Establishment of protective immunity against cerebral cryptococcosis by means of an avirulent, non melanogenic Cryptococcus neoformans strain.

The opportunistic fungal pathogen, Cryptococcus neoformans, shows a marked predilection for the central nervous system (CNS). This can be partially explained by its ability to synthesize melanin starting from the catecholamines, highly concentrated at the CNS level. Two cryptococcal strains, the avirulent non-melanogenic strain Sb26 and the virulent melanogenic revertant strain Sb26Rev, were used in a murine model of intracerebral (i.c.) infection, in order to evaluate their virulence and immunomodulating properties at the cerebral level. We found that, unlike Sb26Rev, Sb26 i.c. infection was never lethal regardless of the challenging dose. Sb26Rev infection resulted in massive CNS tissue damage, associated with little or no cytokine response, as established by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Differently, Sb26 infection failed to alter CNS structure, while inducing IL-12 p40, TNF-alpha, IL-1beta, IFN-gamma and iNOS specific-gene expression as well as IL-12, TNF-alpha and IL-1beta cytokine production. Interestingly, all Sb26 infected mice survived a subsequent lethal challenge with Sb26Rev. The phenomenon was associated with enhanced IL-12, TNF-alpha and IL-1beta production and was strictly specific, as shown by heterologous challenges and delayed type of hypersensitivity assay. Overall, we provide evidence that protective immunity against cerebral cryptococcosis is established by means of an avirulent strain of C. neoformans.

A new azole derivative of 1,4-benzothiazine increases the antifungal mechanisms of natural effector cells.

The most widely used drug for treatment of candidiasis is fluconazole (FCZ). Recently, a new derivative of 1,4-benzothiazine, compound FS5, was developed. FS5 had an appreciable protective effect against murine candidiasis. The present study was designed to dissect the antifungal mechanisms triggered by FS5 and to establish whether this compound could enhance the antimicrobial abilities of natural effector cells. The results show that intraperitoneal injection of FS5 in mice (i) induced an increase in circulating neutrophil levels comparable to that observed in FCZ-treated mice; (ii) enhanced phagocytosis and the killing activities of macrophages (Mphis) isolated from the spleen or peritoneal cavity, with the latter effect correlating with induction of nitric oxide synthesis and production by Mphis; and (iii) increased the levels of expression and synthesis of tumor necrosis factor alpha. These results suggest that the compound-induced synthesis of antimicrobial and proinflammatory molecules by heterogeneous Mphi populations is part of the beneficial effect of FS5 exerted against murine candidiasis.

Role of tumor necrosis factor alpha, interleukin-1beta, and interleukin-6 in a mouse model of group B streptococcal arthritis.

Intravenous inoculation of CD1 mice with 10(7) CFU of type IV group B Streptococcus (GBS IV) results in a high incidence of diffuse septic arthritis. In this study the roles of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in articular pathology were evaluated. Cytokine levels were quantified in the serum and joints by enzyme-linked immunosorbent assay in mice injected with GBS IV and tested or not tested with pentoxifylline (PTF), a methylxanthine that affects cytokine production. PTF was administered intraperitoneally at a dose of 1 mg/mouse (50 mg/kg of body weight) 1 h after GBS infection and then at 24-h intervals for 4 days. High levels of IL-1beta and IL-6, but not TNF-alpha, were detected in the joints of mice injected with GBS IV from 5 to 15 days after infection, when articular lesions were most frequent and severe. IL-1beta and IL-6 concentrations in the joints significantly (P < 0.001) exceeded those detected in the serum, confirming a strong local production. PTF treatment resulted in a strong reduction of cytokine production and in a marked decrease in both the incidence and severity of arthritis. Inoculation of exogenous murine recombinant IL-1beta or IL-6 in mice treated with GBS IV plus PTF resulted in an incidence and severity of articular lesions similar to those obtained with inoculation of GBS IV alone. No significant effect was obtained with TNF-alpha administration. These data show a strong involvement of IL-1beta and IL-6, but not TNF-alpha, in the pathogenesis of GBS arthritis.

Role of the capsule in microglial cell-Cryptococcus neoformans interaction: impairment of antifungal activity but not of secretory functions.

