Michal A. Olszewski

Urease Expression by Cryptococcus neoformans Promotes Microvascular Sequestration, Thereby Enhancing Central Nervous System Invasion

Our objective was to determine the role of the cryptococcal virulence factor urease in pulmonary-to-central nervous system, dissemination, invasion, and growth. C. neoformans H99, the urease knockout strain (ure1) derived from H99, and the urease restored strain ure1+URE1-1 were used for the studies. The absence of cryptococcal urease (ure1infection) resulted in significant protection from the high mortality observed in H99-infected mice. All H99-infected mice had extremely high cryptococcal loads in their brains at the time of death, whereas only two of six animals that died of ure1 infection had detectable C. neoformans in the brain. Histological analysis of the blood-to-brain invasion by C. neoformans H99 demonstrated wedging of the yeasts in small capillaries, altered structure of microvessel walls, formation of mucoid cysts initiated in the proximity of damaged microcapillaries, and the absence of an inflammatory response. Direct inoculation of H99, ure1, and ure1+URE1-1 into the brain demonstrated that urease was not required to grow in the brain. However, the dissemination patterns in the brain, spleen, and other organs after intravenous inoculation indicated that cryptococcal urease contributes to the central nervous system invasion by enhancing yeast sequestration within microcapillary beds (such as within the brain) during hematogenous spread, thereby facilitating blood-to-brain invasion by C. neoformans.

Macrophage M1/M2 Polarization Dynamically Adapts to Changes in Cytokine Microenvironments in Cryptococcus neoformans Infection

ABSTRACT The outcome of cryptococcal pneumonia correlates with local macrophage polarization status, as M1 and M2 polarization marks protective and nonprotective responses, respectively. Overall, pulmonary macrophage polarization status changes over time during a cryptococcal infection. This could have been caused by repolarization of individual macrophages or by a replacement of M2-polarized cells by new M1-polarized cells. To explore the ability of macrophages to change between polarization states, we conducted a series of experiments using in vitro macrophages. Coculture of macrophages with Cryptococcus neoformans resulted in development of a weak M1-like phenotype, with modestly increased inducible nitric oxide synthase (iNOS) but lacking interleukin 6 (IL-6) induction. The C. neoformans-induced M1-like polarization state was plastic, as macrophages stimulated first with C. neoformans and then with gamma interferon (IFN-γ) or IL-4 expressed mRNA polarization patterns similar to those stimulated with cytokines alone. To further evaluate macrophage polarization plasticity, cytokine stimulatory conditions were established which fully polarized macrophages. IFN-γ and IL-4 stimulation differentially induced complete M1 and M2 polarization, defined by differential expression of marker mRNA panels, surface marker expression, and tumor necrosis factor alpha (TNF-α) protein production. Switching IFN-γ- to IL-4-stimulating conditions, and vice versa, resulted in uniform changes in profiles of polarization marker genes consistent with the most recent cytokine environment. Furthermore, the ability of sequentially stimulated macrophages to inhibit C. neoformans reflected the most recent polarizing condition, independent of previous polarization. Collectively, these data indicate that M1/M2 macrophage polarization phenotypes are highly plastic to external signals, and interventions which therapeutically repolarize macrophages could be beneficial for treatment of cryptococcosis. IMPORTANCE Our studies reveal how a major opportunistic fungal pathogen, Cryptococcus neoformans, interacts with macrophages, immune cells which can ingest and kill invading pathogens. Macrophages play a crucial role in the pathogenesis of cryptococcal infection, as their polarization phenotype determines the outcome of the battle between the infected host and C. neoformans. This study suggests that dynamic changes in polarization of macrophages at the level of individual cells are an important characteristic of in vivo cryptococcosis, as they occur throughout the natural course of infection. We demonstrate that macrophages can rapidly and uniformly reverse their polarization phenotype in response to dynamic signaling conditions and lose or regain their fungicidal function. Demonstrating importance of these pathways may become a cornerstone for novel therapeutic strategies for treatment of cryptococcosis in both immunocompromised and immunocompetent patients. Our studies reveal how a major opportunistic fungal pathogen, Cryptococcus neoformans, interacts with macrophages, immune cells which can ingest and kill invading pathogens. Macrophages play a crucial role in the pathogenesis of cryptococcal infection, as their polarization phenotype determines the outcome of the battle between the infected host and C. neoformans. This study suggests that dynamic changes in polarization of macrophages at the level of individual cells are an important characteristic of in vivo cryptococcosis, as they occur throughout the natural course of infection. We demonstrate that macrophages can rapidly and uniformly reverse their polarization phenotype in response to dynamic signaling conditions and lose or regain their fungicidal function. Demonstrating importance of these pathways may become a cornerstone for novel therapeutic strategies for treatment of cryptococcosis in both immunocompromised and immunocompetent patients.

