DAP12 Inhibits Pulmonary Immune Responses to Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that is inhaled into the lungs and can lead to life-threatening meningoencephalitis in immunocompromised patients. Currently, the molecular mechanisms that regulate the mammalian immune response to respiratory cryptococcal challenge remain poorly defined. DAP12, a signaling adapter for multiple pattern recognition receptors in myeloid and natural killer (NK) cells, has been shown to play both activating and inhibitory roles during lung infections by different bacteria and fungi. In this study, we demonstrate that DAP12 plays an important inhibitory role in the immune response to C. neoformans. Infectious outcomes in DAP12^−/− mice, including survival and lung fungal burden, are significantly improved compared to those in C57BL/6 wild-type (WT) mice. We find that eosinophils and macrophages are decreased while NK cells are increased in the lungs of infected DAP12^−/− mice. In contrast to WT NK cells, DAP12^−/− NK cells are able to repress C. neoformans growth in vitro. Additionally, DAP12^−/− macrophages are more highly activated than WT macrophages, with increased production of tumor necrosis factor (TNF) and CCL5/RANTES and more efficient uptake and killing of C. neoformans. These findings suggest that DAP12 acts as a brake on the pulmonary immune response to C. neoformans by promoting pulmonary eosinophilia and by inhibiting the activation and antifungal activities of effector cells, including NK cells and macrophages.     

Human neutrophil-mediated nonoxidative antifungal activity against Cryptococcus neoformans

It has long been appreciated that polymorphonuclear leukocytes (PMN) kill Cryptococcus neoformans, at least in part via generation of fungicidal oxidants. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing of C. neoformans. Treatment of human PMN with inhibitors and scavengers of respiratory burst oxidants only partially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nonoxidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. Incubation of C. neoformans with these fractions for 18 h resulted in percent inhibition of growth of 67.4 +/- 3.4, 84.6 +/- 4.4, and 29.2 +/- 10.5 (mean +/- standard error, n = 3), respectively. Anticryptococcal activity of the cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by pronase treatment, boiling, high ionic strength, and magnesium but not calcium. Fractionation of the primary granules by reverse phase high-pressure liquid chromatography on a C(4) column over an acetonitrile gradient revealed multiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had substantial fungistatic and fungicidal activity. Peak 1 was identified by acid-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxidative anticryptococcal activity residing principally in their cytoplasmic and primary granule fractions. Calprotectin mediates the cytoplasmic activity, whereas multiple proteins, including defensins, are responsible for activity of the primary granules.     

Cryptococcus neoformans CAP10 Gene Regulates the Immune Response in Mice

The capsule associated protein 10 gene (CAP10) is indispensable to the formation of the polysaccharide capsule, and is closely associated with Cryptococcus (C.) neoformans virulence. In this study, we designed the shRNA expression plasmid to interfere with the synthesis of CAP10 gene. We infected mice with yeast cells in the respiratory tract, and monitored the development of infections in lung tissues. Results showed that the cap10-shRNA group may alleviate pathological lesions in pulmonary C. neoformans infection, and a lower degree of inflammatory cells was observed in the cap10-shRNA group. Moreover, the fungal burden was significantly lower in the cap10-shRNA group, indicating that the clearance towards C. neoformans was somehow affected. Down-regulation of CAP10 was beneficial to the balance of Th1/Th2 and Th17/Treg ratios. Collectively, our results showed that the expression of CAP10 was associated with an antifungal immune response in mice, suggesting that CAP10 regulates the inflammatory response. Therefore, we expect that the CAP10 gene will become a new molecular therapeutic target in cryptococcosis treatment.     

Immune response to Cryptococcus neoformans soluble polysaccharide: immunological unresponsiveness.

