Distinct characteristics of hippocampal pathogenic TH17 cells in a mouse model of depression

Abstract

Increasing evidence indicates that multiple actions of the immune system are closely intertwined with the development of depression and subsequent recovery processes. One of these interactions is substantial evidence that the T_H17 subtype of CD4⁺ T cells promotes susceptibility to depression-like behaviors in mice. Comparing subtypes of CD4⁺ T cells, we found that administration of T_H17 cells, but not T_H1 cells or T_REGS, promoted susceptibility to learned-helplessness depressive-like behavior and accumulated in the hippocampus of learned helpless mice. Adoptively transferred T_H17 cells into Rag2^-/- mice that are devoid of endogenous T cells increased susceptibility to learned helplessness, demonstrating that increased peripheral T_H17 cells are capable of modulating depression-like behavior. Moreover, in wild-type mice, adoptively transferred T_H17 cells accumulated in the hippocampus of learned-helpless mice and induced endogenous T_H17 cell differentiation. Hippocampal T_H17 cells from learned-helpless mice expressed markers of pathogenic T_H17 cells (CCR6, IL-23R) and of follicular cells (CXCR5, PD-1), indicating that the hippocampal cells are T_FH-17-like cells. Knockout of CCR6 blocked T_H17 cells from promoting learned helplessness, which was associated with increased expression of PD-1 in CCR6-deficient T_H17 cells. In summary, these results reinforce the conclusion that depression-like behaviors are selectively facilitated by T_H17 cells, and revealed that these cells in the hippocampus of learned helpless mice display characteristics of T_FH17-like cells, which may contribute to their pathogenic actions in promoting depression.

Keywords: Depression; Th17.

Figure 1:

T_H17 cells promote learned-helplessness and accumulate in the hippocampus and PFC. (A) CD4⁺ T cells were differentiated toward either T_H17, T_H1 or undifferentiated CD4⁺ cells for 4 days in vitro and adoptively transferred to male and female Rag2^−/− (B) or male wild-type (C) mice. In (B) 18% of the CD4⁺ cells were T_H17 cells in mice receiving T_H17 cells, and 39% of the CD4⁺ cells were T_H1 cells in mice receiving T_H1 cells. In mice receiving CD4⁺ cells, less than 1% of CD4⁺ cells were T_H17 cells or T_H1 cells. 48 h after transfer mice were subjected to the reduced paradigm of learned-helplessness. The number of escape failures was recorded. Each symbol represents an individual mouse. Mean ± SEM, one-way ANOVA, F(2,36)=4.037, Bonferroni post hoc test *p<0.05 (n=7-14). (C) Mice were subjected or not to the learned-helplessness (LH) paradigm, and were separated into 3 groups: non-shocked (NS), shocked and exhibiting >15/30 escape failures (D), or shocked and not learned-helpless (<15 escape failures, ND). (D-F) Mice were perfused, and hippocampus and PFC were processed to obtain dissociated cells. Cells were stained for CD4 and IL-17A (D), IFNγ (E) or Foxp3 (F), and gated on CD4⁺ cells and analyzed by flow cytometry. Data represent mean ± SEM, one-way ANOVA, F(5, 30)=10.26 (E), F(5,39)=14.64 (F), Bonferroni post hoc test *p<0.05 (n=4-11).

Figure 2:

Adoptively transferred T_H17 cells accumulate in the hippocampus after the learned-helplessness paradigm. (A) CD45.2 wild-type recipient mice received donor CD45.1⁺CD4⁺ T cells or CD45.1⁺ T_H17 cells, and 48 h later were subjected to the learned-helplessness paradigm. 86% of the CD4⁺T cells transferred in the T_H17 cells preparation were T_H17 cells, whereas less than 1% T_H17 cells were detected in the CD4 preparation, and 99.3% of the injected CD4 cells were CD45.1. (B) CD4⁺ CD45.1⁺ (donor cells) or CD4⁺CD45.2⁺ (recipient cells) present in the hippocampus of mice adoptively transferred with CD4 cells or T_H17 cells were analyzed by flow cytometry after gating on CD4⁺ cells (C) CD45.1⁺CD4⁺ (donor cells) or CD45.2⁺IL-17A⁺CD4⁺ (recipient cells) present in the hippocampus of mice adoptively transferred with CD4 cells or T_H17 cells were analyzed by flow cytometry after gating on CD45⁺CD4⁺ cells. Each symbol represents an individual mouse. Data represent mean±SEM, one-way ANOVA, F(3, 16) = 9.522, Bonferroni post-hoc test *p<0.05 (n=5). (D) CD45.1⁺IL-17A⁺CD4⁺ (donor cells) or CD45.2⁺IL-17A⁺CD4⁺ (recipient cells) present in the hippocampus of mice adoptively transferred with CD4 cells or T_H17 cells were analyzed by flow cytometry after gating on CD45⁺IL-17A⁺CD4⁺ cells. Each symbol represents an individual mouse. Data represent mean±SEM, one-way ANOVA, F(3, 14)=9,795, Bonferroni post-hoc test *p<0.05 (n=4-5).