Robust Th1 and Th17 Immunity Supports Pulmonary Clearance but Cannot Prevent Systemic Dissemination of Highly Virulent Cryptococcus neoformans H99

The present study dissected the role of a Th2 bias in pathogenesis of Cryptococcus neoformans H99 infection by comparing inhalational H99 infections in wild-type BALB/c and IL-4/IL-13 double knockout mice. H99-infected wild-type mice showed all major hallmarks of Th2 but not Th1/Th17 immunity in the lungs and lung-associated lymph nodes. In contrast, the IL-4/13(-/-) mice developed robust hallmarks of Th1 and Th17 but not Th2 polarization. The IL-4/IL-13 deletion prevented pulmonary eosinophilia, goblet cell metaplasia in the airways and resulted in elevated serum IgE, and a switch from alternative to classical activation of macrophages. The development of a robust Th1/Th17 response and classical activation of macrophages resulted in significant containment of H99 in the lungs of IL-4/13(-/-) mice compared with unopposed growth of H99 in the lungs of wild-type mice. However, IL-4/13(-/-) mice showed only 1-week longer survival compared with wild-type mice. The comparison of brain and spleen cryptococcal loads at weeks 2, 3, and 4 postinfection revealed that the systemic dissemination in IL-4/13(-/-) mice occurred with an approximate 1-week delay but subsequently progressed with similar rate as in the wild-type mice. Furthermore, wild-type and IL-4/13(-/-) mice developed equivalently severe meningitis/encephalitis at the time of death. These data indicate that the Th2 immune bias is a crucial mechanism for pulmonary virulence of H99, whereas other mechanisms are largely responsible for its central nervous system tropism and systemic dissemination.

Accumulation of CD11b+ Lung Dendritic Cells in Response to Fungal Infection Results from the CCR2-Mediated Recruitment and Differentiation of Ly-6Chigh Monocytes

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans is associated with the CCR2-mediated accumulation of lung dendritic cells (DC) and the development of a T1 adaptive immune response. The objective of this study was to identify the circulating DC precursor(s) responsible for this large increase in lung DC numbers. An established murine model was used to evaluate putative DC precursors in the blood, bone marrow, and lungs of CCR2+/+ mice and CCR2−/− mice throughout a time course following infection with C. neoformans. Results demonstrate that numbers of Ly-6Chigh monocytes increased in parallel in the peripheral blood and lungs of CCR+/+ mice, whereas CD11c+ MHC class II+ pre-DC were 10-fold less prevalent in the peripheral blood and did not differ between the two strains. Accumulation of Ly-6Chigh monocytes correlated with a substantial increase in the numbers of CD11b+ DC in the lungs of infected CCR2+/+ mice. Comparative phenotypic analysis of lung cells recovered in vivo suggests that Ly-6Chigh monocytes differentiate into CD11b+ DC in the lung; differentiation is associated with up-regulation of costimulatory molecules and decreased Ly-6C expression. Furthermore, in vitro experiments confirmed that Ly-6Chigh monocytes differentiate into CD11b+ DC. Accumulation of Ly-6Chigh monocytes and CD11b+ DC was not attributable to their proliferation in situ. We conclude that the CCR2-mediated accumulation of CD11b+ DC in the lungs of Cryptococcus-infected mice is primarily attributable to the continuous recruitment and differentiation of Ly-6Chigh monocytes.

Effect of cytokine interplay on macrophage polarization during chronic pulmonary infection with Cryptococcus neoformans.

