Dynamical regulation of Foxp3 depletion is mediated by DT treatment in Foxp3 cKO mice.
Here, we investigated the regional distribution of mRNA expression of Foxp3 in the PBMC, spleen, and brain to estimate abundance of Foxp3 expression using Foxp3 cKO mice. As shown in Fig. 1A, Foxp3 mRNA expression in PBMC and spleen were approximately two order of magnitude higher compared to the expression in the brain (*p < 0.05).
We utilized a Foxp3 cKO mice, expressing DTR-eGFP transgene under control of Foxp3 promoter allowing depletion of Foxp3 expression by DT treatment to transiently deplete Foxp3 expression (Fig. 1B). After treatment of DT with different concentrations (100 ng and 300 ng) for 6 weeks, we observed a dose-dependent decrease of Foxp3-dependent GFP expression compared to the vehicle treated group (*p < 0.05, Fig. 1C). Based on titration of study, we selected the dose of 300ng of DT to deplete Foxp3 expression for subsequent experiments (Fig. 1B). We next examined whether DT treatment induces depletion of Foxp3 expression in the brain, spleen, and PBMC of Foxp3-depleted mice (Fig. 1D-F). We found no difference in Foxp3 mRNA expression in the brain of Foxp3-depleted mice compared to age matched control mice (Fig. 1D). However, Foxp3 mRNA expression was significantly decreased by 46% in spleen of Foxp3-depleted mice compared to age matched control (*p < 0.05, Fig. 1E).
We also observed that Foxp3-dependant GFP expression was significantly reduced by approximately 60% in PBMC of Foxp3-depleted mice (*p < 0.05, Fig. 1F). Moreover, reduction of Foxp3-dependant GFP expression in PBMC returned to baseline level in Foxp3-restored mice following withdrawal of DT for 8 weeks as assessed by flow cytometry (Fig. 1G).
Taken together, these results indicate that administration of DT in Foxp3 cKO mice induced transient depletion of Foxp3 expression selectively in PBMC after treatment with 300 ng of DT for 6 weeks and its expression was restored to the baseline level following withdrawal of DT.
Transient depletion of Foxp3 expression in PBMC is causally associated to promotion of anxiety or depression-like behaviors in Foxp3 cKO mice.
To investigate causal association between peripheral Foxp3 expression and anxiety/depression-like behaviors in Foxp3 ckO mice, we performed LD, FST and OF to assess locomotion activity, anxiety and depression-like behavior, respectively. No significant change was observed in body weight between treatment with DT and vehicle in Foxp3 cKO mice for treatment period (Fig. 1B and Fig. 2A). Changes in locomotor activity after depletion or restoration of Foxp3 expression in Foxp3 cKO mice was assessed by total distance traveled; no change between Foxp3-depleted, Foxp3-restored mice, and age matched control mice was found (Fig. 2D). These control studies suggest that DT-mediated depletion of Foxp3 expression for 6 weeks was well tolerated in Foxp3 cKO mice at 300ng regimen.
In the LD, Foxp3-depleted mice spent significantly more time in dark area after depletion of Foxp3 expression (***p < 0.001, Fig. 2B, left panel). In addition, we also observed that Foxp3-depleted mice also exhibited increased immobility time in FST after depletion of Foxp3 expression (**p < 0.01, Fig. 2C, left panel). Interestingly, these neurobehavioral impairments were ameliorated after restoration of Foxp3 expression in Foxp3-restored mice compared to age matched control mice (Fig. 2B and 2C, right panels). In particular, we find that Foxp3 expression in PBMC were negatively correlated with anxiety (*p < 0.05, Fig. 2E) and depression-like behavior (*p < 0.05, Fig. 2F).
These results support that transient depletion of Foxp3 expression in PBMC may casually influence anxiety or depression-like behaviors.
Transient Foxp3 depletion is causally associated to differentially expressed genes (DEGs) related with inflammatory response and immune cells trafficking in the hippocampal formation of Foxp3-depleted mice
To understand a potential molecular mechanism of the association between peripheral Foxp3 expression and neurobehavioral changes, RNA sequencing was performed in the hippocampal formation of Foxp3-depleted and Foxp3-restored mice. Hippocampal formation is one of crucial brain regions strongly involved in anxiety and depression-like behaviors in rodents 2.
As shown Fig. 3A, a total of 17,237 genes DEGs were identified in a volcano plot (Fig. 3A, upper panel) and among these genes, 401 DEGs were up-regulated and 422 DEGs were down-regulated significantly in Foxp3-depleted mice compared to age matched control mice (*p < 0.05, Fig. 3A, bottom panel). To explore the involvement of 823 annotated DEGs in canonical pathways and diseases/bio functions, functional prediction analysis was performed according to IPA software (Fig. 3B and Fig. 3C). For canonical pathways analysis, a total of 7 enriched canonical pathways were identified by applying absolute z scores greater than 2 and statistical significance (*p < 0.05). The top 3 canonical pathways, ‘Role of hypercyotokinemia /hyperchemokinemia in the pathogenesis of influenza’, ‘Role of pattern recognition receptors in recognition of bacteria and viruses’ and ‘Interferon signaling’ were identified among 7 other canonical pathways (Fig. 3B, left panel). We also found the expression of 13 DEGs in the top 3 canonical pathways where significantly altered in Foxp3-depleted mice compared to age matched control mice (Fig. 3B, right panel). In addition, diseases and bio functions analysis, by applying absolute z scores greater than 2 and statistical significance, revealed activation of functional categories related to ‘Cell-mediated immune response’ and ‘Immune cells trafficking’ in Foxp3-depleted mice (Fig. 3C).
To investigate whether identified canonical pathways and diseases/bio functions in Foxp3-depleted mice were transiently regulated in Foxp3-restored mice as found in neurobehavioral changes, a comparison analysis was performed by IPA applying absolute z scores greater than 2 and statistical significance (Fig. 3D and 3E). As shown in Fig. 3D, we found the top 9 canonical pathways being enriched in Foxp3-depleted mice were recovered in Foxp3-restored mice. Among these 9 canonical pathways, 5 of them are associated to inflammatory response such as ‘Role of hypercyotokinemia/ hyperchemokinemia in the pathogenesis of influenza’, ‘Role of pattern recognition receptors in recognition of bacteria and viruses’, ‘Interferon signaling’, ‘Role of OKR in interferon induction and antiviral response’ and ‘Neuroinflammation signaling’ (Fig. 3D). Moreover, significant activated cellular function, which were related to ‘Innate immune response’, and ‘Immune cells movement’ were observed in Foxp3-depleted mice and these changes were significantly mitigated in Foxp3-restored mice (Fig. 3E).
Taken together, our results suggest that transient depletion of Foxp3 expression promote inflammatory responses such as immune cell trafficking and inflammasome activation in the hippocampal formation which causally coincided with the transient anxiety and depression-like behaviors.
Transient depletion of Foxp3 expression results in elevated Cxcl10 expression and inflammasome activation in Foxp3 cKO mice.
In a validation study of the transcriptomic investigation and IPA analysis, we assessed mRNA expression of four candidate genes, which are associated with inflammatory responses, in the hippocampal formation of Foxp3-depleted and Foxp3-restored mice as assessed by RT-qPCR. We found that mRNA expression of Cxcl10 (Fig. 4A), a chemokine that plays a key role in controlling leukocyte trafficking into brain, was significantly elevated in the Foxp3-depleted mice compared to age matched control mice (**p < 0.01, Fig. 4A, left panel), while no significant difference in Cxcl10 mRNA expression was identified in Foxp3-restored mice compared to age matched control mice (Fig. 4A, right panel). We also measured mRNA expression of Nlrp3, Caspase-1 and lL-1β to confirm inflammasome activation in the hippocampal formation of Foxp3-depleted mice and Foxp3-restored mice (Fig. 4B-D). The levels of Nlrp3, Caspase-1 and IL-1β mRNA were significantly increased 2, 1.4, and 1.3-fold in Foxp3-depleted mice, respectively (*p < 0.05, **p < 0.01, Fig. 4B-D, left panels). Interestingly, no differences in Foxp3-restored mice was found compared to age matched control mice (Fig. 4B-D, right panels).
In a further confirmatory study, the protein expression of Pro-caspase-1, Caspase-1 and IL-1β and inflammasome activation markers were assessed by gel-blot assay (western blotting, Fig. 4E-4H). As shown in Fig. 4E, we confirmed that cleaved Caspase-1 expression (Fig. 4E) and IL-1β expression (Fig. 4F) were significantly increased in Foxp3-depleted mice compared to those of expression in age matched control mice (*p < 0.05, **p < 0.01, Fig. 4E and 4F). Interestingly, non-detectable changes in Pro-caspase-1, Caspase-1 (Fig. 4G) and IL-1β (Fig. 4H) in the Foxp3-restored mice compared to the age matched control mice were observed.
Overall, these results support the hypothesis that transient depletion of Foxp3 expression may casually and transiently influence brain innate immunity changes which coincided with transient anxiety and depression-like behaviors.
Depletion of Foxp3 expression increases the number of peripheral innate immune cells coincidentally with transient increase of MMP-9 expression in the brain of Foxp3 cKO mice.
Foxp3 is a specific marker of Treg cells influencing immune tolerance through suppression of immune responses 9. Moreover, Treg cells may also promote innate and adaptive immunity 21, 22. We hypothesized that depletion of Foxp3 expression may modulate innate or adaptive immune cell populations in PBMC. Using CyTOF analysis, we assessed the immune profiles of PBMC in Foxp3-depleted mice compared to age matched control mice. To visualize clustering of each cell types, t-SNE analysis was performed based on gate strategy (Fig. 5A and Fig. 5C). All CyTOF were pre-processed and living single immune cells (CD45+) were retained after gating for further analysis (Fig. 5C). Each immune cell coordinates according to their expression of the 6 measure parameters including CD3e, CD4, CD8, CD11, CD19 and Ly-6G (Fig. 5A and C). As shown in Fig. 5B, we identified 5 major immune cell populations, including B lymphocytes (CD3e−CD19+), CD4+ T lymphocytes (CD3e+CD4+), CD8+ T lymphocytes (CD3e+CD8+), macrophages/monocytes (CD3−CD19−CD11b+LY-6G−) and granulocytes (CD3−CD19−CD11b+LY-6G+) based on the canonical cell markers. The t-SNE analysis revealed that depletion of Foxp3 expression increased the proportion of granulocytes or macrophages/monocytes, while other subtypes of immune cells (B lymphocytes, T lymphocytes) showed a similar pattern in Foxp3-depleted mice compared to age matched control (Fig. 5B). Next, we quantified the sub-population of each immune cell type in Foxp3-depleted mice compared to age matched control (Fig. 5D-5F). There were no significant differences between Foxp3-depleted mice and age matched control mice in respect with the percentage of CD3e−CD19+cells, CD3e+CD19− cells, CD3e+CD4+ cells and CD3e+CD8+ cells (Fig. 5E and 5F). Interestingly, the percentage of CD3−CD19−CD11b+LY-6G+ cells (granulocytes, Fig. 5D, left panel) and CD3−CD19−CD11b+LY-6G− cells (macrophages/monocytes, Fig. 5D, right panel) were significantly higher by 84% and 23%, respectively compared to age matched control mice (*p < 0.05, Fig. 5D).
