Claudia Monari

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.

Glucuronoxylomannan, a microbial compound, regulates expression of costimulatory molecules and production of cytokines in macrophages.

Glucuronoxylomannan (GXM) is a microbial compound that can modulate the immune response. We investigated (1) the receptors involved in uptake of GXM on monocyte-derived macrophages (MDMs) from healthy donors, (2) the effects of GXM on expression of specific receptors, (3) the effects of GXM mediated by pattern-recognition receptors, and (4) GXM modulation of MDM accessory and secretory functions. Cellular receptors involved in uptake of GXM included Fc gamma RII, CD18, Toll-like receptor (TLR) 4, and CD14. Some biological functions of MDMs were profoundly affected by treatment with GXM, resulting in (1) increased expression of CD40 and CD86 via perturbation of TLR4, (2) decreased expression of major histocompatibility complex class II, (3) induction of interleukin-10 but not of tumor necrosis factor-alpha, and (4) decreased lipopolysaccharide (LPS)-induced production of cytokines. GXM represents an attractive compound to limit inflammatory processes and induce an LPS-tolerant state.

Cryptococcus neoformans Capsular Glucuronoxylomannan Induces Expression of Fas Ligand in Macrophages

The major component of capsular material of Cryptococcus neoformans is glucuronoxylomannnan (GXM), a polysaccharide that exhibits potent immunosuppressive properties in vitro and in vivo. The results reported here show that 1) soluble purified GXM induces a prompt, long-lasting, and potent up-regulation of Fas ligand (FasL) on macrophages, 2) the up-regulation of FasL is related to induced synthesis and increased mobilization to the cellular surface, 3) this effect is largely mediated by interaction between GXM and TLR4, 4) FasL up-regulation occurs exclusively in GXM-loaded macrophages, 5) macrophages that show up-regulation of FasL induce apoptosis of activated T cells expressing Fas and Jurkat cells that constitutively express Fas, and 6) anti-Fas Abs rescue T cells from apoptosis induced by GXM. Collectively our results reveal novel aspects of the immunoregulatory properties of GXM and suggest that this nontoxic soluble compound could be used to dampen the immune response, to promote or accelerate the death receptor, and to fix FasL expression in a TLR/ligand-dependent manner. In the present study, we delineate potential new therapeutic applications for GXM that exploit death receptors as key molecular targets in regulating cell-mediated cytotoxicity, immune homeostasis, and the immunopathology of diseases.

Cryptococcus neoformans capsular polysaccharide component galactoxylomannan induces apoptosis of human T-cells through activation of caspase-8

The major virulence factor of Cryptococcus neoformans is its polysaccharide capsule composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM) and mannoproteins. A variety of immunomodulating activities have been described for GXM and mannoproteins but little is known about possible interactions of GalXM with the immune system. In the present article, we investigate the effect of purified soluble GalXM on human T lymphocytes. The results indicate that, GalXM (i) can affect selected immune responses; (ii) causes significant impairment of T cell proliferation and increases interferon‐γ and interleukin‐10 production; and (iii) induces apoptosis of T lymphocytes through activation of caspase‐8 that terminates with fragmentation of DNA. These results are the first to suggest a role for GalXM in C. neoformans virulence by demonstrating that it can target human T cells, and that it may impair the development of an effective specific T cell response.

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.

Typing of Nosocomial Outbreaks of Acinetobacter baumannii by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been evaluated for the identification of multidrug-resistant Acinetobacter baumannii nosocomial outbreaks in comparison with the repetitive sequence-based PCR DiversiLab system. The results suggest that MALDI-TOF MS can be used for real-time detection of Acinetobacter outbreaks before results from DNA-based systems are available.

Neutrophils from Patients with Advanced Human Immunodeficiency Virus Infection Have Impaired Complement Receptor Function and Preserved Fcγ Receptor Function

Interleukin (IL)-8 production by human polymorphonuclear leukocytes (PMNL) to Cryptococcus neoformans is related to complement activation. Generation of the bioactive fragments C3a and C5a is responsible for IL-8 release. IL-8 production was analyzed in response to C. neoformans by PMNL from persons with early- and late-stage (>400 and <200 CD4 cells/mm3, respectively) human immunodeficiency virus (HIV) infection who were at high risk for cryptococcosis. IL-8 release by PMNL from persons with early-stage infection and from healthy donors was similar; however, PMNL from persons with late-stage HIV infection had significantly impaired IL-8 production, which correlated with reduced IL-8 response to C3a and C5a proteins and decreased CD88 expression. Addition of murine monoclonal antibody (MAb) 18B7 promoted phagocytosis and restored IL-8 release consistent with integrity of FcgammaRIII. These results provide evidence for a selective defect in CD88 expression on PMNL from persons with late-stage HIV infection. However, Fcgamma receptor expression in PMNL appears to be intact and allows MAb to glucuronoxylomannan to positively influence PMNL function.

Microbial Immune Suppression Mediated by Direct Engagement of Inhibitory Fc Receptor

A microbial polysaccharide (glucuronoxylomannan (GXM)) exerts potent immunosuppression by direct engagement to immunoinhibitory receptor FcγRIIB. Activation of FcγRIIB by GXM leads to the recruitment and phosphorylation of SHIP that prevents IκBα activation. The FcγRIIB blockade inhibits GXM-induced IL-10 production and induces TNF-α secretion. GXM quenches LPS-induced TNF-α release via FcγRIIB. The addition of mAb to GXM reverses GXM-induced immunosuppression by shifting recognition from FcγRIIB to FcγRIIA. These findings indicate a novel mechanism by which microbial products can impair immune function through direct stimulation of an inhibitory receptor. Furthermore, our observations provide a new mechanism for the ability of specific Ab to reverse the immune inhibitory effects of certain microbial products.