Using two isogenic strains of Cryptococcus neoformans, we studied the influence of the capsule in C. neoformans microglial-cell interaction. We demonstrate that the acapsular mutant yeasts (CAP67) are more susceptible to phagocytosis and killing than encapsulated yeasts (B3501) by the murine microglial cells, BV-2. RT-PCR analysis showed that the pattern of gene transcripts for tumour necrosis factor alpha (TNF-a), interleukin (IL)-1beta, IL-6, IL-12p40 and granulocyte macrophage colony stimulating factor remains unchanged following BV-2 cell infection with CAP67 or B3501 yeasts. Moreover, no induction of TNF-alpha secretion occurs in BV-2 cells infected with either B3501 or CAP67 yeasts or exposed to glucuronoxylomannan (GXM) or galactoxylomannan (GalXM). Nevertheless, lipopolysaccharide-induced TNF-alpha secretion is downregulated by cell infection with B3501 or CAP67 yeasts or exposure to GXM or GalXM. Overall, by means of a continuous cell line, it appears that the C. neoformans capsule is detrimental to microglial cell antifungal activity, while no effect can be attributed to the capsule as trend of cytokine gene expression and TNF-alpha secretion.

A low virulent strain of Candida albicans enhances brain anticryptococcal defenses: characterization of the local immune reaction by RT-PCR and histochemical analysis.

Here we studied the involvement of PCA-2, a low-virulent strain of Candida albicans known to act as a potent stimulating agent in the development of cryptococcal meningoencephalitis. To this purpose, mice received saline or PCA-2 intracerebrally 7 days before lethal local challenge with Cryptococcus neoformans. We found that, following C. neoformans challenge, PCA-2-treated but not saline-treated mice exhibited (a) delayed brain colonization, (b) enhanced median survival times, (c) massive local immune reaction consisting of abundant astrocytes, microglial and inflammatory cells, and (d) a peculiar trend of cytokine gene expression, including high steady-state levels of interleukin (IL)-1 beta and tumor necrosis factor alpha transcripts, fluctuating levels of interferon gamma and inducible nitric oxide synthase mRNA and lately detectable IL-6 gene expression. PCA-2-mediated immunostimulating properties were partially impaired by aminoguanidine or pentoxifylline treatment, further strengthening the conclusion that soluble mediators, including proinflammatory cytokines and nitric oxide, are important defense elements against cryptococcal meningoencephalitis.

Enhanced resistance to Cryptococcus neoformans infection induced by chloroquine in a murine model of meningoencephalitis.

Although the pathogenesis of cerebral cryptococcosis is poorly understood, local immune cells, such as microglia and astrocytes, likely play a critical role in containing infection. Chloroquine (CQ) is a weak base that accumulates within acidic vacuoles and increases their pH. Consequently, proteolytic activity of lysosomal enzymes and intracellular iron release/availability are impaired, resulting in decreased availability of nutrients crucial to microorganism survival and growth in the host. We found that CQ enhances BV2 microglial-cell-mediated anticryptococcal activity in vitro. The phenomenon is (i) evident when both unopsonized and opsonized microorganisms are used and (ii) mimicked by NH4Cl, another weak base, and by bafilomycin A1, an inhibitor of vacuolar-type H+-ATPases. In vivo, intracerebral administration of CQ before lethal local challenge with Cryptococcus neoformans results in a significant augmentation of median survival time and a marked reduction of yeast growth in the brain and is associated with the enhancement of local interleukin 1beta (IL-1beta) and IL-6 mRNA transcripts. Overall, these results provide the first evidence that CQ enhances anticryptococcal host defenses.

Iron regulates microglial cell-mediated secretory and effector functions.

Iron homeostasis and macrophage physiology are tightly intertwined. In the present study, we evaluated the influence of iron loading on the constitutive and interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-induced functional and secretory properties of microglial cells, using the in vitro established murine cell line BV-2. We demonstrate that iron augments the basal and IFN-gamma plus LPS-enhanced anti-Candida albicans activity exerted by BV-2 cells and that the phenomenon occurs with no enhancement of phagocytic activity. Furthermore, when the secretory properties of IFN-gamma plus LPS-treated BV-2 cells were assessed, we found that tumor necrosis factor remains unchanged while nitric oxide production is significantly reduced in iron-loaded cells. The addition of the iron chelator deferiprone (L1) reverts the effects of iron on BV-2 functional and secretory properties. These data suggest that iron differently affects secretory and effector functions of BV-2 microglial cells, thus implying that iron interferes with murine microglial cell physiology.

Biomolecular events involved in the establishment of brain anticandidal resistance.

