Sporotrichosis is often manifested as a chronic granulomatous infection and the monocytes/macrophages play a central role in the host defense system. Surface components of Sporothrix schenckii have been characterized and suggestions have been made as to their possible role in pathogenicity. Ergosterol peroxide, cell-wall compounds (alkali-insoluble fraction-F1 and lipid extract-LEY), and exoantigen from the yeast form of the fungus have been characterized as virulence factors, activating both innate, by cytotoxins linked to the activation of reactive oxygen and nitrogen species (H2O2 and NO), and adaptive immune response to produce cytokines Th1 and Th2 profile. In this study, preliminary results have demonstrated that, in systemic sporotrichosis, TLR-4 triggers the innate immune response, activating an oxidative burst. These data represent the first report of the participation of TLR-4 in murine sporotrichosis, in the presence of lipids from the cell wall of S. schenckii. These results taken together may open new perspectives of study leading to an antifungal agent that could be used to benefit the entire population.

In an attempt to elucidate the effects of Sporothrix schenckii infection on the immune response, our laboratory has developed a murine model of disseminated sporotrichosis. Helper T cells can be further subdivided into Th1 and Th2 phenotypes. The differentiation of two subsets of T lymphocytes is driven by IL-12 and IL-4 cytokines, respectively. Th1 cells produce IFN-gamma that activate macrophages and promote cell-mediated immunity. In addition, we found low levels of iNOS and NO production in the initial (1st and 2nd weeks) and final (9th and 10th weeks) periods of the infection, in contrast with the period of week 4 to 7 of elevated values. The determination of IFN-gamma and IL-12 are in agreement with NO/iNOS detection, showing the presence of cellular immune response throughout the infectious process. However, the production of IL-4 shows an increase in levels after the 5th and 6th weeks suggesting a participation of Th2 response in this period as well. Regarding these results, the study demonstrated that in experimental sporotrichosis infection the cellular immune response participated throughout the period analyzed as a nitric oxide dependent mechanism. In contrast, the presence of Th2 response began in the 5th week, suggesting the participation of humoral immune response in advanced stages of sporotrichosis.

SummaryToll-like receptors have been implicated in the recognition of various pathogens, including bacteria, viruses, protozoa and fungi. However, no information is available about Toll-like receptor 4 (TLR4) participation in Sporothrix schenckii recognition and the consequent triggering of the immune response to this fungal pathogen. Following activation of TLRs by ligands of microbial origin, several responses are provoked, including reactions in immune cells that may lead them to produce signalling factors that trigger inflammation. The present study was designed to elucidate the role of TLR4 during the host response to S. schenckii. TLR4-deficient (C3H/HeJ) and control mice (C3H/HePas) were infected with S. schenckii yeast cells and immune response was assessed over 10 weeks by assaying production of pro-inflammatory mediator (nitric oxide and tumour necrosis factor-a) and anti-inflammatory cytokine (interleukin-10) by peritoneal macrophages and their correlation with apoptosis in peritoneal exudate cells. We found that both pro-inflammatory and anti-inflammatory mediators are reduced in TLR4-deficient mice, suggesting the involvement of this receptor in the recognition of this infectious agent. Translocation into the nucleus of nuclear transcription factor, nuclear factor-jB, was also evaluated and showed higher levels in TLR-4 normal mice, consistent with the results found for cytokine production. We are showing here, for the first time, the involvement of TLR4 in S. schenckii recognition. Taken together, our results demonstrate that the activation of peritoneal macrophages in response to S. schenckii lipid extracts has different responses in these two mouse strains which differ in TLR4 expression, suggesting an important role for TLR4 in governing the functions of macrophages in this fungal infection.

For many fungal diseases, macrophages are the major cell population implicated in host protection, primarily by their ability to eliminate the invading fungal pathogen through phagocytosis. In sporotrichosis, this remains true, because of macrophages’ ability to recognize Sporothrix schenckii through specific receptors for some of the fungus’ cellular surface constituents. Further confirmation for macrophages’ pivotal role in fungal diseases came with the identification of toll-like receptors, and the subsequent numerous associations found between TLR-4 deficiency and host susceptibility to diverse fungal pathogens. Involvement of TLR-4 in immune response against sporotrichosis has been conducted to investigate how TLR-4 signaling could affect inflammatory response development through evaluation of H2O2 production and IL-1β, IL-6 and TGF-β release during the course of S. schenckii infection on TLR-4-deficient mice. The results showed that macrophages are largely dependent on TLR-4 for inflammatory activation and that in the absence of TLR-4 signaling, increased TGF-β release may be one of the contributing factors for the abrogated inflammatory activation of peritoneal exudate cells during mice sporotrichosis.