Real-time monitoring of the spatial and temporal progression of infection/gene expression in animals will contribute greatly to our understanding of host-pathogen interactions while reducing the number of animals required to generate statistically significant data sets. Sensitive in vivo imaging technologies can detect low levels of light emitted from luciferase reporters in vivo, but the existing reporters are not optimal for fungal infections. Therefore, our aim was to develop a novel reporter system for imaging Candida albicans infections that overcomes the limitations of current luciferase reporters for this major fungal pathogen. This luciferase reporter was constructed by fusing a synthetic, codon-optimized version of the Gaussia princeps luciferase gene to C. albicans PGA59, which encodes a glycosylphosphatidylinositol-linked cell wall protein. Luciferase expressed from this PGA59-gLUC fusion (referred to as gLUC59) was localized at the C. albicans cell surface, allowing the detection of luciferase in intact cells. The analysis of fusions to strong (ACT1 and EFT3), oxidative stress-induced (TRX1, TRR1, and IPF9996), and morphogenesis-dependent (HWP1) promoters confirmed that gLUC59 is a convenient and sensitive reporter for studies of gene regulation in yeast or hyphal cells, as well as a flexible screening tool. Moreover, the ACT1-gLUC59 fusion represented a powerful tool for the imaging of disease progression in superficial and subcutaneous C. albicans infections. gLUC59 and related cell surfaceexposed luciferase reporters might find wide applications in molecular biology, cell biology, pathobiology, and high-throughput screens.Candida albicans is responsible for a large fraction of fungal infections in humans (5) and, as such, has received considerable attention from the research community over the last two decades. C. albicans now represents an invaluable model for dissecting the interplay between fungal pathogens and their hosts at the molecular level (31,32,43,45,50). Studies of host-pathogen interactions have been greatly facilitated by the use of ex vivo infection models where isolated microorganisms and host cells or reconstituted tissues are brought into contact and the kinetics of pathogen and host cell responses are monitored (12,14,23,36,45). Yet, animal models remain necessary complements to ex vivo infection models, because none of these models fully reflect the development of clinical infections. Animal models allow researchers to monitor the behavior of mutant microorganisms or the expression of reporter genes in the complex environments of organs and in the presence of a fully functional or debilitated immune system (3,20,24).A current limitation of animal models is the need to sacrifice animals in order to image microorganisms at the site of infection. In particular, studies aimed at evaluating whether conditions known to trigger the expression of a specific C. albicans gene in vitro are encountered at sites of infection have often relied on the detection of a reporter in tissue section...

In this study we tested the in vitro and in vivo anti-Cryptococcus neoformans activity of an antilaminarin (anti-␤-glucan) monoclonal antibody (MAb 2G8) (immunoglobulin G2b) which was previously shown to inhibit the growth of ␤-glucan-exposing Candida albicans cells. Here we show that MAb 2G8 binds to the cell wall of C. neoformans and inhibits its growth to an extent comparable to that observed for C. albicans. Binding and growth inhibition were detected almost equally for encapsulated and acapsular C. neoformans strains. In addition, at subinhibitory concentrations, MAb 2G8 reduced the capsule thickness without affecting protease or phospholipase production. Acapsular fungal cells, but not encapsulated fungal cells, were opsonized by the antibody and more efficiently phagocytosed and killed by human monocytes and by murine peritoneal macrophages. A single administration of MAb 2G8 resulted in a reduction in the fungal burden in the brains and livers of mice systemically infected with a highly virulent, encapsulated C. neoformans strain. This protective effect was also detected in neutropenic mice. Overall, these findings demonstrate that cell wall ␤-glucan of encapsulated C. neoformans is accessible to antibodies which can exert remarkable anticryptococcal activities in vitro and in vivo.Deep-seated mycoses are a severe clinical problem because of well-known diagnostic difficulties and the partial inability of antifungal drugs to eradicate the infections in immunocompromised hosts, often resulting in toxicity, drug resistance, and associated high costs of supportive treatment. As a consequence, the mortality rate for invasive fungal infections remains high, particularly in severely immunocompromised pa-

