language of the articles was predominantly English, although the studies were conducted in different countries (Brazil, United States and Serbia).The analytical method used in two studies was the ELISA method. 18,20Real-time polymerase chain reaction (RT-PCR) was used in three studies, 14,15,21 flow cytometry in two studies, 19,20 confocal microscopy in one study, 19 and immunohistochemistry was used in four studies. 16,17,18,20The entire description of studies characteristics is shown in Table 2.

Risk of bias within studies
The cross-sectional studies 16,17,19,20 were rated between two and three.The case-controls studies 14,15,18,21 between six and nine.

Relationship between Treg/Th17 in periapical lesions
All immunohistochemical studies employed FoxP3 and cytokine IL-17, the hallmarks of Treg and Th17, respectively, to characterize the Treg/Th17 response in periapical lesions.
In these studies, differences were found between periapical granulomas and radicular cysts.A higher number of FoxP3 cells were found in granulomas than in cysts, 16,17 although in another study this difference was demonstrated only when comparing lesions and controls. 18Studies failed to demonstrate an association between intensity of inflammation and

Granulomas: 20
Greater expression of FoxP3 was seen in periapical granulomas.The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas.
FoxP3 or IL-17, 16,17 but one study describe the presence of neutrophils linked to a high expression of IL-17. 18mploying different methods, a study also found a higher level of IL-17 in symptomatic lesions; IL-17 was also correlated with neutrophils proportion. 20he differences observed between cysts and granulomas in relation to frequency of Treg/Th17 was suggested to contribute to the growth of cysts that are not regulated by the Treg and are influenced by other cytokines. 17ll studies discussed their results considering the influence of other immunological markers (IL-4, IL-9, IL-10, IL-17, IL-21, IL-22, TGF-ß, TNF-α, IFNγ) in the modulation of the Treg/Th17 response and progression of periapical lesions.The effects of this influence are showed in Table 2.
Since Tregs were first characterized in human periapical lesions, 19 knowledge has advanced and molecular studies have contributed to the understanding of the role of Treg/Th17 in periapical disease.The mRNA of multi Th subsets markers, including FoxP3 and IL-17, were evaluated in active and inactive periapical granuloma, determined by the RANKL/OPG analysis.The lesions showed a widespread cytokine expression, however TNFα, IL-17, IL-21 and IFN-γ were associated with active lesions, while FoxP3, IL-9, IL-4 and IL-22 with lesions inactivity. 15 higher expression of IL-10, T-bet and FoxP3 evaluated by RT-PCR was found in periapical granulomas compared with radicular cysts, and was associated with osteoclastic activity. 21In a more sophisticated study, the DNA methylation profiles of 22 genes were investigated in a large sample of periapical inflammatory lesions, including periapical granulomas and radicular cysts.FOXP3 gene presented the highest DNA methylation, inversely associated with the mRNA levels. 14aken together, the studies suggest that the balance between Treg and Th17 is critical to the outcome of periapical inflammation determining the expansion or healing of chronic lesions.

Synthesis of results and risk of bias across studies
Although initially planned, the meta-analysis of the data was not possible due to the high degree of heterogeneity across the included studies.Heterogeneous factors were study design [case control studies 14,15,18,21 and cross-sectional studies 16,17,19,20 ], method of molecular analysis used [ELISA method 18,20 , RT-PCR 14,15,21 , flow citometry 19,20 , confocal microscopy 19 and immunohistochemistry 16,17,18,20 ] and type of data analysis done to assess the presence of Th17 and Treg in the progression of periapical lesions [p-value and r-value 14,19 , p-value, mean and standard deviation 15,20 , p-value, r-value, mean, standard error of mean and median 16 , p-value, median and interquartile range 17 , p-value, median, interquartile range and z-value 18 , p-value 21 ].Moreover, the studies in which immunohistochemical analysis was used varied in terms of methods adopted [computerized program 18 , three 16 or one 17 microscopic fields of each specimen].Therefore, we carried out a qualitative summary, with the data organized in Table 2 to facilitate a narrative synthesis of this systematic review.

Risk of bias across studies
The objective of this systematic review was to identify studies and not publications.Nevertheless, multiple publications 19,20 using the same data pool were selected because they measured the presence of Th17 and Treg in the progression of periapical lesions using different strategies.One article used FoxP3 as the main target of the study 19 , while the second assessed Th17 markers 20 .

