Glycosaminoglycan profile in macrophages exposed to Candida albicans and interleukins

Glycosaminoglycans (GAG), are extracellular matrix macromolecules that affect the phagocytic properties of macrophages. In order to assess whether the interaction between macrophages and Candida albicans (iCa) provokes changes in the phenotype, we analyzed the GAG profiles in two macrophage lines, ANA-1 (from murine bone-marrow) and BV-2 (from murine brain). We also investigated GAG modulation by interleukin-1alpha (IL-1alpha) and interleukin-6 (IL-6). During iCa treatment and even after the addition of ILs, ANA-1 accumulated less total GAG compared to controls. IL-1 treatment, combined with iCa exposure, induced a decrease in heparan sulfate and chondroitin sulfate chains, and an increase in the hyaluronic acid percentage. IL-6 treatment, with or without iCa, decreased the hyaluronic acid/sulfated GAG ratio. The GAG pattern in BV-2 appears to be different to ANA-1 and iCa exposure does not induce any difference in total GAG. The inhibitory effect induced by ILs on GAG synthesis is less than that observed in ANA-1 and the GAG elution profile is modulated to a lesser extent by treatment with ILs and/or iCa compared to the ANA-1. We suggest that the observed changes in the expression of the individual GAG classes may be responsible for the macrophage functional heterogeneity

Genetic susceptibility to aspergillosis in allogeneic stem-cell transplantation

Invasive aspergillosis (IA) is a major threat to positive outcomes for allogeneic stem-cell transplantation (allo-SCT) patients. Despite presenting similar degrees of immunosuppression, not all individuals at-risk ultimately develop infection. Therefore, the traditional view of neutropenia as a key risk factor for aspergillosis needs to be accommodated within new conceptual advances on host immunity and its relationship to infection. Polymorphisms in innate immune genes, such as those encoding TLRs, cytokines and cytokine receptors, have recently been associated with susceptibility to IA in allo-SCT recipients. This suggests that understanding host-pathogen interactions at the level of host genetic susceptibility will allow the formulation of new targeted and patient-tailored antifungal therapeutics, including improved donor screening.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/65962/2009, SFRH/BPD/46292/2008Specific Targeted Research Projects MANASP (LSHE-CT-2006), contract number 037899 (FP6), Italian Project PRIN2007KLCKP8_004

Non-hematopoietic cells contribute to protective tolerance to Aspergillus fumigatus via a TRIF pathway converging on IDO

Innate responses combine with adaptive immunity to generate the most effective form of anti-Aspergillus immune resistance. Whereas the pivotal role of dendritic cells in determining the balance between immunopathology and protective immunity to the fungus is well established, we determined that epithelial cells (ECs) also contributes to this balance. Mechanistically, EC-mediated protection occurred through a Toll-like receptor 3/Toll/IL-1 receptor domain-containing adaptor-inducing interferon (TLR3/TRIF)-dependent pathway converging on indoleamine 2,3-dioxygenase (IDO) via non-canonical nuclear factor-?B activation. Consistent with the high susceptibility of TRIF-deficient mice to pulmonary aspergillosis, bone marrow chimeric mice with TRIF unresponsive ECs exhibited higher fungal burdens and inflammatory pathology than control mice, underexpressed the IDO-dependent T helper 1/regulatory T cell (Th1/Treg) pathway and overexpressed the Th17 pathway with massive neutrophilic inflammation in the lungs. Further studies with interferon (IFN)-?, IDO or IL-17R unresponsive cells confirmed the dependency of immune tolerance to the fungus on the IFN-?/IDO/Treg pathway and of immune resistance on the MyD88 pathway controlling the fungal growth. Thus, distinct immune pathways contribute to resistance and tolerance to the fungus, to which the hematopoietic/non-hematopoietic compartments contribute through distinct, yet complementary, roles.We thank Cristina Massi Benedetti for digital art and editing. This work was supported by the Specific Targeted Research Project 'Sybaris' (LSHE-CT-2006), contract number 037899 (FP7) and by the Italian Projects PRIN 2007KLCKP8_004 (to LR) and 2007XYB9T9_001 (to SB). CC and AC were financially supported by fellowships from Fundacao para a Ciencia e Tecnologia, Portugal (contracts SFRH/BD/65962/2009 and SFRH/BPD/46292/2008, respectively)

Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.National Institute of Dental & Craniofacial ResearchFundação para a Ciência e Tecnologia (FCT) - SFRH/BD/28222/2006National Institute of Allergy and Infectious Disease

TLR9 activation dampens the early inflammatory response to paracoccidioides brasiliensis, Impacting host survival

Background: Paracoccidioides brasiliensis causes paracoccidioidomycosis, one of the most prevalent systemic mycosis in Latin America. Thus, understanding the characteristics of the protective immune response to P. brasiliensis is of interest, as it may reveal targets for disease control. The initiation of the immune response relies on the activation of pattern recognition receptors, among which are TLRs. Both TLR2 and TLR4 have been implicated in the recognition of P. brasiliensis and regulation of the immune response. However, the role of TLR9 during the infection by this fungus remains unclear.J.F. Menino was supported by a grant from Fundacao para a Ciencia e Tecnologia (FCT), Portugal (SFRH/BD/33446/2008). This work was supported by a grant from FCT (PTDC/BIA-MIC/108309/2008). M. Saraiva is a Ciencia 2007 fellow and M. Sturme is a Ciencia 2008 fellow. We would also like to thank FAPESP (Fundacao para Amparo a Pesquisa do Estado de Sao Paulo) and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Coexpression of vesicular glutamate transporters 1 and 2, glutamic acid decarboxylase and calretinin in rat entorhinal cortex

We studied the distribution and coexpression of vesicular glutamate transporters (VGluT1, VGluT2), glutamic acid decarboxylase (GAD) and calretinin (CR, calcium-binding protein) in rat entorhinal cortex, using immunofluorescence staining and multichannel confocal laser scanning microscopy. Images were computer processed and subjected to automated 3D object recognition, colocalization analysis and 3D reconstruction. Since the VGluTs (in contrast to CR and GAD) occurred in fibers and axon terminals only, we focused our attention on these neuronal processes. An intense, punctate VGluT1-staining occurred everywhere in the entorhinal cortex. Our computer program resolved these punctae as small 3D objects. Also VGluT2 showed a punctate immunostaining pattern, yet with half the number of 3D objects per tissue volume compared with VGluT1, and with statistically significantly larger 3D objects. Both VGluTs were distributed homogeneously across cortical layers, with in MEA VGluT1 slightly more densely distributed than in LEA. The distribution pattern and the size distribution of GAD 3D objects resembled that of VGluT2. CR-immunopositive fibers were abundant in all cortical layers. In double-stained sections we noted ample colocalization of CR and VGluT2, whereas coexpression of CR and VGluT1 was nearly absent. Also in triple-staining experiments (VGluT2, GAD and CR combined) we noted coexpression of VGluT2 and CR and, in addition, frequent coexpression of GAD and CR. Modest colocalization occurred of VGluT2 and GAD, and incidental colocalization of all three markers. We conclude that the CR-containing axon terminals in the entorhinal cortex belong to at least two subpopulations of CR-neurons: a glutamatergic excitatory and a GABAergic inhibitory

Neutrophil responses to Aspergillosis : new roles for old players

Neutrophils are professional phagocytic cells that play a crucial role in innate immunity. Through an assortment of antifungal effector mechanisms, neutrophils are essential in controlling the early stages of fungal infection. These mechanisms range from the production of reactive oxygen intermediates and release of antimicrobial enzymes to the formation of complex extracellular traps that aid in the elimination of the fungus. Their importance in antifungal immunity is supported by the extreme susceptibility to infection of patients with primary (e.g., chronic granulomatous disease) or acquired (e.g., undergoing immunosuppressive therapy) neutrophil deficiency. More recently, common genetic variants affecting neutrophil antifungal capacity have also been disclosed as major risk factors for aspergillosis in conditions of generalized immune deficiency. The present review revisits the role of neutrophils in the host response against Aspergillus and highlights the consequences of their deficiency in susceptibility to aspergillosis.This work was supported by a Research Grant from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Cristina Cunha was supported by the Fundacao para a Ciencia e Tecnologia, Portugal (contract SFRH/BPD/96176/2013)

Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically-modified animal models, together with biochemical and pharmacological approaches, we provide a high resolution expression map and a detailed functional characterization of A2AR-CB1R heteromers in the dorsal striatum. Specifically, our data unveil that the A2AR-CB1R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington’s disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases