TNF-α-Producing Cryptococcus neoformans Exerts Protective Effects on Host Defenses in Murine Pulmonary Cryptococcosis.

Tumor necrosis factor alpha (TNF-α) plays a critical role in the control of cryptococcal infection, and its insufficiency promotes cryptococcal persistence. To explore the therapeutic potential of TNF-α supplementation as a booster of host anti-cryptococcal responses, we engineered a C. neoformans strain expressing murine TNF-α. Using a murine model of pulmonary cryptococcosis, we demonstrated that TNF-α-producing C. neoformans strain enhances protective elements of host response including preferential T-cell accumulation and improved Th1/Th2 cytokine balance, diminished pulmonary eosinophilia and alternative activation of lung macrophages at the adaptive phase of infection compared to wild type strain-infected mice. Furthermore, TNF-α expression by C. neoformans enhanced the fungicidal activity of macrophages in vitro. Finally, mice infected with the TNF-α-producing C. neoformans strain showed improved fungal control and considerably prolonged survival compared to wild type strain-infected mice, but could not induce sterilizing immunity. Taken together, our results support that TNF-α expression by an engineered C. neoformans strain while insufficient to drive complete immune protection, strongly enhanced protective responses during primary cryptococcal infection.

Victors: a web-based knowledge base of virulence factors in human and animal pathogens.

Virulence factors (VFs) are molecules that allow microbial pathogens to overcome host defense mechanisms and cause disease in a host. It is critical to study VFs for better understanding microbial pathogenesis and host defense mechanisms. Victors (http://www.phidias.us/victors) is a novel, manually curated, web-based integrative knowledge base and analysis resource for VFs of pathogens that cause infectious diseases in human and animals. Currently, Victors contains 5296 VFs obtained via manual annotation from peer-reviewed publications, with 4648, 179, 105 and 364 VFs originating from 51 bacterial, 54 viral, 13 parasitic and 8 fungal species, respectively. Our data analysis identified many VF-specific patterns. Within the global VF pool, cytoplasmic proteins were more common, while adhesins were less common compared to findings on protective vaccine antigens. Many VFs showed homology with host proteins and the human proteins interacting with VFs represented the hubs of human-pathogen interactions. All Victors data are queriable with a user-friendly web interface. The VFs can also be searched by a customized BLAST sequence similarity searching program. These VFs and their interactions with the host are represented in a machine-readable Ontology of Host-Pathogen Interactions. Victors supports the 'One Health' research as a vital source of VFs in human and animal pathogens.

Exploitation of Scavenger Receptor, Macrophage Receptor with Collagenous Structure, by Cryptococcus neoformans Promotes Alternative Activation of Pulmonary Lymph Node CD11b+ Conventional Dendritic Cells and Non-Protective Th2 Bias.

Macrophage receptor with collagenous structure (MARCO) contributes to fungal containment during the early/innate phase of cryptococcal infection; however, its role in adaptive antifungal immunity remains unknown. Using a murine model of cryptococcosis, we compared host adaptive immune responses in wild-type and MARCO-/- mice throughout an extended time course post-infection. Unlike in early infection, MARCO deficiency resulted in improved pulmonary fungal clearance and diminished cryptococcal dissemination during the efferent phase. Improved fungal control in the absence of MARCO expression was associated with enhanced hallmarks of protective Th1-immunity, including higher frequency of pulmonary TNF-α-producing T cells, increased cryptococcal-antigen-triggered IFN-γ and TNF-α production by splenocytes, and enhanced expression of M1 polarization genes by pulmonary macrophages. Concurrently, we found lower frequencies of IL-5- and IL-13-producing T cells in the lungs, impaired production of IL-4 and IL-10 by cryptococcal antigen-pulsed splenocytes, and diminished serum IgE, which were hallmarks of profoundly suppressed efferent Th2 responses in MARCO-deficient mice compared to WT mice. Mechanistically, we found that MARCO expression facilitated early accumulation and alternative activation of CD11b+ conventional DC (cDC) in the lung-associated lymph nodes (LALNs), which contributed to the progressive shift of the immune response from Th1 toward Th2 at the priming site (LALNs) and local infection site (lungs) during the efferent phase of cryptococcal infection. Taken together, our study shows that MARCO can be exploited by the fungal pathogen to promote accumulation and alternative activation of CD11b+ cDC in the LALN, which in turn alters Th1/Th2 balance to promote fungal persistence and dissemination.