The ability of encapsulated and acapsular strains of Cryptococcus neoformans to activate dendritic cells (DC) derived from monocytes stimulated with granulocyte macrophage-colony stimulating factor and interleukin-4 was evaluated. Profound differences in DC response to encapsulated and acapsular C. neoformans strains were observed. In particular, (i) the acapsular strain was easily phagocytosed by immature DC, and the process induced several molecular markers, such as major histocompatibility complex (MHC) class I and class II, CD40, and CD83, which are characteristic of mature DC; (ii) the encapsulated strain did not up-regulate MHC class I and class II and CD83 molecules; (iii) the soluble capsular polysaccharide glucuronoxylomannan (GXM) is unable to regulate MHC class I and class II molecules; (iv) the addition of monoclonal antibody to GXM (anti-GXM) to the encapsulated strain facilitated antigen-presenting cell maturation by promoting ingestion of C. neoformans via Fc receptor for immunoglobulin G (FcgammaR)II (CD32) and FcgammaRIII (CD16); (v) pertubation of FcRgammaII or FcgammaRIII was insufficient to promote DC maturation; and (vi) optimal DC maturation permitted efficient T cell activation and differentiation, as documented by the enhancement of lymphoproliferation and interferon-gamma production. These results indicate that the C. neoformans capsule interferes with DC activation and maturation, indicating a new pathway by which the fungus may avoid an efficient T cell response.

BackgroundTh17 cells play a major role in coordinating the host defence in oropharyngeal candidiasis. In this study we investigated the involvement of the Th17 response in an animal model of vulvovaginal candidiasis (VVC).MethodsTo monitor the course of infection we exploited a new in vivo imaging technique.Resultsi) The progression of VVC leads to a strong influx of neutrophils in the vagina soon after the challenge which persisted despite the resolution of infection; ii) IL-17, produced by vaginal cells, particularly CD4 T cells, was detected in the vaginal wash during the infection, reaching a maximum 14 days after the challenge; iii) The amount and kinetics of IL-23 in vaginal fluids were comparable to those in vaginal cells; iv) The inhibition of Th17 differentiation led to significant inhibition of IL-17 production with consequent exacerbation of infection; v) An increased production of βdefensin 2 was manifested in cells of infected mice. This production was strongly reduced when Th17 differentiation was inhibited and was increased by rIL-17 treatment.ConclusionsThese results imply that IL-17 and Th17, along with innate antimicrobial factors, have a role in the immune response to vaginal candidiasis.

In a recent report, we demonstrated that distinct members of the secreted aspartic protease (Sap) family of Candida albicans are able to induce secretion of proinflammatory cytokines by human monocytes, independently of their proteolytic activity and specific pH optima. In particular, C. albicans Sap2 and Sap6 potently induced IL-1β, TNF-α, and IL-6 production. Here, we demonstrate that Sap2 and Sap6 proteins trigger IL-1β and IL-18 production through inflammasome activation. This occurs via NLRP3 and caspase-1 activation, which cleaves pro-IL-1β into secreted bioactive IL-1β, a cytokine that was induced by Saps in monocytes, in monocyte-derived macrophages and in dendritic cells. Downregulation of NLRP3 by RNA interference strongly reduced the secretion of bioactive IL-1β. Inflammasome activation required Sap internalization via a clathrin-dependent mechanism, intracellular induction of K + efflux, and ROS production. Inflammasome activation of monocytes induced by Sap2 and Sap6 differed from that induced by LPS-ATP in several aspects. Our data reveal novel immunoregulatory mechanisms of C. albicans and suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity.Keywords: Aspartic proteases r C. albicans r IL-1β r Inflammasome r Virulence factor IntroductionCandida albicans is a commensal fungus that colonizes human mucosal surfaces such as the vaginal and gastrointestinal tracts without causing harm. However, under conditions of primary or secondary immunodeficiency, this yeast can cause opportunistic infections such as mucosal inflammation and systemic sepsis [1]. The mortality rate associated with invasive candidiasis Correspondence: Dr. Anna Vecchiarelli e-mail: vecchiar@unipg.it has been reported to be as high as 40-50% [2]. Candida species are the fourth most common pathogens isolated from nosocomial bloodstream infections in the USA and Europe [3]. Although the immune status of the host plays a key role in the prevention or pathogenesis of C. albicans infections, a number of virulence attributes of C. albicans, such as factors that mediate adhesion, enzyme secretion, or hyphal formation, contribute to the disease process [4]. Particularly, the secretion of aspartic proteases (Saps), * These authors contributed equally to this work.C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 680Donatella Pietrella et al. Eur. J. Immunol. 2013. 43: 679-692 which are encoded by a gene family with ten members, has long been recognized as a virulence-associated trait of this pathogenic yeast [5].We recently reported that various members of the Sap family, including Sap1, Sap2, Sap3, and Sap6, have different abilities to induce secretion of pro-inflammatory cytokines by human monocytes via Akt/NF-κB activation. Sap1, Sap2, and Sap6 potently induced IL-1β, TNF-α, and IL-6 production. Importantly, Sapinduced cytokine production was independent of the proteolytic activity and of the optimal pH for the individual Sap activities [6]. These data suggest ...