ABSTRACT The immune response to Cryptococcus neoformans following pulmonary infection of C57BL/6 wild-type (WT) mice results in the development of persistent infection with characteristics of allergic bronchopulmonary mycosis (ABPM). To further clarify the role of Th1/Th2 polarizing cytokines in this model, we performed kinetic analysis of cytokine responses and compared cytokine profiles, pathologies, and macrophage (Mac) polarization status in C. neoformans-infected WT, interleukin-4-deficient (IL-4−/−), and gamma interferon-deficient (IFN-γ−/−) C57BL/6 mice. Results show that cytokine expression in the infected WT mice is not permanently Th2 biased but changes dynamically over time. Using multiple Mac activation markers, we further demonstrate that IL-4 and IFN-γ regulate the polarization state of Macs in this model. A higher IL-4/IFN-γ ratio leads to the development of alternatively activated Macs (aaMacs), whereas a higher IFN-γ/IL-4 ratio leads to the generation of classically activated Macs (caMacs). WT mice that coexpress IL-4 and IFN-γ during fungal infection concurrently display both types of Mac polarization markers. Concurrent stimulation of Macs with IFN-γ and IL-4 results in an upregulation of both sets of markers within the same cells, i.e., formation of an intermediate aaMac/caMac phenotype. These cells express both inducible nitric oxide synthase (important for clearance) and arginase (associated with chronic/progressive infection). Together, our data demonstrate that the interplay between Th1 and Th2 cytokines supports chronic infection, chronic inflammation, and the development of ABPM pathology in C. neoformans-infected lungs. This cytokine interplay modulates Mac differentiation, including generation of an intermediate caMac/aaMac phenotype, which in turn may support chronic “steady-state” fungal infection and the resultant ABPM pathology.

The role of macrophage inflammatory protein-1α/CCL3 in regulation of T cell-mediated immunity to Cryptococcus neoformans infection.

Macrophage inflammatory protein-1α (MIP-1α/CCL3) is a CC chemokine required for optimal recruitment of leukocytes in response to cryptococcal Ags. MIP-1α is expressed in the lungs by day 6 post Cryptococcus neoformans infection and could play a role in the development of cell-mediated immunity. To address this possibility, wild-type (MIP-1α+/+) mice and MIP-1α knockout (MIP-1α−/−) mice were infected intratracheally with a highly virulent strain of C. neoformans (145A). MIP-1α message was detected in the lungs on days 3, 7, and 14 in MIP-1α+/+ mice, but it was undetectable in MIP-1α−/− mice. On day 16, MIP-1α−/− mice had a 7-fold increase in C. neoformans burden in the lungs, but no decrease in pulmonary leukocyte recruitment. MIP-1α+/+ and MIP-1α−/− mice had similar numbers of recruited lymphocytes and monocytes/macrophages. Notably, MIP-1α−/− mice had a significantly greater number of eosinophils. MIP-1α−/− mice had extremely high levels of serum IgE. This switch of immune response to a T2 phenotype was associated with enhanced IL-4 and IL-13 expression in the lungs of MIP-1α−/− mice compared with MIP-1α +/+ mice. Progression of pulmonary cryptococcosis in the presence of nonprotective T2 immunity resulted in profound lung damage in MIP-1α−/− mice (eosinophilic crystal deposition, destruction of lung parenchyma, and pulmonary hemorrhage). Twelve-week survival was dramatically decreased in MIP-1α−/− mice. These studies, together with our previous studies, demonstrate that MIP-1α plays a role in both the afferent (T1/T2 development) and efferent (T1-mediated leukocyte recruitment) phases of cell-mediated immunity to C. neoformans.

Cryptococcal Urease Promotes the Accumulation of Immature Dendritic Cells and a Non-Protective T2 Immune Response within the Lung

Urease, a major virulence factor for Cryptococcus neoformans, promotes lethal meningitis/encephalitis in mice. The effect of urease within the lung, the primary site of most invasive fungal infections, is unknown. An established model of murine infection that utilizes either urease-producing (wt and ure1::URE1) or urease-deficient (ure1) strains (H99) of C. neoformans was used to characterize fungal clearance and the resultant immune response evoked by these strains within the lung. Results indicate that mice infected with urease-producing strains of C. neoformans demonstrate a 100-fold increase in fungal burden beginning 2 weeks post-infection (as compared with mice infected with urease-deficient organisms). Infection with urease-producing C. neoformans was associated with a highly polarized T2 immune response as evidenced by increases in the following: 1) pulmonary eosinophils, 2) serum IgE levels, 3) T2 cytokines (interleukin-4, -13, and -4 to interferon-gamma ratio), and 4) alternatively activated macrophages. Furthermore, the percentage and total numbers of immature dendritic cells within the lung-associated lymph nodes was markedly increased in mice infected with urease-producing C. neoformans. Collectively, these data define cryptococcal urease as a pulmonary virulence factor that promotes immature dendritic cell accumulation and a potent, yet non-protective, T2 immune response. These findings provide new insights into mechanisms by which microbial factors contribute to the immunopathology associated with invasive fungal disease.

