Participación de los receptores tipo Toll (TLRs) y efecto de la inmunosenescencia en la respuesta inmunitaria frente a Candida Albicans

RESUMEN Candida albicans es la especie más frecuentemente asociada a las infecciones fúngicas invasivas. Dichas infecciones representan una de las principales causas de mortalidad en pacientes inmunocomprometidos, y son también cada vez más frecuentes y graves entre la población anciana, debido, al menos en parte, a alteraciones del sistema inmunitario innato y adaptativo asociadas a la edad (inmunosenescencia). Los receptores tipo Toll (TLRs) son una familia de receptores que están implicados en el reconocimiento inicial del patógeno y en el consiguiente desarrollo de la respuesta inmunitaria innata, encaminada a eliminar al microorganismo en los primeros estadios de la infección, aunque también participan en la generación y regulación de la respuesta adaptativa. Los objetivos de la presente tesis doctoral han sido (1) determinar la participación de los TLRs en la respuesta inmunitaria frente a C. albicans, y (2) estudiar el efecto del envejecimiento (inmunosenescencia) en la respuesta inmunitaria frente al hongo, así como en la expresión y función de los TLRs. Estudios previos de nuestro grupo habían mostrado que TLR2 es esencial en la defensa frente a las candidiasis, por lo que en la primera parte de este trabajo se ha estudiado la participación de TLR4 (receptor implicado en el reconocimiento de otros hongos) en la respuesta frente a C. albicans. Los resultados obtenidos in vivo e in vitro utilizando un modelo murino de infección y ratones deficientes para este receptor muestran que TLR4 no es esencial en la defensa frente a la candidiasis. Además, empleando células de ratones TLR2-/-, se comprobó que la producción in vitro de citocinas proinflamatorias y Th1 está mediada por TLR2, tanto en respuesta a celulas de C. albicans viables como inactivadas por diferentes métodos (calor, fijación química y tratamiento antifúngico). Resultados adicionales obtenidos empleando linfocitos NK altamente purificados muestran que C. albicans es capaz de inhibir la producción in vitro de IFN- inducida por ligandos de los TLRs, lo que puede representar un mecanismo de evasión del sistema inmunitario que contribuya a la virulencia de C. albicans. Los estudios sobre inmunosenescencia se abordaron empleando una doble aproximación experimental: modelo murino (ratones ancianos) y humano (sangre completa de donantes sanos). Los resultados mostraron que los ratones ancianos son más susceptibles a la candidiasis experimental, lo que se correlaciona con defectos en la producción de citocinas proinflamatorias, una disminución en la expresión de TLR2 en la superficie de macrófagos y una respuesta Th1 disminuida. Por otra parte, empleando sangre humana completa, no se han encontrado deficiencias en la expresión o función de los receptores tipo Toll en células sanguíneas de individuos ancianos sanos que puedan explicar la mayor susceptibilidad a la candidiasis descrita en personas ancianas. __________________________________________________________________________________________________Candida albicans is the species most frequently associated with systemic fungal infections. These infections represent one of the main causes of mortality in the immunocompromised population, and their incidence and severity is also increasing in the aged population due, at least in part, to age-related alterations in the innate and adaptive immune system (immunosenescence). Toll-like receptors (TLRs) are a family of receptors involved in the initial recognition of the pathogen and the subsequent generation of the immune responses. The objectives of this PhD thesis were (1) to determine the participation of TLRs in the immune response against C. albicans, and (2) to study of the effects of aging (immunosenescence) on the immune response against the fungus, as well as on the expression and function of TLRs. First, we have studied the possible participation of TLR4 in the response against C. albicans. In vivo and in vitro results show that TLR4 is not essential in the defence against candidiasis. Second, we have confirmed the role of TLR2 in protection against C. albicans: the in vitro production of proinflammatory and Th1 cytokines is mediated by TLR2, both in response to viable and non-viable C. albicans cells inactivated by different treatments (heat-killed, formaldehyde-fixed or antimycotic-treated). Additional results obtained using highly-purified NK lymphocytes show that C. albicans is able to inhibit the in vitro production of IFN- induced by TLR ligands, an effect that may represent a novel mechanism of immune evasion that contributes to the virulence of C. albicans. The immunosenescence studies were performed using a double experimental approach: a murine model (aged mice) and a human model (whole blood from healthy donors). The results show that aged mice are more susceptible to experimental candidiasis, and this correlates with defects in the production of proinflammatory cytokines, a diminished TLR2 expression in the macrophages cell surface and an impaired Th1 response. However, no deficiencies were found in neither TLR expression or function in blood cells from aged healthy human donors, that could account for the described increased susceptibility to C. albicans infections in the elderly

Can we promote neural regeneration through microbiota-targeted strategies? Introducing the new concept of neurobiotics

Depto. de Biodiversidad, Ecología y EvoluciónFac. de Ciencias BiológicasTRUETempleton World Charity Foundationpu

Candida albicans Yeast and Hyphae are Discriminated by MAPK Signaling in Vaginal Epithelial Cells