Based on this evidence suggesting a role of Tregs in the regulation of innate immune cells, next we explored alteration of cytokines or chemokine production released by innate immune cells in plasma of Foxp3-depleted mice using a proteome profiler array. A total of 6 cytokines were altered in Foxp3-depleted mice with significantly 4 cytokines upregulated (interferon-gamma, IFN-γ; macrophage-colony stimulating factor, M-CSF; triggering receptor expressed on myeloid cells-1, TREM-1; IL-13), 1 cytokine downregulated (tissue inhibitor of metallopeptidase-1, TIMP-1) and a tendency toward 1 cytokine upregulated (IL-17) compared to age matched control mice (Fig. 5G). These results suggest the potential alteration of peripheral innate immunity through depletion of Foxp3 expression.
Several studies have reported that granulocytes derived MMP-9 activity may lead to damage of the BBB23. Based on this evidence and our immune profiling outcome showing elevated levels of granulocyte and monocytes/macrophages, we continued to explore MMP-9 expression in the brain. As shown in Fig. 5H, Foxp3-depleted mice showed significant transient elevation of MMP-9 expression in the hippocampal formation (*p < 0.05, Fig. 5H) compared to age matched control mice which we found being restored to baseline level in Foxp3-restored mice (Fig. 5I). These results suggest that depletion of Foxp3 expression in PBMC may lead to an increase of innate immune cells, possibly influencing transient MMP-9 expression in the brain.
Depletion of Foxp3 expression induces inflammasome activation coinciding with cognitive impairment and Aβ peptide burden in the hippocampal formation in the 5XFAD mice.
Further IPA-derived functional network mapping analysis was performed to identify direct and indirect relationships among DEGs and DEGs’ regulators in Foxp3-depleted mice, which suggested the ‘Developmental disorder, hereditary disorder, and metabolic disease’ network being highly predicated among 31 molecules (score 48). Among them, we found amyloid precursor protein (APP) which is associated with pathogenesis of AD being predicted as a key molecule in Foxp3-depleted mice (Fig. 6A). To examine the relevance of this mechanism in AD pathogenesis, we generated a double transgenic 5xFAD/Foxp3 cKO mouse model to investigate whether depletion of Foxp3 expression in 5xFAD mice would accelerate cognitive impairment and AD neuropathology compared to 5xFAD mice as assessed by NOR (Fig. 6B, upper panel). In a control study, we found no significant change of locomotion activity between all experimental mice groups (Fig. 6B, bottom panel). Interestingly, discrimination index as a measure of memory function significantly decreased in 5.5-month-old 5xFAD/Foxp3-depleted mice which are pre-symptomatic at this age. (*p < 0.05, Fig. 6B, bottom panel). We next evaluated the levels of Aβ peptide and Aβ burden, the hallmark neuropathological characteristic of AD, assessed by ELISA and immunohistochemistry, respectively. We find that statistical significance for reduction of Aβ1−40 isoform and increase of Aβ1−42 isoform, with resulting increase of Aβ1−42 / Aβ1−40 ratio in hippocampal formation of 5xFAD/Foxp3-depleted mice (*p < 0.05, Fig. 6C). In addition, elevation of Aβ plaque burden was observed in dentate gyrus layer of hippocampal formation in 5xFAD/Foxp3-depleted mice compared to age matched 5xFAD mice as assessed by 6E10 immunostaining (Fig. 6D).
This evidence was of high interests in view of the fact that inflammasome activation is currently implicated in mechanisms associated with Aβ pathology and neurodegeneration 24, 25. Based on this, we next examined the potential role of caspase-1 activation and IL-1β generation in 5xFAD/Foxp3-depleted mice (Fig. 6E and 6F). We found the protein expression of the active form of Caspase-1 in the hippocampal formation was significantly increased in 5xFAD/Foxp3-depleted mice compared to age matched 5xFAD mice (*p < 0.05, Fig. 6E). In addition, 5xFAD/Foxp3-depleted mice showed an increasing trend of elevation of IL-1β compared to age matched 5xFAD mice (p = 0.0611, Fig. 6E).
Taken together, these results demonstrate that depletion of Foxp3 expression may lead to inflammasome activation, AD-type neuropathology, and acceleration of the onset of cognitive impairment.