Pulmonary Infection with an Interferon-γ-Producing Cryptococcus neoformans Strain Results in Classical Macrophage Activation and Protection

Alternative macrophage activation is associated with exacerbated disease in murine models of pulmonary cryptococcosis. The present study evaluated the efficacy of interferon-gamma transgene expression by Cryptococcus neoformans strain H99gamma in abrogating alternative macrophage activation in infected mice. Macrophage recruitment into the lungs of mice after infection with C. neoformans strain H99gamma was comparable with that observed in mice challenged with wild-type C. neoformans. However, pulmonary infection in mice with C. neoformans strain H99gamma was associated with reduced pulmonary fungal burden, increased pulmonary Th1-type and interleukin-17 cytokine production, and classical macrophage activation as evidenced by increased inducible nitric oxide synthase expression, histological evidence of enhanced macrophage fungicidal activity, and resolution of inflammation. In contrast, progressive pulmonary infection, enhanced Th2-type cytokine production, and the induction of alternatively activated macrophages expressing arginase-1, found in inflammatory zone 1, Ym1, and macrophage mannose receptor were observed in the lungs of mice infected with wild-type C. neoformans. These alternatively activated macrophages were also shown to harbor highly encapsulated, replicating cryptococci. Our results demonstrate that pulmonary infection with C. neoformans strain H99gamma results in the induction of classically activated macrophages and promotes fungal clearance. These studies indicate that phenotype, as opposed to quantity, of infiltrating macrophages correlates with protection against pulmonary C. neoformans infection.

Development of immune response that protects mice from viral pneumonitis after a single intranasal immunization with influenza A virus and nanoemulsion.

Nanoemulsion, a water-in-oil formulation stabilized by small amounts of surfactant, is non-toxic to mucous membranes and produces biocidal activity against enveloped viruses. We evaluated nanoemulsion as an adjuvant for mucosal influenza vaccines. Mice (C3H/HeNHsd strain) were vaccinated intranasally with 5 x 10(5) plaque forming units (pfu) of influenza A virus (Ann Arbor/6/60 strain) and a nanoemulsion mixture. The mice were challenged on day 21 after immunization with an intranasal lethal dose of 2 x 10(5) pfu of virus. Animals vaccinated with the influenza A/nanoemulsion mixture were completely protected against infection, while animals vaccinated with either formaldehyde-killed virus or nanoemulsion alone developed viral pneumonitis and died by day 6 after the challenge. Mice vaccinated with virus/nanoemulsion mixture had rapid cytokine responses followed by high levels of specific anti-influenza immunoglobulin G (IgG) and immunoglobulin A (IgA) antibodies. Specificity of the immune response was confirmed by assessment of the proliferation and cytokine production in splenocytes. This paper demonstrates that nanoemulsion can be employed as a non-toxic mucosal adjuvant for influenza virus vaccine.

Role of Dendritic Cells and Alveolar Macrophages in Regulating Early Host Defense against Pulmonary Infection with Cryptococcus neoformans

ABSTRACT Successful pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires a T1 adaptive immune response. This response takes up to 3 weeks to fully develop. The role of the initial, innate immune response against the organism is uncertain. In this study, an established model of diphtheria toxin-mediated depletion of resident pulmonary dendritic cells (DC) and alveolar macrophages (AM) was used to assess the contribution of these cells to the initial host response against cryptococcal infection. The results demonstrate that depletion of DC and AM one day prior to infection results in rapid clinical deterioration and death of mice within 6 days postinfection; this effect was not observed in infected groups of control mice not depleted of DC and AM. Depletion did not alter the microbial burden or total leukocyte recruitment in the lung. Mortality (in mice depleted of DC and AM) was associated with increased neutrophil and B-cell accumulation accompanied by histopathologic evidence of suppurative neutrophilic bronchopneumonia, cyst formation, and alveolar damage. Collectively, these data define an important role for DC and AM in regulating the initial innate immune response following pulmonary infection with C. neoformans. These findings provide important insight into the cellular mechanisms which coordinate early host defense against an invasive fungal pathogen in the lung.