Cryptococcus gattii evades CD11b-mediated fungal recognition by coating itself with capsular polysaccharides.

Cryptococcus gattii is a capsular pathogenic fungus causing life-threatening cryptococcosis. Although the capsular polysaccharides (CPs) of C. gattii are considered as virulence factors, the physiological significance of CP biosynthesis and of CPs themselves is not fully understood, with many conflicting data reported. First, we demonstrated that CAP gene deletant of C. gattii completely lacked capsule layer and its virulence, and that the strain was susceptible to host-related factors including oxidizing, hypoxic, and hypotrophic conditions in vitro. Extracellular CPs recovered from culture supernatant bound specifically to C. gattii acapsular strains, not to other fungi and immune cells, and rendered them the immune escape effects. In fact, dendritic cells (DCs) did not efficiently uptake the CP-treated acapsular strains, which possessed no visible capsule layer, and a decreased amount of phosphorylated proteins and cytokine levels after the stimulation. DCs recognized C. gattii acapuslar cells via an immune receptor CD11b- and Syk-related pathway; however, CD11b did not bind to CP-treated acapsular cells. These results suggested that CPs support immune evasion by coating antigens on C. gattii and blocking the interaction between CD11b and C. gattii cells. Here, we describe the importance of CPs in pathogenicity and immune evasion mechanisms of C. gattii.

Neutrophil-mediated antifungal activity against highly virulent Cryptococcus gattii strain R265.

Vaccine-induced immune responses, including neutrophil, macrophage, and T-cell responses, ameliorate cryptococcosis caused by Cryptococcus gattii. However, whether neutrophils can exert fungicidal activity against C. gattii remains to be elucidated. Therefore, in this study, we investigated the neutrophil-mediated fungicidal effect against C. gattii R265 in vitro and compared it to the related fungal pathogen, Cryptococcus neoformans standard strain H99. We found that neutrophils recognized, phagocytosed, and killed C. gattii R265 in the presence of fresh mouse serum. This antifungal effect required phagocytosis and serine protease activity but not nicotinamide adenine dinucleotide phosphate oxidase activity. We also demonstrated that C. gattii R265 was more resistant to oxidative and nitrosative stress than C. neoformans H99. Together, these findings indicate that neutrophils can exert fungicidal activity against highly virulent C. gattii, at least under in vitro conditions.

Epidemiology of Coronavirus Disease 2019 in Yamagata Prefecture, Japan, January-May 2020: the Importance of Retrospective Contact Tracing.

Public health interventions have played an important role in controlling coronavirus disease 2019 (COVID-19), which is a rapidly spreading infectious disease. To contribute to future COVID-19 countermeasures, we aimed to verify the results of the countermeasures employed by public health centers (PHCs) against the first wave of COVID-19 in Yamagata Prefecture, Japan (Yamagata). Between January and May 2020, 1,253 patients suspected of SARS-CoV-2 infection were invited for testing. Simultaneously, based on retrospective contact tracings, PHCs investigated the infection sources and transmission routes of laboratory-confirmed COVID-19 cases and tested 928 contacts. Consequently, 69 cases were confirmed between March 31 and May 4, 58 of whom were from among the contacts (84.1%; 95% confidence interval [CI] 75.5-92.7). The spread of infection was triggered in cases harboring epidemiological links outside Yamagata. Subsequently, the number of cases rapidly increased. However, PHCs identified epidemiological links in 61 (88.4%; 95% CI 80.8-96.0) of the 69 cases, and transmission chains up to the fifth generation. Finally, the spread of infection ended after approximately one month. Our results indicate that the identification of infection sources and active case finding from contacts based on retrospective contact tracing was likely to be an effective strategy in ending the first wave of COVID-19 in Yamagata.

Cryptococcus gattii alters immunostimulatory potential in response to the environment.

Cryptococcus gattii is a capsular fungal pathogen, which causes life-threatening cryptococcosis in immunocompetent individuals. This emerging pathogen is less likely to be recognized by innate immunity compared to traditional Cryptococcus neoformans strains. Previous studies indicate that C-type lectin receptors (CLRs), including dectin-1 and dectin-2, play a role in recognizing cryptococcal cells; however, it remains to be elucidated whether the receptors physically associate with C. gattii yeast cell surfaces. Based on the previous findings, we hypothesized that culture conditions influence the expression or exposure of CLR ligands on C. gattii. Therefore, in the present study, we first investigated the culture conditions that induce exposure of CLR ligands on C. gattii yeast cells via the binding assay using recombinant fusion proteins of mouse CLR and IgG Fc, Fc dectin-1 and Fc dectin-2. Common fungal culture media, such as yeast extract-peptone-dextrose (YPD) broth, Sabouraud broth, and potato dextrose agar, did not induce the exposure of dectin-1 ligands, including ß-1,3-glucan, on both capsular and acapsular C. gattii strains, in contrast to Fc dectin-1 and Fc dectin-2 bound to C. gattii cells growing in the conventional synthetic dextrose (SD) medium [may also be referred to as a yeast nitrogen base with glucose medium]. The medium also induced the exposure of dectin-1 ligands on C. neoformans, whereas all tested media induced dectin-1 and dectin-2 ligands in a control fungus Candida albicans. Notably, C. gattii did not expose dectin-1 ligands in SD medium supplemented with yeast extract or neutral buffer. In addition, compared to YPD medium-induced C. gattii, SD medium-induced C. gattii more efficiently induced the phosphorylation of Syk, Akt, and Erk1/2 in murine dendritic cells (DCs). Afterwards, the cells were considerably engulfed by DCs and remarkably induced DCs to secrete the inflammatory cytokines. Overall, the findings suggest that C. gattii alters its immunostimulatory potential in response to the environment.