We previously reported that a bi-phasic innate immune MAPK response, constituting activation of the mitogen-activated protein kinase (MAPK) phosphatase MKP1 and c-Fos transcription factor, discriminates between the yeast and hyphal forms of Candida albicans in oral epithelial cells (ECs). Since the vast majority of mucosal Candida infections are vaginal, we sought to determine whether a similar bi-phasic MAPK-based immune response was activated by C. albicans in vaginal ECs. Here, we demonstrate that vaginal ECs orchestrate an innate response to C. albicans via NF-κB and MAPK signaling pathways. However, unlike in oral ECs, the first MAPK response, defined by c-Jun transcription factor activation, is delayed until 2 h in vaginal ECs but is still independent of hypha formation. The ‘second’ or ‘late’ MAPK response, constituting MKP1 and c-Fos transcription factor activation, is identical to oral ECs and is dependent upon both hypha formation and fungal burdens. NF-κB activation is immediate but independent of morphology. Furthermore, the proinflammatory response in vaginal ECs is different to oral ECs, with an absence of G-CSF and CCL20 and low level IL-6 production. Therefore, differences exist in how C. albicans activates signaling mechanisms in oral and vaginal ECs; however, the activation of MAPK-based pathways that discriminate between yeast and hyphal forms is retained between these mucosal sites. We conclude that this MAPK-based signaling pathway is a common mechanism enabling different human epithelial tissues to orchestrate innate immune responses specifically against C. albicans hyphae

Gut microbiota and neuroplasticity

The accumulating evidence linking bacteria in the gut and neurons in the brain (the microbiota–gut–brain axis) has led to a paradigm shift in the neurosciences. Understanding the neurobiological mechanisms supporting the relevance of actions mediated by the gut microbiota for brain physiology and neuronal functioning is a key research area. In this review, we discuss the literature showing how the microbiota is emerging as a key regulator of the brain’s function and behavior, as increasing amounts of evidence on the importance of the bidirectional communication between the intestinal bacteria and the brain have accumulated. Based on recent discoveries, we suggest that the interaction between diet and the gut microbiota, which might ultimately affect the brain, represents an unprecedented stimulus for conducting new research that links food and mood. We also review the limited work in the clinical arena to date, and we propose novel approaches for deciphering the gut microbiota–brain axis and, eventually, for manipulating this relationship to boost mental wellness

Pupillary behavior in relation to wavelength and age

Pupil light reflex can be used as a non-invasive ocular predictor of cephalic autonomic nervous system integrity. Spectral sensitivity of the pupil´s response to light has, for some time, been an interesting issue. It has generally, however, only been investigated with the use of white light and studies with monochromatic wavelengths are scarce. This study investigates the effects of wavelength and age within three parameters of the pupil light reflex (amplitude of response, latency, and velocity of constriction) in a large sample of younger and older adults (N=97), in mesopic conditions. Subjects were exposed to a single light stimulus at four different wavelengths: white (5600 ºK), blue (450 nm), green (510 nm) and red (600 nm). Data was analysed appropriately, and, when applicable, using the General Linear Model (GLM), Randomised Complete Block Design (RCBD), Student´s t test and/or ANCOVA. Across all subjects, pupillary response to light had the greatest amplitude and shortest latency in white and green light conditions. In regards to age, older subjects (46-78 years) showed an increased latency in white light and decreased velocity of constriction in green light compared to younger subjects (18-45 years old). This study provides data patterns on parameters of wavelength-dependent pupil reflexes to light in adults and it contributes to the large body of pupillometric research. It is hoped that this study will add to the overall evaluation of cephalic autonomic nervous system integrity

Evaluation of the Role of Candida albicans Agglutinin-Like Sequence (Als) Proteins in Human Oral Epithelial Cell Interactions

The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans Δals mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight Δals mutants tested, only the Δals3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the Δals3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans

Pathogen identification and control of sooty spot caused by Cladosporium ramotenellum, appearing on fresh easy peeler mandarins from Perú

During the 2018 season, superficial dry and firm black spots, where sometimes an aerial mycelium developed, appeared on the rind of easy peeler mandarins causing high economic losses in fresh citrus exports from Perú. In this work, we have identified the causal agent, a species of Cladosporium not previously reported as a citrus pathogen. The pathogen was isolated from rind lesions of affected fruit and was identified by sequencing as Cladosporium ramotenellum; and fulfilment of Koch postulates was proven. This species was present on the surface of immature fruit in the groves, indicating that the infection is likely initiated before harvest. Cladosporium ramotenellum is resistant to the postharvest fungicides imazalil, pyrimethanil, and thiabendazole, but sensitive to propiconazole, prochloraz, and ortho-phenylphenol. We designed a postharvest industrial treatment to decrease the Cladosporium sp. load on the fruit surface that limited the incidence of infection and reduced the postharvest losses caused by the fungus. Although this species is quite ubiquitous, this is the first description of C. ramotenellum causing decay of citrus fruit, being the symptoms of this disease similar to the ones described previously and caused by Cladosporium cladosporoides in cv. Satsuma mandarins from Japan

Killed Candida albicans Yeasts and Hyphae Inhibit Gamma Interferon Release by Murine Natural Killer Cells

Killed yeasts and hyphae of Candida albicans inhibit gamma interferon secretion by highly purified murine NK cells in response to the Toll-like receptor ligands lipopolysaccharide and zymosan. This effect, which is also observed in the presence of NK-activating cytokines (interleukin-2 [IL-2], IL-12, and IL-15), may represent a novel mechanism of immune evasion that contributes to the virulence of C. albicans