RNA helicase signaling is critical for type I interferon production and protection against rift valley fever virus during mucosal challenge

Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV

IL-17 Receptor Signaling in Oral Epithelial Cells Is Critical for Protection against Oropharyngeal Candidiasis

Signaling through the IL-17 receptor (IL-17R) is required to prevent oropharyngeal candidiasis (OPC) in mice and humans. However, the IL-17-responsive cell type(s) that mediate protection are unknown. Using radiation chimeras we were able to rule out a requirement for IL-17RA in the hematopoietic compartment. We saw remarkable concordance of IL-17-controlled gene expression in C. albicans-infected human oral epithelial cells (OECs) and in tongue tissue from mice with OPC. To interrogate the role of the IL-17R in OECs, we generated mice with conditional deletion of IL-17RA in superficial oral and esophageal epithelial cells (Il17ra(ΔK13)). Following oral Candida infection, Il17ra(ΔK13) mice exhibited fungal loads and weight loss indistinguishable from Il17ra(−/−) mice. Susceptibility in Il17ra(ΔK13) mice correlated with expression of the antimicrobial peptide β-defensin 3 (BD3, Defb3). Consistently, Defb3(−/−) mice were susceptible to OPC. Thus, OECs dominantly control IL-17R-dependent responses to OPC through regulation of BD3expression

A Novel Role for the NLRC4 Inflammasome in Mucosal Defenses against the Fungal Pathogen Candida albicans

Candida sp. are opportunistic fungal pathogens that colonize the skin and oral cavity and, when overgrown under permissive conditions, cause inflammation and disease. Previously, we identified a central role for the NLRP3 inflammasome in regulating IL-1β production and resistance to dissemination from oral infection with Candida albicans. Here we show that mucosal expression of NLRP3 and NLRC4 is induced by Candida infection, and up-regulation of these molecules is impaired in NLRP3 and NLRC4 deficient mice. Additionally, we reveal a role for the NLRC4 inflammasome in anti-fungal defenses. NLRC4 is important for control of mucosal Candida infection and impacts inflammatory cell recruitment to infected tissues, as well as protects against systemic dissemination of infection. Deficiency in either NLRC4 or NLRP3 results in severely attenuated pro-inflammatory and antimicrobial peptide responses in the oral cavity. Using bone marrow chimeric mouse models, we show that, in contrast to NLRP3 which limits the severity of infection when present in either the hematopoietic or stromal compartments, NLRC4 plays an important role in limiting mucosal candidiasis when functioning at the level of the mucosal stroma. Collectively, these studies reveal the tissue specific roles of the NLRP3 and NLRC4 inflammasome in innate immune responses against mucosal Candida infection

Aspergillus fumigatus Stimulates the NLRP3 Inflammasome through a Pathway Requiring ROS Production and the Syk Tyrosine Kinase

Invasive aspergillosis (IA) is a life-threatening disease that occurs in immunodepressed patients when infected with Aspergillus fumigatus. This fungus is the second most-common causative agent of fungal disease after Candida albicans. Nevertheless, much remains to be learned about the mechanisms by which A. fulmigatus activates the innate immune system. We investigated the inflammatory response to conidia and hyphae of A. fumigatus and specifically, their capacity to trigger activation of an inflammasome. Our results show that in contrast to conidia, hyphal fragments induce NLRP3 inflammasome assembly, caspase-1 activation and IL-1β release from a human monocyte cell line. The ability of Aspergillus hyphae to activate the NLRP3 inflammasome in the monocytes requires K+ efflux and ROS production. In addition, our data show that NLRP3 inflammasome activation as well as pro-IL-1β expression relies on the Syk tyrosine kinase, which is downstream from the pathogen recognition receptor Dectin-1, reinforcing the importance of Dectin-1 in the innate immune response against fungal infection. Furthermore, we show that treatment of monocytes with corticosteroids inhibits transcription of the gene encoding IL-1β. Thus, our data demonstrate that the innate immune response against A. fumigatus infection involves a two step activation process, with a first signal promoting expression and synthesis of pro-IL-1β; and a second signal, involving Syk-induced activation of the NLRP3 inflammasome and caspase-1, allowing processing and secretion of the mature cytokine

Stage- and Gender-Specific Proteomic Analysis of Brugia malayi Excretory-Secretory Products

To succeed in infection, parasites must have ways to reach the host, penetrate its tissues and escape its defense systems. As they are not necessarily fatal, most helminth parasites remain viable within their host for many years, exerting a strong influence over the host immune function. Many of these functions are performed by products that are released from the parasite. We exploited the remarkable sensitivity of modern proteomics tools together with the availability of a sequenced genome to identify and compare the proteins released in vitro by adult males, adult females and the microfilariae of the filarial nematode Brugia malayi. This parasite is one of the etiological agents of lymphatic filariasis, a disease that poses continuing and significant threats to human health. The different forms of the parasite inhabit different compartments in the mammalian host. We found that the set of proteins released by each form is unique; they must reflect particular developmental processes and different strategies for evasion of host responses. The identification of these proteins will allow us to illuminate the biology of secretory processes in this organism and to establish a path for developing an understanding of how these parasite proteins function in immune evasion events