Mice injected with 100 to 800 microgram of Cryptococcus neoformans soluble polysaccharide showed a reduced ability to produce antibody after a challenge immunization with polysaccharide emulsified in Freund incomplete adjuvant. These animals were considered immunologically unresponsive. Animals given an initial injection of 25 or 50 microgram of polysaccharide responded to a challenge immunization in the same manner as control animals. Reversion of unresponsive mice to antibody production without further antigenic stimulation did not occur during a 12-week experimental period. These animals exhibited a partial response to challenge immunization 8 weeks after induction of unresponsiveness, and they were fully responsive to challenge immunization at 12 weeks. Animals given a single dose of 0.1, 0.4, or 1.6 microgram of polysaccharide produced a marked anamnestic response after challenge immunization. Repeated injections of subimmunogenic doses of polysaccharide did not produce a marked anamnestic response and would induce unresponsiveness only when the cumulative dose reached 100 to 400 microgram of polysaccharide, suggesting that injected cryptococcal polysaccharide might be sequestered in some manner until an amount of antigen sufficient for induction of unresponsiveness is accumulated. This possibility was confirmed by immunofluorescence studies that revealed a long-term deposition of polysaccharide in the tubular epithelial cells of the kidney.     

Humoral response against Cryptococcus neoformans mannoprotein antigens in HIV-infected patients

Twenty-four sera from healthy donors, 18 from HIV-positive patients (< 200 CD4+/mm3) and 18 sera collected before and during cryptococcosis from HIV-positive patients were analysed for the presence of humoral response to C. neoformans mannoproteins. Our results show that samples from healthy subjects and from HIV-positive patients had one of three antibody response profiles: (i) presence of reactive antibodies against both 105 and 80 kilodalton mannoproteins; (ii) presence of reactive antibodies against one of the two mannoproteins; or (iii) absence of reactive antibodies. Importantly the percentage of unreactive sera increased 6-fold in HIV-positive patients and more than 10-fold in patients with cryptococcosis. In addition, in the latter patients no variation of humoral response before and during cryptococcosis was observed. These results suggest that HIV-positive patients show a marked difficulty in mounting or maintaining antibody response to mannoprotein and this could contribute to predisposition to cryptococcosis.     

Differential Regulation of Immune Responses by Highly and Weakly Virulent Cryptococcus neoformans Isolates

Early inflammatory responses, delayed-type hypersensitivity (DTH) responses, and cytokine profiles were studied in mice infected by the pulmonary route with either a highly virulent isolate (NU-2) or a weakly virulent isolate (184A) of Cryptococcus neoformans. After infection, NU-2 remained in the lungs and the capsule became more pronounced during the first 24 h, whereas 184A induced an immediate inflammatory reaction and was rapidly cleared from the lungs. Cryptococcal antigen (GXM) appeared in sera early after infection with NU-2 and increased over the entire observation period. There was no detectable GXM in sera from 184A-infected mice. Both C. neoformans isolates induced anticryptococcal cell-mediated immune responses, but the responses had different profiles. DTH in NU-2-infected mice appeared at day 15 after infection and waned by day 21, whereas DTH in 184A-infected mice was present by day 5 and continued to increase. T helper 1 (Th1) cytokines (interleukin 2 [IL-2] and gamma interferon) were made by spleen cells early after infection with either isolate. NU-2-infected mice lost their ability to produce these cytokines, but 184A-infected mice retained it. IL-4, a Th2 cytokine, was not detected in infected mice. The regulatory cytokine IL-10 was made by spleen cells early but not later after infection with the highly virulent isolate and was not produced by spleen cells from 184A-infected mice. IL-10-deficient mice survived an NU-2 infection significantly longer than wild-type mice, suggesting that IL-10 is important in down-regulating the protective immune response. The induction of anergy appears to be responsible for the inability of NU-2-infected mice to control a C. neoformans infection.     

Antifungal activity of cerebrospinal fluid against Cryptococcus neoformans and Candida species.

