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Elevation of arginase enzyme activity in the lung contributes to the pathogenesis of various chronic inflammatory diseases and infections. Inhibition of arginase expression and activity is able to alleviate those effects. Here, we investigated the immunomodulatory effect of arginase inhibitor in C. neoformans infection. In the pulmonary cryptococcosis model that was shown to recapitulate human infection, we found arginase expression was excessively induced in the lung during the late stage of infection. To inhibit the activity of arginase, we administered a specific arginase inhibitor, nor-NOHA, during C. neoformans infection. Inhibition of arginase reduced eosinophil infiltration and level of IL-13 secretion in the lungs. Whole lung transcriptome RNA-sequencing analysis revealed that treatment with nor-NOHA resulted in shifting the Th2-type gene expression patterns induced by C. neoformans infection to the Th1-type immune profile, with higher expression of cytokines Ifng, Il6, Tnfa, Csf3, chemokines Cxcl9 and Cxcl10 and transcription factor Stat1. More importantly, mice treated with arginase inhibitor had more infiltrating brain leukocytes and enhanced gene expression of Th1-associated cytokines and chemokines that are known to be essential for protection against C. neoformans infection. Inhibition of arginase dramatically attenuated spleen and brain infection, with improved survival. Taken together, these studies demonstrated that inhibiting arginase activity induced by C. neoformans infection can modulate host immune response by enhancing protective type-1 immune response during C. neoformans infection. The inhibition of arginase activity could be an immunomodulatory target to enhance protective anti-cryptococcal immune responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cryptococcus is recognized as one of the emerging fungal pathogens that have major impact on diverse populations worldwide. Because of the high mortality rate and limited antifungal therapy options, there is an urgent need to understand the impact of dynamic processes between fungal pathogens and hosts that influence cryptococcal pathogenesis and disease outcomes. With known common limitations in human studies, experimental murine cryptococcosis models that can recapitulate human disease provide a valuable tool for studying fungal virulence and the host interaction, leading to development of better treatment strategies. Infection with Cryptococcus in mice via intranasal inhalation is mostly used because it is noninvasive and considered to be the most common mode of infection, strongly correlating with cryptococcal disease in humans. The protocols described in this article provide the procedures of establishing a murine model of Cryptococcus infection by intranasal inhalation and assessing the host immune response and disease progression during Cryptococcus infection. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Murine model of pulmonary cryptococcal infection via intranasal inhalation Basic Protocol 2: Assessment of the pulmonary immune response during Cryptococcus infection Support Protocol: Evaluation of pulmonary gene expression by real-time PCR Basic Protocol 3: Enumeration of survival rate and organ fungal burden., (© 2024 Wiley Periodicals LLC.)

The epithelial cell-derived cytokines IL-33 and IL-25 are important mediators in driving type-2 inflammation during C. neoformans infection. Nevertheless, the impact of these cytokines in regulating host T helper cell response during C. neoformans infection is still unclear. We observed that C. neoformans infection promoted a predominant increase of T helper cells that co-expressed IL-25 and IL-33 receptors within the lung during the late infection phase. A comparative transcriptomic analysis of effector T helper cells co-treated with IL-25 and IL-33 revealed a cooperative effect of these cytokines in promoting IL-13 gene expression. Without IL-25 receptor signaling, IL-33 treatment upregulated Th1-associated genes and genes associated with nucleotide metabolism. By contrast, IL-25 had a unique effect in enhancing type-2 cytokines IL-5 and IL-9 and chemokine CCL24, as well as genes in the pathways that are associated with L-arginine metabolisms. Interestingly, this pathogenic T helper cell population that expressed IL-25 and IL-33 receptors was greatly enriched in mice that were infected with high cryptococcal virulence and associated with fungal burdens in the brain. Therefore, our data further provide the additional function of IL-25 and IL-33 in potentiating cryptococcal brain dissemination., (© 2023. The Author(s).)

Cryptococcal meningoencephalitis caused by Cryptococcus neoformans infection is the most common cause of death in HIV/AIDS patients. Macrophages are pivotal for the regulation of immune responses to cryptococcal infection by either playing protective function or facilitating fungal dissemination. However, the mechanisms underlying macrophage responses to C. neoformans remain unclear. To analyze the transcriptomic changes and identify the pathogenic factors of macrophages, we performed a comparative transcriptomic analysis of alveolar macrophage responses during C. neoformans infection. Alveolar macrophages isolated from C. neoformans-infected mice showed dynamic gene expression patterns, with expression change from a protective M1 (classically activated)-like to a pathogenic M2 (alternatively activated)-like phenotype. Arg1, the gene encoding the enzyme arginase 1, was found as the most upregulated gene in alveolar macrophages during the chronic infection phase. The in vitro inhibition of arginase activity resulted in a reduction of cryptococcal phagocytosis, intracellular growth, and proliferation, coupled with an altered macrophage response from pathogenic M2 to a protective M1 phenotype. In an in vitro model of the blood-brain barrier, macrophage-derived arginase was found to be required for C. neoformans invasion of brain microvascular endothelium. Further analysis of the degree of virulence indicated a positive correlation between arginase 1 expression in macrophages and cryptococcal brain dissemination in vivo. Thus, our data suggest that a dynamic macrophage activation that involves arginase expression may contribute to the cryptococcal disease by promoting cryptococcal growth, proliferation, and the invasion to the brain endothelium., (Copyright © 2023 by The American Association of Immunologists, Inc.)