Summary of evidence
This systematic review collected contributions from different studies enabling researchers to understand the key role of Treg and Th17 in inflammatory periapical lesions.Research on the pathogenesis of autoimmune and inflammatory diseases has made significant progress in the past few years, and Th17 and Treg have emerged as major players in autoimmunity.Moreover, the present study allows investigators to be aware of the influence of different immunological markers, such as FoxP3 and IL-17 in this condition. 22Treg and Th17 are both present in inflammation sites, but they seem to have opposing roles in the progression of inflammatory periapical lesions.While Th17 stimulate the immune response, Treg have a regulatory activity and, therefore, play a very important role in the maintenance of tolerance and in the control of the expansion and activation of the immune system. 5The information provided by the studies included in this systematic review might be also relevant for clinical purposes.The Treg/Th17 relationship might help in the understanding of the progressive bone resorption related to periapical lesions. 7tudies have demonstrated 14,15,16,17,19,21 that FoxP3 is a master switch governing the progression and function of Treg, whose functions include the inhibition of the immune response.The inflammatory response featured by FoxP3, IL-10, IL-9, IL-4 and IL-22 contributes to lesions inactivity 15.On the other hand, Th17 immune response seems to play a dominant role in exacerbating inflammation 20 .In addition, different cytokines such as IL-1, IL-6 and TNF-α alongside TGF-β contribute to the production of IL-17 18 .Moreover, Treg can produce IL-17 in accordance with the environment 15 .Thus, the participation of Treg and Th17 has been associated with the modulation of human periapical lesions and bone resorption.However, only the simultaneous analysis of a broad cytokine panel can better elucidate the immune regulatory scenario of the periapical inflammatory disease.
Di fferent met hods of a nalysis (RT-PCR, immunohistochemistry, confocal microscopy, flow cytometry, ELISA) were used to evaluate the role of cellular immune response in the progression of periapical lesions.Each of these approaches has advantages and disadvantages. 23Confocal microscopy displays cell images in high resolution, but the absolute fluorescence sensitivity is substantially lower than other techniques.Flow cytometry compromises image quality in favor of fluorescence sensitivity and the method has the advantage of performing rapid analysis of a great amount of cells. 24RT-PCR is a quantitative method that enables researchers to identify many types of cytokines in small samples.However, the presence of RNA may reflect protein levels and decrease accuracy levels. 25ELISA, on the other hand, allows the quantitative detection of cytokines at protein level.While the afore-mentioned protocols cannot identify the cytokine-producing cell types, immunohistochemistry overcomes this deficiency.The identification of these cells may be done in tissues that do not produced sufficient cytokines to be detected by the other approaches.On the other hand, immunohistochemistry does not provide a precise quantitative analysis, as the other methods, and although IL-17 is a marker of Th17 lymphocytes, other cells can express this cytokine. 23he role of Treg/Th17 response in inflammatory immune bone resorption has been explored in other diseases. 26In rheumatoid arthritis (RA), studies describe an excessive production of IL-17 followed by bone and cartilage destruction.Moreover, Treg in RA are not entirely efficient to control inflammation.A similar mechanism is found in periodontal disease. 5,27ith respect to the periapical lesion progression, clinical and experimental studies suggest that the balance between Treg and Th17 is critical to the outcomes of periapical lesions in terms of activity (lesion expansion), or inactivity (healing). 15,18Higher IL-17 levels were also observed in cases of patients with sinus tract. 20Chronic periapical lesions might experience an exacerbation process, with increased presence of IL-17.These data are shown in Table 2.
Caution should also be taken in the interpretation of the findings presented herein, as although the study focused on reliable and specific biomarkers to detect Treg and Th17 other biomarkers are known to be involved in the Treg/Th17 imbalance. 14,15ggestions for future research In conclusion, this systematic review confirmed the presence of Treg and Th17 in the progression of periapical lesions.The relationship between the inhibitory characteristic of FoxP3 and the proinflammatory feature of IL-17 in the periapical lesions was also highlighted.However, some of the existing articles 33,34 addressing this issue were excluded from this systematic review because they used animal experiments in their methodology, which limits the extension of the results to humans.
Few scientific reports with humans are available in research databases.Therefore, we suggest that further studies evaluating the influence of Treg/Th17 in the progression of human periapical lesions and bone resorption are conducted to generate comparable data allowing a quantitative analysis of the findings. 35dditionally, strong future research should also be carried out to overcome the limitations of the included studies and to translate research findings into clinical practice. 30ppendix 3: Quality assessment of included case control studies based on the Newcastle-Ottawa scale

Figure 2 .
Figure 2. Flowchart of study selection of for this systematic review.
1 a) yes, with independent validation ✯, b) yes, eg record linkage or based on self reports, c) no description 2 a) consecutive or obviously representative series of cases ✯, b) potential for selection biases or not stated 3 a) community controls ✯, b) hospital controls, c) no complete description 4 a) no history of disease (endpoint) ✯, b) no description of source 5 a) study control for periapical lesion ✯, b) study control for 2 or more confounding variables ✯✯ 6 a) secure record (eg Real-time polymerase chain reaction, Immunohistochemical analysis, Confocal microscopy ,Flow cytometry , ELISA) ✯, b) written self report or medical record only, c) no description 7 a) yes ✯, b) no 8 a) same rate for both groups ✯, b) non respondents described, c) rate different and no designation

Table 1 .
Search strategy for each each electronic database

Table 2 .
Summary of characteristics of the included studies.
* a maximum of 2 points for each item; *** a maximum of 1 point for each item *a maximum of 1 point for each item; *