Sandro Rogério de Almeida

Two physically, functionally, and developmentally distinct peritoneal macrophage subsets

The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (MØ) are commonly drawn for functional studies. Here we define two MØ subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal MØ (LPM), contains approximately 90% of the PerC MØ in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical MØ surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal MØ (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of MØ heterogeneity and shed new light on PerC MØ diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC MØ.

Restoration of Pattern Recognition Receptor Costimulation to Treat Chromoblastomycosis, a Chronic Fungal Infection of the Skin

Summary Chromoblastomycosis is a chronic skin infection caused by the fungus Fonsecaea pedrosoi. Exploring the reasons underlying the chronic nature of F. pedrosoi infection in a murine model of chromoblastomycosis, we find that chronicity develops due to a lack of pattern recognition receptor (PRR) costimulation. F. pedrosoi was recognized primarily by C-type lectin receptors (CLRs), but not by Toll-like receptors (TLRs), which resulted in the defective induction of proinflammatory cytokines. Inflammatory responses to F. pedrosoi could be reinstated by TLR costimulation, but also required the CLR Mincle and signaling via the Syk/CARD9 pathway. Importantly, exogenously administering TLR ligands helped clear F. pedrosoi infection in vivo. These results demonstrate how a failure in innate recognition can result in chronic infection, highlight the importance of coordinated PRR signaling, and provide proof of the principle that exogenously applied PRR agonists can be used therapeutically.

Immunology of Dermatophytosis

The immune response to infection by dermatophytes ranges from a non-specific host mechanism to a humoral and cell-mediated immune response. The currently accepted view is that a cell-mediated immune response is responsible for the control of dermatophytosis. Indeed, some individuals develop a chronic or recurrent infection mediated by the suppression of a cell-mediated immune response. The immune response to Trichophyton is unusual in that this fungus can elicit both immediate hypersensitivity (IH) and delayed-type hypersensitivity (DTH) in different individuals when they are submitted to a skin test reaction. Understanding the nature and function of the immune response to dermatophytes is an exciting challenge that might lead to novel approaches in the treatment and immunological prophylaxis of dermatophytosis.

Characterization of gp70 and Anti-gp70 Monoclonal Antibodies in Paracoccidioides brasiliensis Pathogenesis

ABSTRACT Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis whose agent is Paracoccidioides brasiliensis. In the culture supernatant, the fungus expresses glycoproteins of from 13 to 148 kDa. A cell surface glycoprotein of 43 kDa is the major antigenic component of P. brasiliensis. Another expressed glycoprotein, gp70, is recognized by 96% of sera from PCM patients and is able to induce lymphoproliferation. Since, little is known about this glycoprotein, we produced monoclonal antibodies (MAbs) against gp70 to isolate the molecule from total fungus extracts and to investigate its possible role in the pathogenesis of PCM. Using these MAbs, it was observed by confocal microscopy that gp70 is located mainly in the intracellular compartment of the fungus, although it was also detected in the culture supernatant. Based on observations showing that gp43 has a down-regulatory effect on mouse peritoneal macrophages, we tested the effects of gp70 on their phagocytic ability. Purified gp70 was able to inhibit the activity of macrophages through the mannose receptors and also through the Fc receptors; the latter effect was not observed with gp43. gp70 inhibits NO and H2O2 liberation by peritoneal macrophages in vitro, as does gp43. Results obtained with gp43 led us to hypothesize that gp70 could act as an escape mechanism for fungal establishment in primary infections. To corroborate this hypothesis, we analyzed the effect of passive immunization of mice during infection with P. brasiliensis using anti-gp70 MAbs. This treatment almost completely abolished granuloma formation in the lungs, suggesting that the protein facilitates fungal establishment and progression of lesions in primary infection.

Interaction between Paracoccidioides brasiliensis and Pulmonary Dendritic Cells Induces Interleukin-10 Production and Toll-Like Receptor–2 Expression: Possible Mechanisms of Susceptibility

Dendritic cells (DCs) are the most effective antigen-presenting cells for inducing cell-mediated immune responses; it is thus important to investigate the role played by lung DCs in the pathogenesis of paracoccidioidomycosis (PCM) and their potential to initiate an immune response in mice susceptible (B10.A) and resistant (A/J) to PCM. Initially, we observed that lung DCs from susceptible mice were more phagocytic than cells from resistant mice, and we observed that phagocytosis in the presence of laminarin was inhibited only in DCs from susceptible mice. DCs from resistant mice produced a low concentration of interleukin (IL)-10, IL-12, and tumor necrosis factor (TNF)- alpha . In contrast, DCs from susceptible mice produced high concentrations of TNF- alpha and IL-10, but IL-10 production was significantly inhibited in the presence of laminarin. We also observed that DCs from Toll-like receptor (TLR)-2 knockout mice displayed defective production of IL-10. After 15 days of Paracoccidioides brasiliensis infection, DCs from susceptible mice produced IL-10 and expressed costimulatory molecules at a low level. We found that expression of the gene for TLR-2 is increased after infection in susceptible, but not resistant, mice. In conclusion, our data suggest that P. brasiliensis induces regulatory DCs in susceptible mice, which promotes IL-10 production and contributes to the susceptibility of mice to P. brasiliensis infection.

Differences in Cell Morphometry, Cell Wall Topography and Gp70 Expression Correlate with the Virulence of Sporothrix brasiliensis Clinical Isolates

Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.

