Cyclic GMP-AMP synthase (cGAS) is a newly identified DNA sensor that recognizes foreign DNA, including the genome of herpes simplex virus 1 (HSV-1). Upon binding of viral DNA, cGAS produces cyclic ...GMP-AMP, which interacts with and activates stimulator of interferon genes (STING) to trigger the transcription of antiviral genes such as type I interferons (IFNs), and the production of inflammatory cytokines. HSV-1 UL24 is widely conserved among members of the herpesviruses family and is essential for efficient viral replication. In this study, we found that ectopically expressed UL24 could inhibit cGAS-STING-mediated promoter activation of IFN-β and interleukin-6 (IL-6), and UL24 also inhibited interferon-stimulatory DNA-mediated IFN-β and IL-6 production during HSV-1 infection. Furthermore, UL24 selectively blocked nuclear factor κB (NF-κB) but not IFN-regulatory factor 3 promoter activation. Coimmunoprecipitation analysis demonstrated that UL24 bound to the endogenous NF-κB subunits p65 and p50 in HSV-1-infected cells, and UL24 was also found to bind the Rel homology domains (RHDs) of these subunits. Furthermore, UL24 reduced the tumor necrosis factor alpha (TNF-α)-mediated nuclear translocation of p65 and p50. Finally, mutational analysis revealed that the region spanning amino acids (aa) 74 to 134 of UL24 UL24(74-134) is responsible for inhibiting cGAS-STING-mediated NF-κB promoter activity. For the first time, UL24 was shown to play an important role in immune evasion during HSV-1 infection.
NF-κB is a critical component of the innate immune response and is strongly induced downstream of most pattern recognition receptors (PRRs), leading to the production of IFN-β as well as a number of inflammatory chemokines and interleukins. To establish persistent infection, viruses have evolved various mechanisms to counteract the host NF-κB pathway. In the present study, for the first time, HSV-1 UL24 was demonstrated to inhibit the activation of NF-κB in the DNA sensing signal pathway via binding to the RHDs of the NF-κB subunits p65 and p50 and abolishing their nuclear translocation.
Approximately 1.2 billion people suffer from fungal diseases worldwide. Arguably, the most serious manifestation occurs when pathogenic fungi infect the brain, often causing fatal ...meningoencephalitis. For most fungi, infection occurs via the vascular route. The organism must first be arrested in the brain microvasculature and transmigrate into the brain parenchyma across the blood-brain barrier. As a result, host immune cells are recruited into the brain to contain the fungi. However, it remains poorly understood how fungi traffic to, and migrate into the brain and how immune cells interact with invading fungi in the brain. A new era of intravital fluorescence microscopy has begun to provide insights. We are able to employ this powerful approach to study dynamic interactions of disseminating fungi with brain endothelial cells as well as resident and recruited immune cells during the brain infection. In this review, with a focus on Cryptococcus neoformans, we will provide an overview of the application of intravital imaging in fungal infections in the brain, discuss recent findings and speculate on possible future research directions.
With increasing numbers of immune-compromised patients with malignancy, hematologic disease, and HIV, as well as those receiving immunosupressive drug regimens for the management of organ ...transplantation or autoimmune inflammatory conditions, the incidence of fungal infections has dramatically increased over recent years. Definitive diagnosis of pulmonary fungal infections has also been substantially assisted by the development of newer diagnostic methods and techniques, including the use of antigen detection, polymerase chain reaction, serologies, computed tomography and positron emission tomography scans, bronchoscopy, mediastinoscopy, and video-assisted thorascopic biopsy. At the same time, the introduction of new treatment modalities has significantly broadened options available to physicians who treat these conditions. While traditionally antifungal therapy was limited to the use of amphotericin B, flucytosine, and a handful of clinically available azole agents, current pharmacologic treatment options include potent new azole compounds with extended antifungal activity, lipid forms of amphotericin B, and newer antifungal drugs, including the echinocandins. In view of the changing treatment of pulmonary fungal infections, the American Thoracic Society convened a working group of experts in fungal infections to develop a concise clinical statement of current therapeutic options for those fungal infections of particular relevance to pulmonary and critical care practice. This document focuses on three primary areas of concern: the endemic mycoses, including histoplasmosis, sporotrichosis, blastomycosis, and coccidioidomycosis; fungal infections of special concern for immune-compromised and critically ill patients, including cryptococcosis, aspergillosis, candidiasis, and Pneumocystis pneumonia; and rare and emerging fungal infections.
Infectious meningitis and encephalitis is caused by invasion of circulating pathogens into the brain. It is unknown how the circulating pathogens dynamically interact with brain endothelium under ...shear stress, leading to invasion into the brain. Here, using intravital microscopy, we have shown that Cryptococcus neoformans, a yeast pathogen that causes meningoencephalitis, stops suddenly in mouse brain capillaries of a similar or smaller diameter than the organism, in the same manner and with the same kinetics as polystyrene microspheres, without rolling and tethering to the endothelial surface. Trapping of the yeast pathogen in the mouse brain was not affected by viability or known virulence factors. After stopping in the brain, C. neoformans was seen to cross the capillary wall in real time. In contrast to trapping, viability, but not replication, was essential for the organism to cross the brain microvasculature. Using a knockout strain of C. neoformans, we demonstrated that transmigration into the mouse brain is urease dependent. To determine whether this could be amenable to therapy, we used the urease inhibitor flurofamide. Flurofamide ameliorated infection of the mouse brain by reducing transmigration into the brain. Together, these results suggest that C. neoformans is mechanically trapped in the brain capillary, which may not be amenable to pharmacotherapy, but actively transmigrates to the brain parenchyma with contributions from urease, suggesting that a therapeutic strategy aimed at inhibiting this enzyme could help prevent meningitis and encephalitis caused by C. neoformans infection.
