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.

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.

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.

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.

The secretion of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, IL-5, and IL-10 by antigen-stimulated lymph node cells, eosinophil maturation, and the antibody isotypes produced were examined during intraperitoneal infection of susceptible (B10.A) and resistant (A/Sn) mice with Paracoccidioides brasiliensis. Lymph node cells from resistant mice produced early and sustained levels of IFN-gamma and IL-2, whereas susceptible animals secreted low to undetectable amounts of these type 1 cytokines. Both mouse strains presented late and transient production of IL-4, whereas IL-10 was produced constantly throughout the course of disease. Resistant animals produced increasing levels of IL-5 in the chronic phase of the infection (from the eighth week on), whereas susceptible mice showed two peaks of IL-5 production, at the first and twelfth weeks after infection. Only the susceptible strain presented medullary and splenic eosinophilia concomitant with the raised IL-5 production. In resistant mice, the levels of IgG2a antibodies were significantly higher than those observed in susceptible mice, which preferentially secreted IgG2b and IgA isotypes. Taken together, these results demonstrate that a sustained production of IFN-gamma and IL-2 and a predominant secretion of IgG2a antibodies are associated with resistance to P. brasiliensis. In contrast, the production of low levels of IFN-gamma, early secretion of high levels of IL-5 and IL-10, eosinophilia, and a preferential secretion of IgG2b and IgA isotypes characterize the progressive disease in susceptible animals.

Plasmacytoid dendritic cells (pDCs) were initially considered as critical for innate immunity to viruses. However, our group has shown that pDCs bind to and inhibit the growth of Aspergillus fumigatus hyphae and that depletion of pDCs renders mice hypersusceptible to experimental aspergillosis. In this study, we examined pDC receptors contributing to hyphal recognition and downstream events in pDCs stimulated by A. fumigatus hyphae. Our data show that Dectin-2, but not Dectin-1, participates in A. fumigatus hyphal recognition, TNF-α and IFN-α release, and antifungal activity. Moreover, Dectin-2 acts in cooperation with the FcRγ chain to trigger signaling responses. In addition, using confocal and electron microscopy we demonstrated that the interaction between pDCs and A. fumigatus induced the formation of pDC extracellular traps (pETs) containing DNA and citrullinated histone H3. These structures closely resembled those of neutrophil extracellular traps (NETs). The microarray analysis of the pDC transcriptome upon A. fumigatus infection also demonstrated up-regulated expression of genes associated with apoptosis as well as type I interferon-induced genes. Thus, human pDCs directly recognize A. fumigatus hyphae via Dectin-2; this interaction results in cytokine release and antifungal activity. Moreover, hyphal stimulation of pDCs triggers a distinct pattern of pDC gene expression and leads to pET formation.

Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and presents a wide spectrum of clinical manifestations. We established a genetically controlled murine model of PCM, where A/Sn mice develop an infection which mimics the benign disease (immune responses which favor cellular immunity) and B10.A animals present the progressive disseminated form of PCM (preferential activation of B cells and impairment of cellular immune responses). To understand the immunoregulatory phenomena associated with resistance and susceptibility in experimental PCM, A/Sn and B10.A mice were studied regarding antigen-elicited secretion of monokines (TNF-alpha and TGF-beta) and type-1 (IL-2 and IFN-gamma) and type-2 (IL-4,5,10) cytokines. Total lymph node cells from resistant mice infected i.p. with P. brasiliensis produced early and sustained levels of IFN-gamma and IL-2; type-2 cytokines (IL-4 and IL-5) started to appear 8 weeks after infection. In contrast, susceptible mice produced low levels of IFN-gamma concomitant with significant levels of IL-5 and IL-10 early in the infection. In the chronic phase of the disease, susceptible animals presented a transitory secretion of IL-2, and IL-4. In the pulmonary infection IL-4, IL-5 and IL-10 were preferentially detected in the lung cells washings of susceptible animals. After in vitro challenge with fungal antigens, normal peritoneal macrophages from B10.A mice secreted high levels of TGF-beta and low levels of TNF-alpha. In contrast, macrophages from A/Sn animals released high levels of TNF-alpha associated with a small production of TGF-beta. The in vivo depletion of IFN-gamma not only abrogated the resistance of A/Sn mice but also diminished the relative resistance of B10.A animals. The in vivo depletion of IL-4 did not alter the disease outcome, whereas administration of rIL-12 significantly enhanced resistance in susceptible animals. Taken together, these results suggest that an early secretion of high levels of TNF-alpha and IFN-gamma followed by a sustained secretion of IL-2 and IFN-gamma plays a dominant role in the resistance mechanisms to P. brasiliensis infection. In contrast, an early and ephemeral secretion of low levels of TNF-alpha and IFN-gamma associated with production of IL-5, IL-10 and TGF-beta characterizes the progressive disease of susceptible animals.

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-␣), 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.