Passive immunization with monoclonal antibody against a 70-kDa putative adhesin of Sporothrix schenckii induces protection in murine sporotrichosis

Cell‐mediated and innate immunity are considered the most important mechanisms of host defense against fungus infections. However, recent studies demonstrated that specific antibodies show different degrees of protection against mycosis. In a previous study, antigens secreted by Sporothrix schenckii induced a specific humoral response in infected animals, mainly against the 70‐kDa molecule, indicating a possible participation of antibodies to this antigen in infection control. In the present study, an IgG1 mAb was produced against a 70‐kDa glycoprotein of S. schenckii in order to better understand the effect of passive immunization of mice infected with S. schenckii. Results showed a significant reduction in the number of CFU in organs of mice when the mAb was injected before and during S. schenckii infection. Similar results were observed when T‐cell‐deficient mice were used. Moreover, in a second schedule treatment, the mAb was injected after infection was established, and again we observed a significant reduction in CFU associated with an increase of IFN‐γproduction. Also, the 70‐kDa antigen is shown to be a putative adhesin present on the surface of this fungus. In conclusion, we report for the first time the protective effect of a specific antibody against S. schenckii.

Structure, Cellular Distribution, Antigenicity, and Biological Functions of Fonsecaea pedrosoi Ceramide Monohexosides

ABSTRACT Monohexosylceramides (CMHs, or cerebrosides) have been reported as membrane and cell wall constituents of both pathogenic and nonpathogenic fungi, presenting remarkable differences in their ceramide moiety compared to mammalian CMHs. Current evidence suggests that CMHs are involved in fungal differentiation and growth and contribute to host immune response. Here we describe a structural diversity between cerebrosides obtained from different forms of the human pathogen Fonsecaea pedrosoi. The major CMH species produced by conidial forms displayed the same structure previously demonstrated by our group for mycelia, an N-2′-hydroxyhexadecanoyl-1-β-d-glucopyranosyl-9-methyl-4,8-sphingadienine. However, the major cerebroside species purified from sclerotic cells carries an additional hydroxyl group, bound to its long-chain base. The structural difference between cerebrosides from mycelial and sclerotic cells was apparently not relevant for their antigenicity, since they were both recognized at similar levels by sera from individuals with chromoblastomycosis and a monoclonal antibody to a conserved cerebroside structure. Preincubation of fungal cells with anti-CMH monoclonal antibodies had no effect on the interaction of F. pedrosoi sclerotic cells with murine macrophages. In contrast to what has been described for other fungal species, sclerotic bodies are resistant to the antifungal action of anti-CMH antibodies. Immunofluorescence analysis showed that recognition of sclerotic cells by these antibodies only occurs at cell wall regions in which melanization is not evident. Accordingly, melanin removal with alkali results in an increased reaction of fungal cells with anti-CMH antibodies. Our results indicate that cerebroside expression in F. pedrosoi cells is associated with dimorphism and melanin assembly on the fungal cell wall.

Nitric oxide-dependent killing of Cryptococcus neoformans by B-1-derived mononuclear phagocyte

The role of B lymphocytes in protecting the host against pulmonary Cryptococcus neoformans infection is until now, uncertain. A recent study using B lymphocyte‐deficient mice suggests that B lymphocytes play a protective role in cryptococcal infection. It has been well established that at least three B cell subsets, B‐1a, B‐1b, and B‐2, are present in the mouse periphery. B‐1 cells constitute a minor fraction of the B cell population in the spleen and are not detected in lymph nodes of mice. We demonstrated that B‐1 cells migrate to a nonspecific, inflammatory focus and differentiate into macrophage‐like cells. However, the role these cells might play on the kinetics and evolution of the inflammatory response and on fungal infection has not yet been established. Based on these data, we decided to investigate the interaction of B‐1‐derived mononuclear phagocytes (BDMP) with C. neoformans to elucidate the possible influence of this cell in the progression of the disease. In this study, we demonstrated that the BDMP cell internalized C. neoformans and that this process was mediated by complement receptor 3. Thus, our results showed that the BDMP cell was more fungicidal than a macrophage and up‐regulated major histocompatibility complex type II and the CD86 costimulatory molecule with the production of proinflammatory cytokines. The phagocytosis of C. neoformans results in the nitric oxide (NO)‐mediated death of the fungus, as demonstrated by experiments using NO synthase 2 knockout and aminoguanidine‐treated, wild‐type mice.

Cell surface expression of adhesins for fibronectin correlates with virulence in Sporothrix schenckii

The virulence of four Sporothrix schenckii isolates was compared in a murine model of sporotrichosis, together with the protein pattern of the yeast cell surface and the capacity to bind the extracellular matrix protein fibronectin. Virulence was determined by the mortality rate, fungal burden and histopathology. Two clinical isolates were more virulent for C57BL/6 mice, but no direct correlation was seen between virulence and the clinical or environmental origin of the isolates. The lowest virulence was observed for an isolate recovered from a patient with meningeal sporotrichosis. Although all isolates could effectively disseminate, the dissemination patterns were not similar. Using flow cytometry analysis, we investigated the interaction of all the strains with fibronectin, and showed that the binding capacity correlated with virulence. Western blot analysis of S. schenckii cell wall extracts revealed positive bands for fibronectin in the range of 37-92 kDa. The 70 kDa adhesin was also recognized by a protective monoclonal antibody raised against a gp70 antigen of S. schenckii (mAb P6E7). Confocal microscopy confirmed the co-localization of fibronectin and mAb P6E7 on the yeast cell surface. To our knowledge, this is the first report identifying adhesins for fibronectin on the surface of this human pathogen.