Pattern recognition receptors in antifungal immunity

We thank the Wellcome Trust for funding this study.Peer reviewedPublisher PD

Fonsecaea pedrosoi-induced Th17-cell differentiation in mice is fostered by Dectin-2 and suppressed by Mincle recognition

Funded by NIH. Grant Number: R01 AI093553 Wellcome TrustPeer reviewedPublisher PD

Fatal Disseminated Cryptococcus gattii Infection in New Mexico

We report a case of fatal disseminated infection with Cryptococcus gattii in a patient from New Mexico. The patient had no history of recent travel to known C. gattii-endemic areas. Multilocus sequence typing revealed that the isolate belonged to the major molecular type VGIII. Virulence studies in a mouse pulmonary model of infection demonstrated that the strain was less virulent than other C. gattii strains. This represents the first documented case of C. gattii likely acquired in New Mexico

Cryptococcus neoformans hyperfilamentous strain is hypervirulent in a murine model of cryptococcal meningoencephalitis.

Cryptococcus neoformans is a human fungal pathogen that causes lethal infections of the lung and central nervous system in immunocompromised individuals. C. neoformans has a defined bipolar sexual life cycle with a and α mating types. During the sexual cycle, which can occur between cells of opposite mating types (bisexual reproduction) or cells of one mating type (unisexual reproduction), a dimorphic transition from yeast to hyphal growth occurs. Hyphal development and meiosis generate abundant spores that, following inhalation, penetrate deep into the lung to enter the alveoli, germinate, and establish a pulmonary infection growing as budding yeast cells. Unisexual reproduction has been directly observed only in the Cryptococcus var. neoformans (serotype D) lineage under laboratory conditions. However, hyphal development has been previously associated with reduced virulence and the serotype D lineage exhibits limited pathogenicity in the murine model. In this study we show that the serotype D hyperfilamentous strain XL280α is hypervirulent in an animal model. It can grow inside the lung of the host, establish a pulmonary infection, and then disseminate to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans, and reveal the virulence potential of serotype D as broader and more dynamic than previously appreciated

Role of IL-17A on Resolution of Pulmonary C. neoformans Infection

The current studies evaluated the role of interleukin (IL)-17A in the induction of protective immunity against pulmonary cryptococcosis in mice. Protection against pulmonary infection with C. neoformans strain H99c was associated with increased IL-17A production. Signaling through the IFN-c receptor (R) was required for increased IL-17A production, however, a Th17-type cytokine profile was not observed. Neutrophils were found to be the predominant leukocytic source of IL-17A, rather than T cells, suggesting that the IL-17A produced was not part of a T cell-mediated Th17-type immune response. Depletion of IL-17A in mice during pulmonary infection with C. neoformans strain H99c resulted in an initial increase in pulmonary fungal burden, but had no effect on cryptococcal burden at later time points. Also, depletion of IL-17A did not affect the local production of other cytokines. IL-17RA 2/2 mice infected with C. neoformans strain H99c survived the primary infection as well as a secondary challenge with wild-type cryptococci. However, dissemination of the wild-type strain to the brain was noted in the surviving IL-17RA 2/2 mice. Altogether, our results suggested that IL-17A may be important for optimal protective immune responsiveness during pulmonary C. neoformans infection, but protective Th1typ

Interleukin-17 Is Not Required for Classical Macrophage Activation in a Pulmonary Mouse Model of Cryptococcus neoformans Infection▿

Cryptococcus neoformans is an opportunistic fungal pathogen that causes disease in individuals with suppressed cell-mediated immunity. Recent studies in our laboratory have shown that increases in pulmonary Th1-type and interleukin-17A (IL-17A) cytokine production, classical macrophage activation, and sterilizing immunity are elicited in response to infection with a gamma interferon (IFN-γ)-producing C. neoformans strain, H99γ. IL-17A-treated macrophages, compared to IL-4-treated macrophages, have been demonstrated to exhibit increased microbicidal activity in vitro, a characteristic consistent with classical macrophage activation. The purpose of these studies is to determine the role of IL-17A in the induction of classically activated macrophages following infection with C. neoformans. Immunohistochemistry and real-time PCR were used to characterize the macrophage activation phenotype in lung tissues of mice treated with isotype control or anti-IL-17A antibodies and given an experimental pulmonary infection with C. neoformans strain H99γ. The pulmonary fungal burden was resolved, albeit more slowly, in mice depleted of IL-17A compared to the fungal burden in isotype control-treated mice. Nonetheless, no difference in classical macrophage activation was observed in IL-17A-depleted mice. Similarly, classical macrophage activation was evident in mice deficient in IL-17A or the IL-17 receptor A, which mediates IL-17A signaling, following pulmonary infection with wild-type C. neoformans strain H99 or H99γ. These studies suggest that IL-17A may play a role in the early immune response to C. neoformans but is not required for classical macrophage activation in mice experimentally infected with C. neoformans