RIPK3/Fas-Associated Death Domain Axis Regulates Pulmonary Immunopathology to Cryptococcal Infection Independent of Necroptosis.

Fas-associated death domain (FADD) and receptor interacting protein kinase 3 (RIPK3) are multifunctional regulators of cell death and immune response. Using a mouse model of cryptococcal infection, the roles of FADD and RIPK3 in anti-cryptococcal defense were investigated. Deletion of RIPK3 alone led to increased inflammatory cytokine production in the Cryptococcus neoformans-infected lungs, but in combination with FADD deletion, it led to a robust Th1-biased response with M1-biased macrophage activation. Rather than being protective, these responses led to paradoxical C. neoformans expansion and rapid clinical deterioration in Ripk3-/- and Ripk3-/-Fadd-/- mice. The increased mortality of Ripk3-/- and even more accelerated mortality in Ripk3-/-Fadd-/- mice was attributed to profound pulmonary damage due to neutrophil-dominant infiltration with prominent upregulation of pro-inflammatory cytokines. This phenomenon was partially associated with selective alterations in the apoptotic frequency of some leukocyte subsets, such as eosinophils and neutrophils, in infected Ripk3-/-Fadd-/- mice. In conclusion, our study shows that RIPK3 in concert with FADD serve as physiological "brakes," preventing the development of excessive inflammation and Th1 bias, which in turn contributes to pulmonary damage and defective fungal clearance. This novel link between the protective effect of FADD and RIPK3 in antifungal defense and sustenance of immune homeostasis may be important for the development of novel immunomodulatory therapies against invasive fungal infections.

Methane-rich saline alleviates cerulein-induced acute pancreatitis by inhibiting inflammatory response, oxidative stress and pancreatic apoptosis in mice.

BACKGROUND: Acute pancreatitis (AP) is a potentially life-threatening gastrointestinal disease involving intracellular activation of digestive enzymes and pancreatic acinar cell injury. The present study was performed to investigate whether methane-rich saline (MS) was involved in the regulation of AP. METHODS: MS (16ml/kg) was administered at different dosing frequencies on mice with cerulein-induced AP. Serum amylase, lipase and histopathological changes in the pancreas tissue were measured. Serum cytokine TNFα, IL-6, IFNγ and IL-10 were detected by ELISA. The mRNA levels of these inflammatory cytokines in the pancreas were detected by real time-PCR. Myeloperoxidase (MPO) and superoxide dismutase (SOD) were determined using commercial kits. Apoptosis was assessed by immunohistochemistry and Western blot. RESULTS: MS treatment reversed the increased serum level of amylase and lipase, alleviated the pathological damage in the pancreas, and decreased the expression of TNFα, IL-6, IFNγ and IL-10 in cerulean-induced AP mice. In addition, MPO was down-regulated and SOD was up-regulated in the MS treated pancreas, indicating that MS had an anti-oxidant effect against AP. Furthermore, MS protected pancreatic cells against cerulean-induced apoptosis and abolished cleaved caspase-3. CONCLUSION: MS exerted anti-inflammatory, anti-oxidant and anti-apoptotic effects on cerulein-induced AP in mice and may proved to be a promising therapeutic agent for the clinical treatment of pancreatitis.

Scavenger Receptor MARCO Orchestrates Early Defenses and Contributes to Fungal Containment during Cryptococcal Infection.

The scavenger receptor macrophage receptor with collagenous structure (MARCO) promotes protective innate immunity against bacterial and parasitic infections; however, its role in host immunity against fungal pathogens, including the major human opportunistic fungal pathogen Cryptococcus neoformans, remains unknown. Using a mouse model of C. neoformans infection, we demonstrated that MARCO deficiency leads to impaired fungal control during the afferent phase of cryptococcal infection. Diminished fungal containment in MARCO-/- mice was accompanied by impaired recruitment of Ly6Chigh monocytes and monocyte-derived dendritic cells (moDC) and lower moDC costimulatory maturation. The reduced recruitment and activation of mononuclear phagocytes in MARCO-/- mice was linked to diminished early expression of IFN-γ along with profound suppression of CCL2 and CCL7 chemokines, providing evidence for roles of MARCO in activation of the CCR2 axis during C. neoformans infection. Lastly, we found that MARCO was involved in C. neoformans phagocytosis by resident pulmonary macrophages and DC. We conclude that MARCO facilitates early interactions between C. neoformans and lung-resident cells and promotes the production of CCR2 ligands. In turn, this contributes to a more robust recruitment and activation of moDC that opposes rapid fungal expansion during the afferent phase of cryptococcal infection.