Using a murine model, we have demonstrated the establishment of cerebral resistance to local lethal challenge with Candida albicans strain CA-6, by previous intracerebral (i.c.) infection with the low-virulent strain PCA-2. Here we show that i.c. infection with PCA-2 is effective in drastically reducing brain colonization following secondary infection with CA-6. As assessed by colony forming unit assay and histopathological analysis, microbial counts are impaired, granuloma formation and hyphal growth are also reduced in brains of PCA-2- and CA-6-infected mice with respect to CA-6-challenged mice. Furthermore, using PCR studies, we found that, while PCA-2 (i.e. healing infection) induces transient cytokine gene expression in the mouse brain, CA-6 lethal challenge results in long-lasting (until mouse death) high levels of all cytokine gene transcripts assessed. Finally brains from mice that will resist CA-6 challenge, because of previous infection with PCA-2, also exhibit a transient induction of all cytokine genes. Only IL-1 beta remains highly expressed at all time- points tested. Overall, these results provide evidence that healing and non-healing C. albicans i.c. infections differ in the immune reaction(s) locally evoked, at least in terms of cytokine gene expression, strongly suggesting cytokine involvement in the establishment of brain anticandidal resistance.

Influence of the Bcg locus on macrophage response to the dimorphic fungus Candida albicans.

The Bcg/Ity/Lsh gene (candidate Nramp) controls natural resistance to several parasites, such as Mycobacterium bovis, Leishmania donovani, and Salmonella typhimurium. Using two macrophage (M phi) cell lines (B10R and B10S) derived from mouse strains congenic at Bcg, we found that M phi s from resistant mice (B10R M phi s) act more effectively against the two morphogenetic forms of the dimorphic fungus Candida albicans compared with M phi s from susceptible mice (B10S M phi s). Moreover, when assessed for tumor necrosis factor secretion in response to the hyphal form of C. albicans, B10R M phi s are significantly more effective at expressing this secretory function than are B10S M phi s, closely resembling the trend of response to lipopolysaccharide. Overall, these results provide insight into the influence of the Bcg locus on the M phi response to C. albicans.

Differential susceptibility of yeast and hyphal forms of Candida albicans to macrophage-derived nitrogen-containing compounds.

Candida albicans is a dimorphic fungus capable of transition from the yeast form (Y-Candida) to the hyphal form (H-Candida). Both Y-Candida and H-Candida are known to be growth inhibited by murine macrophages (M phi) in vitro. In the present report, we demonstrate that M phi exposed to interferon gamma (IFN-gamma) plus lipopolysaccharide (LPS) show enhanced anti-Y-Candida and anti-H-Candida activities. To further investigate the phenomenon, Y-Candida and H-Candida were assessed for susceptibilities to M phi-derived supernatants. Only the growth of H-Candida, and not that of Y-Candida, is impaired by cell-free supernatants from M phi treated with IFN-gamma plus LPS. In contrast, no H-Candida growth inhibition occurs when supernatants from M phi exposed to IFN-gamma plus LPS in the presence of NG-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthesis, are employed. Finally, supernatants from M phi incubated with sodium nitroprusside, an NO-generating agent, also show anti-H-Candida activity. In conclusion, these results indicate that H-Candida but not Y-Candida is susceptible to extracellular antifungal mechanisms employed by M phi, which likely involve stable nitrogen-containing compounds.

Role of nitric oxide and melanogenesis in the accomplishment of anticryptococcal activity by the BV-2 microglial cell line.

In the present paper, we investigated the involvement of cryptococcal melanogenesis and macrophage nitric oxide (NO) production in the accomplishment of anticryptococcal activity by microglial effector cells, using the murine cell line BV-2. We demonstrate that the constitutive levels of anticryptococcal activity exerted by BV-2 cells is significantly enhanced upon interferon gamma plus lipopolysaccharide treatment. The phenomenon, which occurs with no enhancement of phagocytic activity, is associated with the production of high levels of NO and is abolished by addition of NG-monomethyl-L-arginine. Comparable patterns of results are observed employing either unopsonized or opsonized microbial targets, the latter microorganisms being markedly more susceptible to BV-2 cell antimicrobial activity. Furthermore, melanization of Cryptococcus neoformans significantly reduces its susceptibility to BV-2 antimicrobial activity, regardless of the fact that activated macrophages or opsonized microorganisms have been employed. In conclusion, our results provide evidence that NO-dependent events are involved in the fulfillment of anticryptococcal activity by activated microglial cells and that fungal melanization is a precious escamotage through which C. neoformans overcomes host defenses.

Biomolecular events involved in anticryptococcal resistance in the brain.

