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Cryptococcus neoformans is a fungal pathogen that is found throughout the environment and can cause life-threatening infections of the lung and central nervous system in severely immunocompromised individuals. Caspase recruitment domain-containing protein 9 (CARD9) is a critical molecule that is activated after interactions of C-type lectin receptors (CLRs) found on the surfaces of specific immune cells, with carbohydrate structures associated with fungi. Patients with defects in CARD9 are significantly more susceptible to a multitude of fungal infections. C. neoformans contains several carbohydrate structures that interact with CLRs on immune cells and activate CARD9. Consequently, these studies evaluated the necessity of CARD9 for the induction of protective immunity against C. neoformans infection. These results are important, as they advance our understanding of cryptococcal pathogenesis and host factors necessary for the induction of protective immunity against C. neoformans., Caspase recruitment domain-containing protein 9 (CARD9) is a critical adaptor molecule triggered by the interaction of C-type lectin receptors (CLRs) with carbohydrate motifs found in fungi. Consequently, clinical and animal studies indicate that CARD9 is an important regulator of protective immunity against fungal pathogens. Previous studies suggest that CARD9 is important for the induction of protection against Cryptococcus neoformans, an opportunistic fungal pathogen that causes life-threatening infections of the central nervous system in immunocompromised patients. However, the effect of CARD9 deficiency on the induction of protective immune responses against C. neoformans is unknown. Immunization with a C. neoformans mutant that overexpresses the transcription factor zinc finger 2, denoted LW10, results in protection against an otherwise lethal challenge with wild-type (WT) C. neoformans. Our results showed that CARD9 is essential for the induction of vaccine-mediated immunity against C. neoformans infection. We observed significant decreases in interleukin-17 (IL-17) production and significant increases in Th2-type cytokine (IL-4, IL-5, and IL-13) production in CARD9-deficient mice after inoculation with strain LW10. While leukocyte infiltration to the lungs of CARD9-deficient mice was similar in LW10 and WT C. neoformans-infected mice, macrophages derived from CARD9-deficient mice inherently skewed toward an M2 activation phenotype, were unable to contain the growth of LW10, and failed to produce nitric oxide in response to infection with LW10 or stimulation with lipopolysaccharide. These results suggest that CARD9-mediated signaling is required for M1 macrophage activation and fungicidal activity necessary for the induction of vaccine-mediated immunity against C. neoformans.

Conventional dendritic cells (cDCs) are critical for protection against pulmonary infection with the opportunistic fungal pathogen Cryptococcus neoformans ; however, the role of plasmacytoid dendritic cells (pDCs) is unknown. We show for the first time that murine pDCs have direct activity against C. neoformans via reactive oxygen species (ROS), a mechanism different from that employed to control Aspergillus fumigatus infections. The anticryptococcal activity of murine pDCs is independent of opsonization but appears to require the C-type lectin receptor Dectin-3, a receptor not previously evaluated during cryptococcal infections. Human pDCs can also inhibit cryptococcal growth by a mechanism similar to that of murine pDCs. Experimental pulmonary infection of mice with a C. neoformans strain that induces protective immunity demonstrated that recruitment of pDCs to the lungs is CXCR3 dependent. Taken together, our results show that pDCs inhibit C. neoformans growth in vitro via the production of ROS and that Dectin-3 is required for optimal growth-inhibitory activity.

Development of vaccines against opportunistic infections is difficult as patients most at risk of developing disease are deficient in aspects of the adaptive immune system. Here, we utilized an experimental immunization strategy to induce innate memory in macrophages in vivo. Unlike current trained immunity models, we present an innate memory-like phenotype in macrophages that is maintained for at least 70 days post-immunization and results in complete protection against secondary challenge in the absence of adaptive immune cells. RNA-seq analysis of in vivo IFN-γ primed macrophages revealed a rapid up-regulation of IFN-γ and STAT1 signaling pathways following secondary challenge. The enhanced cytokine recall responses appeared to be pathogen-specific, dependent on changes in histone methylation and acetylation, and correlated with increased STAT1 binding to promoter regions of genes associated with protective anti-fungal immunity. Thus, we demonstrate an alternative mechanism to induce macrophage innate memory in vivo that facilitates pathogen-specific vaccine-mediated immune responses., Author summary Fungal infections are a significant global health problem that can affect anyone, however, individuals with a weakened immune system are most at risk. Cryptococcus neoformans infections can progress to meningitis in immune compromised individuals accounting for nearly 220,000 new cases annually, resulting in 181,000 deaths. Vaccine strategies tend to target CD4+ T cells for the generation of protective memory responses. However, immune compromised individuals have decreased numbers of these adaptive cells, providing a challenge for anti-fungal vaccine design. Here, we define a cellular mechanism by which macrophages, an innate cell population, generate protective immune responses against C. neoformans following initial exposure to a C. neoformans strain that secretes IFN-γ. We determined that the macrophages primed in vivo have heightened proinflammatory cytokine responses upon secondary exposure to C. neoformans in a manner that is mTOR-independent, yet dependent on histone modification dynamics. We show that IFN-γ primed macrophages can maintain STAT1 binding to the promoter regions of key proinflammatory genes long after the initial exposure. Remarkably, our studies show long-lived, cryptococcal-specific protective immunity in vivo. The results presented herein demonstrate that innate cell populations, namely macrophages, can be utilized as vaccine targets to protect against cryptococcal infections in immune compromised populations.

