Sporotrichosis is a subcutaneous mycosis caused by several closely related thermo-dimorphic fungi of the Sporothrix schenckii species complex, affecting humans and other mammals. In the last few years, new strategies have been proposed for controlling sporotrichosis owning to concerns about its growing incidence in humans, cats, and dogs in Brazil, as well as the toxicity and limited efficacy of conventional antifungal drugs. In this study, we assessed the immunogenicity and protective properties of two aluminum hydroxide (AH)-adsorbed S. schenckii cell wall protein (ssCWP)-based vaccine formulations in a mouse model of systemic S. schenckii infection. Fractioning by SDS-PAGE revealed nine protein bands, two of which were functionally characterized: a 44kDa peptide hydrolase and a 47kDa enolase, which was predicted to be an adhesin. Sera from immunized mice recognized the 47kDa enolase and another unidentified 71kDa protein, whereas serum from S. schenckii-infected mice recognized both these proteins plus another unidentified 9.4kDa protein. Furthermore, opsonization with the anti-ssCWP sera led to markedly increased phagocytosis and was able to strongly inhibit the fungus' adhesion to fibroblasts. Immunization with the higher-dose AH-adjuvanted formulation led to increased ex vivo release of IL-12, IFN-γ, IL-4, and IL-17, whereas only IL-12 and IFN-γ were induced by the higher-dose non-adjuvanted formulation. Lastly, passive transference of the higher-dose AH-adjuvanted formulation's anti-ssCWP serum was able to afford in vivo protection in a subsequent challenge with S. schenckii, becoming a viable vaccine candidate for further testing.

Sporotrichosis is a mycosis caused by fungi from the Sporothrix schenckii species complex, whose prototypical member is Sporothrix schenckii sensu stricto. Pattern recognition receptors (PRRs) recognize and respond to pathogen-associated molecular patterns (PAMPs) and shape the following adaptive immune response. A family of PRRs most frequently associated with fungal recognition is the nucleotide-binding oligomerization domain-like receptor (NLR). After PAMP recognition, NLR family pyrin domain-containing 3 (NLRP3) binds to apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 to form the NLRP3 inflammasome. When activated, this complex promotes the maturation of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 and cell death through pyroptosis. In this study, we aimed to evaluate the importance of the NLRP3 inflammasome in the outcome of S. schenckii infection using the following three different knockout (KO) mice: NLRP3 , ASC and caspase-1 . All KO mice were more susceptible to infection than the wild-type, suggesting that NLRP3-triggered responses contribute to host protection during S. schenckii infection. Furthermore, the NLRP3 inflammasome appeared to be critical for the ex vivo release of IL-1β, IL-18 and IL-17 but not interferon-γ. Additionally, a role for the inflammasome in shaping the adaptive immune response was suggested by the lower frequencies of type 17 helper T (Th17) cells and Th1/Th17 but not Th1 cells in S. schenckii-infected KO mice. Overall, our results indicate that the NLRP3 inflammasome links the innate recognition of S. schenckii to the adaptive immune response, so contributing to protection against this infection.

Little is known about the differences in the CD4þ T-cell response induced by Sporothrix schenckii and Sporothrix brasiliensis, the most virulent species that cause sporotrichosis. Here, the helper (Th) and regulatory T cells (Tregs) responses were evaluated comparatively in a murine model of sporotrichosis on days 7, 21 and 35 after subcutaneous infection with either S. schenckii or S. brasiliensis conidia. The fungal load was measured at the site of infection, as well as in the liver and spleen. The Th1/Th17/Tregs responses were analyzed in the spleen, while the level of IL-2, IL-4, IL-6, TNF-alpha, IFN-ɣ, IL-17A and IL-10 cytokines were measured at the local site of infection on 24 h postinfections and in sera on the indicated days. S. brasiliensis caused a longer-lasting infection in the skin and chronic systemic dissemination associated to more severe granulomatous lesions. Similar Th1/Th1-Th17/Tregs responses were induced by both S. brasiliensis and S. schenckii on 7th and 21st d.p.i but on 35 d.p.i a reduction of Th1 and Th1-Th17 cells, associated to higher values of Th17/Tregs cells was observed only in S. brasiliensis-infected mice. In summary, S. brasiliensis caused a more severe disease associated with sustained Th17/Tregs responses than S. schenckii in mice.

The available information about the role of Dectin-1 in sporotrichosis is scarce. Hence, we aimed to assess Dectin-1 expression by macrophages and the activation of some related antifungal mechanisms during the Sporothrix schenckii sensu stricto infection as a first attempt to elucidate the role of this receptor in sporotrichosis. Balb/c mice were intraperitoneally infected with S. schenckii sensu stricto yeast ATCC 16345 and euthanized on days 5, 10 and 15 post-infection, when the following parameters were evaluated: fungal burden in spleen, Dectin-1 expression and nitric oxide (NO) production by peritoneal macrophages, as well as IL-1β, TNF-α and IL-10 ex vivo secretion by these same cells. Peritoneal macrophages were ex vivo challenged with either the alkali-insoluble fraction (F1) extracted from the S. schenckii cell wall, a commercially available purified β-1,3-glucan or whole heat-killed S. schenckii yeasts (HKss). Additionally, a Dectin-1 antibody-mediated blockade assay was performed on day 10 post-infection to assess the participation of this receptor in cytokine secretion. Our results showed that Dectin-1 expression by peritoneal macrophages was augmented on days 10 and 15 post-infection alongside elevated NO production and ex vivo secretion of IL-10, TNF-α and IL-1β. The antibody-mediated blockade of Dectin-1 inhibited cytokine production in both infected and non-infected mice, mainly after β-1,3-glucan stimulation. Our results suggest a role for Dectin-1 in triggering the immune response during S. schenckii infection.

