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Beyond their canonical roles in hemostasis and thrombosis, platelets function in the innate immune response by interacting directly with pathogens and by regulating the recruitment and activation of immune effector cells. Thrombocytopenia often coincides with neutropenia in patients with hematologic malignancies and in allogeneic hematopoietic cell transplant recipients, patient groups at high risk for invasive fungal infections. While neutropenia is well established as a major clinical risk factor for invasive fungal infections, the role of platelets in host defense against human fungal pathogens remains understudied. Here, we examined the role of platelets in murine Aspergillus fumigatus infection using two complementary approaches to induce thrombocytopenia without concurrent neutropenia. Thrombocytopenic mice were highly susceptible to A. fumigatus challenge and rapidly succumbed to infection. Although platelets regulated early conidial phagocytosis by neutrophils in a spleen tyrosine kinase (Syk)-dependent manner, platelet-regulated conidial phagocytosis was dispensable for host survival. Instead, our data indicated that platelets primarily function to maintain hemostasis and lung integrity in response to exposed fungal antigens, since thrombocytopenic mice exhibited severe hemorrhage into the airways in response to fungal challenge in the absence of overt angioinvasion. Challenge with swollen, heat-killed, conidia was lethal in thrombocytopenic hosts and could be reversed by platelet transfusion, consistent with the model that fungus-induced inflammation in platelet-depleted mice was sufficient to induce lethal hemorrhage. These data provide new insights into the role of platelets in the anti-Aspergillus host response and expand their role to host defense against filamentous molds.

Cryptococcus neoformans is a leading cause of invasive fungal infections among immunocompromised patients. However, the cellular constituents of the innate immune response that promote clearance versus progression of infection upon respiratory acquisition of C. neoformans remain poorly defined. In this study, we found that during acute C. neoformans infection, CCR2+ Ly6Chi inflammatory monocytes (IM) rapidly infiltrate the lungs and mediate fungal trafficking to lung-draining lymph nodes. Interestingly, this influx of IM is detrimental to the host, since ablating IM or impairing their recruitment to the lungs improves murine survival and reduces fungal proliferation and dissemination. Using a novel conditional gene deletion strategy, we determined that MHC class II expression by IM did not mediate their deleterious impact on the host. Furthermore, although ablation of IM reduced the number of lymphocytes, innate lymphoid cells, and eosinophils in the lungs, the effects of IM were not dependent on these cells. We ascertained that IM in the lungs upregulated transcripts associated with alternatively activated (M2) macrophages in response to C. neoformans, consistent with the model that IM assume a cellular phenotype that is permissive for fungal growth. We also determined that conditional knockout of the prototypical M2 marker arginase 1 in IM and deletion of the M2-associated transcription factor STAT6 were not sufficient to reverse the harmful effects of IM. Overall, our findings indicate that C. neoformans can subvert the fungicidal potential of IM to enable the progression of infection through a mechanism that is not dependent on lymphocyte priming, eosinophil recruitment, or downstream M2 macrophage polarization pathways. These results give us new insight into the plasticity of IM function during fungal infections and the level of control that C. neoformans can exert on host immune responses.

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.

Aspergillus fumigatus forms ubiquitous airborne conidia that humans inhale on a daily basis. Although respiratory fungal infection activates the adaptor proteins CARD9 and MyD88 via C-type lectin, Toll-like, and interleukin-1 family receptor signals, defining the temporal and spatial pattern of MyD88- and CARD9-coupled signals in immune activation and fungal clearance has been difficult to achieve. Herein, we demonstrate that MyD88 and CARD9 act in two discrete phases and in two cellular compartments to direct chemokine- and neutrophil-dependent host defense. The first phase depends on MyD88 signaling because genetic deletion of MyD88 leads to delayed induction of the neutrophil chemokines CXCL1 and CXCL5, delayed neutrophil lung trafficking, and fatal pulmonary damage at the onset of respiratory fungal infection. MyD88 expression in lung epithelial cells restores rapid chemokine induction and neutrophil recruitment via interleukin-1 receptor signaling. Exogenous CXCL1 administration reverses murine mortality in MyD88-deficient mice. The second phase depends predominately on CARD9 signaling because genetic deletion of CARD9 in radiosensitive hematopoietic cells interrupts CXCL1 and CXCL2 production and lung neutrophil recruitment beyond the initial MyD88-dependent phase. Using a CXCL2 reporter mouse, we show that lung-infiltrating neutrophils represent the major cellular source of CXCL2 during CARD9-dependent recruitment. Although neutrophil-intrinsic MyD88 and CARD9 function are dispensable for neutrophil conidial uptake and killing in the lung, global deletion of both adaptor proteins triggers rapidly progressive invasive disease when mice are challenged with an inoculum that is sub-lethal for single adapter protein knockout mice. Our findings demonstrate that distinct signal transduction pathways in the respiratory epithelium and hematopoietic compartment partially overlap to ensure optimal chemokine induction, neutrophil recruitment, and fungal clearance within the respiratory tract.

