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Rescue of maternal immune activation-induced behavioral abnormalities in adult mouse offspring by pathogen-activated maternal Treg cells

Nature Neuroscience volume 24, pages 818–830 (2021)Cite this article

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Abstract

Maternal immune activation (MIA) induced by lipopolysaccharides or polyinosinic:polycytidylic acid injections can induce behavioral abnormalities in adult mouse offspring. Here, we used the soluble tachyzoite antigen from Toxoplasma gondii, a parasite that infects approximately two billion people, to induce MIA in mice. The adult male offspring showed autism-relevant behaviors and abnormal brain microstructure, along with a pro-inflammatory T-cell immune profile in the periphery and upregulation of interleukin-6 in brain astrocytes. We show that adoptive transfer of regulatory T (Treg) cells largely reversed these MIA-induced phenotypes. Notably, pathogen-activated maternal Treg cells showed greater rescue efficacy than those from control donors. Single-cell RNA sequencing identified and characterized a unique group of pathogen-activated Treg cells that constitute 32.6% of the pathogen-activated maternal Treg population. Our study establishes a new preclinical parasite-mimicking MIA model and suggests therapeutic potential of adoptive Treg cell transfer in neuropsychiatric disorders associated with immune alterations.

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Fig. 1: STAg-elicited MIA leads to ASD-like behaviors in the offspring.
Fig. 2: STAg-MIA altered peripheral CD4+ T-cell differentiation in adult offspring.
Fig. 3: STAg-MIA-induced upregulation of astrocytic IL-6 in the adult offspring.
Fig. 4: Adoptive transfer of Treg cells restores CD4+ T-cell profile in MIA offspring.
Fig. 5: Therapeutic effects of adoptive transfer of Treg cells in adult MIA offspring.
Fig. 6: Adoptive transfer of Treg cells suppresses pro-inflammatory IL-6 expression in ASD-like offspring brain.
Fig. 7: Identification and characterization of unique subsets of MIATreg cells.
Fig. 8: Enhanced MIATreg cell infiltration into the brain parenchyma in adult MIA offspring.

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Data availability

The scRNA-sequencing dataset is available in the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/) under accession code GSE132803. Source data are provided with this paper. All other data presented in this study are available from the corresponding authors upon request.

Code availability

The R code for scRNA-seq analysis can be found in the Supplementary Software.

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Acknowledgements

We thank H.T. Wu for technical assistance with the USV experiments in pups. We thank all members of the laboratories of M.J. and Z.Z. for technical assistance and discussion. This work was supported by the National Natural Science Foundation of China (81971022 to Z.Z.), Program of Shanghai Academic Research Leader (19XD1423300 to Z.Z.), Natural Science Foundation of Jiangsu Province (BK20171049 to Z.X.), Science and Technology Commission of Shanghai Municipality (201409002600 to Z.Z.), Foundation of Jiangsu Province Key Laboratory of Modern Pathogen Biology (JX218GSP20171003 to Z.Z.), Shanghai Key Laboratory of Psychotic Disorders Open Grant (19-K01 to M.J.), Shanghai Mental Health Center-Clinical Research Center (CRC2019ZD01 to Z.Z.) and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20191835 to Z.Z.).

Author information

Author notes

  1. These authors contributed equally: Zhipeng Xu, Xiaoyun Zhang, Hao Chang.

Authors and Affiliations

  1. State Key Laboratory of Reproductive Medicine, Department of Pathogen Biology, Jiangsu Province Key Laboratory of Modern Pathogen Biology, Nanjing Medical University, Nanjing, China

    Zhipeng Xu, Hao Chang, Yangyue Ni, Ran Liu, Min Hou, Wen-Tao Liu & Minjun Ji

  2. Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Xiaoyun Zhang, Yue Kong, Xiaolin Zhang, Yang Hu, Zhi Yang, Rui Mao, Yasong Du, Shunying Yu & Zikai Zhou

  3. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Xiaoyun Zhang, Zhen Wang & Zikai Zhou

  4. Zhongshan Institute for Drug Discovery, the Institutes of Drug Discovery and Development, Chinese Academy of Sciences, Zhongshan, China

    Xiaoyun Zhang & Zikai Zhou

  5. Jiangsu Province Engineering Research Center of Antibody Drug, Nanjing, China

    Minjun Ji

  6. Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China

    Zikai Zhou

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Contributions

Conceptualization: Z.Z. and M.J.; STAg preparation and injection: Z.X., R.L., Y.N. and M.H.; behavioral tests and analysis: Xiaoyun Zhang, Xiaolin Zhang, Y.K. and Z.W.; flow cytometry: Z.X., H.C., Y.N. and W.-T.L.; immunohistochemistry: Xiaoyun Zhang, Y.K., Z.X. and S.Y.; two-photon imaging: Y.K. and Xiaoyun Zhang; Treg cell preparation and adoptive transfer: Z.X., H.C. and Y.K.; scRNA-seq and analysis: Xiaolin Zhang and Xiaoyun Zhang; MRI and analysis: Y.H., Z.Y. and R.M.; western blot, qPCR and ELISA: Xiaoyun Zhang, H.C. and Y.D.; funding acquisition: Z.Z., M.J. and Z.X.; supervision: Z.Z. and M.J.; Z.Z. wrote the paper with input from the co-authors.

Corresponding authors

Correspondence to Minjun Ji or Zikai Zhou.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Neuroscience thanks Staci Bilbo, Qizhi Tang, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Pro-inflammatory CD4+ T cell profile in the pregnant mother mice indicates STAg-elicited MIA.

a, Gating schemes for analysis of the percentages of CD3+CD4+ IFN-γ+ (TH1), CD3+CD4+IL-4+(TH2), and CD3+CD4+IL-17+(TH17) cells from R1 and R2. The expression of CD4+CD25+Foxp3+ (Treg) cells were gated from R1 and R3. Single-cell suspensions of splenic cells were labeled with CD3-APC and CD4-FITC, and then intracellularly labeled with PE-conjugated antibodies against IFN-γ, IL-4 or IL-17 for flow cytometric analysis of CD3+CD4+ IFN-γ+ (TH1), CD3+CD4+IL-4+ (TH2), and CD3+CD4+IL-17+ (TH17) cells, respectively. Cells were stained with CD4-FITC and CD25-APC, and then intracellularly labeled with PE-conjugated antibodies against Foxp3 for FACS analysis of CD4+CD25+Foxp3+ Treg cells. b-d, Flow cytometric analysis of splenic lymphocytes from pregnant mother mice prepared at three days after STAg injection. b, Bar chart shows the percentages of TH1, TH2, TH17 and Treg cells in CD4+ splenic lymphocytes. c, The absolute numbers of total splenic cells and (d) CD4+ T cells in PBS- or STAg-treated pregnant mother mice. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 2 Further behavioral analysis on the MIA offspring.

a-l, Maternal isolation-induced USV profiles. (a and b) Representative USV spectrograms from PBS and STAg pups on P12 and P14. The peak frequency at start (c), end (d) and the mean peak frequency (e); the peak amplitude at start (f), end (g) and the mean peak amplitude (h) of calls made by PBS and STAg pups at P5, P7, P9, P12 and P14. i-j, The sonograms of USVs emitted by the pups are classified into ten distinct categories and analyzed for the number of calls (i) and the duration of the calls (j) during the 5-min recording period at P7. (k and l) Ten syllable categories of 5-min recording were analyzed at P14 for the number of calls (k) and the duration of the calls (l). m-n, Open filed test. Total distance traveled (m) and mean speed (n) in 10 min of open field test. o-p, Elevated plus maze test. Number of entries into open arms (o) and percentage of entries into open arms (p). q, Olfactory function test. PBS and STAg offspring displayed comparable investigation when exposed to three non-social cues (water, banana, orange) and two social cues. r-s, The social interaction ratio in reciprocal social interactions. The ratio of experimental mouse and stimulus mouse during the test with age-matched peer (r) or a juvenile target (s). t-v, Three-chamber social interaction test. (t) Time spent in the left and right chambers during the habituation stage by PBS and STAg-MIA offspring. (u) Total time spent in the empty chamber and the chamber with stranger mouse 1 (S1) at social approach stage. (v) Total time spent in the chamber with S1 and the chamber with stranger mouse 2 (S2) at social novelty stage. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 3 STAg-elicited MIA did not alter learning and memory in the offspring.

