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

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.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)National Institute of Health (US NIH)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fed Univ Alfenas UNIFAL MG, Inst Biomed Sci, Dept Microbiol & Immunol, Alfenas, MG, BrazilFed Univ Alfenas UNIFAL MG, Inst Biomed Sci, Dept Biochem, Alfenas, MG, BrazilState Univ Campinas UNICAMP, Inst Biol, Dept Struct & Funct Biol, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, São Paulo, SP, BrazilMassachusetts Gen Hosp, Wellman Ctr Photomed, Boston, MA 02114 USAHarvard Univ, Sch Med, Dept Dermatol, Boston, MA 02115 USAMIT, Harvard Mit Div Hlth Sci & Technol, Cambridge, MA 02139 USAFed Univ Alfenas UNIFAL MG, Inst Biomed Sci, Dept Pathol & Parasitol, Alfenas, MG, BrazilFed Univ São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, São Paulo, SP, BrazilCNPq: 486135/2012-8CNPq: 304827/2012-6FAPEMIG: CBB-PPM-00119-14National Institute of Health (US NIH): R01AI050875CAPES: AEX-9765-14-0Web of Scienc

PMN mitochondrial activity after co-cultivation.

<p>Mitochondrial activity of light irradiated and non-irradiated PMN recruited by either <i>Pb</i> or Zymosan stimuli and co-cultivated with <i>Pb</i> in vitro. p = 0.0029 (*) between the irradiated and non-irradiated PMN of the <i>Pb</i> group; p = 0.0012 (*) between the irradiated PMN of <i>Pb</i> group and irradiated PMN of the Zy group; p = 0.0004 (*) between the irradiated and non-irradiated PMN of the Zy group; p = 0.0001 (*) between the non-irradiated PMN of the <i>Pb</i> group and the non-irradiated PMN of the Zy group.</p

Extraction of PMN from the air-pouch.

<p>Absolute number of PMN at extraction time for both irradiated and non-irradiated mice of <i>Pb</i> and Zymosan (Zy) groups; p = 0.0001 (*) between non-irradiated and irradiated PMN of <i>Pb</i> groups; p = 0.0001 (*) between non-irradiated and irradiated PMN of Zy groups; p = 0.0001 (*) between PMN from non-irradiated <i>Pb</i> and Zy groups; p = 0.0399 (*) between PMN from irradiated <i>Pb</i> and Zy groups.</p

Kinetic behavior of PMN viability.

<p>Kinetics of PMN cell viability in irradiated and non-irradiated mice after stimuli with: <b>A</b>—<i>Pb</i>: higher viability for the irradiated PMN at 6 hours—p = 0.0278 (*); <b>B</b>—Zymosan: higher viability for the irradiated PMN at 2 hours—p = 0.0274 (*); <b>C</b>—<i>Pb</i> stimulated and co-cultivated with <i>Pb</i>; <b>D—</b>Zymosan stimulated and co-cultivated with <i>Pb</i>.</p

Fungicidal capacity of PMN.

<p><b>A—</b>Colony forming units (CFU) of <i>Pb</i> at 7 and 12 days evaluation for non-irradiated and irradiated PMN. p = 0.0002 (*) between non-irradiated and irradiated PMN after 12 days of fungal growth; p = 0.0118 (*) between the irradiated groups at 7 and 12 days of fungal growth and p = 0.0003 (*) between the non-irradiated groups at 7 and 12 days of fungal growth. The colony forming units of <i>Pb</i> after co-cultivation with PMN recruited by <i>Pb</i> infection or Zy inoculation are shown after: <b>B—</b>7 days: p = 0.0369 (*) between the irradiated and non-irradiated PMN of the <i>Pb</i> group; p = 0.0232 (*) between the irradiated and non-irradiated PMN of the Zy group; or <b>C—</b>12 days: p = 0.0193 (*) between the irradiated and non-irradiated PMN of the <i>Pb</i> group; p = 0.0492 (*) between the irradiated and non-irradiated PMN of the Zy group.</p

Subcutaneous air-pouch.

<p>Clinical appearance of the air-pouch on the dorsum of the mouse after skin flap procedure.</p

Quantification of reactive oxygen species (ROS) produced by PMN.

<p>Luminol chemiluminescence of ROS (photon counts x 10⁴ per second) by light irradiated and non-irradiated PMN recruited by <b>A—</b><i>Pb</i> or Zymosan (Zy): p = 0.0425 (*) between non-irradiated and irradiated PMN of <i>Pb</i> groups; p = 0.0325 (*) between non-irradiated and irradiated PMN of Zy groups; and <b>B—</b><i>Pb</i> or Zy co-cultivated with <i>Pb</i>: p = 0.0356 (*) between the irradiated and non-irradiated PMN of the <i>Pb</i> group; p = 0.0325 (*) between the irradiated and non-irradiated PMN of the Zy group; p = 0.0406 (*) between the non-irradiated PMN of the <i>Pb</i> and Zy groups.</p