Type II Natural Killer T Cells Contribute to Protection Against Systemic Methicillin-Resistant Staphylococcus aureus Infection

Abstract

Methicillin-resistant Staphylococcus aureus (SA) bacteremia is responsible for over 10,000 deaths in the hospital setting each year. Both conventional CD4⁺ T cells and γδ T cells play protective roles in SA infection through secretion of IFN-γ and IL-17. However, the role of other unconventional T cells in SA infection is largely unknown. Natural killer T (NKT) cells, a subset of innate-like T cells, are activated rapidly in response to a wide range of self and microbial lipid antigens presented by MHC I-like molecule CD1d. NKT cells are divided into two groups, invariant NKT (iNKT) and type II NKT cells, based on TCR usage. Using mice lacking either iNKT cells or both types of NKT cells, we show that both NKT cell subsets are activated after systemic SA infection and produce IFN-γ in response to SA antigen, however type II NKT cells are sufficient to control bacterial burden and inflammatory infiltrate in infected organs. This protective capacity was specific for NKT cells, as mice lacking mucosal associated invariant T (MAIT) cells, another innate-like T cell subset, had no increased susceptibility to SA systemic infection. We identify polar lipid species from SA that induce IFN-γ production from type II NKT cells, which requires both CD1d-TCR engagement and IL-12 production by antigen presenting cells. We also demonstrate that a population of T cells enriched for type II NKT cells are increased in PBMC of SA bacteremic patients compared to healthy controls. Therefore, type II NKT cells perform effector functions that enhance control of SA infection prior to conventional T cell activation and recognize SA-derived lipid antigens. As CD1d is highly conserved in humans, these CD1d-restricted SA lipid antigens could be used in the design of next generation SA vaccines targeting cell-mediated immunity.

Keywords: CD1; Staphylococcus aureus; cytokine; knockout mice; lipid antigens; natural killer T cells.

Figure 1

Mice lacking NKT cells have increased bacterial burdens and neutrophilic infiltrate in SA infected organs. (A, B) Colony forming units (CFU) quantified in liver (A) and kidneys (B) of B6 and CD1d^-/- mice at various times post infection (N=6–8 mice/genotype/timepoint). (C, D) CFU quantified in liver (C) and kidneys (D) of MR1^+/+ and MR1^-/- mice at various times post infection (N=4-7 mice/genotype/timepoint). (E, F) Total number of neutrophils in the liver (E) and kidney (F) of infected mice (liver: N=3 naïve, N=14 4 dpi/genotype, kidney: N=4 naïve, N=11-12 4 dpi/genotype). Statistical analysis: (A–D) Mann-Whitney test; (E, F) 2-way ANOVA.

Figure 2

Type II NKT cells are sufficient to control SA growth and mediate cytokine production in infected organs. (A, B) CFU quantified in the liver (A) and kidney (B) of indicated mice at 4 dpi: liver (N=6–13 mice/genotype), kidney (N=6–8 mice/genotype) (X=below limit of detection). (C, D) H&E staining of kidney sections at 4 dpi, inflammatory foci area quantified in (D), (N=6-7 mice/genotype). (E, F) IFN-γ ELISA of liver (E) and spleen (F) lymphocytes from 4 dpi mice pulsed with HKSA, unstimulated not graphed (undetectable) (N=2–4 naïve, N=7-10 infected mice/genotype). Statistical analysis: (A, B, D) Mann-Whitney test, (E, F) 2-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3

iNKT and type II NKT cells are expanded and activated after SA infection. (A, B) Total number of iNKT and type II NKT cells in liver at various times post infection (N=4–5 naïve, N=4–8 infected mice/timepoint). (C, D) CD69 mean fluorescence intensity (MFI) of iNKT cells (C) and type II NKT cells (D) from the liver of SA-infected B6 mice or Jα18^-/- mice, respectively (N=4 naïve, N=4–6 infected mice/timepoint). (E–G) Ki67 expression in iNKT and type II NKT cells from SA-infected B6 liver. Representative Ki67 dot plot (E), quantified as % of iNKT cells (F) and type II NKT cells (G) (N=4 naïve, N=3–7 infected mice/timepoint). Statistical analysis: (A, C–G) one-way ANOVA, (B) 2-way ANOVA. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

