Low-Level Laser Therapy Reduces Lung Inflammation in an Experimental Model of Chronic Obstructive Pulmonary Disease Involving P2X7 Receptor

Chronic obstructive pulmonary disease (COPD) is a progressive disease characterized by irreversible airflow limitation, airway inflammation and remodeling, and enlargement of alveolar spaces. COPD is in the top five leading causes of deaths worldwide and presents a high economic cost. However, there are some preventive measures to lower the risk of developing COPD. Low-level laser therapy (LLLT) is a new effective therapy, with very low cost and no side effects. So, our objective was to investigate if LLLT reduces pulmonary alterations in an experimental model of COPD. C57BL/6 mice were submitted to cigarette smoke for 75 days (2x/day). After 60 days to smoke exposure, the treated group was submitted to LLLT (diode laser, 660 nm, 30 mW, and 3 J/cm(2)) for 15 days and euthanized for morphologic and functional analysis of the lungs. Our results showed that LLLT significantly reduced the number of inflammatory cells and the proinflammatory cytokine secretion such as IL-1 beta, IL-6, and TNF-alpha in bronchoalveolar lavage fluid (BALF). We also observed that LLLT decreased collagen deposition as well as the expression of purinergic P2X7 receptor. On the other hand, LLLT increased the IL-10 release. Thus, LLLT can be pointed as a promising therapeutic approach for lung inflammatory diseases as COPD.Sao Paulo Research Foundation (FAPESP) [2012/16498-5, 2012/15165-2]FAPESP [2015/23152-6, 2014/14604-8, 2015/13486-4]Univ Nove Julho UNINOVE, Post Grad Program Biophoton Appl Hlth Sci, Sao Paulo, SP, BrazilBrazilian Inst Teaching & Res Pulm & Exercise Imm, Sao Jose Dos Campos, SP, BrazilUniv Nove Julho UNINOVE, Masters Degree & PhD Program Rehabil Sci, Expt Cardioresp Physiol Lab, Sao Paulo, SP, BrazilUniv Calif San Diego UCSD Hlth Sci, Div Trauma Surg Crit Care Burns & Acute Care Surg, Dept Surg, San Diego, CA USAFed Univ Sao Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilUniv Brasil, Postgrad Program Bioengn, Sao Paulo, SP, BrazilFed Univ Sao Paulo UNIFESP, Postgrad Program Sci Human Movement & Rehabil, Santos, SP, BrazilFed Univ Sao Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilFed Univ Sao Paulo UNIFESP, Postgrad Program Sci Human Movement & Rehabil, Santos, SP, BrazilFAPESP [2012/16498-5, 2012/15165-2]FAPESP [2015/23152-6, 2014/14604-8, 2015/13486-4]Web of Scienc

Effects of periodontitis on the development of asthma: The role of photodynamic therapy

<div><p>To evaluate whether periodontitis modulates lung inflammation in an experimental model of asthma as well as the photodynamic therapy (PDT) is associated with a reduction of lung inflammation. Seventy-two BALB/c male mice (~2 months) were randomly divided into 8 groups (n = 9): Basal, Periodontitis (P), P+PT, P+PT+PDT, Asthma (A), A+P, A+P+PT, and A+P+PT+PDT. Periodontitis was induced by using the ligature technique and asthma was induced by ovalbumin (OVA). PT was performed with curettes and PDT with methylene blue (0.005%), λ = 660nm, with a radiant exposure of 318J/cm². After 43 days, euthanasia was carried out prior to lung and mandible morphological analyzes. All of the manipulations of the animals were performed by only one operator. The total and differential cell counts and cytokines IL-4, IL-5, IL-10, IFN-γ, TNF-α, IL-1β, and IL-6 were evaluated in the bronchoalveolar lavage (BAL) and in the serum. Mucus and alkaline phosphatase were also quantified. Statistical analyzes were performed by a blinded statistician. One-way analysis of variance (ANOVA) was employed, followed by the Student-Newman-Keuls test. Periodontitis group (P) increased alkaline phosphatase and bone resorption (p<0.05), validating the experimental model of periodontitis. The A group and the P group increased the total amount of cells (p <0.05) in the BAL. However, in the A+P group, there was a decrease in these cells, except for in the A+P+PT+PDT group (p<0.05). The asthma group increased the Th2 cytokines and P group increased the Th1 cytokine profile, and A+P+PT+PDT group increased IL-10 cytokine. Mucus was increased for the A and P groups. In conclusion, periodontitis in the asthmatic mice reduced the inflammatory migrated cells in the BAL (eosinophils, lymphocytes, macrophages). In addition, it reduced the levels of the IL-4 and TNF-α cytokines, which was also accompanied by a decreased mucus production. After PDT treatment the total cell count increased however, this increase was not accompanied by a pro-inflammatory cytokines release. Only in PDT group the anti-inflammatory IL-10 was increased. Further studies are needed to understand this mechanism of action.</p></div

Reduced mucus secretion in the lungs of periodontitis induced animals.