A multifunctional glycoprotein, osteopontin (OPN), can modulate the function of macrophages, resulting in either protective or deleterious effects in various inflammatory diseases and infection in the lungs. Although macrophages play the critical roles in mediating host defenses against cryptococcosis or cryptococcal pathogenesis, the involvement of macrophage-derived OPN in pulmonary infection caused by fungus Cryptococcus has not been elucidated. Thus, our current study aimed to investigate the contribution of OPN to the regulation of host immune response and macrophage function using a mouse model of pulmonary cryptococcosis. We found that OPN was predominantly expressed in alveolar macrophages during C. neoformans infection. Systemic treatment of OPN during C. neoformans infection resulted in an enhanced pulmonary fungal load and an early onset of type 2 inflammation within the lung, as indicated by the increase of pulmonary eosinophil infiltration, type 2 cytokine production, and M2-associated gene expression. Moreover, CRISPR/Cas9-mediated OPN knockout murine macrophages had enhanced ability to clear the intracellular fungus and altered macrophage phenotype from pathogenic M2 to protective M1. Altogether, our data suggested that macrophage-derived OPN contributes to the elaboration of C. neoformans -induced type 2 immune responses and polarization of M2s that promote fungal survival and proliferation within macrophages., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Cryptococcal meningitis is one of the most common life-threatening diseases caused by Cryptococcus infection. Increasing evidence indicates that type 2 immunity is associated with disease progression by promoting fungal growth and dissemination. However, factors that govern this pathogenic response during infection are still elusive. In this study, we investigated the role of IL-25, one of the type 2-inducing cytokines produced by epithelial cells, in contributing to the pathogenesis of cryptococcosis. We found that pulmonary but not systemic infection with a high-virulence strain of C. neoformans significantly induced pulmonary IL-25 expression in the lungs but not brains. In response to pulmonary infection, mice deficient in the surface IL-17 receptor B, a component of the IL-25R, exhibited improved survival with a decreased brain fungal burden. The absence of IL-25R signaling diminished the type 2 and enhanced the type 1 immune response that directed macrophage polarization toward M1 macrophages. Interestingly, Cryptococcus -mediated IL-25 signaling suppressed the expression of cytokines and chemokines associated with protection in the brain, including Ifng, Il1b, Ip10, and Nos2 , without affecting brain cellular inflammation and microglia cell activation. Il17rb -/- mice receiving cryptococcal-specific CD4 + T cells from wild-type had a shorter survival time with higher fungal burden within the brain and an elevated expression of M2 macrophage markers than those receiving cryptococcal-specific CD4 + T cells from Il17rb -/- mice. Taken together, our data indicated that IL-25 signaling subverts the induction of protective immunity and amplifies the type 2 immune response that may favor the development of cryptococcal disease and the fungal dissemination to the CNS., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Cryptococcosis is an infectious disease caused by two fungal species, Cryptococcus neoformans and Cryptococcus gattii While C. neoformans affects mainly immunocompromised patients, C. gattii infects both immunocompetent and immunocompromised individuals. Laccase is an important virulence factor that contributes to the virulence of C. neoformans by promoting pulmonary growth and dissemination to the brain. The presence of laccase in C. neoformans can shift the host immune response toward a nonprotective Th2-type response. However, the role of laccase in the immune response against C. gattii remains unclear. In this study, we characterized laccase activity in C. neoformans and C. gattii isolates from Thailand and investigated whether C. gattii that is deficient in laccase might modulate immune responses during infection. C. gattii was found to have higher laccase activity than C. neoformans , indicating the importance of laccase in the pathogenesis of C. gattii infection. The expression of laccase promoted intracellular proliferation in macrophages and inhibited in vitro fungal clearance. Mice infected with a lac1Δ mutant strain of C. gattii had reduced lung burdens at the early but not the late stage of infection. Without affecting type-1 and type-2 responses, the deficiency of laccase in C. gattii induced cryptococcus-specific interleukin-17 (IL-17) cytokine, neutrophil accumulation, and expression of the neutrophil-associated cytokine gene Csf3 and chemokine genes Cxcl1, Cxcl2 , and Cxcl5 in vivo , as well as enhanced neutrophil-mediated phagocytosis and killing in vitro Thus, our data suggest that laccase constitutes an important virulence factor of C. gattii that plays roles in attenuating Th17-type immunity, neutrophil recruitment, and function during the early stage of infection., (Copyright © 2020 American Society for Microbiology.)