Compared to other fungal pathogens, Cryptococcus neoformans is particularly adept at avoiding detection by innate immune cells. To explore fungal cellular features involved in immune avoidance, we characterized cell surface changes of the C. neoformans rim101Δ mutant, a strain that fails to organize and shield immunogenic epitopes from host detection. These cell surface changes are associated with an exaggerated, detrimental inflammatory response in mouse models of infection. We determined that the disorganized strain rim101Δ cell wall increases macrophage detection in a contact-dependent manner. Using biochemical and microscopy methods, we demonstrated that the rim101Δ strain shows a modest increase in the levels of both cell wall chitin and chitosan but that it shows a more dramatic increase in chito-oligomer exposure, as measured by wheat germ agglutinin staining. We also created a series of mutants with various levels of cell wall wheat germ agglutinin staining, and we demonstrated that the staining intensity correlates with the degree of macrophage activation in response to each strain. To explore the host receptors responsible for recognizing the rim101Δ mutant, we determined that both the MyD88 and CARD9 innate immune signaling proteins are involved. Finally, we characterized the immune response to the rim101Δ mutant in vivo, documenting a dramatic and sustained increase in Th1 and Th17 cytokine responses. These results suggest that the Rim101 transcription factor actively regulates the C. neoformans cell wall to prevent the exposure of immune stimulatory molecules within the host. These studies further explored the ways in which immune cells detect C. neoformans and other fungal pathogens by mechanisms that include sensing N-acetylglucosamine-containing structures, such as chitin and chitosan., IMPORTANCE Infectious microorganisms have developed many ways to avoid recognition by the host immune system. For example, pathogenic fungi alter their cell surfaces to mask immunogenic epitopes. We have created a fungal strain with a targeted mutation in a pH response pathway that is unable to properly organize its cell wall, resulting in a dramatic immune reaction during infection. This mutant cell wall is defective in hiding important cell wall components, such as the chito-oligomers chitin and chitosan. By creating a series of cell wall mutants, we demonstrated that the degree of chito-oligomer exposure correlates with the intensity of innate immune cell activation. This activation requires a combination of host receptors to recognize and respond to these infecting microorganisms. Therefore, these experiments explored host-pathogen interactions that determine the degree of the subsequent inflammatory response and the likely outcome of infection.

C-type lectin receptors (CLRs) are diverse, trans-membrane proteins that function as pattern recognition receptors (PRRs) which are necessary for orchestrating immune responses against pathogens. CLRs have been shown to play a major role in recognition and protection against fungal pathogens. Dectin-3 (also known as MCL, Clecsf8, or Clec4d) is a myeloid cell-specific CLR that recognizes mycobacterial trehalose 6,6’-dimycolate (TDM) as well as α-mannans present in the cell wall of fungal pathogens. To date, a potential role for Dectin-3 in the mediation of protective immune responses against C. neoformans has yet to be determined. Consequently, we evaluated the impact of Dectin-3 deficiency on the development of protective immune responses against C. neoformans using an experimental murine model of pulmonary cryptococcosis. Dectin-3 deficiency did not lead to increased susceptibility of mice to experimental pulmonary C. neoformans infection. Also, no significant differences in pulmonary leukocyte recruitment and cytokine production were observed in Dectin-3 deficient mice compared to wild type infected mice. In addition, we observed no differences in uptake and anti-cryptococcal activity of Dectin-3 deficient dendritic cells and macrophages. Altogether, our studies show that Dectin-3 is dispensable for mediating protective immune responses against pulmonary C. neoformans infection.