Sporotrichosis is a subcutaneous mycosis that has re-emerged in several tropical and subtropical regions over the last decades. Growing findings suggest that the interplay of host, pathogen, and environment has a determinant effect on the diversity, local distribution, and virulence of Sporothrix schenckii sensu lato, the etiologic agent. Among the environmental factors, we have studied the potential role of repeated exposures to mercury (Hg), a known immunotoxic xenobiotic that is widely used in gold mining regions where sporotrichosis outbreaks are frequently reported. In this study, male Swiss mice received subcutaneous injections of either 300 or 1200 µg/kg of mercury (II) chloride (HgCl2) for 14 days, three times a week. A control group was injected with the vehicle Phosphate Buffered Saline (PBS). Treatment with HgCl2 impaired several immunologic parameters that are involved in host response to Sporothrix infection, such as the production of TNFα, IL-1, and nitric oxide by macrophages, and Th1/Th2/Th17 populations and their respective cytokines. The consequences of these effects on the host resistance to S. schenckii infection were subsequently evaluated. Hg-exposed mice exhibited a higher fungal load in the fungal inoculation site associated to systemic dissemination to spleen and liver on 14 days post-infection and a higher production of specific IgG1 and mild reduction of IgG2a. These findings suggest that repeated exposition to Hg enhances susceptibility to S. schenckii infection in mice and can be a factor associated to sporotrichosis outbreaks in endemic and highly Hg-polluted areas.

The response of hydrogen peroxide (H 2 O 2) and cytokines during an experimental sporotrichosis in male Swiss mice was assessed over a period of 10 weeks by monitoring macrophage activation challenged with exoantigen (ExoAg) from the fungus Sporothrix schenckii. The studied endpoints were: H 2 O 2 production, fungal burden at spleen, apoptosis in peritoneal macrophages, and IL-1b, IL-6, IL-2, IL-10 production. During the two first weeks of infection was observed low burden of yeast in spleen and high response of H 2 O 2 , IL-2, and IL-1b. The weeks of highest fungal burden (fourth-sixth) coincided with major apoptosis in peritoneal macrophages, normal production of IL-6 and lower production of H 2 O 2 , IL-2, and IL-1b, suggesting a role for these three last in the early control of infection. On the other hand, IL-1b (but not IL-6) was recovered since the sixth week, suggesting a possible role in the late phase of infection, contributing to the fungal clearance in conjunction with the specific mechanisms. The IL-10 was elevated until the sixth, principally in the second week. These results evidences that ExoAg is involved in the host immune modulation, influencing the S. Schenckii virulence, and its role is related with the time of the infection in the model used.

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24Environmental factors modify the physiology of microorganisms, allowing their survival in 25 extreme conditions. However, the influence of chemical contaminants on fungal virulence has 26 been little studied. Sporotrichosis is an emergent fungal disease caused by Sporothrix schenckii, 27 a soil-inhabiting fungus that has been found in polluted environments. Here, we evaluated the 28 adaptive stress response of S. schenckii induced by toluene, a key soil contaminant. The effect 29 on fungal virulence and host immune response was also assessed. The fungus survived up to 30 0.10% toluene in liquid medium. Greater production of melanosomes and enhanced activity 31 superoxide dismutase, associated to increased tolerance to H2O2 were observed in toluene-32 exposed fungi. Intraperitoneal infection of mice with S. schenckii treated with either 0, 0.01 or 33 0.1% of toluene, resulted in greater fungal burden at day 7 post-infection in spleen and liver in 34 the groups infected with fungus treated with toluene 0.1%. A higher production of Il-1β, TNF-35 α, IL-10 and nitric oxyde by peritoneal macrophages and IFNɣ, IL-4 and IL-17 by splenocytes 36 was also observed in that group. Our findings showed that morphological and functional 37 changes induced by toluene leads to increased S. schenckii virulence and antifungal host 38 immune response in our model. 39 40 41 42 43 44 45 78 Aromatic compounds such as benzene, toluene, ethylbenzene and xylene isomers, 79 collectively known as BTEX, are one of the major contributors to environmental pollution. 80 BTEX are extensively used as solvents in many industrial processes and as base reagents for 81 the production of chemicals products. In addition, BTEX are components of gasoline and 82 aviation fuels. They are often released in the environment during production, transport, use and 83 disposal, causing groundwater, surface water, and soil contamination. Toluene has been found 84 in at least 1,012 of the 1,699 National Priorities List sites identified by the Environmental 85 Protection Agency (EPA) and it is included in the Agency for Toxic Substances and Disease 86 Registry (ATSDR) 2017 substance priority list, which is based on a combination of their 87 frequency, toxicity, and potential for human exposure (Agency for Toxic Substances and 88 Disease Registry. ATSDR's Substance Priority List. 2017).89Prenafreta-Boldu isolated several fungi, including S. schenckii, in biofilters exposed to 90 polluted air or hydrocarbon gas streams. The data from this research showed that volatile 91 hydrocarbon-degrading strains are closely related to a very restricted number of pathogenic 92 fungal species that cause severe mycoses, especially neurological infections, in 93 immunocompetent individuals (Prenafeta-Boldú et al., 2006). 94To date, there have been no studies examining the influence of chemical contaminants 95 on the biology of S. schenckii sensu latu. Given that this fungus inhabits soils and this niche is 96 frequently contaminated with oil and their products, the objective of this...