Neurocandidiasis: Experimental models to approach the innate immuneresponse of the glial cells components.

FIGUEREDO CM, VIGEZZI C, MAYOL G, PERALTA-RAMOS JM, MIRÓ MS, RODRIGUEZ E, ICELY PA, SOTOMAYOR CE.

Santiago de Chile

Congreso; XIV Forum on Fungal Infection in the Clinical Practice, INFOCUS 2016 and XIX Immunocompromised Host Society Symposium; 2016

Introduction.Neurocandidiasis is a pathology with considerable incidence.This form of mycosis is classically associated to acute disseminated candidiasis, immunosuppressive states, neurosurgery and catheter use. Although considerably works have been done to identify the receptors, fungal moieties, and responses involved in anti-Candida immunity, little is known about these interactions in CNS.Goals.The aim of the present work was to develop different in vivo and in vitro model in order to explore the innate immune mechanisms involved during C.albicans-host interaction in CNS, focusing our studies in the role of Astrocytes and Microglia.Material and method.For explore the in vivo host-fungus interaction we developed two models on C57CL/6 mice. MODEL I: Mice were inoculated intravenously (i.v) with 2.5x106C. albicans (ATCC 36801) (Ca group) or PBS alone (N groups). On time 4h, 12h, 24h, 48h post infection (pi) animals were killed. The brain was removed to evaluate thr fungal burden(CFU), histological changes(PAS and HE stain), immunostain with Amb anti-GFAP and anti-CD11b (Astrocytes and Microglia marker), and in situ production of cytokines (ELISA). MODEL II: For intracerebral (i.c) inoculation the infusion cannula was stereotaxically lowered into the Caudate Putamen using coordinates according to the atlas of Franklin and Paxinos. Mice were injected with physiologic solution (N) or 5.103C. albicans (ATCC 36801) (Ca). After 48h post i.c infection, animals were killed and brain removed and processed for histological studies, IF assay and flow cytometric(FC) analysis with anti-GFAP and anti-CD11b.In vitro MODEL: For mixed glial cultures (Mx), cerebral cortices from 1- 2 day-old neonatal mice were dissected and digested, resuspended in growth-glial-culture medium and cultured at 37°C in humidified 5% CO2. For Highly-enriched Astroglial (As) cultures, on day 8, Mx culture monolayers were first treated with 8M cytosine-d-arabinofuranoside and later with 60mM Leucyne Methyl Ester to eliminate remanent Microglia, and allowed to recover prior to experimentation. The composition of Mx culture and purity of As- culture was strictly evaluated by FC using specific mAb. Both cultures where exposed to different stimuli: C.albicans(5:1 ratio); Zymozan (Zym), PGN (TLR2 agonist) and LPS (TLR4 agonist). After 24h, supernatants were collected to study cytokines (ELISA) and cells for TLR2 and TLR4 detection (FC and IFI).Results:C. albicans was able to quickly invade the brain tissue after i.v infection and CFU were recovered after 4h. The most obvious histopathological changes were observed after 24h pi with meningitis and microabscesses. In Ca group, an early local production of proinflammatory cytokines IL-1β, IL-6 and TNF-α (p