The effect of human cerebrospinal fluid (CSF) on the growth of Cryptococcus neoformans and Candida species was tested in RPMI-1640. CSF alone was highly fungistatic for both yeasts and inhibited growth in a concentration-dependent manner. Unlike human serum, CSF did not collaborate with fluconazole for killing C. neoformans. Molecular sieve fractionation of CSF on a G-200 Sephadex column yielded a highly antifungal fraction with a molecular weight around 66 kDa. On SDS-PAGE this fraction migrated as a major and a minor band corresponding to the mobility of bovine serum albumin. These novel findings suggest that CSF contains a factor(s) that provides resistance to the growth of C. neoformans or Candida species.     

Cryptococcus neoformans variety gattii.

Cryptococcus neoformans var. gattii is emerging as a primary human pathogen which is distinct genetically and biochemically from C. neoformans var. neoformans. There is increasing evidence that it should be reclassified as a separate species within the Tremellales. In nature, C. n. var. gattii has been consistently isolated from decaying wood in hollows of species of the red gum group of eucalyptus trees (Eucalyptus ser. Exsertae Blakely). The role that trees play in the life-cycle of C. n. var. gattii is not known, but its association with decaying wood is suggestive of an endophytic existence, in common with other wood-rot fungi. Despite the demonstration in the laboratory of sexual reproduction between mating types oc and a of F. neoformans var. gattii, this has not been demonstrated in nature. Human cryptococcosis develops following environmental exposure and inhalation of the infectious propagule. Whether this is the basidiospore or dessicated yeast form is uncertain. The major risk factor for development of disease appears to be exposure, though there is indirect evidence that unidentified host factors may contribute to the relatively high incidence of cryptococcosis in Australian Aboriginals. The rarity of cryptococcosis due to C. n. var. gattii in immunocompromised patients remains unexplained. Virulence determinants of C. neoformans are currently the subject of intensive investigation, especially in C. n. var. neoformans. The best-characterized, major, virulence determinants in this variety, the polysaccharide capsule, products of the laccase enzyme pathway and ability to grow at physiological temperatures, contribute to its survival in the host. They are also present in C. n. var. gattii. A potential determinant of tissue invasion, secreted phospholipase B, is produced in vitro and in vivo by C. n. var. gattii. This enzyme has now been confirmed to play a role in the virulence of C. neoformans serotype A. Disease caused by C. n. var. gattii is distinguished from that due to C. n. var. neoformans by an increased incidence of cryptococcomas in lung and brain, increased neurological morbidity and a slower response to antifungal therapy. The difference in clinical presentation is predominantly due to the effect of underlying immunocompromise in patients infected with C. n. var. neoformans.     

Galactoxylomannans of Cryptococcus neoformans.

Galactoxylomannans (GalXMs) from single isolates of Cryptococcus neoformans serotypes A, B, and D were isolated from culture supernatants and then purified by affinity, ion-exchange, and gel-filtration chromatography. GalXMs are a group of closely related complex polysaccharides. GalXMs from serotypes A (9759 A) and C (3183 C) and an acapsular mutant of serotype D (Cap67 D) have similar galactose, xylose, and mannose molar ratios, but each has some unique structural features. GalXM9759 A and GalXM 3183 C were associated with a starchlike glucan that was removed during purification. Only a trace of glucose was detected in the Cap67 D GalXM. Gas-liquid chromatography-mass spectroscopy of per-O-methylated polysaccharides and 13C nuclear magnetic resonance spectroscopy showed that GalXM is a complex branched polysaccharide. The main chain consists of mannose or galactose or alternating mannose and galactose residues. Xylose is present only as nonreducing termini. Galactofuranose occurs only in 3183 C and Cap67 D, and it is always present as nonreducing termini.     

