Allergy Asthma Immunol Res.  2012 Sep;4(5):295-304. 10.4168/aair.2012.4.5.295.

TLR4, 5, and 9 Agonists Inhibit Murine Airway Invariant Natural Killer T Cells in an IL-12-Dependent Manner

• Shim JU
• Rhee JH
• Koh YI

• KMID: 2167049
• DOI: http://doi.org/10.4168/aair.2012.4.5.295

Abstract

PURPOSE
Invariant natural killer T (iNKT) cells may play an important role in the pathogenesis of asthma in mice and humans. Thus, an agent that modulates the function of iNKT cells may have therapeutic potential to control asthma. We hypothesized that lipopolysaccharide (LPS)-, flagellin-, or CpG-induced changes in the cytokine milieu may modify and even inhibit the function of airway iNKT cells in asthma.
METHODS
Because increased alpha-galactosylceramide (GalCer)-induced airway hyperreactivity (AHR) reflects the presence of airway iNKT cells, alpha-GalCer-induced AHR, as well as inflammatory cells and cytokines in bronchoalveolar lavage (BAL) fluid, were determined 24 hours after in vivo treatment with LPS, flagellin, or CpG in naive BALB/c mice. Intracellular IL-4 and IFN-gamma were measured in spleen iNKT cells after in vitro treatment with LPS, flagellin, or CpG. A role for IL-12 following the treatments was determined.
RESULTS
Intranasal administration of LPS, flagellin, or CpG reduced development of alpha-GalCer-induced AHR, eosinophilic airway inflammation, and Th1 and Th2 cytokine responses in BAL fluid, while producing IL-12 in BAL fluid. Intraperitoneal administration of IL-12 mAb blocked the suppressive effect of LPS, flagellin, or CpG. In vitro treatment with LPS, flagellin, or CpG reduced production of IL-4 and IFN-gamma from alpha-GalCer-stimulated spleen iNKT cells; these effects were ameliorated by addition of anti-IL-12 mAb.
CONCLUSIONS
TLR4, 5, and 9 agonists may suppress the function of airway and spleen iNKT cells via IL-12-dependent mechanisms. Anergy of iNKT cells by IL-12 might play a role in suppression by these TLR agonists.

Keyword

CpG; flagellin; lipopolysaccharide; natural killer T cells

MeSH Terms

Administration, Intranasal
Animals
Asthma
Bronchoalveolar Lavage
Cytokines
Eosinophils
Flagellin
Galactosylceramides
Humans
Inflammation
Interleukin-12
Interleukin-4
Mice
Natural Killer T-Cells
Spleen
Cytokines
Flagellin
Galactosylceramides
Interleukin-12
Interleukin-4

Figure

• 

  Fig. 1 Effect of lipopolysaccharide (LPS) on α-galactosylceramide (GalCer)-induced airway hyperreactivity (AHR), airway inflammation, and cytokine production. Naïve mice were intranasally administered α-GalCer or phosphate-buffered saline (PBS). Twenty-four hours later, AHR to methacholine (A), inflammatory cells (B), and cytokines (C, D, and E) in bronchoalveolar lavage (BAL) fluid were measured. LPS was intranasally administered 24 hr before α-GalCer administration. LPS alone denotes administration of LPS without α-GalCer. Enhanced pause (Penh) was expressed as the percentage increase in Penh, where Penh after PBS represented 100%. Total cells, macrophages (Mac), eosinophils (Eos), neutrophils (Neut), and lymphocytes (Lymph) were expressed as the number per mL BAL fluid. Data are expressed as the mean±standard error of the mean (SEM; n=5). *P<0.01 for α-GalCer vs. PBS; †P<0.05 and ‡P<0.01 for LPS + α-GalCer vs. α-GalCer; and §P<0.05 and ∥P<0.01 for LPS alone vs. α-GalCer.

• 

  Fig. 2 Effect of flagellin B (FlaB) on α-GalCer-induced AHR, airway inflammation, and cytokine production. Naïve mice were intranasally administered α-GalCer or PBS. Twenty-four hours later, AHR to methacholine (A), inflammatory cells (B), and cytokines (C, D, and E) in BAL fluid were measured. FlaB was intranasally administered 24 hr before α-GalCer administration. FlaB alone denotes the administration of FlaB without α-GalCer. Penh was expressed as the percentage increase in Penh, where Penh after PBS represented 100%. Total cells, Mac, Eos, Neut, and Lymph were expressed as the number per mL BAL fluid. Data are expressed as the mean±SEM (n=5). *P<0.05 and †P<0.01 for α-GalCer vs. PBS; ‡P<0.05 and §P<0.01 for FlaB + α-GalCer vs. α-GalCer; and ∥P<0.05 and ¶P<0.01 for FlaB alone vs. α-GalCer.