We have recently shown that intracerebral (i.c.) administration of heat-killed Cryptococcus neoformans (HCN) enhances mouse resistance to a subsequent local challenge with lethal doses of viable yeast cells. Here we show that i.c. administration of HCN is also effective in significantly delaying brain colonization of mice intravenously infected with viable C. neoformans. PCR analysis revealed that interleukin 6 (IL-6) and IL-1 beta gene expression occurs in brain of HCN-treated mice but not in brains of saline-treated controls. In contrast, no differences are observed in terms of tumor necrosis factor alpha and IL-1 alpha gene transcripts, which are slightly and highly detectable, respectively, in saline-treated mice and which remain such also following HCN treatment. Furthermore, i.c. administration of exogenous IL-6 or IL-1 beta, but not tumor necrosis factor alpha, before local challenge with viable C. neoformans results in significantly reduced microbial counts in the brain and blood and in increased mouse survival. Taken together, these observations provide initial evidence that brain anticryptococcal resistance involves elicitation of a local cytokine response, involving primarily IL-6 and IL-1 beta.

Differential susceptibility of yeast and hyphal forms of Candida albicans to proteolytic activity of macrophages.

The dimorphic transition of Candida albicans from the yeast (Y-Candida) to the hyphal (H-Candida) form is a complex event whose relevance in fungal pathogenicity is still poorly understood. Using a cloned macrophage (M phi) cell line (ANA-1), we have previously shown that a M phi can discriminate between the two fungal forms, eliciting different secretory responses. In the present study, we investigated the susceptibility of Y-Candida and H-Candida to M phi proteolytic activity. In particular, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) techniques were employed to analyze the patterns of lyticase proteinaceous extracts from cell walls of Y-Candida and H-Candida which had been unexposed or exposed to ANA-1 M phis for 3 h. Silver staining allowed detection of a complex protein pattern in both forms of C. albicans, qualitatively and quantitatively differing from each other, mainly at molecular masses below 106 kDa. Western blot staining with anti-C. albicans mannan antibodies and convalescent-phase sera of mice previously infected systemically or intracerebrally with C. albicans showed that, after contact with M phis, Y-Candida but not H-Candida proteinaceous cell wall components are profoundly modified, with substantial reduction and/or disappearance of many bands. Our experimental approach provides initial insights into the differential susceptibility of Y-Candida and H-Candida to the proteolytic activity of M phis.

Different events involved in the induction of macrophage tumor necrosis factor by Candida albicans and lipopolysaccharide.

Using an in vitro experimental model, we have recently demonstrated that Candida albicans in its hyphal form (H-Candida), similarly to lipopolysaccharide (LPS), enhances tumor necrosis factor (TNF) secretory response in the cloned macrophage (M phi) population ANA-1. Here we show that H-Candida and LPS each differ in their requirements for intact protein kinase functions, susceptibility to 0.4-microns micropore-size membranes, and sensitivity to polymyxin B. These results, together with the synergistic effect occurring between H-Candida and LPS in inducing TNF response, indicate the existence of different receptor(s) and/or signal-transduction pathway(s) through which the two stimuli act.

Comparative studies on functional and secretory properties of macrophage cell lines derived from different anatomical sites.

In the present study, we compared four macrophage (M phi) cell lines from different anatomical origins for functional and secretory activities against the two morphogenetic forms of the fungus Candida albicans. We show that all the cell lines actively phagocytize the yeast and exert antimicrobial activity against both forms of Candida, although M phi of microglial origin are the most effective. When assessed for secretory properties, microglial M phi exhibit a peculiar pattern with respect to other M phi populations under either basal or stimulated conditions. In particular, only microglial M phi fail to respond to the hyphal form of the fungus (H-Candida), which instead acts as a potent tumor necrosis factor inducer in the other M phi cell lines. When exposed to H-Candida, microglial M phi are indistinguishable from other M phi in their ability to modulate specific surface adhesion molecules. In addition to strengthening the knowledge on functional heterogeneity among M phi, our data provide evidence on the peculiar behavior of microglial M phi. To what extent M phi heterogeneity may be related to tissue homeostasis is discussed.

Anticryptococcal resistance in the mouse brain: beneficial effects of local administration of heat-inactivated yeast cells.

Using a murine model, we have previously shown that brain resistance to local infection with opportunistic fungi is affected by manipulation of the host myelomonocytic compartment. Here, we demonstrate that intracerebral administration of heat-inactivated Cryptococcus neoformans (H-CN) yeast cells results in a consistent enhancement of mouse survival to subsequent local challenge with lethal doses of C. neoformans. The phenomenon, more pronounced upon double H-CN treatment, is associated with (i) massive local inflammatory response, (ii) reduced growth of the fungus within the brain, and (iii) induction of delayed-type hypersensitivity. Moreover, H-CN treatment confers protection against local heterologous challenges. Our data provide initial evidence that intracerebral administration of H-CN results in the establishment of aspecific and specific immune responses; the mechanisms of elicitation and relative contributions to host antimicrobial resistance remains to be elucidated.

