Clearance of Staphylococcus aureus nasal carriage is T cell dependent and mediated through interleukin-17A expression and neutrophil influx

Abstract

The anterior nares of humans are the major reservoir for Staphylococcus aureus colonization. Approximately 20% of the healthy human population is persistently and 80% is intermittently colonized with S. aureus in the nasal cavity. Previous studies have shown a strong causal connection between S. aureus nasal carriage and increased risk of nosocomial infection, as well as increased carriage due to immune dysfunction. However, the immune responses that permit persistence or mediate clearance of S. aureus on the nasal mucosa are fundamentally undefined. In this study, we developed a carriage model in C57BL/6J mice and showed that clearance begins 14 days postinoculation. In contrast, SCID mice that have a deficient adaptive immune response are unable to eliminate S. aureus even after 28 days postinoculation. Furthermore, decolonization was found to be T cell mediated but B cell independent by evaluating carriage clearance in T-cell receptor β/δ (TCR-β/δ) knockout (KO) and IgH-μ KO mice, respectively. Upregulation of the cytokines interleukin 1β (IL-1β), KC (also termed CXC ligand 1 [CXCL1]), and IL-17A occurred following inoculation with intranasal S. aureus. IL-17A production was crucial for clearance, since IL-17A-deficient mice were unable to effectively eliminate S. aureus carriage. Subsequently, cell differential counts were evaluated from nasal lavage fluid obtained from wild-type and IL-17A-deficient colonized mice. These counts displayed IL-17A-dependent neutrophil migration. Antibody-mediated depletion of neutrophils in colonized mice caused reduced clearance compared to that in isotype-treated controls. Our data suggest that the Th17-associated immune response is required for nasal decolonization. This response is T cell dependent and mediated via IL-17A production and neutrophil influx. Th17-associated immune responses may be targeted for strategies to mitigate distal infections originating from persistent S. aureus carriage in humans.

Fig 1

S. aureus nasal colonization is intermittent in C57BL/6J mice. C57BL/6J mice were inoculated intranasally with S. aureus clinical isolate SA1108. (A) CFU count/nose for the indicated days postinoculation. Nasal tissue was harvested at various days postinoculation, homogenized, and plated on CHROMagar-S. aureus to determine CFU counts. Detection limit = 100 CFU. (B) Colonization rate for the indicated days postinoculation. Colonization rate was determined by number of mice positive for S. aureus/total number of mice. n = 5 or 6 C57BL/6J mice per time point.

Fig 2

Decolonization is a T-cell-mediated, B-cell-independent response. C57BL/6J WT, SCID, IgH-μ KO, and TCR-β/δ KO mice were inoculated intranasally with S. aureus clinical isolate SA1108. (A) CFU count/nose for the indicated days postinoculation. Nasal tissue was harvested at 28 d.p.i., homogenized, and plated on CHROMagar-S. aureus to determine CFU counts. Detection limit = 100 CFU. (B) Colonization rate 28 days postinoculation. Colonization rate was determined by number of mice positive for S. aureus/total number of mice. n = 7 to 9 C57BL/6J mice per group. Data were combined from two independent experiments. *, P < 0.05.

Fig 3

S. aureus nasal carriage upregulates proinflammatory and Th17-associated cytokines. C57BL/6J mice were inoculated intranasally with S. aureus clinical isolate SA1108. Nasal tissue was harvested at various time points and homogenized. (A) A multiplex enzyme-linked immunosorbent assay was used to determine the cytokine response from noninoculated control and 2-, 7-, and 28-d.p.i. nasal tissue homogenates. (B to D) Levels of IL-1β, KC, and IL-17A were significantly upregulated during colonization. n = 3 to 6 C57BL/6J mice per time point. The detection range of the assay is 3.2 to 10,000 pg/ml. *, P < 0.05.

Fig 4

Clearance is IL-23 independent and relies on IL-17A. C57BL/6J WT, IL-23p19 KO, and IL-17A KO mice were inoculated intranasally with S. aureus clinical isolate SA1108. (A) CFU count/nose 28 days postinoculation. Nasal tissue was harvested at 28 d.p.i., homogenized, and plated on CHROMagar-S. aureus to determine CFU counts. Detection limit = 100 CFU. (B) Colonization rate 28 days postinoculation. Colonization rate was determined by number of mice positive for S. aureus/total number of mice. n = 7 to 9 C57BL/6J mice per group. Data were combined from two independent experiments. *, P < 0.05.

Fig 5

Neutrophil migration into the nasal lumen is IL-17A dependent and crucial for decolonization. C57BL/6J mice were inoculated intranasally with S. aureus clinical isolate SA1108. (A) Nasal lavages were performed 0, 4, 7, and 14 days postinoculation. Cell differential counts were done after cytospin of nasal lavage fluid. Cells were counted on 10 random fields of view at a magnification of ×200. Day 0 mice served as noninoculated controls. (B) C57BL/6J WT and IL-17A KO mouse nasal lavages were performed 0 and 7 days postinoculation, and cell differential counts were done to enumerate neutrophil influx. Day 0 mice served as noninoculated controls. (C and D) C57BL/6J mice were injected i.p. with either 200 μg rat anti-mouse Ly-6G or rat IgG2a isotype control antibody 1 day prior to inoculation and every 3 days after. (C) CFU count/nose 14 days postinoculation. Nasal tissue was harvested at 14 d.p.i., homogenized, and plated on CHROMagar-S. aureus to determine CFU counts. Detection limit = 100 CFU. (D) Colonization rate 14 days postinoculation. Colonization rate was determined by number of mice positive for S. aureus/total number of mice. n = 6 to 8 C57BL/6J mice per group. Data were combined from two independent experiments. *, P < 0.05; ns, not significant.