Tumor Induction by Cryptococcus neoformans

A human isolate of Cryptococcus neoformans strain CIA, which was originally obtained in 1963, produced fatal disease in mice. Postmortem examinations showed extensive central nervous system disease. After using this yeast for research for 7 years, we found that it began to produce large tumors after intraperitoneal inoculation in mice. The present study showed that the tumors consisted of large agglomerates of yeast cells, capillaries, and reticular stroma. In concomitant experiments, the same C. neoformans strain, introduced into the lungs of mice in an aerosol, produced asymptomatic infections. A definition for the term "cryptococcoma" is proposed.     

Immune Response to Cryptococcus neoformans Soluble Polysaccharide I. Serological Assay for Antigen and Antibody

Chromium chloride was used as a coupling agent for the conjugation of purified cryptococcal polysaccharide to sheep erythrocytes. Sensitized erythrocytes were used in a passive hemagglutination (PHA) assay for antibody to cryptococcal polysaccharide and a passive hemagglutination inhibition (PHI) assay for antigen. The PHA assay was more sensitive than complement fixation, agglutination, or precipitation tests for antibody. The PHI assay could detect submicrogram quantities of soluble polysaccharide. Antigen or antibody could be detected in serum or spinal fluid from seven of eight patients with cryptococcosis. Tests for antigen or antibody were negative with sera from patients with histoplasmosis, blastomycosis, coccidioidomycosis, aspergillosis, or allescheriosis. A low frequency (3%) of positive reactors for antibody was found among sera from normal persons and from persons with unrelated diseases; whereas, all tests for antigen were negative. The assay showed a high degree of sensitivity for immunoglobulins of the immunoglobulin M class; however, cryptococcal antibody of the immunoglobulin G class was also detected. The immunological specificity of the polysaccharide preparation was due to carbohydrate rather than to protein associated with the polysaccharide.     

IL-12 protects mice against pulmonary and disseminated infection caused by Cryptococcus neoformans

We examined the role of IL-12 in host resistance to Cryptococcus neoformans using a murine model of pulmonary and disseminated infection. In this model, mice were infected intratracheally with viable yeast cells. Mice untreated with IL-12 allowed an uncontrolled multiplication of yeast cells in the lung with infiltrations of few inflammatory cells, and a cryptococcal dissemination to the brain and meningitis by 3 weeks, resulting in death of all animals within 4–6 weeks. IL-12, when administered from the day of tracheal infection for 7 days, induced a marked infiltration of inflammatory cells, consisting mostly of mononuclear cells, and significantly reduced the number of viable yeast cells in the lung. The treatment suppressed brain dissemination, as shown by a marked reduction of yeast cells in the brain and prevention of meningitis. These effects resulted in a significant increase in the survival rate of infected mice. In contrast, late administration of IL-12 commencing on day 7 after instillation of yeast cells failed to protect the mice against infection with C. neoformans. In further experiments, early administration of IL-12 markedly induced interferon-gamma (IFN-γ) mRNA in the lungs of infected mice, while no IFN-γ mRNA was detected without this treatment. Our results indicate that IL-12 is effective when administered in the early period of pulmonary cryptococcal infection.     

In vitro synergistic effect of minocycline combined with antifungals against Cryptococcus neoformans