Cryptococcus neoformans and C. gattii are fungal pathogens that cause life-threatening disease. These fungi commonly enter their host via inhalation into the lungs where they encounter resident phagocytes, including macrophages and dendritic cells, whose response has a pronounced impact on the outcome of disease. Cryptococcus has complex interactions with the resident and infiltrating innate immune cells that, ideally, result in destruction of the yeast. These phagocytic cells have pattern recognition receptors that allow recognition of specific cryptococcal cell wall and capsule components. However, Cryptococcus possesses several virulence factors including a polysaccharide capsule, melanin production and secretion of various enzymes that aid in evasion of the immune system or enhance its ability to thrive within the phagocyte. This review focuses on the intricate interactions between the cryptococci and innate phagocytic cells including discussion of manipulation and evasion strategies used by Cryptococcus, anti-cryptococcal responses by the phagocytes and approaches for targeting phagocytes for the development of novel immunotherapeutics.

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an encapsulated fungal pathogen that can cause fungal pneumonia and life-threatening infections of the central nervous system (CNS) [1]. C. neoformans can be found ubiquitously throughout the environment [1]. Inhalation of airborne yeast or desiccated basidiospores typically results in asymptomatic disease or dormant infections; however, progression towards clinical disease commonly occurs in persons with severely compromised immune responses. Global estimates suggest that 1 million cases of cryptococcal meningitis occur each year, resulting in approximately 625,000 deaths [2]. Morbidity and mortality rates due to cryptococcosis are significantly higher in resource-limited settings and in individuals with impaired CD4+ T cell-mediated immune responses (reviewed in [3–5]). Current therapies are often rendered ineffective because of the development of drug resistance by C. neoformans, drug toxicity, and treatment cost. Thus, a need remains for a cost-effective approach to prevent cryptococcosis.

Nonprotective immune responses to highly virulent Cryptococcus neoformans strains, such as H99, are associated with Th2-type cytokine production, alternatively activated macrophages, and inability of the host to clear the fungus. In contrast, experimental studies show that protective immune responses against cryptococcosis are associated with Th1-type cytokine production and classical macrophage activation. The protective response induced during C. neoformans strain H99γ (C. neoformans strain H99 engineered to produce murine IFN-γ) infection correlates with enhanced phosphorylation of the transcription factor STAT1 in macrophages; however, the role of STAT1 in protective immunity to C. neoformans is unknown. The current studies examined the effect of STAT1 deletion in murine models of protective immunity to C. neoformans. Survival and fungal burden were evaluated in wild-type and STAT1 knockout (KO) mice infected with either strain H99γ or C. neoformans strain 52D (unmodified clinical isolate). Both strains H99γ and 52D were rapidly cleared from the lungs, did not disseminate to the CNS, or cause mortality in the wild-type mice. Conversely, STAT1 KO mice infected with H99γ or 52D had significantly increased pulmonary fungal burden, CNS dissemination, and 90–100% mortality. STAT1 deletion resulted in a shift from Th1 to Th2 cytokine bias, pronounced lung inflammation, and defective classical macrophage activation. Pulmonary macrophages from STAT1 KO mice exhibited defects in NO production correlating with inefficient inhibition of fungal proliferation. These studies demonstrate that STAT1 signaling is essential not only for regulation of immune polarization but also for the classical activation of macrophages that occurs during protective anticryptococcal immune responses.

Cryptococcus gattii is a fungal pathogen that can cause life-threatening respiratory and disseminated infections in immune-competent and immune-suppressed individuals. Currently, there are no standardized vaccines against cryptococcosis in humans, underlying an urgent need for effective therapies and/or vaccines. In this study, we evaluated the efficacy of intranasal immunization with C. gattii cell wall associated (CW) and/or cytoplasmic (CP) protein preparations to induce protection against experimental pulmonary C. gattii infection in mice. BALB/c mice immunized with C. gattii CW and/or CP protein preparations exhibited a significant reduction in pulmonary fungal burden and prolonged survival following pulmonary challenge with C. gattii. Protection was associated with significantly increased pro-inflammatory and Th1-type cytokine recall responses, in vitro and increased C. gattii-specific antibody production in immunized mice challenged with C. gattii. A number of immunodominant proteins were identified following immunoblot analysis of C. gattii CW and CP protein preparations using sera from immunized mice. Immunization with a combined CW and CP protein preparation resulted in an early increase in pulmonary T cell infiltrates following challenge with C. gattii. Overall, our studies show that C. gattii CW and CP protein preparations contain antigens that may be included in a subunit vaccine to induce prolonged protection against pulmonary C. gattii infection.