Low-level Laser Therapy to the Mouse Femur Enhances the Fungicidal Response of Neutrophils against Paracoccidioides brasiliensis

Gérsika B Santos,Maísa R P L Brigagão,Luiz Cosme C Malaquias,Eva Burger,Zoilo P De Camargo,Giulia M A C Bani,Michael R Hamblin,Jorge Kleber Chavasco,Felipe F Sperandio,Liana M Verinaud,Ana Carolina S C Mendes

doi:10.1371/journal.pntd.0003541

Abstract

Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. The goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. The aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. The number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. The highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. The kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.

Highlights

Paracoccidioides brasiliensis (Pb) is a non-sexual thermodimorphic fungus that exists in either a mycelium or a yeast form; the latter being pathogenic to humans and can cause an important and neglected systemic infection called paracoccidioidomycosis (PCM)
The animals inoculated with saline showed no neutrophils at the site of infection even after 10 days (S1 Fig.), which clearly showed that neither the air-pouch procedure alone nor the laser irradiation alone was responsible for the polymorphonuclear cells (PMN) recruitment
The PMN produced by the inflammatory stimuli were harvested from the subcutaneous air-pouches (Fig. 1), and whilst the total number of PMN recruited to these air pouches was significantly diminished (p = 0.0001) when level laser therapy (LLLT) was used after the Pb infection, the number of PMN was significantly increased (p = 0.0001) when LLLT was used after mice were inoculated with Zymosan (Fig. 2)

Summary

Introduction

Paracoccidioides brasiliensis (Pb) is a non-sexual thermodimorphic fungus that exists in either a mycelium or a yeast form; the latter being pathogenic to humans and can cause an important and neglected systemic infection called paracoccidioidomycosis (PCM). Patients with immune suppression or defects in immune cell activation are more susceptible to PCM [2,3]. PCM presents as a primary acute infection that is later transformed to a chronic phase. Besides the production of several direct antimicrobial factors, PMN may secrete cytokines, chemokines and growth factors [5] that promote the host response against the infection. PMN are critical for the innate immune response, but can help the adaptive immune response by interacting with B lymphocytes [6], T cells [7] and dendritic cells [8]

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