Figure 3:

Induction of learned-helplessness increases the induction of GFP in RORγT^GFP/+ mice. (A-B) RORγT^GFP/+ mice adoptively transferred with undifferentiated CD4 cells or T_H17 cells 48 h before being subjected to the reduced learned-helplessness paradigm, and the number of escape failures was recorded. Each symbol represents an individual mouse. Data represent mean±SEM, Mann Whitney, U=37.5, *p<0.05, (n=11-18). (C-F) Unshocked (NS) mice or mice transferred with T_H17 cells (Th17), or mice exhibiting learned-helplessness (D) or non-learned-helpless mice (ND) that received T_H17 cells were perfused after the last shock and hippocampal (C-D) or splenic (E-F) cells were dissociated. CD4⁺ T cells were stained for IL-17A, and both IL-17A⁺CD4⁺ T cells (C, E) and GFP⁺CD4⁺ T cells (D, F) were evaluated by flow cytometry after gating on CD4⁺ cells as described in panel (A). Each symbol represents an individual mouse. Data represent mean ±SEM, one-way ANOVA, F(3, 26)= 9,492 (B), F(3,25)=15.75 (C), Bonferroni post hoc test *p<0.05 (n=4-12).

Figure 4:

Follicular and pathogenic markers are elevated in the hippocampus of learned-helpless mice. (A) Mice were subjected or not to the learned-helplessness paradigm, and mice were separated into 3 groups: learned-helpless (D), non-learned-helpless (ND) and non-shocked (NS) as described in Fig 1D. Immediately after the last foot shocks, hippocampi were collected and mRNA extracted to measure by qRT-PCR: IL-17A mRNA (A), an array of T_H17 cell-related genes (B), ICOS mRNA (C), c-maf mRNA (D), IL-27 mRNA (E) and Bcl6 mRNA (F). Genes upregulated or downregulated more than 4-fold are reported in (B). Each symbol represents an individual mouse. Data represent mean±SEM, One-way ANOVA, F(2, 12)=6.212 (A), F(2, 25) = 20.21 (C), F(2,18)=57.66 (D), Bonferroni post-hoc test *p<0.05 (n=4-10). (G-I) Mice were subjected to the learned-helplessness paradigm, or not, and mice were separated into 3 groups: unshocked (NS), learned-helpless (D) and non-learned-helpless (ND) mice. Immediately after the last foot shocks, mice were perfused and hippocampi were collected. Dissociated cells were stained for CD4, IL-17A, and either CCR6 (G) or IL-23R (I), and gated on IL-17A⁺ CD4⁺ cells. (H) mRNA was extracted from hippocampus and CCL20 expression was analyzed by qRT-PCR. Each symbol represents an individual mouse. Data represent mean±SEM, one-way ANOVA, F(2, 23)= 11.59 (A), F(2, 15)=16.50 (B), F(2,33)=8.034 (C), Bonferroni post-hoc test *p<0.05 (n=8-17).

Figure 5:

T_H17 cells associated with learned helplessness present a follicular phenotype. Mice were subjected to the learned-helplessness paradigm, or not, and mice were separated into 3 groups: unshocked (NS), learned-helpless (D) and non-learned-helpless (ND) as described in Fig 1D. Immediately after the last foot shocks, mice were perfused and hippocampi were collected. Dissociated cells were stained for CD4, CXCR5 and gated on CD4⁺ cells (A-B) or cells were stained for CD4, CXCR5, IL-17A and gated (C-D) or not (E-F) on CXCR5⁺CD4⁺ cells; or cells were stained for CD4, CXCR5 and either IFNγ (G-H), or ICOS (I-J), or IL-21 (M-N), and gated on CXCR5⁺CD4⁺ cells (G-L); or cells were stained for CD4, ICOS and gated on CD4⁺ cells (K-L) or CD4, IL-21 and gated on CD4⁺ICOS⁺ cells (O-P). Representative plots of flow cytometry are presented in A, C, E, G, I, K, M and O, and quantifications are presented in B, D, F, H, J, L, N and P. Each symbol represents an individual mouse. Data represent mean± SEM, one-way ANOVA, F(2,32)=13.33 (B), F(2,38)= 11.20 (D), Bonferroni post-hoc test and Mann-Whitney, U=14 (J), *p<0.05 (n=4-20).