Host genetic signatures of susceptibility to fungal disease

Our relative inability to predict the development of fungal disease and its clinical outcome raises fundamental questions about its actual pathogenesis. Several clinical risk factors are described to predispose to fungal disease, particularly in immunocompromised and severely ill patients. However, these alone do not entirely explain why, under comparable clinical conditions, only some patients develop infection. Recent clinical and epidemiological studies have reported an expanding number of monogenic defects and common polymorphisms associated with fungal disease. By directly implicating genetic variation in the functional regulation of immune mediators and interacting pathways, these studies have provided critical insights into the human immunobiology of fungal disease. Most of the common genetic defects reported were described or suggested to impair fungal recognition by the innate immune system. Here, we review common genetic variation in pattern recognition receptors and its impact on the immune response against the two major fungal pathogens Candida albicans and Aspergillus fumigatus. In addition, we discuss potential strategies and opportunities for the clinical translation of genetic information in the field of medical mycology. These approaches are expected to transfigure current clinical practice by unleashing an unprecedented ability to personalize prophylaxis, therapy and monitoring for fungal disease.This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013), the Fundação para a Ciência e Tecnologia (FCT) (IF/00735/2014 to AC, and SFRH/BPD/96176/2013 to CC), the Institut Mérieux (Mérieux Research Grant 2017 to CC), and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID Research Grant 2017 to AC)

Multiplicity of cerebrospinal fluid functions: New challenges in health and disease

This review integrates eight aspects of cerebrospinal fluid (CSF) circulatory dynamics: formation rate, pressure, flow, volume, turnover rate, composition, recycling and reabsorption. Novel ways to modulate CSF formation emanate from recent analyses of choroid plexus transcription factors (E2F5), ion transporters (NaHCO3 cotransport), transport enzymes (isoforms of carbonic anhydrase), aquaporin 1 regulation, and plasticity of receptors for fluid-regulating neuropeptides. A greater appreciation of CSF pressure (CSFP) is being generated by fresh insights on peptidergic regulatory servomechanisms, the role of dysfunctional ependyma and circumventricular organs in causing congenital hydrocephalus, and the clinical use of algorithms to delineate CSFP waveforms for diagnostic and prognostic utility. Increasing attention focuses on CSF flow: how it impacts cerebral metabolism and hemodynamics, neural stem cell progression in the subventricular zone, and catabolite/peptide clearance from the CNS. The pathophysiological significance of changes in CSF volume is assessed from the respective viewpoints of hemodynamics (choroid plexus blood flow and pulsatility), hydrodynamics (choroidal hypo- and hypersecretion) and neuroendocrine factors (i.e., coordinated regulation by atrial natriuretic peptide, arginine vasopressin and basic fibroblast growth factor). In aging, normal pressure hydrocephalus and Alzheimer's disease, the expanding CSF space reduces the CSF turnover rate, thus compromising the CSF sink action to clear harmful metabolites (e.g., amyloid) from the CNS. Dwindling CSF dynamics greatly harms the interstitial environment of neurons. Accordingly the altered CSF composition in neurodegenerative diseases and senescence, because of adverse effects on neural processes and cognition, needs more effective clinical management. CSF recycling between subarachnoid space, brain and ventricles promotes interstitial fluid (ISF) convection with both trophic and excretory benefits. Finally, CSF reabsorption via multiple pathways (olfactory and spinal arachnoidal bulk flow) is likely complemented by fluid clearance across capillary walls (aquaporin 4) and arachnoid villi when CSFP and fluid retention are markedly elevated. A model is presented that links CSF and ISF homeostasis to coordinated fluxes of water and solutes at both the blood-CSF and blood-brain transport interfaces

Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis of Aspergillus fumigatus Keratitis

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1β and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that β-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1−/− corneas have impaired IL-1β and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high β-glucan. In contrast to Dectin 1−/− mice, cellular infiltration into infected TLR2−/−, TLR4−/−, and MD-2−/− mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4−/− mice, but not TLR2−/− or MD-2−/− mice. We also found that TRIF−/− and TIRAP−/− mice exhibited no fungal-killing defects, but that MyD88−/− and IL-1R1−/− mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which β-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1β, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing

Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis of Aspergillus fumigatus Keratitis

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1β and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that β-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1−/− corneas have impaired IL-1β and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high β-glucan. In contrast to Dectin 1−/− mice, cellular infiltration into infected TLR2−/−, TLR4−/−, and MD-2−/− mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4−/− mice, but not TLR2−/− or MD-2−/− mice. We also found that TRIF−/− and TIRAP−/− mice exhibited no fungal-killing defects, but that MyD88−/− and IL-1R1−/− mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which β-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1β, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing