Invariant Natural Killer T Cells as Key Players in Host Resistance against Paracoccidioides brasiliensis

Abstract

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.

Figure 1

The invariant Natural Killer T cells drive P. brasiliensis killing by macrophages and respond for the innate source of IFN-γ upon fungal infection. Thioglycolate-elicited peritoneal MΦ from C57Bl/6 mice were infected with Pb18 yeast (1 : 10) for 2 h. After washing, splenocytes (Spln) from WT, CD1d^−/−, or Jα18^−/− C57BL/6 mouse strains were added to the MΦ culture (10 : 1) for 48 h. After this period, supernatant was collected, and adherent cells were lysed with distilled water for fungal recovery. (a) Number of viable cell yeast obtained by colony-forming units assay (CFU). (b) NO levels in culture supernatants. (c) IFN-γ levels in the culture supernatants (ND = not detected). Data represent the mean ± SD of quintuplicate samples from 1 of 2 independent experiments (a, b) and of 1 independent experiment (c). ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗∗p < 0.0001.

Figure 2

Deficiency in invariant Natural Killer T lymphocytes impairs the acute inflammatory response against P. brasiliensis infection. WT and Jα18^−/− C57BL/6 mouse strains were infected with 1 x 10⁶ Pb18 yeasts, and 72 h later, the BAL content was analyzed for: (a) total number of cells; (b) presence of mononuclear cells and neutrophils; BAL levels of (c) IFN-γ, (d) KC, and (e) NO. Data represent the mean ± SD from 1 of 2 independent experiments (n = 4 − 5/group). ^∗p < 0.05; ^∗∗p < 0.005.

Figure 3

Deficiency in invariant Natural Killer T cells impairs early CD8 T lymphocyte activation during P. brasiliensis infection. WT and Jα18^−/− C57BL/6 mouse strains were infected with 1 x 10⁶ Pb18 yeasts, and 72 h later, the lung parenchyma was analyzed for: (a) frequency of CD4⁺ and CD8⁺ T lymphocytes and (b) frequency of CD69-expressing CD4 and CD8 T cells. Data represent the mean ± SD from 1 of 2 independent experiments (n = 5/group). ^∗∗∗∗p < 0.0001.

Figure 4

iNKT cells deficiency is associated with the accumulation of suppressor myeloid-derived cells in the lungs of P. brasiliensis infected mice. WT and Jα18^−/− C57BL/6 mouse strains were infected with 1 x 10⁶ Pb18 yeasts, and 72 h later, the lung parenchyma was analyzed for: Frequency of (a) GR1⁺CD11b⁺SCC^high and (b) GR1⁺CD11b⁺SCC^low myeloid-derived cells. Frequency of MHC-II-expressing cells within (b) GR1⁺CD11b⁺SCC^high and (e) GR1⁺CD11b⁺SCC^low subsets and MHC-II surface expression level (c, f, respectively). Expression levels of CD11b molecules within the (g) GR1⁺CD11b⁺SCC^high and (h) GR1⁺CD11b⁺SCC^low myeloid-derived cells subsets. Data represent the mean ± SD from 1 of 2 independent experiments (n = 5/group). ^∗p < 0.05.

Figure 5

Invariant Natural Killer T cells restrain P. brasiliensis growth and dissemination. WT and Jα18^−/− C57BL/6 mouse strains were infected with 1 x 10⁶ Pb18 yeast, and 45 days later, the fungal loads were determined in the lungs (a), spleen (b), and liver (c). Data represent the mean ± SD from 1 of 2 independent experiments (n = 5/group). ^∗p < 0.05.

Figure 6

Treatment of P. brasiliensis infected mice with α-GalCer, an iNKT specific agonist, enhances host resistance to P. brasiliensis infection. WT C57BL/6 mice were infected with 1 x 10⁶ Pb18 yeasts and treated four weeks later with 10 μg of α-GalCer by the i.v. route. At week 8 after infection, the number of inflammatory cells in the BAL and the number of viable yeasts in the lung parenchyma of α-GalCer-treated and untreated mice were determined. (a) Total number of inflammatory cells in BAL. (b) Mononuclear and neutrophil cell counts in the airways. (c) Fungal loads in lung parenchyma. Data represent the mean ± SD from 1 independent experiment (n = 5/group). ^∗p < 0.05.