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

FIGUEREDO, MAURICIO; VIGEZZI, CECILIA; MAYOL, GONZALO; PERALTA-RAMOS, JAVIER; MIRÓ, MARÍA SOLEDAD; RODRIGUEZ, EMILSE; ICELY, PAULA ALEJANDRA; SOTOMAYOR, CLAUDIA ELENA

Congreso; XIV Forum on Fungal Infection in the Clinical Practice; 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<0,001), as well as anti-inflammatory IL-10 (p<0,001), was observed (12h pi). IF assay showed the presence of astrogliosis after 12h pi, with a high expression at 48h. In the Caudate Putamen of Ca i.c group, reactive Astrocytes also were detected, with large infiltrating of CD11d + cells. FC analysis of cells recovered from Caudado Putamen confirmed the activation of both gial populations. TNF-α also was detected in supernatants of Mx cultures stimulated with C. albicans (p<0,01) but not in As culture, beside the expression of innate receptor involved in the fungal recognition such as TLR2 and TLR4 (IFI and FC). Astrocytes produces significant levels of TNF- and IL-6 against microbial component such as PGN and LPS (p<0,01), interestingly Zym (fungal wall component) showed a similar profile that observed against C.albicans.Conclusions:The in vivo model reveals a very early colonization by C.albicans of the brain which histopathological findings similar to those found in humans, characterized by the presence of microabscesses. Early changes in cytokines production assigned an important role to CNS resident glial cell population. Astrogliosis observed indicate the participation of Astrocytes response during infection. The in vitro model shows a responsiveness of glial cells against the fungus, mainly microglia.The differences in the production of proinflammatory cytokines found against other microbial stimuli indicate an intrinsic property of C. albicans. We successfully developed experimental in vivo and in vitro models for the study of early immune response in brain against C.albicans and the glial cells contribution.