a-e, Novel object recognition test. (a) Representative heatmaps of mouse movement at the training session (top) and test session (bottom) of PBS and STAg offspring. (b) Time spent exploring the identical objects A1 and A2 and discrimination index (c) in training session. (d) Time spent exploring the familiar object A and a novel object B and discrimination index (e) in test session. f-j, Barnes maze test. (f) The experimental paradigm. (g) The image of probe test on day 5 and representative heatmaps of PBS and STAg offspring movement. (h) The latency time to enter the hiding box in the Barnes maze during acquisition days 1–4. (i and j) The number of visits to the target hole (i) and time in target quadrant (j) in probe test. Data are presented as mean ± s.e.m. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 4 Immune cell profiles in MIA offspring.

a-e, Pregnant C57BL/6 mice were i.p. injected with STAg or vehicle (PBS) on E14.5. At 2 and 4 weeks of age, 2×106 bone marrow cells were stained with CD3-PerCP-Cy5.5, CD19-APC, and Gr1-FITC, respectively. a, Gating schemes for analysis of T cells (CD19−CD3+), B cells (CD19+CD3−) and granulocytes (Gr-1+CD19−) were gated from R1. b, Flow cytometric analysis of proportion of T cells (CD19−CD3+), B cells (CD19+CD3−) (b and c), and granulocytes (Gr-1+CD19−) (d and e) in bone marrow cells. f-j, (f) Gating schemes for analysis of the percentages of CD3+CD4+ (CD4+ T cells) and CD3+CD4+ CXCR5+PD1+ (Tfh) cells in the offspring. Single-cell suspensions of splenic cells were labeled with CD3-APC and CD4-FITC for flow cytometric analysis of CD3+CD4+ cells (g), and the percentage of CD3+CD4+ cells were analyzed (h). i, Cells were labeled with CD3-APC, CD4-FITC, CXCR5-Percp-Cy5.5, and PD1-PE for flow cytometric analysis of TFH cells (j). Data are presented as mean ± s.e.m. ****P < 0.0001. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 5 STAg-MIA specifically upregulated IL-6 expression in multiple brain regions in adult offspring.

a-c, The mRNA levels of IL-12, TNF-α, IFN-γ, IL-10, and IL-4 in the mouse hippocampi (a), striatum (b), cortices (c) were determined by qPCR in offspring from PBS and STAg treated mother mice. The gene mRNA levels were normalized to Gapdh. d, IL-6 protein level in serum measured by ELISA. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ****P < 0.0001. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 6 Further analysis of therapeutic effects of adoptive transfer of Treg cells in adult MIA offspring.

a and b, Open fields test. At 8 weeks of age, 5×105 of CTreg or MIATreg cells were transferred via intravenous injection into each adult PBS and STAg-MIA offspring. One week later, these mice were subjected to behavioral tests. Total distance traveled (a) and mean speed (b) in 10 min of open field test. c-e, Elevated plus maze test. Total time (c) and the percentage of time spent (d) in open arms, and the percentage of entries into open arms (e). f-g, The social interaction ratio in reciprocal social interactions. The ratio of experimental mouse and stimulus mouse during the test with age-matched mouse (f) or a juvenile target (g) measured after adoptive transfer of CTreg or MIATreg cells in PBS or STAg-MIA offspring. h-j, Three-chamber social interaction test. (h) Time spent in the left and right chambers during the habituation stage. i, Total time spent in the empty chamber and the chamber with stranger mouse 1 (S1) in social approach stage. j, Total time spent in the chamber with stranger mouse 1 (S1) and the chamber with stranger mouse 2 (S2) in social novelty stage. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 7 Long-term maintenance of transferred EGFP-Treg cells in MIA offspring.

a, Pregnant EGFP or WT C57BL/6 mice were i.p. injected with STAg or vehicle (PBS) on E14.5. CTreg or MIATreg cells (5×105) from EGPF mice were purified and transferred via intravenous injection into each adult STAg-MIA offspring. At 3 weeks and 8 weeks after adoptive transfer, 2×106 cells were stained with CD4-BV21and CD25-APC in the spleen and blood from the STAg-MIA offspring. The EGFP+ cells were gated from CD4+CD25+ cells. Statistical results of the percentage of CD4+CD25+ cells in EGFP+ cells in the blood (b) and spleen (c). Data are presented as mean ± s.e.m. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 8 Distribution of adoptively transferred Treg cells in the brain regions in adult MIA offspring.

a, Coronal plane of the mouse left hemisphere combined with immunohistology image of the right hemisphere section with matching stereotaxic coordinates (bregma −0.82 mm and interaural 2.98 mm). Adoptively transferred Treg cells are labeled with CMTMR (red), nuclei are labeled with DAPI (blue). Scale bar = 500 μm. b, Zoom-in images of representative brain regions: medial habenular nucleus (MHb) (region 1), cingulate/retrosplenial cortex (Cg/RS) (region 2), and primary somatosensory cortex, barrel field 0 (S1BF) (region 3). Scale bars = 25 μm. c, Coronal plane of the mouse left hemisphere combined with immunohistology image of the right hemisphere section with matching stereotaxic coordinates (bregma −2.46 mm and interaural 1.34 mm). Scale bar = 500 μm. d, Zoom-in images of representative brain regions: pre-commissural nucleus (PrC) (region 4), retrosplenial granular cortex (RSG) (region 5), and field CA2 of hippocampus (CA2) (region 6). The distribution pattern of transferred Tregs is highly similar in three independently repeated experiments.

Extended Data Fig. 9 Flow cytometric analysis of cell surface proteins in Treg cells.

a-f, Pregnant C57BL/6 mice were i.p. injected with STAg or vehicle (PBS) on E14.5. Splenic cells (2×106) were stained with CD4-FITC and CD25-APC, and then labeled with PD1-PE, CCR5-Percp-cy5.5, ICOS-BV421, KLRG-1-PE, or CCR4-PE for flow cytometric analysis of cell surface protein levels of the suppressive markers in CTreg or MIATreg cells. The percentages of PD1, CCR5, ICOS, KLRG-1, and CCR4 in CTreg or MIATreg cells were gated from CD4+CD25+ cells (a). Statistical comparation of the surface levels of PD1 (b), ICOS (c), KLRG1 (d), CCR4 (e) and CCR5 (f) are presented respectively. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ****P < 0.0001. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 10 MRI analysis of neuronal connectivity in the adult brain.

a, Different layers of T2 weighted whole brain images of P60 control (PBS) and STAg-MIA offspring mice. Scale bars, 5 mm. b, Summary graph showing unaltered brain area (mm2) and cortical thickness (mm) (c). d, Fiber tractography images of adult mouse brains. The color schemes are: red, left-right; green, ventral-dorsal; and blue, caudal-rostral. e, Fractional anisotropy (FA) values of major white matter tracts. f, Average axial diffusivity (AD), radial diffusivity (RD) (g), and mean diffusivity (MD) (h) of major white matter tracts. Summary results showing fiber density of whole brain (i), hippocampus (Hip) (j), corpus callosum (CC) (k) and cortex (Ctx) (l). Data are presented as mean ± s.e.m. *P < 0.05, ***P < 0.001. For detailed statistics information, see Supplementary Table 1.

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table for statistical details.

Supplementary Software

The R code for scRNA-seq analysis.

Source data

Source Data Fig. 3

Unprocessed western blot for Fig. 3.

Source Data Fig. 6

Unprocessed western blot for Fig. 6.

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Xu, Z., Zhang, X., Chang, H. et al. Rescue of maternal immune activation-induced behavioral abnormalities in adult mouse offspring by pathogen-activated maternal Treg cells. Nat Neurosci 24, 818–830 (2021). https://doi.org/10.1038/s41593-021-00837-1

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