Cinzia Retini

Encapsulation of Cryptococcus neoformans regulates fungicidal activity and the antigen presentation process in human alveolar macrophages

Our previous studies have shown that unstimulated alveolar macrophages (AM) play a predominant role as antigen‐presenting cells in Cryptococcus neoformans infections, while the function as effector cells seems to be of minor relevance. The present study focuses on the role of encapsulation of C. neoformans on fungicidal activity and the antigen presentation process of AM. Fungicidal activity in unstimulated AM occurs to a higher degree when the acapsular strain is employed, but this is impaired compared with other natural effectors, such as peripheral blood monocytes (PBM) and polymorphonuclear (PMN) cells. Cryptococcus‐laden AM also induce a higher proliferative response in autologous CD4+ lymphocytes when the acapsular strain is used compared with encapsulated yeast. The enhanced blastogenic response is, in part, ascribed to an augmented IL‐2 production by T cells. In addition, higher levels of interferon‐gamma (IFN‐γ). but not 1L‐4, are produced by the responding T cells, when the acapsular strain is used compared with the encapsulated yeast. Moreover, IFN‐γ) is able to induce fungicidal activity in AM against the encapsulated yeast and augments killing activity of the acapsular strain. This phenomenon is not mediated by nitric oxide production, but is correlated with an enhancement of fungicidal activity of cytoplasmic cationic proteases. We speculate that encapsulation of C. neoformans could down‐regulate the development of the immune response mediated by Cryptocaccus‐laden AM al lung level.

Differences in outcome of the interaction between Cryptococcus neoformans glucuronoxylomannan and human monocytes and neutrophils

Disseminated infections by the opportunistic yeast Cryptococcus neoformans are characterized by accumulation in tissues of glucuronoxylomannan (GXM), the major component of the capsular polysaccharide. We investigated binding, uptake, and disposal of GXM by peripheral blood neutrophils and monocytes, and the effect of GXM uptake on phagocytic cell function. GXM was efficiently bound and internalized by both types of phagocytic cells, with maximal loading at 50 μg/ml, a GXM concentration found in serum and cerebrospinal fluid of some cryptococcosis patients. However, substantial differences were noted in the kinetics for uptake by macrophages and neutrophils. Whereas neutrophils rapidly ingested limited amounts of GXM and then expelled or degraded it after 1 h of incubation, macrophages demonstrated continuous intracellular accumulation for up to 1 week of incubation. Accumulation of GXM by neutrophils was accompanied by reduced anticryptococcal activity, suggesting one more mechanism for virulence enhancement by the major capsular component of C. neoformans.

Interdependency of interleukin-10 and interleukin-12 in regulation of T-cell differentiation and effector function of monocytes in response to stimulation with Cryptococcus neoformans

ABSTRACT We previously demonstrated that the principal component of capsular material of Cryptococcus neoformans, glucuronoxylomannan (GXM), induces interleukin-10 (IL-10) secretion from human monocytes. Here we report that encapsulation of the yeast with GXM is able to down-regulate interleukin-12 (IL-12) production by monocytes that would normally occur in the absence of encapsulation. This phenomenon appeared to be the result of inhibition of the phagocytic process by encapsulation with GXM as well as of negative signals such as IL-10 secretion produced by interaction of GXM with leukocytes. Decreased secretion of IL-12 correlated with decreased release of gamma interferon (IFN-γ) from T cells, suggesting a role for encapsulation with GXM in hindering a T helper type 1 (Th1) response. This is supported by the ability of encapsulation with GXM to limit increased expression of B7-1 costimulatory molecules that otherwise might limit IL-10 secretion. Endogenous IL-10 played a critical role in modulatory activity associated with encapsulation with GXM. Blocking IL-10 with monoclonal antibody to IL-10 resulted in increased (i) IL-12 secretion, (ii) IFN-γ release from T cells, and (iii) killing of C. neoformans by monocytes. These results suggest that encapsulation with GXM limits development of a protective Th1-type response, an inhibitory process in which IL-10 plays a critical role. Scavengers of GXM and/or IL-10 could be useful in a protective Th1-type response in patients with cryptococcosis.