Hypoxia inducible factor 1α (HIF1α) is the mammalian transcriptional factor that controls metabolism, survival, and innate immunity in response to inflammation and low oxygen. Previous work established that generation of hypoxic microenvironments occurs within the lung during infection with the human fungal pathogen Aspergillus fumigatus. Here we demonstrate that A. fumigatus stabilizes HIF1α protein early after pulmonary challenge that is inhibited by treatment of mice with the steroid triamcinolone. Utilizing myeloid deficient HIF1α mice, we observed that HIF1α is required for survival and fungal clearance early following pulmonary challenge with A. fumigatus. Unlike previously reported research with bacterial pathogens, HIF1α deficient neutrophils and macrophages were surprisingly not defective in fungal conidial killing. The increase in susceptibility of the myeloid deficient HIF1α mice to A. fumigatus was in part due to decreased early production of the chemokine CXCL1 (KC) and increased neutrophil apoptosis at the site of infection, resulting in decreased neutrophil numbers in the lung. Addition of recombinant CXCL1 restored neutrophil survival and numbers, murine survival, and fungal clearance. These results suggest that there are unique HIF1α mediated mechanisms employed by the host for protection and defense against fungal pathogen growth and invasion in the lung. Additionally, this work supports the strategy of exploring HIF1α as a therapeutic target in specific immunosuppressed populations with fungal infections.

Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients. Although -CC-chemokine receptor-2 (CCR2) and Ly6C-expressing inflammatory monocytes (CCR2⁺Mo) and their derivatives initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain poorly defined. Using conditional and antibody-mediated cell ablation strategies, we found that CCR2⁺Mo and monocyte-derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent Aspergillus reporter (FLARE) conidia that report fungal cell association and viability in vivo, we identify two mechanisms by which CCR2⁺Mo and Mo-DCs exert innate antifungal activity. First, CCR2⁺Mo and Mo-DCs condition the lung inflammatory milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2⁺Mo temporally coincided with their differentiation into Mo-DCs, a process that resulted in direct conidial killing. Our findings illustrate both indirect and direct functions for CCR2⁺Mo and their derivatives in innate antifungal immunity in the lung.

Currently, our knowledge of how pathogenic fungi grow in mammalian host environments is limited. Using a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA) and (1)H-NMR metabolomics, we detected ethanol in the lungs of mice infected with Aspergillus fumigatus. This result suggests that A. fumigatus is exposed to oxygen depleted microenvironments during infection. To test this hypothesis, we utilized a chemical hypoxia detection agent, pimonidazole hydrochloride, in three immunologically distinct murine models of IPA (chemotherapeutic, X-CGD, and corticosteroid). In all three IPA murine models, hypoxia was observed during the course of infection. We next tested the hypothesis that production of ethanol in vivo by the fungus is involved in hypoxia adaptation and fungal pathogenesis. Ethanol deficient A. fumigatus strains showed no growth defects in hypoxia and were able to cause wild type levels of mortality in all 3 murine models. However, lung immunohistopathology and flow cytometry analyses revealed an increase in the inflammatory response in mice infected with an alcohol dehydrogenase null mutant strain that corresponded with a reduction in fungal burden. Consequently, in this study we present the first in vivo observations that hypoxic microenvironments occur during a pulmonary invasive fungal infection and observe that a fungal alcohol dehydrogenase influences fungal pathogenesis in the lung. Thus, environmental conditions encountered by invading pathogenic fungi may result in substantial fungal metabolism changes that influence subsequent host immune responses.

