Fig 1.
T. gondii chronic infection induces anxiety-like behavior in mice.
(A-F) showed the results of OFT. (A) Representative track plots of OFT. (B) Time in the central zone. (C) Percentage of time in the central zone to total time. (D) Distance in the central zone. (E) Percentage of distance in the central zone to total distance. (F) Entries into the central zone. (G-K) showed the results of EMPT. (G). Representative track plots of EPMT. (H) Frequencies of head entries in open arms. (I) Time of head entries in open arms. (J) Time in open arms. (K) Percentage of time in open arms to total time. (L) 5-HT level in serum of mice. Values are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the Con group. (M) Pearson Correlation of 5-HT level with the time spent in the central zone (TCZ) and open arms (TOZ). * P < 0.05, ** P < 0.01. n = 10 for each group.
Fig 2.
T. gondii chronic infection alters the expression pattern of anxiety- and neuroinflammation- related genes in the amygdala of mice.
(A) The numbers of differentially expressed genes (DEGs) in the amygdala. (B) The biological processes associated with behavior change. (C) The relative change of DEGs associated with anxiety and neurotransmitters. (D) The biological processes of DEGs enriched in the activation of astrocytes and microglia. (E) The pathways of DEGs associated with pro-inflammatory cytokine production. (F) The expression of M1 microglia related markers. n =3 for each group.
Fig 3.
T. gondii chronic infection activates microglia and upregulates Acod1 expression in the amygdala of mice.
(A) The immunofluorescent staining of Iba-1 in the amygdala (n = 3, 10 images per mouse, scale bar: 50 μm). The image captured from the box was marked with a dotted line (scale bar 10 μm). (B) The quantification of Iba-1+ cells number in amygdala. (C, D) The solidity and circularity of Iba-1+ cells in the amygdala. (E-G) The mRNA expression levels of TNF-α, IL-6 and IL-1β in amygdala. (H) The Acod1expression of amygdala in the transcriptome. (I) The protein expression level of Acod1 in the amygdala (n=3). (J) The protein expression level of Nrf2 in the amygdala (n = 3). (K) The protein expression level of Keap1 in the amygdala (n = 3) Values are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the Con group.
Fig 4.
Acod1/itaconate regulates anxiety-like behavior in mice. (A) Representative track plots of OFT.
(B) Time in the central zone. (C) Percentage of time in the central zone to total time. (D) Distance in the central zone. (E) Percentage of distance in the central zone to total distance. (F) Entries into the central zone. (G). Representative track plots of EPMT. (H) Frequencies of head entries in open arms. (I) Time of head entries in open arms. (J) Time in open arms. (K) Percentage of time spent in open arms to total time. Values are mean ± SEM. vs WT, * P < 0.05, ** P < 0.01, *** P < 0.001. vs Acod1-/-, # P < 0.05, ## P < 0.01, ### P < 0.001.
Fig 5.
DI prevents anxiety induced by T. gondii chronic infection.
(A) The schematic timeline of experiment 3, in which DI administration started one week before T. gondii infection and lasted until the end of the experiment. The aim of experiment 3 was to investigate the preventive effect of DI on T. gondii-induced anxiety-like behavior. (B) Time in the central zone. (C) Percentage of time in the central zone to total time. (D) Distance in the central zone. (E) Percentage of distance in the central zone to total distance. (F) Entries into the central zone. (G) Frequencies of head entries in open arms. (H) Time of head entries in open arms. (I) Time in open arms. (J) Percentage of time in open arms to total time. (K-M) The mRNA expression levels of TNF-α, IL-6 and IL-1β in the amygdala. Values are mean ± SEM. vs Con + Veh, * P < 0.05, ** P < 0.01, *** P < 0.001. vs Tg + Veh, # P < 0.05, ## P < 0.01, ### P < 0.001.
Fig 6.
DI therapeutically reverses anxiety induced by T. gondii chronic infection.
(A) The schematic timeline of experiment 4, in which DI administration started four weeks after infection and lasted until the end of the experiment. The aim of experiment 4 was to evaluate the therapeutic effect of DI on T. gondii-induced anxiety. (B) Representative track plots of OFT. (C) Time in the central zone. (D) Percentage of time in the central zone to total time. (E) Distance in the central zone. (F)Percentage of distance in the central zone to total distance. (G) Entries in the central zone. (H) Representative track plots of EPMT. (I) Frequencies of head entries in open arms. (J) Time of head entries in open arms. (I) Time in open arms. (J) Percentage of time in open arms to total time. Values are mean ± SEM. vs Con + Veh, * P < 0.05, ** P < 0.01, *** P < 0.001. vs Tg + Veh, # P < 0.05, ## P < 0.01, ### P < 0.001.
Fig 7.
DI attenuates anxiety-related neuropathological alterations induced by T. gondii chronic infection.
(A) The immunofluorescent staining of Iba-1 in the amygdala (n = 3, 10 images per mouse, scale bar: 50 μm). The image captured from the box was marked with a dotted line (scale bar 10 μm). (B) The quantification of Iba-1+ cells number in amygdala. (C, D) The solidity and circularity of Iba-1+ cells in amygdala. (E-G) The mRNA expression levels of TNF-α, IL-6 and IL-1β in amygdala. (H, I) The mRNA expression levels of IDO and 5-HT1AR in the amygdala. (J, K) The IDO and 5-HT levels in the serum. Values are mean ± SEM. Tg + Veh vs Con + Veh, * P < 0.05, ** P < 0.01, *** P < 0.001. Tg + DI vs Tg + Veh, # P < 0.05, ## P < 0.01, ### P < 0.001.
Fig 8.
DI activates the Nrf2-Keap1 pathway to suppress pro‑inflammatory responses in microglia.
(A) The schematic timeline of in-vitro experiment 1 and 2, in which BV2 cells were pretreated with DI or SFN for 3h prior to T. gondii infection. The aim of experiments was to evaluate the mechanistic link underlying the anti-inflammatory function of DI. (B) The protein expression level of Nrf2 in DI pretreated group. (C) The protein expression level of Keap1 in DI pretreated group. (D-F) The mRNA expression levels of TNF-α, IL-6, and IL-1β in DI pretreated group. (G, H) The mRNA expression levels of CD86 and iNOS in DI pretreated group. (I-K) The mRNA expression levels of TNF-α, IL-6, and IL-1β in SFN pretreated group. (L, M) The mRNA expression levels of CD86 and iNOS in SFN pretreated group. Values are mean ± SEM. Tg + Veh vs Con + Veh, * P < 0.05, ** P < 0.01, *** P < 0.001. Tg + DI or Tg + SFN vs Tg + Veh, # P < 0.05, ## P < 0.01, ### P < 0.001.
Fig 9.
Acod1/itaconate regulates neuroinflammation and anxiety induced by T. gondii chronic infection.
(A) In mice, T. gondii infection upregulates the expression of pro-inflammatory cytokines in microglia, which increases IDO and decreases 5-HT levels, and consequently, leading to anxiety. Dimethyl itaconate (DI) ameliorates T. gondii- induced anxiety via suppressing neuroinflammation. (B) Acod1 loss induces anxiety in mice, which can be restored by supplementing DI. IDO, indoleamine 2,3-dioxygenase; 5-HT, 5-hydroxytryptamine; DI, dimethyl itaconate. Created in BioRender. Zhou, Y. (2026) https://BioRender.com/7xh71r1.