CRYPTOCOCCUS NEOFORMANS DIFFERENTLY REGULATES B7-1 (CD80) AND B7-2 (CD86) EXPRESSION ON HUMAN MONOCYTES

To induce a specific response in primary resting T cells, two signals must be provided by antigen‐presenting cells (APC). The first antigen‐specific signal is mediated by formation of the T cell receptor major histocompatibility complex molecule ternary complexes. The second signal is delivered by interaction of either B7‐1 or B7‐2 expressed by APC with CD28 or CTLA‐4 on T cells. In this study, we examined the modulation of B7‐1 and B7‐2 molecules on human monocytes exposed to encapsulated or acapsular Cryptococcus neoformans or Candida albicans. In our experimental system, C. albicans or acapsular C. neoformans are able to induce B7‐1 expression while the encapsulated yeast is a poor stimulator. A modest increase of B7‐2 expression was also observed after monocyte treatment with acapsular C. neoformans or C. albicans, while the encapsulated yeast was ineffective in inducing B7‐2 molecules. Kinetic analysis showed the maximum expression of B7‐1 after 24 to 48 h. Addition of the opsonic IgG1 mAb 2H1 to monocytes and C. neoformans significantly increased B7‐1, but not B7‐2, expression. The contribution of B7‐1 and B7‐2 co‐stimulatory (CS) molecules to cryptococcal‐specific T cell activation was analyzed and a substantial inhibition of T cell proliferation was observed. In this study we provide the first demonstration of fungal interference in the regulation of CS molecules. Our results suggest a potential mechanism for poor inflammatory responses observed in C. neoformans infections.

Inhibition of fungicidal activity of polymorphonuclear leukocytes from HIV-infected patients by interleukin (IL)-4 and IL-10

Objective:To investigate the effect of human recombinant interleukin (hrIL)-4 or hrIL-10 on the functional status of polymorphonuclear leukocytes (PMNL) from normal subjects and HIV-infected patients. Design:In an in vitro system we studied the effect of hrIL-4 and hrIL-10 on phagocytosis, fungicidal activity and superoxide anion production by PMNL. Methods:PMNL were treated in vitro with hrIL-4 or hrIL-10 or their combination for 6 h and then candidacidal activity was evaluated in a colony-forming unit inhibition assay. Superoxide anion generation by PMNL was measured in the presence or absence of preopsonized zymosan or Candida albicans. Results:Treatment in vitro with hrIL-4 or hrIL-10 of PMNL for 6 h was able to impair candidacidal activity of neutrophils in both normal or HIV-infected patients. The inhibitory effect was time- and dose-dependent and was more evident in PMNL from HIV-infected subjects, and reflected in these latter cells a decrease of superoxide anion generation. The impairment of candidacidal activity in PMNL from HIV-infected patients was accompanied by survival of the yeasts shown by budding formation into phagosomic organelles of cytokine-treated PMNL. Conclusions:Our data highlight new biological effects of IL-4 and IL-10 evidenced by suppressed effector function of neutrophils; this phenomenon is emphasized in HIV-infected patients suggesting a role for these cytokines in mediating increased susceptibility to microbial infection during AIDS progression.

T lymphocyte and monocyte interaction by CD40/CD40 ligand facilitates a lymphoproliferative response and killing of Cryptococcus neoformans in vitro

This study explored the role of CD40 / CD40 ligand (CD40L) in the induction of a lymphoproliferative response and killing of Cryptococcus neoformans in vitro. In our experimental system, monocytes exposed to C. neoformans were used as antigen‐presenting cells (APC) and co‐cultured with autologous T cells. The results showed that CD40 / CD40L strongly regulated the blastogenic response of T cells to C. neoformans. The fungus up‐regulated CD40 expression on APC. An acapsular strain appeared to be a better inducer than an encapsulated strain. Time course experiments showed optimal regulation of CD40 expression at 48 h of incubation. Blocking the interaction of CD40 on APC with CD40L on T cells using mAb to CD40L resulted in a significant inhibition of IFN‐γ production. The anti‐cryptococcal activity of monocytes was greatly influenced by the CD40 / CD40L interaction, and a positive correlation was found between nitric oxide secretion and enhanced killing of C. neoformans. Finally, the CD40 / CD40L interaction was critical for induction of optimal secretion of pro‐inflammatory cytokines such as TNF‐α  and IL‐1β. These results indicate an important role for CD40 / CD40L interaction in inducing activation of T cells. Such cell‐to‐cell contact promotes anti‐cryptococcal activity as well as secretion of pro‐inflammatory cytokines by monocytes.

Regulatory role of exogenous IL-10 in the development of immune response versus Cryptococcus neoformans.

