Vera L. G. Calich

Macrophage Interactions with Neutrophils Regulate Leishmania major Infection

Macrophages are host cells for the pathogenic parasite Leishmania major. Neutrophils die and are ingested by macrophages in the tissues. We investigated the role of macrophage interactions with inflammatory neutrophils in control of L. major infection. Coculture of dead exudate neutrophils exacerbated parasite growth in infected macrophages from susceptible BALB, but killed intracellular L. major in resistant B6 mice. Coinjection of dead neutrophils amplified L. major replication in vivo in BALB, but prevented parasite growth in B6 mice. Neutrophil depletion reduced parasite load in infected BALB, but exacerbated infection in B6 mice. Exacerbated growth of L. major required PGE2 and TGF-β production by macrophages, while parasite killing depended on neutrophil elastase and TNF-α production. These results indicate that macrophage interactions with dead neutrophils play a previously unrecognized role in host responses to L. major infection.

A new fluorescent viability test for fungi cells

The fluorescein diacetate-ethidium bromide (FDA-EB) fluorescence method, primarily used to determine viability of mammalian cells, was applied to several fungi species. Living fungi cells produced fluorochromasia, i.e., an intracellular accumulation of fluorescein which could be easily visualized as a green color under the U.V. microscope. Dead cells showed a red bright color due to ethidium bromide penetration. The FDA-EB test can be successfuly employed to assay yeast and yeast like cells viability since a good correlation was observed between this assay and the colony count technique. The main advantages of FDA-EB test are its speed, high sensitivity and simplicity.

Dual Role for Nitric Oxide in Paracoccidioidomycosis: Essential for Resistance, but Overproduction Associated with Susceptibility

Using a murine model of susceptibility and resistance to paracoccidioidomycosis, we have previously demonstrated that immunosuppression occurs in susceptible (B10.A), but not in resistant (A/Sn), mouse strains. Accumulating evidence shows that NO is involved in the induction of T cell immunosuppression during infection as well as in the killing of Paracoccidioides brasiliensis. In the present work, we focused on NO and other macrophage products that could be associated with resistance or susceptibility to paracoccidioidomycosis. A striking difference was related to NO and TNF production. Macrophages from B10.A mice produced high and persistent NO levels, while in A/Sn animals, TNF production predominated. In in vitro cultures, P. brasiliensis-infected macrophages from A/Sn mice also produced large amounts of TNF, while B10.A macrophages only produced NO. TNF production by B10.A macrophages appeared to be suppressed by NO, because the addition of aminoguanidine sulfate, an inducible NO synthase (NOS2) inhibitor, resulted in TNF production. These results suggested that enhanced TNF or NO production is associated with resistance and susceptibility, respectively. However, regardless of the mouse strain, NOS2-deficient or aminoguanidine sulfate-treated mice presented extensive tissue lesions with increased fungal load in lungs and liver compared with their controls. We conclude that NOS2-derived NO is essential for resistance to paracoccidioidomycosis, but overproduction is associated with susceptibility.

The source of the growth-promoting factor(s) affects the plating efficiency of Paracoccidioides brasiliensis

We studied the influence of the growth factor (GF) source, concentration and production time on the plating efficiency of Paracoccidioides brasiliensis yeast cells. The highest plating efficiencies were achieved when the GF was derived from a fast growing P. brasiliensis isolate which was not homologous to the plated samples.

TLR2 Is a Negative Regulator of Th17 Cells and Tissue Pathology in a Pulmonary Model of Fungal Infection

To study the role of TLR2 in a experimental model of chronic pulmonary infection, TLR2-deficient and wild-type mice were intratracheally infected with Paracoccidioides brasiliensis, a primary fungal pathogen. Compared with control, TLR2−/− mice developed a less severe pulmonary infection and decreased NO synthesis. Equivalent results were detected with in vitro-infected macrophages. Unexpectedly, despite the differences in fungal loads both mouse strains showed equivalent survival times and severe pulmonary inflammatory reactions. Studies on lung-infiltrating leukocytes of TLR2−/− mice demonstrated an increased presence of polymorphonuclear neutrophils that control fungal loads but were associated with diminished numbers of activated CD4+ and CD8+ T lymphocytes. TLR2 deficiency leads to minor differences in the levels of pulmonary type 1 and type 2 cytokines, but results in increased production of KC, a CXC chemokine involved in neutrophils chemotaxis, as well as TGF-β, IL-6, IL-23, and IL-17 skewing T cell immunity to a Th17 pattern. In addition, the preferential Th17 immunity of TLR2−/− mice was associated with impaired expansion of regulatory CD4+CD25+FoxP3+ T cells. This is the first study to show that TLR2 activation controls innate and adaptive immunity to P. brasiliensis infection. TLR2 deficiency results in increased Th17 immunity associated with diminished expansion of regulatory T cells and increased lung pathology due to unrestrained inflammatory reactions.

Experimental murine paracoccidioidomycosis: relationship among the dissemination of the infection, humoral and cellular immune responses

The dissemination of Paracoccidioides brasiliensis cells to the heart, omentum/pancreas, spleen, liver and lungs, assessed by colony forming unit (CFU) counts, the levels of specific antibodies to this fungal agent (by ELISA), and the specific DTH reaction were studied in susceptible (B10.A) and resistant (A/Sn) mice. The animals were infected intraperitoneally with P. brasiliensis yeast cells and were evaluated 2, 4, 12 and 16 weeks later. The mosl remarkable differences between the two mouse strains were observed 16 weeks after infection, when B10.A mice displayed high numbers ofCFU in all examined organs, except the heart, high antibody titres, and depressed DTH response. At this point. A/Sn mice presented low or absent CFU in all organs, low antibody litres and expressive DTH response. The CFU counts were shown to be a reliable parameter to discriminate susceptible from resistant animals. The fungal load in the most affected organs correlated wilh the antibody litres and was inversely correlated with the intensity of the DTH reaction. The patterns of immune response in this model mimic human paracoccidioidomycosis, in which high specific antibody levels and depressed DTH reactions are found in multifocal and severe forms of the disease.