Complex Roles of Annexin A2 in Host Blood-Brain Barrier Invasion by Cryptococcus neoformans.

INTRODUCTION: Fungal transversal across the brain microvascular endothelial cells (BMECs) is the essential step for the development of cryptococcal meningoencephalitis. Annexin A2 (AnxA2) is an important signaling protein involved in several intracellular processes such as membrane trafficking, endocytosis, and exocytosis. AIM: To investigate the roles and mechanism of AnxA2 during cryptococcal transversal of BMECs. RESULTS: Cryptococcus neoformans infection initiated upregulation of AnxA2 in mouse BMECs. Blockade with anti-AnxA2 antibody led to a reduction in fungal transcytosis activity but no change in its adhesion efficiency. Intriguingly, AnxA2 depletion caused a significant increase in fungal association activity but had no effect on their transcytosis. AnxA2 suppression resulted in marked reduction in its partner protein S100A10, and S100A10 suppression in BMECs significantly reduced the cryptococcal transcytosis efficiency. Furthermore, AnxA2 dephosphorylation at Tyr23 and dephosphorylation of downstream cofilin were required for cryptococcal transversal of BMECs, both of which might be primarily involved in the association of C. neoformans with host cells. CONCLUSIONS: Our work indicated that AnxA2 played complex roles in traversal of C. neoformans across host BMECs, which might be dependent on downstream cofilin to inhibit fungal adhesion but rely on its partner S100A10 to promote cryptococcal transcytosis.

microRNA-625 inhibits tumorigenicity by suppressing proliferation, migration and invasion in malignant melanoma.

Dysregulated microRNA (miR)-625 expression has been observed in several kinds of cancer. MicroRNAs are important factors in the development and progression of malignant melanoma, though the clinical significance and function of miR-625 in human malignant melanoma remain unclear. Levels of miR-625 expression were therefore determined in 36 pairs of malignant melanoma and adjacent non-tumor tissue using qPCR. The effects of miR-625 dysregulation on malignant melanoma cell proliferation, wound healing, migration and invasion in vitro and tumorigenicity in vivo were investigated using CCK-8, transwell assays, and a nude mouse subcutaneous tumor model. Bioinformatics analysis and luciferase reporter system were used to predict and confirm the target gene of miR-625. miR-625 levels were frequently decreased in malignant melanoma. Ectopic expression of miR-625 suppressed proliferation, wound healing, migration, and tumorgenicity in malignant melanoma. Moreover, miR-625 acted, at least in part, by suppressing potential target SOX2. These results show that miR-625 is a tumor suppressor that inhibits the development and progression of malignant melanoma, which suggests miR-625 is potentially a new diagnostic marker and therapeutic target of malignant melanoma.

Pd@Ag Nanosheets in Combination with Amphotericin B Exert a Potent Anti-Cryptococcal Fungicidal Effect.

Silver nanoparticles have received considerable interest as new "nanoantibiotics" with the potential to kill drug-resistant microorganisms. Recently, a class of new core-shell nanostructures, Pd@Ag nanosheets (Pd@Ag NSs), were created using deposition techniques and demonstrated excellent inhibitory effects on various bacteria in vitro. In this study, we evaluated the antifungal activity of Pd@Ag NSs against common invasive fungal pathogens. Among these organisms, Cryptococcus neoformans complex species was most susceptible to Pd@Ag NSs, which exhibited potent antifungal activity against various molecular types or sources of cryptococcal strains including fluconazole-resistant isolates. The anticryptococcal activity of Pd@Ag NSs was significantly greater than fluconazole and similar to that of amphotericin B (AmB). At relatively high concentrations, Pd@Ag NSs exhibited fungicidal activity against Cryptococcus spp., which can likely be attributed to the disruption of cell integrity, intracellular protein synthesis, and energy metabolism. Intriguingly, Pd@Ag NSs also exhibited strong synergistic anti-cryptococcal fungicidal effects at low concentrations in combination with AmB but exhibited much better safety in erythrocytes than AmB, even at the minimal fungicidal concentration. Therefore, Pd@Ag NSs may be a promising adjunctive agent for treating cryptococcosis, and further investigation for clinical applications is required.