•Novel immune-based intervention strategies might be developed which, instead of targeting a class of pathogen (bacteria, virus, parasite, or fungus), target a shared infectious process. We suggest ...three novel approaches to activating phagocytic cells prior to or at the time of infection by phagosomal pathogens. Preventing the formation of the pathogen niche within the phagolysosome might be crucial for preventing infectious disease; a greater emphasis on the timing of phagocyte activation might generate more effective prophylactic treatment options.
Prophylactic vaccination strategies designed to prevent diseases caused by phagosomal pathogens have been largely unsuccessful.We propose that trained immunity, natural killer cell activation, and maintenance of circulating T helper 1 effector cells may represent three novel intervention strategies to target a shared infectious process rather than a specific pathogen.These novel approaches might be effective based on the inherent cellular ability to activate phagocytes prior to, or at the time of, infection.Failure to activate phagocytes prior to, or at the time of, infection may not provide protection due to the establishment of the phagosomal pathogen niche.
Prophylactic vaccination strategies designed to prevent diseases caused by pathogens using the phagolysosome of innate immune cells as a site of intracellular replication and survival have been largely ineffective. These include Mycobacterium tuberculosis (Mtb), Leishmania spp., and Cryptococcus spp. These failed strategies have traditionally targeted CD4+ T helper (Th) 1 cell-mediated immune memory, deeming it crucial for vaccine efficacy. This failure warrants an investigation of alternative mediators of protection. Here, we suggest three novel approaches to activate phagocytic cells prior to or at the time of infection. We hypothesize that preventing the formation of the pathogen niche within the phagolysosome is essential for preventing disease, and a greater emphasis on the timing of phagocyte activation should generate more effective prophylactic treatment options.
Cryptococcal Lung Infections Skolnik, Kate; Huston, Shaunna; Mody, Christopher H
Clinics in chest medicine,
09/2017, Letnik:
38, Številka:
3
Journal Article
Recenzirano
Cryptococcus is among the most common invasive fungal pathogens globally and is one of the leading causes of acquired immunodeficiency virus-related deaths. Cryptococcus neoformans and Cryptococcus ...gattii are the most clinically relevant species and account for most cryptococcal disease. Pulmonary manifestations can range from mild symptoms to life-threatening infection. Treatment is tailored based on the severity of pulmonary infection, the presence of disseminated or central nervous system disease, and patient immune status. Amphotericin B and flucytosine followed by fluconazole remain the standard agents for the treatment of severe cryptococcal infection.
Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural ...killer (NK) cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co-culture of YT cells, a human NK cell line, and GFP-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process.
Natural killer (NK) cells use the activating receptor NKp30 as a microbial pattern-recognition receptor to recognize, activate cytolytic pathways, and directly kill the fungi Cryptococcus neoformans ...and Candida albicans. However, the fungal pathogen-associated molecular pattern (PAMP) that triggers NKp30-mediated killing remains to be identified. Here we show that β-1,3-glucan, a component of the fungal cell wall, binds to NKp30. We further demonstrate that β-1,3-glucan stimulates granule convergence and polarization, as shown by live cell imaging. Through Src Family Kinase signaling, β-1,3-glucan increases expression and clustering of NKp30 at the microbial and NK cell synapse to induce perforin release for fungal cytotoxicity. Rather than blocking the interaction between fungi and NK cells, soluble β-1,3-glucan enhances fungal killing and restores defective cryptococcal killing by NK cells from HIV-positive individuals, implicating β-1,3-glucan to be both an activating ligand and a soluble PAMP that shapes NK cell host immunity.
Microbial killing by NK cells Mody, Christopher H.; Ogbomo, Henry; Xiang, Richard F. ...
Journal of leukocyte biology,
June 2019, Letnik:
105, Številka:
6
Journal Article
Recenzirano
It is now evident that NK cells kill bacteria, fungi, and parasites in addition to tumor and virus‐infected cells. In addition to a number of recent publications that have identified the receptors ...and ligands, and mechanisms of cytotoxicity, new insights are reflected in the reports from researchers all over the world at the 17th Meeting of the Society for Natural Immunity held in San Antonio, TX, USA from May 28 through June 1, 2018. We will provide an overview of the field and discuss how the presentations at the meeting might shape our knowledge and future directions in the field.
Mechanisms of microbial killing by NK cells is rapidly providing novel insights in host defense as well as informing strategies for tumor cytotoxicity.
Natural killer (NK) cells are a subset of immune effectors that directly bind and kill fungi via a perforin-dependent mechanism. The receptor mediating this activity and its potential role in disease ...remain unknown. Using an unbiased approach, we determined that NKp30 is responsible for recognition and killing of the fungal pathogens Cryptococcus and Candida. NKp30 was required for NK cell-fungal conjugate formation, phosphatidylinositol 3-kinase (PI3K) signaling, and perforin release. Because fungal infections are a leading cause of death in AIDS patients, we examined NKp30 expression in HIV-infected patients. NK cells from these patients had diminished NKp30 expression, defective perforin release, and blunted microbicidal activity. Surprisingly, interleukin-12 (IL-12) restored NKp30 expression and fungal killing. Thus, the NKp30 receptor plays a critical role in NK cell antifungal cytotoxicity, and diminished expression of NKp30 is responsible for defective antifungal activity of NK cells from HIV-infected patients, which can be corrected with IL-12.
•An unbiased approach identifies NKp30 as a NK cell fungicidal receptor•NKp30 mediates direct binding, signaling, and perforin release in response to fungi•NK cells from HIV patients had diminished NKp30 expression and blunted fungal killing•IL-12 restored NKp30 expression and fungal killing in NK cells from HIV patients
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