XL280α infection results in eosinophil infiltration and goblet cell metaplasia.

<p>Lungs of mice challenged with JEC21α and XL280α were isolated 10 days post infection, fixed in formalin, and processed for histological analysis. Tissue sections were either stained with Hematoxylin and eosin (H&E) stain and photographed with a 10× (A and B), 40× (C and E), or 60× objective (D), or stained with Periodic acid–Schiff (PAS) stain and photographed at 20× (F). JEC21 caused marked inflammation in the lung (<b>A</b>) with widespread growth of <i>Cryptococcus</i> and enlarged macrophages with proliferating intracellular cryptococci (yellow arrows) (<b>C</b>). Airways and vessels exhibit normal goblet cell growth (PAS-negative) (E). In contrast, XL280α yeast cells (red arrows) stimulated a diffuse inflammatory response (<b>B</b>) with pulmonary eosinophilia (green arrows) (<b>D</b>) and PAS-positive goblet cells (<b>F</b>). Internalized yeast cells were not detected in XL280α infected pulmonary tissues. Scale bar = 50 µm.</p

XL280α disseminates to the brain.

<p>(A) Mice were infected intranasally with 5×10⁵ yeast cells. At two and four weeks post infection the lungs and brains were isolated from 10 animals infected with each strain. The tissues were homogenized and serial dilutions were plated to recover CFUs and calculate the fungal tissue burden. XL280α fungal burden was significantly higher in both the lung and brain at four weeks post infection (<i>p</i> = 0.0042 and <i>p</i> = 0.0285). (B) Based on histopathological analysis of pulmonary tissues, XL280α yeast cells were morphologically different from JEC21α. They were significantly larger and exhibited an oval structure. Scale bar = 20 µm.</p

Phenotypes of virulence factors.

<p>In vitro phenotypic assays of virulence associated attributes of XL280α and JEC21α. (A) The strains were grown in liquid YPD, washed with water, and spotted in 10-fold serial dilutions on the following media and conditions: YPD at 30°C and 37°C for 2 days, YPD plus 8 or 16 µg/ml of fluconazole at 30°C for 6 days, YPD plus 1 µg/ml of FK506 at 30°C and 37°C for 3 days, L-Dopa at 30°C for 3 days, and niger seed at 30°C for 6 days. (B) For capsule evaluation, cells were grown in DMEM for 6 days and visualized by light microscopy using India ink and (C) supernatants of the cells containing secreted capsule were isolated, subjected to gel electrophoresis, and then immunoblotted with an anti-GXM antibody. (D) Unisexual reproduction cultures were incubated on V8 agar (pH = 7), for 2–3 weeks in the dark at room temperature. Scale bar = 100 µm.</p

Immune response to XL280α is polarized towards Th2-type immunity.

<p>Mice were infected intranasally with 1×10⁶ yeast cells and cytokine production was assayed at 3, 7, and 10 days post infection. (<b>A</b>) The lungs were isolated, homogenized, serially diluted, and plated for CFUs. (<b>B</b>) The Th1 cytokines IL-2, TNF, and IFN-γ were similar between XL280α and JEC21α infected mice. (<b>C</b>) In contrast the Th2-type associated cytokines IL-4, IL-5, and IL-10 were significantly higher in XL280α infected mice. (<b>D</b>) The ratio of IL-4/IFN-γ was significantly higher in XL280α infected animals at 7 and 10 days post infection. The data represents four independent experiments. The asterisk (*) signifies a <i>p</i><0.05, derived from two-way ANOVA statistical analysis.</p