Blood, gingival crevicular fluid (GCF), and saliva samples will be collected from patients at baseline and 12 weeks after non-surgical periodontal treatment. From fasting blood samples (12h), peripheral blood mononuclear cells (PBMCs) and neutrophil fractions will be isolated using an immunomagnetic method, following the manufacturer's protocol, and plasma and serum will be stored at -80°C until analysis. LUNA-FL will be used to determine cell count and viability (acridine orange and propidium iodide double stain) in cell samples. GCF samples will be collected in duplicate by carefully inserting sterile paper points into the periodontal pocket 1 mm for 30 s. The volume will be determined using the Periotron 8000 and stored at -80°C for future determinations. Unstimulated saliva will be collected in RNase-free tubes by expectoration, and after centrifugation, the supernatant will be stored at -80°C for microRNA (miRNA) analysis.
Clinical periodontal parameters of probing pockets depth (PD), millimetres of clinical attachment level (CAL), bleeding on probing (BOP), simplified plaque index of Silness and Löe, and simplified calculus index of Greene and Vermillion will be determined using a conventional manual periodontal probe (UNC-15 PCP). A periodontal examination will be performed to measure PD, CAL, and BOP at six sites per tooth for all teeth, excluding third molars, as previously described. Patients will be interviewed about their medical history, lifestyle habits (smoking, frequency of tooth brushing, physical activity), and sociodemographic characteristics. Weight, height, and blood pressure will be measured using standardized methods. Biochemical parameters of carbohydrate metabolism - glucose, insulin, glycated hemoglobin (A1c) -, lipid profile - total cholesterol, LDL, HDL, triglycerides (TG), apolipoproteins AI and B -, emerging inflammatory and cardiovascular risk markers - C-Reactive Protein (CRP), C3c, and retinol-binding protein 4 (RBP4) -, and complete blood count will be determined at the hospital's Clinical Analysis Service.
For detection of differences in protein expression, cells will be incubated in lysis buffer with protease and phosphatase inhibitors (RIPA Buffer) for 15 minutes at 4 degrees celsius (°C). The supernatant will be collected after centrifugation for 15 minutes at 16,000g. The total protein concentration will be quantified using a bicinchoninic acid (BCA) protein assay. Aliquots of 25 µg of protein will be resolved on 8-16% gradient SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Target proteins will be detected by incubating the membranes with anti-actin, JNK, NFkB, MCP1, GPX-1, NLRP3, ASC, procaspase 1, caspase 1, NADPH oxidase, catalase, GPX1, SOD1, Beclin, ATG5-ATG12, p62, LC3 I, LC3 II, Pink1, GRP78, eIF2alpha, IRE1 alpha, ATF6, CHOP, PGC1 alpha, mTFA, VDAC, Complex I, II, III, IV and V. The protein signal will be detected by chemiluminescence and analyzed by densitometry.
Inflammasome complex assembly (first stage of activation) through co-localization studies of NLRP3-ASC in PBMCs will be conducted using the confocal and/or fluorescence microscopy. Briefly, PBMCs will be seeded on coverslips coated with Poly-D-Lysine, fixed with paraformaldehyde (PFA) 4% for 20 minutes, permeabilized with Triton X-100 for 20 minutes and blocking with Phosphate-buffered saline buffer-Bovine serum albumn (PBS-BSA) 3% for 1 hour at room temperature. Hybridization with specific primary antibodies (diluted in PBS-BSA 1%) will be carry out overnight at 4 ºC and then, secondary antibodies conjugated with AlexaFluor fluorophores will be incubated for 1 hour in the dark at room temperature. Stained samples will be transferred the coverslip onto microscope slide and conserved in anti-fade fluorescence mounting medium.