• 

  Fig. 3 Effect of CpG on α-GalCer-induced AHR, airway inflammation and cytokine production. Naïve mice were intranasally administered α-GalCer or PBS. Twenty-four hours later, AHR to methacholine (A), inflammatory cells (B), and cytokines (C, D, and E) in BAL fluid were measured. CpG was intranasally administered 24 hr before α-GalCer administration. CpG alone denotes the administration of FlaB without α-GalCer. Penh was expressed as the percentage increase in Penh, where Penh after PBS represented 100%. Total cells, Mac, Eos, Neut, and Lymph were expressed as the number per mL BAL fluid. Data are expressed as the mean±SEM (n=5). *P<0.05 and †P<0.01 for α-GalCer vs. PBS; ‡P<0.05 and §P<0.01 for CpG + α-GalCer vs. α-GalCer; and ∥P<0.05 and ¶P<0.01 for CpG alone vs. α-GalCer.

• 

  Fig. 4 Blocking effect of IL12 mAb on the suppressive effect of lipopolysaccharide (LPS) on α-GalCer-induced AHR, airway inflammation and cytokine production. As described in Fig. 1, AHR to methacholine (A), inflammatory cells (B) and cytokines (C, D, and E) in BAL fluid were measured. IL-12 mAb or the IgG2b isotype was intraperitoneally administered immediately before LPS administration. Penh was expressed as the percentage increase in Penh, where Penh after PBS represented 100%. Total cells, Mac, Eos, Neut, and Lymph were expressed as the number per mL BAL fluid. Data are expressed as the mean±SEM (n=5). *P<0.05 and †P<0.01 for anti-IL-12 or the isotype vs. LPS + α-GalCer.

• 

  Fig. 5 Blocking effect of IL12 mAb on the suppressive effect of FlaB on α-GalCer-induced AHR, airway inflammation, and cytokine production. As described in Fig. 2, AHR to methacholine (A), inflammatory cells (B) and cytokines (C, D, and E) in BAL fluid were measured. IL-12 mAb or the IgG2b isotype was intraperitoneally administered immediately before FlaB administration. Penh was expressed as the percentage increase in Penh, where Penh after PBS represented 100%. Total cells, Mac, Eos, Neut, and Lymph were expressed as the number per mL BAL fluid. Data are expressed as the mean±SEM (n=5). *P<0.05 and †P<0.01 for anti-IL-12 or the isotype vs. FlaB + α-GalCer.

• 

  Fig. 6 Blocking effect of IL12 mAb on the suppressive effect of CpG on α-GalCer-induced AHR, airway inflammation, and cytokine production. As described in Fig. 3, AHR to methacholine (A), inflammatory cells (B), and cytokines (C, D, and E) in BAL fluid were measured. IL-12 mAb or the IgG2b isotype was intraperitoneally administered immediately before CpG administration. Penh was expressed as the percentage increase in Penh, where Penh after PBS represented 100%. Total cells, Mac, Eos, Neut, and Lymph were expressed as the number per mL BAL fluid. Data are expressed as the mean±SEM (n=5). *P<0.05 and †P<0.01 for anti-IL-12 or the isotype vs. CpG + α-GalCer.

• 

  Fig. 7 Effects of LPS, FlaB, or CpG on IL-4 and IFN-γ production from α-GalCer-stimulated spleen invariant natural killer T (iNKT) cells and the blocking effect of IL12 mAb. Spleen cells were stimulated with α-GalCer and, 90 min later, intracellular cytokines of iNKT cells were stained. LPS, FlaB, or CpG was added 16 hr before α-GalCer stimulation. IL-12 or the IgG2b isotype was added immediately before addition of LPS, FlaB, or CpG. CD3+ α-GalCer-loaded CD1d tetramer+ iNKT cells were gated and the proportion of IL-4+ or IFN-γ + iNKT cells was analyzed, in which IgG1 isotypes were used for IL-4 and IFN-γ mAbs (A). IL-4+ (B) and IFN-γ + (C) iNKT cells were measured in cultures treated with LPS. IL-4+ (D) and IFN-γ + (E) iNKT cells were measured in cultures treated with FlaB. IL-4+ (F) and IFN-γ + (G) iNKT cells were measured in cultures treated with CpG. Spleen cells were pooled from three mice, and data are expressed as the mean±SEM of triplicate measurements. *P<0.05.

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