<p>Asthmatic animals were submitted to different protocols as described in the material and methods. Further all animals were euthanized and lungs were sectioned and stained with PAS—Periodic Acid Schiff (400x) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187945#pone.0187945.s002" target="_blank">S2 Fig</a>. Different groups can be observed (A-H): A- Basal, B- Asthma (A), C- Periodontitis (P), D- Induction of Asthma + Induction of Periodontitis (Asthma+PD), E- Induction of Periodontitis + Standard Periodontal Treatment (P+TP), F- Induction of Asthma + Induction of Periodontitis + Standard Periodontal Treatment (Asthma+P+TP), G- Induction of Periodontitis + Standard Periodontal Treatment + Photodynamic Therapy (P+PT+PDT), H-Asthma Induction + Induction of Periodontitis + Standard Periodontal Treatment + Photodynamic Therapy (Asthma+P+TP+PDT). Data representative of 2 experiments. (n = 4–5 animals per group). Bar = 20 μm.</p

Quantification of alkaline phosphatase in the serum.

<p>The quantification of serum alkaline phosphatase was also quantified by using ELISA. The groups were: Basal (unmanaged animals), P (periodontitis), P + PT (periodontitis + periodontal treatment), P + PT + PDT (periodontitis + periodontal treatment + photodynamic therapy), Asthma, Asthma + P (asthmatic animals + periodontitis), Asthma + P + PT (asthmatic animals + periodontitis + periodontal treatment), Asthma + P + PT + PDT + Asthma (periodontitis + periodontal treatment+ photodynamic therapy). The results are shown as mean ± SEM for the 2 experiments (n = 4–5); ANOVA was employed, followed by the Student-Newman-Keuls test. *** p <0.001 compared to the Basal group; Φp <0.01 compared to the P group.</p

Histological analysis of alveolar bone between the 1st and the 2nd molar.

<p>(A-H): A- Basal, B- Periodontitis (P), C- P+PT (Periodontal treatment), D- P+PT+PDT (photodynamic therapy, E- Asthma (A), F- A+P, G- A+P+PT, and H -A+P+PT+PDT. Decalcified periodontal mandibles were sectioned (5μm). Decalcified periodontal tissue sections (256x) were stained with HE (R HEhematoxylin and eosin solution) Representative tissue sections are shown. (*) represents alveolar bone. Bar = 30 μm.</p

Total bone loss.

<p>Total bone loss.</p

Parameters of red laser.

<p>Parameters of red laser.</p

Experiment timeline and design.

<p>Experiment timeline and design.</p

Quantification of the total number of cells that were recovered from the BAL.

<p>The volume of recovered BAL was used for the total cell count with Trypan Blue (x10⁴) with a Neubauer chamber. The groups were: Basal (unmanaged animals), P (periodontitis), P + TP (periodontitis + periodontal treatment), P + TP + PDT (periodontitis + periodontal treatment + photodynamic therapy), Asthma (asthmatic animals + periodontitis), Asthma + P + TP (asthmatic animals + periodontitis + periodontal treatment), Asthma + P + PT + PDT photodynamic therapy). The results are shown as mean ± SEM of the 2 experiments (n = 4–5). ANOVA was employed, followed by the Student-Newman-Keuls test. *p<0.05; *** p <0.001 when compared to the Basal group; ^ϕp <0.05 when compared to the P group; Δp <0.001 relative to the Asthma group; ^θp <0.001 when compared to the Asthma + P group.</p

Analyzes of the cytokines IL-1β (A), IL-6 (B) and TNF-α (C) in the BAL.

<p>For an evaluation of lung inflammation in the BAL the lungs were washed with phosphate buffered saline and after centrifugation, the supernatant was collected for the cytokine analyzes by ELISA. The groups were: Basal (unmanaged animals), P (periodontitis), P + PT (periodontitis + periodontal treatment), P + PT + PDT (periodontitis + periodontal treatment + photodynamic therapy), Asthma, Asthma + P (asthmatic animals + periodontitis), Asthma + P + PT (asthmatic animals + periodontitis + periodontal treatment), Asthma + P + PT + PDT + Asthma (periodontitis + periodontal treatment + photodynamic therapy). The results are shown as mean ± SEM for the 2 experiments (n = 4–5); ANOVA was employed, followed by the Student-Newman-Keuls test. (A) ** p <0.01 related to the Basal group; (B); *** p <0.001 when compared to the basal group; Φ p <0.001 in relation to the P group; (C) ** p <0.05; *** p <0.001 when compared to the basal group; Φp <0.05 for the group P + TP when compared to the P group and φ p <0.01 for the group P +TP + PDT when compared to the P group; Δp <0.05 when compared to the Asthma group.</p