Inhibition of autoimmune Th17 cell responses by pain killer ketamine

Abstract

Ketamine is widely used in animals and humans as a systemic anesthetic. Although several immune-modulatory functions of ketamine have been reported, the effects of ketamine on the differentiation of Th17 cell are unknown. We found that ketamine significantly diminished the frequency of IL-17-producers among CD4⁺ T cells stimulated under Th17-skewing conditions. Mechanistic studies showed that ketamine had little effect on the production of Th17-inducing cytokines by dendritic cells and the proliferation of T cells in response to anti-CD3; however it significantly hampered IL-21 expression as well as STAT3 phosphorylation in T cells upon IL-6 stimulation. Moreover, MOG-reactive CD4⁺ T cells expanded in the presence of ketamine produced reduced amounts of Th17 cytokines, leading to diminished EAE severity when transferred into TCRβ-deficient mice in comparison to those treated with vehicle. These findings demonstrate that ketamine suppresses autoimmune Th17 cell responses by inhibiting the differentiation as well as the reactivation of Th17 cells.

Keywords: IL-21; Immune response; Immunity; Immunology and Microbiology Section; STAT3; Th17 cell; autoimmunity; ketamine.

Figure 1. Ketamine inhibits DC-mediated Th17 cell differentiation

Naïve CD4⁺ T cells and CD11c⁺ bone marrow-derived dendritic cells were stimulated with soluble anti-CD3 and co-cultured under Th17-skewing condition for 3 days. Detection of IL-17 expression cells was conducted using flow cytometry analysis. A., B. The levels of IL-17 in the supernatant were determined by ELISA. C. The expression levels of indicated transcripts were analyzed by quantitative real-time RT-PCR. D. Data represent three independent experiments. Data shown are mean ± SEM. *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 2. Effect of ketamine on DCs and CD4⁺ T cells during Th17 cell differentiation

Bone marrow-derived dendritic cells were stimulated with 100 ng/mL of LPS in the presence of various concentrations of ketamine for 24 hours. The amounts of indicated cytokines in the supernatant were measured by ELISA. A.. FACS-sorted naïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 under Th17-skewing condition for 3 days, and the frequency of IL-17-expressing T cells were analyzed. B., C. IL-17 concentrations of the supernatants were measured by ELISA. D. Data represent at least 3 independent experiments. Data shown are mean ± SEM. *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 3. Effect of ketamine on the proliferation of T cells during Th17 cell differentiation

Naïve CD4⁺ T cells were labeled with CFSE before being stimulated with plate-bound anti-CD3 and anti-CD28 under Th17-skewing condition for 3 days. The dilution of CFSE and the frequency of IL-17-expressing cells were analyzed by flow cytometry. A. The proportion of cells divided more than once was measured. B. Data represent two independent experiments. Data shown are mean ± SEM. *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 4. Ketamine inhibits Th17 cell differentiation induced by IL-6, IL-1β and IL-23 in the absence of exogenous TGF-β

Naïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 in the presence of IL-6 plus TGF-β. A. or IL-1β plus IL-6 plus IL-23 B. for 3 days. The levels of IL-17 and IL-22 in the supernatant as well as the frequency of RORγt-expressing T cells were analyzed by ELISA and flow cytometry, respectively. ND, Not detectable. Data represent two independent experiments. Data shown are mean ± SEM. *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 5. Ketamine negatively regulates the expression of IL-21 and the phosphorylation of STAT3 during Th17 cell differentiation

Naïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 under Th17-skewing condition for 48 hours in the presence or absence of ketamine. The level of Il21 transcript as well as IL-21 in the supernatant was determined. A. The levels of phosphorylated STAT3 in CD4⁺ T cells stimulated under plate-bound anti-CD3 and anti-CD28 plus IL-6 conditions were measured by flow cytometry. B. Shaded, IL-6 unstimulated negative control; closed line, vehicle or ketamine treated; NC. Data represent two independent experiments. Data shown are mean ± SEM. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ####, p < 0.0001.

Figure 6. Ketamine inhibits the reactivation of MOG-reactive Th17 cells

Lymphoid cells from the MOG-immunized mice were re-stimulated with MOG peptide plus IL-23 in the presence of ketamine or vehicle for 5 days before CD4⁺ T cells were sorted by MACS. The expression of IL-17 and IFN-γ was measured. A., B. The sorted CD4⁺ T cells were i.v. transferred into TCRβ-deficient mice and the recipient mice were s.c. injected with MOG in CFA. Body weight and clinical disease score were daily monitored C., and the frequencies and absolute numbers of the indicated population among CD4⁺ T cells in the CNS of the recipients were determined D. & E. Data represent two independent experiments. Data shown are mean ± SEM. *, p < 0.05, **, p < 0.01, ***, p < 0.001.