A dendritic cell-based systemic vaccine induces long-lived lung-resident memory Th17 cells and ameliorates pulmonary mycosis.

Tissue-resident memory T cells (TRMs) are a novel nonvascular memory T cell subset. Although CD8+ TRMs are well-characterized, CD4+ TRMs-especially lung-resident memory Th17 cells-are still being defined. In this study, we characterized lung-resident memory Th17 cells (lung TRM17) and their role in protection against the highly virulent fungus Cryptococcus gattii. We found that intravenously transferred DCs preferentially migrated to lungs and attracted recipient DCs and led to the induction of long-lived Th17 cells expressing characteristic markers. This population could be clearly discriminated from circulating T cells by intravascular staining and was not depleted by the immunosuppressive agent FTY720. The C. gattii antigen re-stimulation assay revealed that vaccine-induced lung Th17 cells produced IL-17A but not IFNγ. The DC vaccine significantly increased IL-17A production and suppressed fungal burden in the lungs and improved the survival of mice infected with C. gattii. This protective effect was significantly reduced in the IL-17A knockout (KO) mice, but not in the FTY720-treated mice. The protective effect also coincided with the activation of neutrophils and multinucleated giant cells, and these inflammatory responses were suppressed in the vaccinated IL-17A KO mice. Overall, these data demonstrated that the systemic DC vaccine induced lung TRM17, which played a substantial role in anti-fungal immunity.

Mouse LIMR3/CD300f is a negative regulator of the antimicrobial activity of neutrophils.

Leukocyte mono-immunoglobulin-like receptor (LMIR)/CD300 proteins comprise a family of immunoglobulin-like receptors that are widely expressed on the immune cell surface in humans and mice. In general, LMIR3/CD300f suppresses the inflammatory response, but it can occasionally promote it. However, the precise roles of LMIR3 in the function of neutrophils remain to be elucidated. In the present study, we investigated LMIR3 expression in mature and immature neutrophils, and evaluated the effects of LMIR3 deficiency in mouse neutrophils. Our results indicated that bone marrow (BM) neutrophils expressed LMIR3 on their cell surface during cell maturation and that surface LMIR3 expression increased in response to Pseudomonas aeruginosa infection in a TLR4/MyD88-dependent manner. LMIR3-knockout (KO) neutrophils displayed significantly increased hypochlorous acid production, and elastase release, as well as significantly augmented cytotoxic activity against P. aeruginosa and Candida albicans; meanwhile, inhibitors of elastase and myeloperoxidase offset this enhanced antimicrobial activity. Furthermore, LMIR3-KO mice were significantly more resistant to Pseudomonas peritonitis and systemic candidiasis, although this may not be entirely due to the enhanced activity of neutrophils. These results demonstrate that LMIR3/CD300f deficiency augments the antimicrobial activity of mouse neutrophils.

The effectiveness of a nurse-initiated intervention to reduce catheter-associated bloodstream infections in an urban acute hospital: an intervention study with before and after comparison.

BACKGROUND: Catheter care is considered to be important for prevention of catheter-associated bloodstream infections (CABSIs) although epidemiological evidence is sparse. OBJECTIVES: To identify problems associated with catheter care and evaluate the effectiveness of nurse-initiated interventions to reduce CABSIs. DESIGN: An intervention study with before and after comparison. SETTINGS: CABSI surveillance was conducted in a 560-bed acute hospital located in a major urban area in Japan. PARTICIPANTS: Patients were enrolled in this study from April 2000 to December 2002 based on the following criteria: (1) adult inpatients; and (2) those in whom central venous lines or Swan-Ganz catheters were inserted for 2 days or longer. METHODS: In the first year, risk factors for CABSI and problems associated with catheter care were identified by inspection of the infection control nurse (ICN) or four trained link nurses, and the laboratory results. In the subsequent 2 years, the following interventions based on the surveillance results were implemented: (1) enhanced skin preparation by scrubbing with regular bathing soap and tap water; (2) a new method for stabilisation of the catheter inserted into the internal jugular vein, where additional dressing was placed over the sterilised dressing; (3) educating the staff on maximal sterile precautions by teaching staff members at their section meetings and displaying posters; (4) use of a check list and observation of catheter insertion by link nurses to monitor compliance; and (5) selection of a disinfectant that requires shorter contact time and has longer residual effect. RESULTS: After these interventions were implemented, the overall bloodstream infection (BSI) rate declined from 4.0/1000 device-days to 1.1/1000 device-days (p<0.005). CONCLUSIONS: We identified four problems-those related to skin preparation, dressing, sterile precautions and disinfectant. We implemented a series of interventions to reduce CABSIs; the overall CABSI rate decreased significantly.