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Invariant Natural Killer T (iNKT) cells are key players in the immunity to several pathogens; however, their involvement in the resistance to Paracoccidioides brasiliensis infection remains unknown. Using splenocytes from CD1d (CD1d-/-) and iNKT-deficient (Jα18-/-) mice, we found that iNKT cells are the innate source of IFN-γ after P. brasiliensis infection and are required to potentiate macrophage oxidative burst and control fungal growth. To determine whether iNKT cells contribute in vivo to host resistance against P. brasiliensis infection, we infected intratracheally wild-type and Jα18-/- C57BL/6 mouse strains with the virulent Pb18 isolate. iNKT cell deficiency impaired the airway acute inflammatory response, resulting in decreased airway neutrophilia and reduced IFN-γ, KC, and nitric oxide (NO) production. The deficient innate immune response of Jα18-/- mice to Pb18 infection resulted in increased fungal burden in the lungs and spleen. Besides, the activation of iNKT cells in vivo by administration of the exogenous iNKT ligand α-galactosylceramide (α-GalCer) improved host resistance to P. brasiliensis infection. Although the mechanisms responsible for this phenomenon remain to be clarified, α-GalCer treatment boosted the local inflammatory response and reduced pulmonary fungal burden. In conclusion, our study is the first evidence that iNKT cells are important for the protective immunity to P. brasiliensis infection and their activation by an exogenous ligand is sufficient to improve the host resistance to this fungal infection.

Granzyme A (GzmA) is secreted by cytotoxic lymphocytes and has traditionally been viewed as a mediator of cell death. However, a growing body of data suggests the physiological role of GzmA is promotion of inflammation. Here, we show that GzmA is significantly elevated in the sera of chikungunya virus (CHIKV) patients and that GzmA levels correlated with viral loads and disease scores in these patients. Serum GzmA levels were also elevated in CHIKV mouse models, with NK cells the likely source. Infection of mice deficient in type I interferon responses with CHIKV, Zika virus, or dengue virus resulted in high levels of circulating GzmA. We also show that subcutaneous injection of enzymically active recombinant mouse GzmA was able to mediate inflammation, both locally at the injection site as well as at a distant site. Protease activated receptors (PARs) may represent targets for GzmA, and we show that treatment with PAR antagonist ameliorated GzmA- and CHIKV-mediated inflammation.

A promising alternative vaccine candidate to reduce the burden of pneumococcal diseases is the protein antigen PspA (Pneumococcal surface protein A). Since concomitant colonization with two or more pneumococcal strains is very common in children, we aimed to determine if immunization with PspA would be able to control co-colonization. We evaluated nasal immunization with recombinant PspA (rPspA) in a model of co-colonization with two strains expressing different PspAs. Mice were immunized intranasally with rPspAs from clades 1 to 4 (rPspA1, rPspA2, rPspA3 or rPspA4) using whole-cell pertussis vaccine (wP) as adjuvant. Mice were then challenged with a mixture of two serotype 6B isolates St491/00 (PspA1) and St472/96 (PspA4). Immunization with rPspA1+wP and rPspA4+wP reduced colonization with both strains and the mixture of rPspA1+rPspA4+wP induced greater reduction than a single antigen. Immunization rPspA1+rPspA4+wP also reduced colonization when challenge experiments were performed with a mixture of isolates of serotypes 6B (PspA3) and 23F (PspA2). Furthermore, none of the tested formulations led to a pronounced increase in colonization of one isolate over the other, showing that the vaccine strategy would not favor replacement. Interestingly, the adjuvant wP by itself already led to some reduction in pneumococcal colonization, indicating the induction of non-specific immune responses. Anti-rPspA IgG was observed in serum, nasal wash (NW) and bronchoalveolar lavage fluid (BALF) samples, whereas animals inoculated with formulations containing the adjuvant wP (with or without rPspA) showed higher levels of IL-6 and KC in NW and increase in tissue macrophages, B cells and CD4+T cells in BALF.

Paracoccidioidomycosis, a primary fungal infection restricted to Latin America, is acquired by inhalation of fungal particles. The immunoregulatory mechanisms that control the severe and mild forms of paracoccidioidomycosis are still unclear. Indoleamine 2,3-dioxygenase (IDO), an IFN-γ induced enzyme that catalyzes tryptophan metabolism, can control host-pathogen interaction by inhibiting pathogen growth, T cell immunity and tissue inflammation.In this study, we investigated the role of IDO in pulmonary paracoccidioidomycosis of susceptible and resistant mice. IDO was blocked by 1-methyl-dl-tryptophan (1MT), and fungal infection studied in vitro and in vivo. Paracoccidioides brasiliensis infection was more severe in 1MT treated than untreated macrophages of resistant and susceptible mice, concurrently with decreased production of kynurenines and IDO mRNA. Similar results were observed in the pulmonary infection. Independent of the host genetic pattern, IDO inhibition reduced fungal clearance but enhanced T cell immunity. The early IDO inhibition resulted in increased differentiation of dendritic and Th17 cells, accompanied by reduced responses of Th1 and Treg cells. Despite these equivalent biological effects, only in susceptible mice the temporary IDO blockade caused sustained fungal growth, increased tissue pathology and mortality rates. In contrast, resistant mice were able to recover the transitory IDO blockade by the late control of fungal burdens without enhanced tissue pathology.Our studies demonstrate for the first time that in pulmonary paracoccidioidomycosis, IDO is an important immunoregulatory enzyme that promotes fungal clearance and inhibits T cell immunity and inflammation, with prominent importance to susceptible hosts. In fact, only in the susceptible background IDO inhibition resulted in uncontrolled tissue pathology and mortality rates. Our findings open new perspectives to understand the immunopathology of paracoccidioidomycosis, and suggest that an insufficient IDO activity could be associated with the severe cases of human PCM characterized by inefficient fungal clearance and excessive inflammation.