BackgroundCryptococcus neoformans infections occur in immunocompromised patients, especially those with HIV infection, chemoradiotherapy after cancer, and organ transplantation. Infection can cause pneumonia and meningoencephalitis in severe cases with a high mortality rate if not treated. Although fluconazole and amphotericin B are the first-line treatments for cryptococcosis, the rate of fluconazole resistance has increased significantly due to long-term use. Minocycline is a derivative of tetracycline that exerts its antibacterial effect through inhibition of bacterial protein synthesis. It is also able to pass the blood-brain barrier to act on the central nervous system. The present study investigates the effects of minocycline in combination with antifungals in treating C. neoformans.ObjectiveTo determine in vitro interactions of minocycline combined with itraconazole, voriconazole, posaconazole, fluconazole and amphotericin B against C. neoformans.MethodsThe minimum inhibitory concentrations (MIC) of the antifungals were determined by the CLSI Clinical and Laboratory Standards Institute M27-A3 microdilution method. The in vitro synergistic effects of minocycline combined with itraconazole, voriconazole, posaconazole, fluconazole, and amphotericin B on C. neoformans were detected by the broth microdilution checkerboard technique and disk diffusion testing.Results and ConclusionThe working concentration ranges were 0.125–4 µg/mL for itraconazole, 0.03–0.125 µg/ml for voriconazole, 0.03–1 µg/ml for posaconazole, 0.25–16 µg/ml for fluconazole, and 0.125–2 µg/ml for amphotericin B. The synergistic rates of minocycline combinations against C. neoformans were 55% with itraconazole, 10% with voriconazole, 85% with posaconazole, 20% with fluconazole, and 70% with amphotericin B. The effective MIC value of minocycline in the synergistic combination decreased to 2–32 µg/ml, while the MIC of itraconazole decreased to 0.03–0.125 µg/ml, voriconazole 0.03–0.125 µg/ml, posaconazole 0.03–0.125 µg/ml, 0.125–4 µg/ml fluconazole, and 0.06–0.50 µg/ml amphotericin B. The disk diffusion assay showed that the plates containing minocycline and antifungal drugs produced inhibition zones with diameters larger than the single drug plates. Minocycline showed no antagonistic effect in the combinations. In conclusion, the combination of minocycline and azoles or amphotericin B has synergistic effects against C. neoformans in vitro.     

Variable efficacy of passive antibody administration against diverse Cryptococcus neoformans strains.

The efficacy of monoclonal antibody (MAb 2H1) against diverse strains of Cryptococcus neoformans was studied in a murine model of intravenous infection. For six of eight strains, administration of MAb prior to infection prolonged survival of mice. For two strains, 371 and SB4A, administration of MAb prior to infection did not prolong survival in multiple experiments with inocula ranging from 10(2) to 10(6) yeast cells per mouse. Mice infected with strains 371 and SB4A had fewer CFU than non-MAb-treated controls, but the CFU reduction was not sufficient to affect survival. Serum glucuronoxylomannan (GXM) levels varied for the different C. neoformans strains. For mice that did not receive MAb 2H1, there was a positive correlation between lung fungal burden and serum GXM levels. MAb 2H1-treated mice had significantly reduced serum GXM levels. The results indicate that the efficacy of MAb 2H1 administration in prolonging survival and/or reducing organ CFU varies with the C. neoformans strain.     

Protection against cryptococcosis by using a murine gamma interferon-producing Cryptococcus neoformans strain

We evaluated cell-mediated immune (CMI) responses in mice given a pulmonary infection with a Cryptococcus neoformans strain engineered to produce the Th1-type cytokine gamma interferon (IFN-gamma). Mice given a pulmonary infection with an IFN-gamma-producing C. neoformans strain were able to resolve the primary infection and demonstrated complete (100%) protection against a second pulmonary challenge with a pathogenic C. neoformans strain. Pulmonary cytokine analyses showed that Th1-type/proinflammatory cytokine and chemokine expression were significantly higher and Th2-type cytokine expression was significantly lower in mice infected with the IFN-gamma-producing C. neoformans strain compared to wild-type-infected mice. This increased pulmonary Th1-type cytokine expression was also associated with significantly lower pulmonary fungal burden and significantly higher pulmonary leukocyte and T-lymphocyte recruitment in mice infected with the IFN-gamma-producing C. neoformans strain compared to wild-type-infected mice. Our results demonstrate that pulmonary infection of mice with a C. neoformans strain expressing IFN-gamma results in the stimulation of local Th1-type anti-cryptococcal CMI responses and the development of protective host immunity against future pulmonary cryptococcal infections. The use of fungi engineered to produce host cytokines is a novel method to study immune responses to infection and may be useful in developing vaccine strategies in humans.     