The incidence of inflammatory bowel disease is increasing all over the world, especially in industrialized countries. The aim of the present work was to verify the anti-inflammatory activity of metabolites. In particular, cell-free supernatants of Lactobacillus acidophilus, Lactobacillus casei, Lactococcus lactis, Lactobacillus reuteri, and Saccharomyces boulardii have been investigated. Metabolites produced by these probiotics were able to downregulate the expression of PGE-2 and IL-8 in human colon epithelial HT-29 cells. Moreover, probiotic supernatants can differently modulate IL-1β, IL-6, TNF-α, and IL-10 production by human macrophages, suggesting a peculiar anti-inflammatory activity. Furthermore, supernatants showed a significant dose-dependent radical scavenging activity. This study suggests one of the mechanisms by which probiotics exert their anti-inflammatory activity affecting directly the intestinal epithelial cells and the underlying macrophages. This study provides a further evidence to support the possible use of probiotic metabolites in preventing and downregulating intestinal inflammation as adjuvant in anti-inflammatory therapy.

Glucuronoxylomannan (GXM) is a microbial compound that can modulate the immune response. We investigated (1) the receptors involved in uptake of GXM on monocyte-derived macrophages (MDMs) from healthy donors, (2) the effects of GXM on expression of specific receptors, (3) the effects of GXM mediated by pattern-recognition receptors, and (4) GXM modulation of MDM accessory and secretory functions. Cellular receptors involved in uptake of GXM included Fc gamma RII, CD18, Toll-like receptor (TLR) 4, and CD14. Some biological functions of MDMs were profoundly affected by treatment with GXM, resulting in (1) increased expression of CD40 and CD86 via perturbation of TLR4, (2) decreased expression of major histocompatibility complex class II, (3) induction of interleukin-10 but not of tumor necrosis factor-alpha, and (4) decreased lipopolysaccharide (LPS)-induced production of cytokines. GXM represents an attractive compound to limit inflammatory processes and induce an LPS-tolerant state.

The regulation by Cryptococcus neoformans encapsulation of interleukin 1␤ (IL-1␤) and tumor necrosis factor alpha (TNF-␣) production by human monocytes was investigated. By using encapsulated and acapsular C. neoformans, we demonstrated that both strains induce cytokine production, although the acapsular strain was a better stimulator than the thinly encapsulated strain. The cytokine levels produced by cells stimulated by the two strains were lower and followed a different kinetic than those stimulated by lipopolysaccharide (LPS). Purified capsular polysaccharide inhibits TNF-␣ secretion induced by LPS or acapsular C. neoformans. In contrast, no regulatory effect on IL-1␤ was observed when LPS was used. The secretory response of these cytokines follows different pathways of macrophage activation; in fact, complete inhibition of TNF-␣ does not affect IL-1␤ production and vice versa. These data indicate that purified capsular polysaccharide of C. neoformans could contribute to the in vivo progress of cryptococcosis by suppressing cytokine production of macrophages and suggest that a therapeutic approach to address the suppressive effect of cryptococcal polysaccharide could be devised.