The most important event involved in host defence against Cryptococcus neoformans is the development of an adequate cell‐mediated immune response. IL‐10, abundantly produced during AIDS progression, could be a negative factor that affects the T cell response through its own immunosuppressive action on antigen‐presenting cells. To determine whether this cytokine affects the course of immune response against C. neoformans, we added exogenous IL‐10 to cultured Cryptococcus‐laden monocytes plus T lymphocytes. The data from this study confirmed the down‐regulatory effect of exogenous IL‐10 on monocytes and expanded the known inhibitory role to include an increase of the deleterious effect due to capsular material of C. neoformans on (i) lymphoproliferation, (ii) down‐regulation of MHC class II molecules, (iii) inhibition of IL‐2 mRNA expression and protein secretion by T lymphocytes. These results indicate that the presence of IL‐10 in AIDS patients, due to the progression of disease, could represent a pivotal problem contributing to augment the pathogenic effect of C. neoformans.

Monocyte dysfunction in patients with acquired immunodeficiency syndrome (AIDS) versus Cryptococcus neoformans

In the present study we investigated the response of monocytes from AIDS patients, susceptible to cryptococcosis (<200 CD4 cells/microl), against Cryptococcus neoformans. Different patterns of response were observed in these cells compared to cells from healthy donors. In particular, fungicidal activity versus this fungus was impaired; this phenomenon could be due to the difficulty of monocytes to internalize C. neoformans in the presence of an intact complement system. Impairment of complement receptor type 3 and direct involvement of this receptor in phagocytosis of C. neoformans were found in monocytes from AIDS patients, which may account for the difficulty in phagocytosis of the fungus. Also, superoxide anion production was dramatically reduced in monocytes from AIDS patients. An increase of spontaneous tumor necrosis factor (TNF) production was evidenced after in vitro addition of C. neoformans. However, this did not activate the antifungal capacity of monocytes from AIDS patients. Moreover, cryptococcus-laden monocytes from AIDS patients were able to induce only a weak response of autologous T-lymphocytes. Hence, monocyte dysfunction could play a part in the progression of cryptococcosis in AIDS.

Human Immunodeficiency Virus Type 1 Envelope Protein gp120 Impairs Intracellular Antifungal Mechanisms in Human Monocytes

The key to success of fungal opportunistic pathogens in the immunocompromised host is related to survival inside phagocytic cells, which represent the first line of defense against microorganisms. The contribution of human immunodeficiency virus-1 recombinant envelope protein gp120 on effector functions of peripheral blood monocytes (PBM) against Candida albicans was investigated. gp120 binds CD4 receptors on PBM while not affecting the access of the fungus into the lysosome compartment. However, gp120 reduces the antifungal capacity of PBM. This phenomenon correlates with impaired oxygen-dependent antimicrobial machinery and reduced ability of phagolysosome acidification. The maintenance of phagolysosomal pH at approximately 6.2 restricts antimicrobial properties of the enzyme that work at a low pH, as evidenced by reduced antifungal capability of lysosomal protein extracted from gp120-treated PBM. These findings highlight gp120 perturbation of intracellular antimicrobial mechanisms of phagocytic cells and suggest a new aspect for gp120 in impairing immune functions.

HIV type 1 envelope glycoprotein gp120 induces development of a T helper type 2 response to Cryptococcus neoformans.

OBJECTIVE To analyse the contribution of HIV type 1 envelope glycoprotein gp120 to regulation of a T-cell response to Cryptococcus neoformans. DESIGN Monocytes treated with recombinant gp120 and exposed to C. neoformans were used as antigen presenting cells (APC) in coculture with autologous T lymphocytes. METHODS Costimulatory and major histocompatibility complex class II molecules were evaluated on APC by flow cytometry analysis. T-cell proliferation was determined as 3H thymidine incorporation. Cytokine production was analysed by enzyme-linked immunosorbent assay. RESULTS gp120 had multiple effects on APC and the T-cell response including: (i) up-regulation of major histocompatibility complex class II antigens on the APC surface resulting from both redistribution of molecules from the intracellular pool and synthesis of new molecules; (ii) up-regulation of B7-2 molecules on the APC surface; (iii) altered T-cell proliferation; and (iv) promotion of interleukin-4 and inhibition of interferon-gamma synthesis and release. CONCLUSIONS These data indicate that gp120 alters the normal T-cell response to C. neoformans, promoting a T-helper type 2 response. The altered T-cell response produced by gp120 may play an important role in the pathogenesis of cryptococcosis in the patient with AIDS.