Figure 6:

T_FH-like 17 cells have increased PD-1 levels and express CCR6 and IL-23R. Mice were subjected to the learned-helplessness paradigm, and were separated into 2 groups: learned-helpless (D) and non-learned-helpless (ND). Immediately after the last foot shocks, mice were perfused and hippocampi were collected. Dissociated cells were stained for CD4, CXCR5, IL-17A, and either PD-1 (A-B), or CCR7 (C-D), or BCL-6 (E-F), and gated on IL-17A⁺CD4⁺ cells (A-F). Each symbol represents an individual mouse. Data represent mean±SEM, t-test, t(10)=2.729 *p<0.05 (n=5-7). (G-N) Mice were subjected to the learned-helplessness paradigm, or not, and mice were separated into 3 groups: unshocked (NS), learned-helpless (D) and non-learned-helpless (ND) mice. Immediately after the last foot shocks, mice were perfused and hippocampi were collected. Dissociated cells were stained for CD4, CXCR5, IL-17A, and either CCR6 (G-H, K-L) or IL-23R (I-J, M-N), and gated on CXCR5⁺IL-17A⁺CD4⁺ cells (G-J) or on CXCR5⁺CD4⁺cells (K-M). Representative plots of flow cytometry are presented in G, I, K and M, and quantifications are presented in H, J, L and N. Each symbol represents an individual mouse. Data represent mean± SEM, Mann-Whitney, U=17 (H), U=20 (J), *p<0.05 (n=8-17).

Figure 7:

T_FH-like 17 cells originate from the host. CD45.2 wild-type recipient mice received donor CD45.1⁺CD4⁺ T cells or CD45.1⁺ T_H17 cells, and 48 h later were subjected to the learned-helplessness paradigm. 86% of the CD4⁺T cells transferred in the T_H17 cells group were T_H17 cells, whereas less than 1% T_H17 cells were detected in the CD4 group, and 99.3% of the CD4 injected cells were CD45.1 as described in Fig 2A. (A) CD4⁺CD45.1⁺ (donor cells) or CD4⁺CD45.2⁺ (recipient cells) present in the hippocampus of mice adoptively transferred with CD4 or T_H17 cells were analyzed by flow cytometry after gating on CD4⁺ cells. CD45.1⁺CXCR5⁺IL-17A⁺CD4⁺ (donor cells) or CD45.2⁺CXCR5⁺IL-17A⁺CD4⁺ (recipient cells) present in the hippocampus (B) or spleen (C) of mice adoptively transferred with CD4 or T_H17 cells were analyzed by flow cytometry after gating on CD45⁺CXCR5⁺IL-17A⁺CD4⁺ cells as described in panel (A). Each symbol represents an individual mouse. Data represent mean±SEM, one-way ANOVA, F(2,14)=22.9 (A), F(3,16)=34.35 (B), Bonferroni post-hoc test *p<0.05 (n=5).

Figure 8:

CCR6 expression is required for T_H17 cells to promote learned-helplessness. Male and female mice received T_H17 cells transduced with either a scrambled siRNA (siSCR) or siRNA targeting CCR6 (siCCR6) and 48 h later were subjected to the reduced paradigm of learned-helplessness. (A) Before transfer, T_H17 cells transduced with either a scrambled siRNA (47.4% CD4 are T_H17 cells) or siRNA targeting CCR6 (43.5% CD4 are T_H17 cells) were stained for CCR6, IL-17A and CD4 for analysis by flow cytometry. Mean ± SEM, Mann-Whitney, U=0, *p<0.05 (n=3-4). (B) The number of escape failures was recorded. Each symbol represents an individual mouse. Mean ± SEM, t-test, t(20)=2.132, *p<0.05 (n=11). Immediately after the last foot shocks, mice were perfused and hippocampi were collected. Dissociated cells were stained for IL-17A and CD4 (C), CD4 (C), Foxp3 and CD4 (E), F4/80 and CD45 (F), F4/80 and CD45 (G), CD11c and CD45 (H) and gated on CD4⁺ cells (C-E). Data represent mean±SEM, (n=5). (I) Mice received T_H17 cells differentiated from wild-type CD4 cells (WT, 33% CD4 are T_H17 cells) or CCR6-deficient CD4 cells (CCR6 KO, 28% CD4 are T_H17 cells) and 48 h later were subjected to the reduced paradigm of learned-helplessness. The number of escape failures was recorded. Each symbol represents an individual mouse. Mean ± SEM, Mann-Whitney, *p<0.05 (n=10). Immediately after the last foot shocks, mice were perfused and hippocampi were collected. Dissociated cells were stained for IL-17A and CD4, and gated on CD4⁺ cells (J) or stained for PD-1, IL-17A, and CD4 and gated on IL-17A⁺ CD4⁺ cells (K). Data represent mean±SEM, Mann-Whitney U=21 (I) and Wilcoxon test, W=−19 (K), *p<0.05 (n=9).