Antibody-mediated protection against Cryptococcus neoformans pulmonary infection is dependent on B cells

The pathogenesis of pulmonary Cryptococcus neoformans infection and the efficacy of passive immunoglobulin G1 (IgG1) administration were investigated in B-cell-deficient and C57BL/6J mice. C57BL/6J mice lived longer than B-cell-deficient mice after both intratracheal and intravenous infections. Administration of IgG1 prior to infection prolonged the survival of C57BL/6J mice but had no effect on the survival or numbers of CFU in the lungs of B-cell-deficient mice. C. neoformans infection in B-cell-deficient mice resulted in significantly higher levels of gamma interferon (IFN-gamma), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha) than in C57BL/6J mice. IgG1 administration reduced IFN-gamma and MCP-1 levels in C57BL/6J mice but not in B-cell-deficient mice. In addition, compared to its effect in C57BL/6J mice, C. neoformans infection in FcRgammaIII-deficient, athymic, and SCID mice significantly increased IFN-gamma and MCP-1 levels. IgG1 administration was associated with reduced IFN-gamma levels in C57BL/6J mice but not in FcRgammaIII-deficient, athymic, and SCID mice. These observations suggest that IgG1-mediated protection in this system is a consequence of alterations in the inflammatory response that translate into less damage to the host without directly reducing the fungal burden. For hosts with impaired immunities, the ineffectiveness of passive antibody (Ab) may reflect an inability to down-regulate inflammation and avoid self-damage. The results indicate an important role for B cells in host defense against C. neoformans infection and demonstrate a surprising dependence of Ab-mediated protection on B cells in this system.     

Monoclonal antibody to fungal glucosylceramide protects mice against lethal Cryptococcus neoformans infection

Glucosylceramides (GlcCer) are involved in the regulation of Cryptococcus neoformans virulence. In the present study, we demonstrate that passive immunization with a monoclonal antibody to GlcCer significantly reduces host inflammation and prolongs the survival of mice lethally infected with C. neoformans, revealing a potential therapeutic strategy to control cryptococcosis.     

In Vitro and In Vivo Stability a Cryptococcus neoformans Glucuronoxylomannan Epitope That Elicits Protective Antibodies

The monoclonal antibody (MAb) 2H1 defines an epitope in Cryptococcus neoformans capsular glucuronoxylomannan (GXM) that can elicit protective antibodies. In murine models of cryptococcosis, MAb 2H1 administration prolongs survival and reduces fungal burden but seldom clears the infection. The mechanism by which C. neoformans persists and escape antibody-mediated clearance is not understood. One possibility is that variants that do not bind MAb 2H1 emerge in the course of infection. Using an agglutination-sedimentation protocol, we recovered a variant of strain 24067 that did not agglutinate, could not be serotyped, and had marked reduction in GXM O-acetyl groups. Binding of MAb 2H1 to 24067 variant cells produced a different immunofluorescence pattern and lower fluorescence intensity relative to the parent 24067 cells. Addition of MAb 2H1 to 24067 variant cells had no effect on cell charge. Phagocytic assays demonstrated that MAb 2H1 was not an effective opsonin for the 24067 variant. The 24067 variant was less virulent than the 24067 parent strain in mice, and MAb 2H1 administration did not prolong survival in animals infected with the variant strain. To investigate whether variants which do not bind MAb 2H1 are selected in experimental infection, three C. neoformans strains were serially passaged in mice given either MAb 2H1 or no antibody. Analysis of passaged isolates by agglutination assay, flow cytometry, and indirect immunofluorescence revealed changes in MAb 2H1 epitope expression but no clear trend with regards to gain or loss of MAb 2H1 epitope. C. neoformans variants with reduced MAb 2H1 epitope content can be isolated in vitro, but persistence of infection in mice given MAb 2H1 does not appear to be a result of selection of escape variants that lack the MAb 2H1 epitope.