Pattern of cytokine gene expression in brains of mice protected by picolinic acid against lethal intracerebral infection with Candida albicans.

Recently, we demonstrated that intracerebral (i.c.) administration of picolinic acid (PLA) confers protection against a lethal local challenge with the opportunistic pathogen Candida albicans. By histopathological studies, we show here that mice receiving PLA treatment survive challenge and no evidence of fungal invasion is found within the brain compartment. In contrast, PLA-untreated mice succumb to infection within 7-10 days and show massive brain colonization with extensive granulomatous reaction. By PCR analysis, we show that, unlike naive brains, PLA-treated brains show transient activation of TNF alpha, IL-1 beta and IL-6 genes. C. albicans infection results in high levels of all cytokine transcripts, the phenomenon being long-lasting in PLA-untreated brains, while gradually declining in PLA-treated brains. The only exception is IL-1 beta, whose levels remain high at the latest time-points tested, also in PLA-treated brains. Finally, IL-1 alpha, constitutively detectable in naive brains, is slightly enhanced by C. albicans challenge, regardless of prior treatment. These findings, together with the knowledge that PLA is a potent co-stimulus for macrophages, suggest the involvement of cytokine circuits, likely of macrophage origin, in anti-Candida resistance established by PLA at the cerebral level.

Protective effect of picolinic acid on mice intracerebrally infected with lethal doses of Candida albicans.

We have studied the effects of picolinic acid (PLA), a product of tryptophan degradation, on mouse susceptibility to intracerebral infection with Candida albicans. We show that intraperitoneal administration of PLA significantly enhances the median survival time of mice inoculated with the lethal challenge. Furthermore, intracerebral administration of this agent induces a protective state against the local lethal infection, the phenomenon depending upon the administration schedule and doses of PLA employed. According to survival data, yeast growth in the brain as well as yeast colonization of the kidneys are drastically reduced in PLA-treated mice compared with those for untreated controls. Northern (RNA) blot analysis of brain tissues demonstrates that mRNA levels specific for tumor necrosis factor and interleukin 1 are augmented and induced, respectively, after inoculation of PLA. These results indicate that PLA has a protective effect likely involving elicitation of a cytokine response in vivo against fungal infections.

Differential host susceptibility to intracerebral infections with Candida albicans and Cryptococcus neoformans.

To investigate the immune defense mechanisms employed against fungi in the brain, mice were experimentally infected by intracerebral inoculation of Candida albicans or Cryptococcus neoformans. Parameters such as median survival time and numbers of yeast cells in the brains were assessed for naive and immunomodulated mice. We found that no mice survived either C. albicans or C. neoformans challenge at doses of > or = 10(6) yeast cells per mouse. However, when the inoculum size was decreased (< or = 10(5) yeast cells per mouse), C. albicans was no longer lethal (100% survival), whereas 100 and 70% of the mice still succumbed to challenge doses of 10(4) and 10(3) C. neoformans yeast cells, respectively. Pharmacological manipulation and transfer experiments revealed that the myelomonocytic compartment had a minor role against C. neoformans but was deeply involved in the control of intracerebral C. albicans infection. By counting the number of yeast cells in the brains of naive and immunomodulated animals, we established that, unlike C. albicans, C. neoformans remained essentially in the brain, where massive colonization and damage occurred whether naive or immunomodulated defense mechanisms were employed by the host. Overall, these data suggest that the differential role of the myelomonocytic compartment, together with the diverse tropisms of the two fungi, can explain the different development and outcome of intracerebral C. albicans and C. neoformans infections.

Experimental model of intracerebral infection with Cryptococcus neoformans: roles of phagocytes and opsonization.

A murine model of intracerebral (i.c.) infection with Cryptococcus neoformans in which naive mice receiving an i.c. fungal inoculation developed a severe disease has been established. The effect was strictly dependent on the number of microorganisms injected and evolved as lethal meningoencephalitis. Murine susceptibility to i.c. infection with C. neoformans was enhanced by treatment with chloroquine and colchicine, agents known to greatly affect the host phagocytic compartment. Furthermore, the life spans of both naive and drug-treated mice were significantly augmented when opsonized fungi were injected. Therefore, phagocyte-mediated mechanisms are likely involved in local resistance to i.c. infection with C. neoformans. Further support for this conclusion was supplied by in vitro data showing that microglial cells were proficient anticryptococcal effectors, provided opsonized microorganisms were used.