Cryptococcus-macrophage interaction is crucial in the development of cryptococcocal diseases. C. neoformans and C. gattii are major pathogenic species that occupy different niches and cause different clinical manifestations. However, the differences of macrophage interaction among these species in affecting different disease outcomes and immune responses have not been clearly addressed. Here, we examined the macrophage uptake rates, intracellular loads and intracellular proliferation rates of C. neoformans and C. gattii clinical isolates from Thailand and analyzed the effect of those interactions on fungal burdens and host immune responses. C. neoformans isolates showed a higher phagocytosis rate but lower intracellular proliferation rate than C. gattii. Indeed, the high intracellular proliferation rate of C. gattii isolates did not influence the fungal burdens in lungs and brains of infected mice, whereas infection with high-uptake C. neoformans isolates resulted in significantly higher brain burdens that associated with reduced survival rate. Interestingly, alveolar macrophages of mice infected with high-uptake C. neoformans isolates showed distinct patterns of alternatively activated macrophage (M2) gene expressions with higher Arg1, Fizz1, Il13 and lower Nos2, Ifng, Il6, Tnfa, Mcp1, csf2 and Ip10 transcripts. Corresponding to this finding, infection with high-uptake C. neoformans resulted in enhanced arginase enzyme activity, elevated IL-4 and IL-13 and lowered IL-17 in the bronchoalveolar lavage. Thus, our data suggest that the macrophage interaction with C. neoformans and C. gattii may affect different disease outcomes and the high phagocytosis rates of C. neoformans influence the induction of type-2 immune responses that support fungal dissemination and disease progression. Abbreviation: Arg1: Arginase 1; BAL: Bronchoalveolar lavage; CCL17: Chemokine (C-C motif) ligand 17; CNS: Central nervous system; CSF: Cerebrospinal fluid; Csf2: Colony-stimulating factor 2; Fizz1: Found in inflammatory zone 1; HIV: Human immunodeficiency virus; ICL: Intracellular cryptococcal load; Ifng: Interferon gamma; Ip10: IFN-g-inducible protein 10; IPR: Intracellular proliferation rate; Mcp1: Monocyte chemoattractant protein 1; Nos2: Nitric oxide synthase 2; PBS: Phosphate-Buffered Saline; Th: T helper cell; Tnfa: Tumor necrosis factor alpha.

• Pulmonary C. gattii infection enhances GM-CSF expression, mainly in T helper cells. • GM-CSF treatment promotes protection against C. gattii and C. neoformans infection • Intranasal GM-CSF alters C. gattii and C. neoformans immune responses differently. • GM-CSF treatment promotes Th17/neutrophil response during C. gattii infection. • Intranasal GM-CSF boosts Th1/M1 macrophage activation in C. neoformans infection. Cryptococcosis, caused by infections with C. neoformans and C. gattii , presents a serious threat to global health and necessitates effective treatment strategies. Granulocyte-Macrophage Colony-Stimulating Factor, GM-CSF, is an immune-modulating cytokine that has been utilized clinically to improve host defense against infection; however, the impact of GM-CSF treatment in C. gattii infection has not been elucidated. Our current study aimed to investigate the effect of GM-CSF treatment on pulmonary immune response during C. gattii infection. In response to C. gattii infection, GM-CSF-expressing T helper cells and CD11b+ myeloid were enhanced in the lungs. The intranasal administration of GM-CSF during C. gattii infection significantly reduced pulmonary cryptococcal load, promoted an increase in pulmonary Th17 cells, as well as neutrophil infiltration in the lungs. Exposure of neutrophils to C. gattii in the presence of GM-CSF resulted in an increased neutrophil phagocytosis and fungal killing capacity, generation of reactive oxygen species (ROS), and upregulation of inflammatory cytokines and anti-microbial peptides. Although GM-CSF treatment in C. neoformans -infected mice had a comparable impact on the reduction of lung fungal burden, it resulted in the enhancement of Th1-type cytokine IFN-γ and the activation of M1 macrophages. Altogether, this study demonstrated that the intranasal delivery of GM-CSF has distinct effects on promoting the protection against C. gattii and C. neoformans by activating neutrophil/type-17 immune response and stimulating M1 macrophage/type-1 immunity, respectively. [ABSTRACT FROM AUTHOR]