Innate immunity to Paracoccidioides brasiliensis infection.

Innate immunity is based in pre-existing elements of the immune system that directly interact with all types of microbes leading to their destruction or growth inhibition. Several elements of this early defense mechanism act in concert to control initial pathogen growth and have profound effect on the adaptative immune response that further develops. Although most studies in paracoccidioidomycosis have been dedicated to understand cellular and humoral immune responses, innate immunity remains poorly defined. Hence, the main purpose of this review is to present and discuss some mechanisms of innate immunity developed by resistant and susceptible mice to Paracoccidioides brasiliensis infection, trying to understand how this initial host-pathogen interface interferes with the protective or deleterious adaptative immune response that will dictate disease outcome. An analysis of some mechanisms and mediators of innate immunity such as the activation of complement proteins, the microbicidal activity of natural killer cells and phagocytes, the production of inflammatory eicosanoids, cytokines, and chemokines among others, is presented trying to show the important role played by innate immunity in the host response to P. brasiliensis infection.

In vivo and in vitro characteristics of six Paracoccidioides brasiliensis strains.

The yeast-like forms of six P. brasiliensis strains were characterized and compared using in vitro (growth curve determination) and in vivo (pathogenicity to sensitive inbred mice) criteria. Strains Pb 18 and Pb 265 which behaved similarly in vitro, showing low counts of fungi and long mean generation times, were respectively the most and the least pathogenic strains. Strains Pb 2052 and IVIC Pb 267, which grow abundantly in vitro were, respectively virulent and avirulent. Strains Pb SN and IVIC Pb 9 behaved similarly both in vitro and in vivo displaying an intermediate pattern of virulence and growing conditions.

The activation of the complement system by Paracoccidioides brasiliensis in vitro: its opsonic effect and possible significance for an in vivo model of infection.

Abstract The yeast phase of Paracoccidioides brasiliensis is poorly phagocytosed by “stimulated” mouse peritoneal macrophages in culture. However, either heterologous or homologous antibodies greatly enhanced the phagocytosis of P. brasiliensis by macrophages. The same effect was observed when the fungus was pretreated with fresh normal sera. The following treatments abolished the opsonic activity of fresh normal serum: heating at 56°C for 30 min, addition of 10 mM EDTA. CoF treatment, and properdin or factor-B depletion. Conversely, EGTA had no effect. These data indicate that the alternative pathway of complement activation is involved in the expression of this phenomenon. When yeast cells of P. brasiliensis were incubated with fresh normal serum, the third component of complement changed its electrophoretic mobility of β1C to β1A. In an attempt to correlate these data with the in vivo conditions, fungi were inoculated into the subcutaneous tissues of normal mice. The resultant lesions reached their maximal size 48 hr after inoculation. Histological studies of these lesions showed a massive influx of polymorphonucleocytes. The histological picture was not modified conclusively when the mice were pretreated with CoF or when the fungi were inoculated in C5-deficient mice. The hypothesis that the fungus might secrete a chemotactic factor was tested in an in vitro system. It was verified that the activation of the complement system by P. brasiliensis elicited the formation of chemotactic substances for mouse polymorphonucleocytes. However, when the supernatant of cultures of the fungus in medium 199 was used as a chemotactic source, no effect could be detected. Thus, neither complement activation nor a fungus-secreted factor seems to explain the initial events of the lesion formation induced by P. brasiliensis.

Toll-Like Receptor 4 Signaling Leads to Severe Fungal Infection Associated with Enhanced Proinflammatory Immunity and Impaired Expansion of Regulatory T Cells

ABSTRACT Toll-like receptors (TLRs) present in innate immune cells recognize pathogen molecular patterns and influence immunity to control the host-parasite interaction. The objective of this study was to characterize the involvement of TLR4 in the innate and adaptive immunity to Paracoccidioides brasiliensis, the most important primary fungal pathogen of Latin America. We compared the responses of C3H/HeJ mice, which are naturally defective in TLR4 signaling, with those of C3H/HePas mice, which express functional receptors, after in vitro and in vivo infection with P. brasiliensis. Unexpectedly, we verified that TLR4-defective macrophages infected in vitro with P. brasiliensis presented decreased fungal loads associated with impaired synthesis of nitric oxide, interleukin-12 (IL-12), and macrophage chemotactic protein 1 (MCP-1). After intratracheal infection with 1 million yeasts, TLR4-defective mice developed reduced fungal burdens and decreased levels of pulmonary nitric oxide, proinflammatory cytokines, and antibodies. TLR4-competent mice produced elevated levels of IL-12 and tumor necrosis factor alpha (TNF-α), besides cytokines of the Th17 pattern, indicating a proinflammatory role for TLR4 signaling. The more severe infection of TLR4-normal mice resulted in increased influx of activated macrophages and T cells to the lungs and progressive control of fungal burdens but impaired expansion of regulatory T cells (Treg cells). In contrast, TLR4-defective mice were not able to clear their diminished fungal burdens totally, a defect associated with deficient activation of T-cell immunity and enhanced development of Treg cells. These divergent patterns of immunity, however, resulted in equivalent mortality rates, indicating that control of elevated fungal growth mediated by vigorous inflammatory reactions is as deleterious to the hosts as low fungal loads inefficiently controlled by limited inflammatory reactions.