iNKT and type II NKT cells produce IFN-γ after SA infection. (A, B) Representative FACS plots of IFN-γ production of iNKT (A) and type II NKT (B) cells at 1 dpi from B6 liver, cultured directly ex vivo. (C, D) Quantified % cytokine production, gated on iNKT cells (C) and type II NKT cells (D). (N=3–6 naïve, N=5–12 infected mice). (E) rtPCR of cytokine mRNA of type II NKT cells sorted from 4 dpi Jα18^-/- mouse pooled liver lymphocytes (N=10 mice) compared to sorted conventional CD4⁺NK1.1^- T cells from Jα18^-/- splenocytes (N=2 mice), data represented as cytokine mRNA levels relative to β-actin. Statistical analysis: (C–E) 2-way ANOVA. *p < 0.05; ***p < 0.001; ****p < 0.0001.

Figure 5

Type II NKT cells require CD1d to recognize total SA lipids. (A, B) IFN-γ (A) and IL-17A (B) ELISPOT of Jα18^-/- total liver lymphocytes collected from 4 dpi livers and co-cultured with MHC-II^-/- or MHC-II^-/-CD1d^-/- DCs, unpulsed or pulsed overnight with 10 µg/ml total SA lipids (SAlip) (N=4 mice/experiment, representative 1 of 3). Statistical analysis: (A, B) 2-way ANOVA.***p < 0.001; ****p < 0.0001.

Figure 6

Type II NKT cells produce MyD88-dependent CD1d-restricted IFN-γ to polar SA lipids. (A) List of SA lipid fractions, isolated from total SA lipids using silica gel column chromatography and chloroform-methanol gradient in order of increasing polarity, PG=phosphatidylglycerol, DPG= diacylphosphatidylglycerol, MGDG= monogalactosyldiacylglycerol. (B–D) ELISPOT of IFN-γ production by T cells enriched from 4 dpi Jα18^-/- liver lymphocytes: (B, C) MHC-II^-/- and MHC-II^-/-CD1d^-/- DCs or (D) B6 and MyD88^-/- DCs +/- IL-12 blocking antibody pulsed with total SA lipids and total SA lipid fractions (FR-1 to FR-9, 10 µg/ml pulsed overnight). (B) N=5–7 pooled mice, 2/3 replicates per condition, representative of 2 experiments. Statistical analysis: (B–D) 2-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 7

Type II NKT cells protect mice from systemic SA infection. (A) IFN-γ ELISPOT of naïve 24α⁺β⁺ lymphocytes from liver co-cultured with B6 DCs unpulsed or pulsed with total SA lipids (SAlip) (10 µg/ml). (B) Schematic of type II NKT cell adoptive transfer experiment. (C) Bacterial CFU in the spleen of recipient mice at 2 dpi, PBS= control group (D, E). Representative FACS plots of adoptively transferred 24α⁺β⁺ NKT cells from spleen of 2 dpi recipient, % IFN-γ production after stimulation with PMA/Ionomycin or unstimulated (2 h + 4 h BFA), quantified in E (N=8–10 mice/group). Statistical analysis: (A) one-way ANOVA, (C) Mann-Whitney test, (E) 2-way ANOVA. *p < 0.05; **p < 0.01.

Figure 8

Type II NKT cells expanded in SA bacteremic mice and human patients. (A) Percentage of iNKT cells in blood of B6 and CD1d^-/- mice. (B) Percentage of type II NKT cells in blood of B6 and CD1d^-/- mice, (A, B) N=7 naïve, three to four infected mice/genotype. (C) Percentage of iNKT cells in SA infected (SA) and healthy control (21) PBMC. (D) Percentage of type II NKT cells in SA and HC PBMC. (E) Percentage of MAIT cells in SA and HC PBMC, (C–E) N=8–9 HC, seven SA patients. Statistical analysis: (A–C, E) 2-way ANOVA, (D) student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.