The Role of Anti-inflammatory Cytokines and Antimicrobial Peptide LL-37 Biomarkers in the Treatment of Periodontal Disease.

'The role of anti-inflammatory cytokines and antimicrobial peptide LL-37 biomarkers in the treatment of periodontal disease.

1. Introduction Periodontal disease is an infectious process that consists of the destruction of the supporting anatomical structures of the tooth, as a consequence of the inflammatory response to the periopathogenic activity of the subgingival bacterial plaque, which leads to an innate, inflammatory and adaptative immune response. The clinical manifestation of the disease depends of host and environmental factors as well as the specific microbiological agent involved. The interrelation amongst bacteria and the immunodefence mechanisms of the host constitute the base of the immunopathological mechanisms that lead to the disease. In healthy conditions, there is a balance between the activity of the periodontal microbiota and the host immune response. In the innate defence system of the oral cavity there are several antimicrobial peptides involved, being one of them, the human cathelicidin LL-37. The activity of antimicrobial peptides such as LL-37 in the gingival sulcus is considered one of the greatest defence mechanisms of the periodontum. The gingival epithelium not only constitutes a physical barrier to avoid bacterial invasion. Neutrophils migrate to the junctional epithelium and create a barrier between the bacterial plaque and the underlaying epithelium. They also produce antimicrobial peptides, one of them the 18 kDa (hCAP18). Once the neutrophils are activated in response to the bacterial aggression the release LL-37, with a similar activity to a broad-spectrum antibiotic. The total absence of this peptide (LL-37) has been associated to aggressive periodontitis, in particular in patients with a neutrophil deficiency such as those who suffer Morfan Kosmann syndrome The nature of this research aims to elucidate the role of LL-37 in periodontal disease by comparing the use of a none invasive technique such as crevicular fluid samples in patients that suffer periodontal disease, before and after receiving a course of basic periodontal treatment (root scaling and planing). In additionto the above, pro and antiinflammatory cytokines (IL-6, IL-4, IL-10 in particular) will also be evaluated before and after periodontal treatment. Samples obtained will be analysed by immunohystochemical analysis (ELISA). The quantification of LL-37 before and after periodontal treatment associated with the clinical findings observed may serve as diagnostic biomarker of the immunopathological response inherent to the periodontal disease.
2. Justification of research question. Current status Over the last years, a great effort by scientific community has been made in order to try to understand the complex immunopathological processes related to periodontal disease and the relationship between the activity of the periodontal microbiota and the host defence mechanisms. The relevance of this research may serve to understand in more detail the role of the antimicrobial peptide LL-37 in periodontal disease not only due to their bactericidal effect but in the modulation of the immunological response of the host and its activity before and after periodontal treatment. The lack of studies on this matter reflects the need for further research on this topic. Ultimately, the findings of this research may form the basis of a new therapeutic approach based on the use of LL-37 to help modulate the immunological response of the host in combination with conventional periodontal treatment. This may be particularly relevant in patients susceptible to develop periodontal or periimplant diseases or in particular cases that are resistant to conventional treatment alternatives.
3. Summary of intended research

The main aim of the study is to investigate the role of LL-37 in the immunopathological response of the periodontum. Subsequent aims are to investigate the possibility of using this microbial peptide to serve as diagnostic, prognostic as well as a therapeutic agent in the treatment of periodontal disease.

Research hypothesis: There are significant quantitative differences in the levels of LL-37 before and after periodontal treatment, with a significant reduction of LL-37 at re-evaluation (4-6 weeks after a course of basic periodontal treatment -root scaling and planing).

Null hypothesis: There are no significant quantitative differences in the levels of LL-37 before and after periodontal treatment

The objectives of the present study are:

1. Determine if healthy patients and patients with periodontal disease have different levels of LL-37, IL-6, IL-4 and IL-10 in crevicular fluid at baseline and related them to the stage/severity of periodontal disease.
2. Evaluate if after a course of basic periodontal treatment these levels of LL-37, IL-6, IL-4 and IL-10 at baseline are similar to the quantified levels on healthy patients at baseline.
3. With respect to crevicular fluid samples, evaluate if levels of LL-37, IL-6, IL-4 and IL-10 in teeth affected by periodontal disease differ from healthy sites on the same individual or if there are no significant differences in the above before and after a course of periodontal treatment.
4. Stablish a non-invasive protocol to evaluate the immunological state of the patient before and after periodontal treatment as prognostic factor.
5. Material and methods

Ethical approval from the Ethical Commite of University Rey Juan Carlos of Madrid (URJC) has been granted. A selection of patients of the "Master in Advanced Implantology, Tissue Regeneration and Implant supported prosthesis" of the FCU-URJC that fulfil the inclusion criteria and that consent to be part of the study will be made.

The criteria for entry were as follows: aged 18 or over, with no previous history of periodontal treatment in the last six months, and no medical or dental history that could contraindicate periodontal treatment. Exclusion criteria included current pregnancy or lactation, immunological disorders, periodontal treatment within the last 6 months, oral contraception and antibiotic usage within the last 3 months, infections (such as HIV, hepatitis, and tuberculosis). In addition, third molar teeth were excluded from GCF sampling for being often impacted or extracted

Smoker patients will not be excluded and will be classified as light smokers (less than 10 cigarettes/day) or heavy smokers (more than 10 cigarettes /day).

Diabetic patients will not be excluded either but will be identified and accounted for.

All patients included in the study will go through the following stages:

1. Complete periodontal examination (probing depth (PD), clinical attachment level (CAL), plaque index (PI), blood on probing (BOP)).
2. Patients will be divided in two groups:

• 30 healthy patients (control)
• 30 periodontal patients (test- periodontitis stage I-II (15 patients), periodontitis stage III- IV (15 patients).

Due to lack of in vivo studies, simple sample size calculation was made using a conservative power analysis (G*Power 3.1.9.4 program, 2009). The simple size (27 subjects) was based on a two-tailed test with an effective size of 0.05, an alphabetical error probability of 0.05, difference of 50% in mean value, standard deviations to be maxium 80% of the mean values and a power of 0.90.

Taking into account these parameters, a simple sample size between 21-30 patients per group is estimated.

Crevicular fluid samples will also be obtained following the manufacturers protocol using perio papers (Periopaper, Applied Biosystems, Inc, Foster City, CA). Teeth to be examined will be isolated with cotton rolls and air dried. Paper strips will be introduced into the gingival sulcus for approximately 30 seconds. Test group patients' samples will be taken from periodontally pathogenic sites and healthy sites. Heavily contaminated samples with saliva or blood will be discarded.

Samples will be stored at -80 C at the laboratory of Departamento de Ciencias Básicas de la Salud, Edificio Departamental I, Fundación Clínica Universitaria, URJC (Alcorcon). Once all samples are obtained, ELISA analysis of all samples will be performed by using a specific ELISA kit commercially available for human LL-37 (HyCult Biotechnology, Uden, the Netherlands) and commercially available kits for IL-4, IL-4, IL-6 and IL-10 ((Diaclone SAS; Besançon, France; 850.890, 950.035 and 850.880, respectively).

Patients of the test group will receive a course of basic periodontal treatment (root scaling and planing) and oral hygiene instructions after the first sample collection.

A re-evaluation appointment at 4-6 weeks will take place where new crevicular fluid samples of both groups will be obtained (test and control), to evaluate any statistically significant difference with respect to the quantity of LL-37 in relation to the baseline measurements recorded. After that, samples will be scrutinised again by performing ELISA assays following the protocol described above.

Once all results are obtained, statistical analysis of the samples will be carried out.