Inhalation of fungal spores (conidia) occurs commonly and, in specific circumstances, can result in invasive disease. We investigated the murine inflammatory response to conidia of Aspergillus fumigatus, the most common invasive mold in immunocompromised hosts. In contrast to dormant spores, germinating conidia induce neutrophil recruitment to the airways and TNF-alpha/MIP-2 secretion by alveolar macrophages. Fungal beta-glucans act as a trigger for the induction of these inflammatory responses through their time-dependent exposure on the surface of germinating conidia. Dectin-1, an innate immune receptor that recognizes fungal beta-glucans, is recruited in vivo to alveolar macrophage phagosomes that have internalized conidia with exposed beta-glucans. Antibody-mediated blockade of Dectin-1 partially inhibits TNF-alpha/MIP-2 induction by metabolically active conidia. TLR-2- and MyD88-mediated signals provide an additive contribution to macrophage activation by germinating conidia. Selective responsiveness to germinating conidia provides the innate immune system with a mechanism to restrict inflammatory responses to metabolically active, potentially invasive fungal spores.

Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the plus ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics in the different proliferative stages are not well understood. Genome-wide shared orthology analysis of Plasmodium spp. revealed the presence of two kinesin-8 motor proteins, kinesin-8X and kinesin-8B. Here we studied the biochemical properties of kinesin-8X and its role in parasite proliferation. In vitro, kinesin-8X has motility and depolymerization activities like other kinesin-8 motors. To understand the role of Plasmodium kinesin-8X in cell division, we used fluorescence-tagging and live cell imaging to define its location, and gene targeting to analyse its function, during all proliferative stages of the rodent malaria parasite P. berghei life cycle. The results revealed a spatio-temporal involvement of kinesin-8X in spindle dynamics and an association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the kinesin-8X gene revealed a defect in oocyst development, confirmed by ultrastructural studies, suggesting that this protein is required for oocyst development and sporogony. Transcriptome analysis of Δkinesin-8X gametocytes revealed modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression, supporting a role for kinesin-8X in cell division. Kinesin-8X is thus required for parasite proliferation within the mosquito and for transmission to the vertebrate host.

Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.

Meeting the increasing food and energy demands of a growing population will require the development of ground-breaking strategies that promote sustainable plant production. Host-induced gene silencing has shown great potential for controlling pest and diseases in crop plants. However, while delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a promising concept, it requires the generation of transgenic crop plants which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species. Using the agronomically important barley-Fusarium graminearum pathosystem, we alternatively demonstrate that a spray application of a long noncoding dsRNA (791 nt CYP3-dsRNA), which targets the three fungal cytochrome P450 lanosterol C-14α-demethylases, required for biosynthesis of fungal ergosterol, inhibits fungal growth in the directly sprayed (local) as well as the non-sprayed (distal) parts of detached leaves. Unexpectedly, efficient spray-induced control of fungal infections in the distal tissue involved passage of CYP3-dsRNA via the plant vascular system and processing into small interfering (si)RNAs by fungal DICER-LIKE 1 (FgDCL-1) after uptake by the pathogen. We discuss important consequences of this new finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential as a new plant protection strategy.

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species.

In man, infection with South American Andes virus (ANDV) causes hantavirus cardiopulmonary syndrome (HCPS). HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-gamma ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3(+)CD8(+) T cells were specific for the single HLA-B*3501-restricted epitope Gn(465-473) years after the acute infection. Remarkably, Gn(465-473)-specific cells readily secreted IFN-gamma, granzyme B and TNF-alpha but not IL-2 upon stimulation and showed a 'revertant' CD45RA(+)CD27(-)CD28(-)CCR7(-)CD127(-) effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T cells are critical for viral control and protective immunity. If so, Gn-derived immunodominant epitopes could be of high value for future ANDV vaccines.

Inhalation of fungal spores (conidia) occurs commonly and, in specific circumstances, can result in invasive disease. We investigated the murine inflammatory response to conidia of Aspergillus fumigatus, the most common invasive mold in immunocompromised hosts. In contrast to dormant spores, germinating conidia induce neutrophil recruitment to the airways and TNF-alpha/MIP-2 secretion by alveolar macrophages. Fungal beta-glucans act as a trigger for the induction of these inflammatory responses through their time-dependent exposure on the surface of germinating conidia. Dectin-1, an innate immune receptor that recognizes fungal beta-glucans, is recruited in vivo to alveolar macrophage phagosomes that have internalized conidia with exposed beta-glucans. Antibody-mediated blockade of Dectin-1 partially inhibits TNF-alpha/MIP-2 induction by metabolically active conidia. TLR-2- and MyD88-mediated signals provide an additive contribution to macrophage activation by germinating conidia. Selective responsiveness to germinating conidia provides the innate immune system with a mechanism to restrict inflammatory responses to metabolically active, potentially invasive fungal spores.