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. 2011 Aug;79(8):3141-8.
doi: 10.1128/IAI.00177-11. Epub 2011 May 31.

CD1d-independent activation of invariant natural killer T cells by staphylococcal enterotoxin B through major histocompatibility complex class II/T cell receptor interaction results in acute lung injury

Sadiye Amcaoglu Rieder  1 , Prakash Nagarkatti, Mitzi Nagarkatti
Affiliations

CD1d-independent activation of invariant natural killer T cells by staphylococcal enterotoxin B through major histocompatibility complex class II/T cell receptor interaction results in acute lung injury

Sadiye Amcaoglu Rieder et al. Infect Immun. 2011 Aug.

Abstract

There are two important mechanisms of activation of invariant natural killer T cells (iNKT cells) by microbes: direct activation of the invariant T-cell receptor (TCR) by microbial glycolipids presented by CD1d and indirect activation, mediated by the responses of antigen-presenting cells to microbes. In this study, we provide evidence for a novel CD1d-independent direct activation of iNKT cells involving a microbial protein superantigen presented in the context of major histocompatibility complex class II (MHC-II), which plays a critical role in pathogenesis, thereby redefining the role of iNKT cells. Intranasal exposure to staphylococcal enterotoxin B (SEB) in C57BL/6 wild-type mice caused acute lung injury (ALI) characterized by vascular leak, cytokine storm, and infiltration of mononuclear cells in the lungs. In contrast, the vascular leak and inflammation were decreased by ~50% in NKT cell-deficient Jα18(-/-) and CD1d(-/-) mice following SEB exposure, which was reversed following adoptive transfer of iNKT cells into CD1d(-/-) mice. In vitro, SEB could directly stimulate iNKT cells in a CD1d-independent manner via MHC-II/TCR interaction, specifically involving Vβ8. These studies not only demonstrate that iNKT cells can be activated directly by a bacterial protein superantigen independent of CD1d but also indicate that in addition to the conventional T cells, iNKT cells play a critical role in SEB-mediated ALI.

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Figures

Fig. 1.
Fig. 1.
NKT cells regulate SEB-induced vascular injury in the lungs of mice. Groups of five C57BL/6 wild-type mice were administered SEB or PBS intranasally, and vascular leak in the lungs was measured as described in Materials and Methods. (a) Analysis of vascular leak in the lungs of mice using different doses of SEB and detection of peak response at 50 μg/mouse. (b) Time course for the vascular injury induced by SEB at 24, 48, and 72 h after SEB administration. (c) Set of representative lungs from each group showing the leakage of Evans blue into the interstitium 48 h after SEB or PBS exposure. (d and e) Quantification of vascular injury in lungs of WT mice compared to Jα18−/− and CD1d −/− mice after 48 h of exposure to SEB. (f) Quantification of vascular injury in lungs of WT mice compared to Jα18−/− mice after intranasal α-GalCer (10 μg/mouse) administration (**, P < 0.01 by a Student t test; ANOVA, P < 0.001 with a Tukey test; ***, P < 0.05 versus WT SEB; ‡, P < 0.05 versus WT SEB; †, P < 0.05 versus CD1dKO SEB).
Fig. 2.
Fig. 2.
Absence of NKT cells leads to decreased infiltration in the lungs upon SEB exposure. Mice were administered 50 μg/mouse SEB through the intranasal route. (a and b) The lung sections from SEB-exposed animals were stained with H&E, and the photographs were taken at magnifications of ×4 (a) and ×100 (b). Cellular infiltration is indicated by arrows. (c) The layers of infiltrating cells were counted around 10 different capillaries from each group and averaged for quantification (*, P < 0.05 versus WT SEB by a Student t test).
Fig. 3.
Fig. 3.
Cytokine storm induced by SEB is alleviated in mice that lack NKT cells. Groups of five mice were exposed to PBS or SEB through the intranasal route, and 48 h later, IL-2, TNF-α, and IFN-γ were measured in the BALF (a, b, c) and in the serum (d) of mice (*, P < 0.05 versus WT SEB by a Student t test).
Fig. 4.
Fig. 4.
Intranasal administration of α-GalCer or SEB results in the infiltration of lymphocytes into the lungs of WT mice. (a) The lung-infiltrating cells were collected 48 h after antigen administration. The cells were then stained with CD1d/PBS-57 tetramer (iNKT cell marker) and antibodies against Vβ8 TCR. The percentages were applied to the total number of cells in each group in order to obtain the total number of each population. (b, c, and d) Absolute cell numbers in the lungs of mice; data are shown for iNKT cells, Vβ8 TCR+ cells, and iNKT cells, as indicated, that are Vβ8 TCR+ positive. (e) Flow cytometry data where the lung-infiltrating cells were stained with CD1d/PBS-57–PE tetramer and anti-Vβ8 TCR-FITC (ANOVA, P < 0.02 with a Tukey test; *, P < 0.05 versus control; ***, P < 0.01 versus control).
Fig. 5.
Fig. 5.
SEB can directly activate iNKT cells through TCR involving MHC-II but independent of CD1d. (a and b) Purity of the dendritic cells and iNKT cells was determined before the cells were placed in cocultures. iNKT cells for these experiments were purified from the spleens of Vα14 Tg mice. (c) Purified BMDCs from WT and CD1d−/− mice were mixed with purified iNKT cells from Vα14 Tg mice in the presence of α-GalCer or SEB for 72 h. Next, the culture supernatants were analyzed for IFN-γ and IL-4 (ANOVA, P < 0.005 with Tukey test *, **, ‡, P < 0.05). (d) WT BMDCs were pulsed with SEB and placed in coculture with iNKT cells in the presence of isotype control antibodies or antibodies against MHC-II or TCR (10 μg/ml) (ANOVA: *, P < 0.003 with a Tukey test; **, P < 0.05).
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