Deubiquitinase Ubp5 Is Required for the Growth and Pathogenicity of Cryptococcus gattii.

Cryptococcus gattii is a resurgent fungal pathogen that primarily infects immunocompetent hosts. Thus, it poses an increasingly significant impact on global public health; however, the mechanisms underlying its pathogenesis remain largely unknown. We conducted a detailed characterization of the deubiquitinase Ubp5 in the biology and virulence of C. gattii using the hypervirulent strain R265, and defined its properties as either distinctive or shared with C. neoformans. Deletion of the C. gattii Ubp5 protein by site-directed disruption resulted in a severe growth defect under both normal and stressful conditions (such as high temperature, high salt, cell wall damaging agents, and antifungal agents), similar to the effects observed in C. neoformans. However, unlike C. neoformans, the C. gattii ubp5Δ mutant displayed a slight enhancement of capsule and melanin production, indicating the evolutionary convergence and divergence of Ubp5 between these two sibling species. Attenuated virulence of the Cg-ubp5Δ mutant was not solely due to its reduced thermotolerance at 37°C, as shown in both worm and mouse survival assays. In addition, the assessment of fungal burden in mammalian organs further indicated that Ubp5 was required for C. gattii pulmonary survival and, consequently, extrapulmonary dissemination. Taken together, our work highlights the importance of deubiquitinase Ubp5 in the virulence composite of both pathogenic cryptococcal species, and it facilitates a better understanding of C. gattii virulence mechanisms.

Epidemiology of Cryptococcus and cryptococcosis in China.

Cryptococcosis is a significant invasive fungal infection with noteworthy morbidity and mortality, primarily caused by Cryptococcus neoformans and Cryptococcus gattii. In China, C. neoformans var. grubii (especially molecular type VNI) is the most common variety in the environment and responsible for the majority of cryptococcal infections. C. gattii infections are quite rare in China and the primary molecular type is VGI, which is closely related to C. gattii isolates in Australia. Interestingly, the majority of cryptococcosis in China were reported in the HIV-uninfected patients (especially immunocompetent hosts). This unique phenomenon may be attributed to multiple polymorphisms in the genes encoding mannose-binding lectin (MBL) and Fc-gamma receptor 2B (FCGR2B) in the Han population, the major ethnic group in China. Compared to immunocompromised patients, immunocompetent patients with cryptococcal meningitis often presented with more intense inflammatory responses and more severe neurological complications, but less fungal burdens and disseminated infection. The overall prognosis, which is independently associated with amphotericin B-based initial therapy, is similar between immunocompetent and immunocompromised patients. In addition, intrathecal administration of amphotericin B has been proved to be an effective adjunctive treatment for cryptococcosis in China.

Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome.

Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen that mainly infects immunocompromised individuals such as AIDS patients. Although cell surface receptors for recognition of C. neoformans have been studies intensively, cytoplasmic recognition of this pathogen remains unclear. As an important detector of pathogen infection, inflammasome can sense and get activated by infection of various pathogens, including pathogenic fungi such as Candida albicans and Aspergillus fumigatus. Our present study showed that acapsular C. neoformans (cap59Δ) activated the NLRP3-, but not AIM2-nor NLRC4- inflammasome. During this process, viability of the fungus was required. Moreover, our in vivo results showed that during the pulmonary infection of cap59Δ, immune cell infiltration into the lung and effective clearance of the fungus were both dependent on the presence of NLRP3 inflammasome. In summary, our data suggest that the capsule of C. neoformans prevents recognition of the fungus by host NLRP3 inflammasome and indicate that manipulation of inflammasome activity maybe a novel approach to control C. neoformans infection.