Circulating levels of cytokines, adhesion molecules and serum oxidative stress markers will be measured in serum samples, but for secretome studies 1x10\^6 PBMCs will be previously incubated in 1 mL RPMI with 10% fetal bovine serum (FBS) for 4 hours (37°C, 5% CO2) to obtain the supernatant. Both serums and supernatants will be analyzed with a Luminex® 200 analyzer system following the Milliplex® MAP Kit manufacturer's procedure. These same determinations will be carried out on GCF and serum/plasma samples from patients.
The quantification of functional autophagosomes in PBMCs will be performed using flow cytometry on our BD Accuri equipment with the CYTO-ID® Autophagy detection kit following manufacturer's procedure. In parallel, leucocyte from whole blood samples will be incubated with redox status detecting fluorescent probes for 15 minutes and then, they will be analysed using flow cytometry. Additionally, a multicolor panels for flow cytometry based on cluster of differentiation (CD) antigens detection, CD3/CD4/CD8/CD45RA/CCR7/CD38 and CD14/CD16, will be designed to analyze the percentage of lymphocyte T subpopulations (naive T cells (TN), central memory (TCM), effector memory (TEM) and terminally differentiated (TEMRA) cells) and, monocytes subpopulations (classical, non-classical, and intermediate), respectively in blood samples. In both procedures 10000 events will be acquired and single staining and FMO controls for all fluorochrome-conjugated antibodies in the panels will be performed to establish adequate compensation and define positive signals.
A parallel plate flow chamber, connected to an inverted microscope, will enable the researchers to measure neutrophil-endothelial cell interactions in vitro. Through this system, the leukocyte suspension obtained from patients will be perfused over a monolayer of immortalized endothelial cells (HUVEC/TERT 2) under conditions simulating blood flow. Videos will be analyzed afterward to determine flow, rolling velocity, and firm adhesion of leukocytes to endothelial cells, as previously described (Antioxidants. 2020 Aug 11;9(8):734).
Changes in gene expression levels will be evaluated using Nanostring® technology for nCounter®. To sum up, total RNA will be extracted from PBMCs using the GeneAllR RibospinTM total kit, and starting from 100-200 ng of total RNA, Diagnostica Longwood, S.L. will analyze the differential gene expression response, thus obtaining the multiplex metabolism panel.
For differential gene expression analysis of miRNAs (DEGs), RNA will be extracted using the miRNeasy Serum/Plasma kit (Qiagen). After short-chain RNA extraction, libraries will be prepared for sequencing using the TruSeq Small RNA library preparation kit (Illumina, Inc). Libraries will be pooled equimolarly and quantified using the KAPA SYBR FAST Universal qPCR kit with Illumina Primer Premix, and group size will be measured using a Bioanalyzer (Agilent). Finally, 2 nanomoles of the group will be sequenced on the Illumina NovaSeq6000 platform with a 1% PhiX control in the FISABIO facilities. Libraries will be sequenced using 2x100 bp chemistry in an SP flow cell (Illumina, Inc). After multiplexing, raw data will be processed using the "COMPSRA: a COMprehensive Platform for Small RNA-Seq data Analysis" pipeline.
DNA will also be extracted from plasma according to the "blood and body fluid protocol" of the QIAamp blood reagent set (QIAgen, Hilden, Germany); 400 μL of plasma will be applied to each column, DNA will be eluted in 200 μL of supplied buffer and will be stored at -20°C until use. mtDNA will be quantified with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using specific primers for obtaining circulating mtDNA levels.
Data analysis will be performed with SPSS 17.0. Groups will be compared using unpaired Student's t-tests or Mann-Whitney U tests for parametric and non-parametric data, respectively. Changes after intervention will be evaluated using paired Student's t-tests or Wilcoxon tests, depending on the variable distribution. Pearson or Spearman correlation coefficients will be used to measure the strength of association between variables. In multivariable regression models, the relationship between two or more explanatory variables (independent variables) and a response variable (dependent variable) will be evaluated by fitting a linear equation to the obtained data. Qualitative data will be expressed in percentages, and proportions will be compared using a Chi-square test. All tests will use a 95% confidence interval, and differences will be considered statistically significant when p \<0.05.
