Inflammatory T helper 17 cells promote depression-like behavior in mice

Abstract

Background: Recognition of substantial immune-neural interactions is revising dogmas about their insular actions and revealing that immune-neural interactions can substantially impact central nervous system functions. The inflammatory cytokine interleukin-6 promotes susceptibility to depression and drives production of inflammatory T helper 17 (Th17) T cells, raising the hypothesis that in mouse models, Th17 cells promote susceptibility to depression-like behaviors.

Methods: Behavioral characteristics were measured in male mice administered Th17 cells, CD4(+) cells, or vehicle and in retinoid-related orphan receptor-γT (RORγT)(+/GFP) mice or male mice treated with RORγT inhibitor or anti-interleukin-17A antibodies.

Results: Mouse brain Th17 cells were elevated by learned helplessness and chronic restraint stress, two common depression-like models. Th17 cell administration promoted learned helplessness in 89% of mice in a paradigm where no vehicle-treated mice developed learned helplessness, and impaired novelty suppressed feeding and social interaction behaviors. Mice deficient in the RORγT transcription factor necessary for Th17 cell production exhibited resistance to learned helplessness, identifying modulation of RORγT as a potential intervention. Treatment with the RORγT inhibitor SR1001, or anti-interleukin-17A antibodies to abrogate Th17 cell function, reduced Th17-dependent learned helplessness.

Conclusions: These findings indicate that Th17 cells are increased in the brain during depression-like states, promote depression-like behaviors in mice, and specifically inhibiting the production or function of Th17 cells reduces vulnerability to depression-like behavior, suggesting antidepressant effects may be attained by targeting Th17 cells.

Figure 1

Th17 cells accumulate in the brains of mice exhibiting learned helplessness. Wild-type mice were subjected to mild inescapable foot shocks, 24 h later lithium chloride (LiCl; 100 mg/kg; i.p.) was administered, and after 24 h mice were tested with escapable foot shocks. (A) Escape failures after standard IES. Each symbol represents total escape failures in 30 trials for an individual mouse, bars represent group means ± sem (n=10–21 mice/group, t-test *p<0.05, t=3.030, df=31). Mice were separated into 3 groups: mice that failed to escape <15 of 30 trials (nondepressed, ND), mice that failed to escape >15 of 30 trials (depressed, D), and non-depressed lithium-treated mice. After behavior measurements, the indicated (B) Th cells or (C) immune cells from the brain, or (D) Th cells from the spleen, were stained and analyzed by flow cytometry. Bars represent mean ± sem. (ANOVA, *p<0.05 compared with values from non-depressed mice, df=10, F=8.466 (Th17 cells), *p<0.05 compared to ND+Li, df=7, F=9.614 (Th1 cells)). (E) Exposure to chronic restraint stress (CS) for two weeks increased the number of Th17 cells and (F) Th1 cells in the brains of mice compared to non-stressed mice (n=5/group, Mann-Whitney test *p<0.05, † p=0.06). (G) GSK3 knockin (KI) mice received escapable shocks only, and 46% of the mice failed to escape >15 of 30 trials (depressed, D). Each symbol represents total escape failures in 30 trials for an individual mouse (n=13). Bars in panels A–G are representative of 3 independent experiments. (H) After behavior measurements, Th cells recovered from 5 brains per group (ND and D) of GSK3 knockin mice were pooled together, stained as indicated, and analyzed by flow cytometry.

Figure 2

Transfer of Th17 cells increased susceptibility to depression-like behavior. Wild-type mice received either PBS, Th17 cells, or CD4⁺ T cells, 48 h later mice were subjected to reduced-intensity inescapable foot shocks that did not induce learned helplessness in vehicle- or CD4⁺ T cell-treated mice, and 24 h later mice were tested with escapable foot shocks. (A) Escape failures after reduced intensity IES. Each symbol represents total escape failures in 30 trials for an individual mouse, bars represent group means±sem (n=3–9 mice/group, ANOVA *p<0.05, df=20, F=58.67). (B) Average escape latency during 5-trial blocks after reduced intensity IES. WT mice injected with PBS or CD4⁺ cells had an average of 5.5±1.4 or 4±0.7 sec escape latency, respectively, whereas mice injected with Th17 cells had an average of 22.7±0.8 sec escape latency. Bars represent group means±sem (n=3–9 mice/group, ANOVA *p<0.05, df=17, F=98.30). After behavior measurements, the indicated (C) Th cells or immune cells were stained and analyzed by flow cytometry. Bars represent mean±sem (n=3–9 mice/group ANOVA *p<0.05 compared with PBS-treated mice, df=21, F=10.84). (D) In the NSF test, Th17-recipient mice displayed significantly longer latencies to feed when placed in a novel arena with food following 24 hr food deprivation compared with control CD4⁺ T cell-treated mice (n=4 mice/group, *p<0.05 compared with CD4-treated mice, paired t-test, df=3, t=3.200). (E) Th17-recipient mice demonstrated significantly fewer nose contacts with a novel conspecific mouse than did CD4⁺ T cell-treated mice but did not differ in the time spent in each chamber of the apparatus (CH1, chamber 1 containing the conspecific mouse; CH2, empty middle chamber 2; CH3, chamber 3, equivalent to CH1 without a mouse) (n=5 mice/group, *p<0.05 compared with CD4-treated mice, paired t-test, df=4, t=3.670).

Figure 3

Depletion of Th17 cells in Rorc(γT)^+/GFP mice reduces acquisition of learned helplessness. (A) Basal activity of wild-type or Rorc(γT)^+/GFP mice was measured in an open field for 30 min. (B–D) Mice were subjected to standard IES, and 24 h later tested with escapable foot shocks. (B) Escape failures after standard IES. Each symbol represents total escape failures in 30 trials for an individual mouse, bars represent group means (n=8–12 mice/group, t-test *p<0.05, df=18, t=2.267). (C) Average escape latency during 5-trial blocks after standard IES. WT and Rorc(γT)^+/GFP mice had an average of 19.4±0.4 and 10.9±1.3 sec escape latency, respectively. (n=8–12 mice/group, Mann Whitney *p<0.05). After behavior measurements, the indicated (D) Th cells or immune cells were stained and analyzed by flow cytometry. Bars represent mean ± sem. (n=8–12 mice/group Mann Whitney *p<0.05).

Figure 4

Inhibiting RORγT or IL-17A has antidepressant effects in Th17-dependent learned helplessness. Wild-type mice pretreated where indicated with anti-IL-17A antibodies or SR1001, a RORγT inhibitor, received Th17 cells. (A–B) Mice were subjected to reduced intensity IES, and 24 h later tested with escapable foot shocks. (A) Escape failures after reduced IES. Each symbol represents total escape failures in 30 trials for an individual mouse, bars represent group means (n=8–10 mice/group, ANOVA *p<0.05 compared with untreated Th17 recipient mice, df=45, F=8.220). (B) Average escape latency during 5-trial blocks after reduced intensity IES. Mice receiving Th17, Th17+ anti-IL17A, and Th17 + SR1001 mice had an average of 21.8±1.6, 13.6±0.6 and 15.1±0.8 sec escape latency, respectively. (n=8–10 mice/group, ANOVA *p<0.05 compared with untreated Th17 recipient mice, df=17, F=15.77).