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Cited by 111Neuroinflammation and Neurodegeneration MechanismsGut microbiota and healthTryptophan and brain disorders

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The gut microbiome modulates the transformation of microglial subtypes

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Huang, Yu, Wu, Jing, Zhang, Hanping, Li, Yifan, Wen, Lu, Tan, Xunmin, Cheng, Ke, Liu, Yiyun, Pu, Juncai, Liu, Lanxiang, Wang, Haiyang, Li, Wenxia, Perry, Seth W., Wong, Ma-Li, Licinio, Julio, Zheng, Peng, Xie, Peng·2023·Molecular Psychiatry
DOI 10.1038/s41380-023-02017-yPMC PMC10208978
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Abstract

Clinical and animal studies have shown that gut microbiome disturbances can affect neural function and behaviors via the microbiota–gut–brain axis, and may be implicated in the pathogenesis of several brain diseases. However, exactly how the gut microbiome modulates nervous system activity remains obscure. Here, using a single-cell nucleus sequencing approach, we sought to characterize the cell type–specific transcriptomic changes in the prefrontal cortex and hippocampus derived from germ-free (GF), specific pathogen free, and colonized-GF mice. We found that the absence of gut microbiota resulted in cell-specific transcriptomic changes. Furthermore, microglia transcriptomes were preferentially influenced, which could be effectively reversed by microbial colonization. Significantly, the gut microbiome modulated the mutual transformation of microglial subpopulations in the two regions. Cross-species analysis showed that the transcriptome changes of these microglial subpopulations were mainly associated with Alzheimer’s disease (AD) and major depressive disorder (MDD), which were further supported by animal behavioral tests. Our findings demonstrate that gut microbiota mainly modulate the mutual transformation of microglial subtypes, which may lead to new insights into the pathogenesis of AD and MDD.

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Fig. 1 Gut microbiota absence changed microglial proportion in the Hip and PFC. a Schematic graph shows the workflow of acquiring hippocampus (Hip) and prefrontal lobe cortex (PFC) samples, nuclei isolation and droplet-based capture (10X Genomics) to produce cell type–specific transcriptomic signatures. b, c UMAP graph-based clustering and visualization of all captured nuclei for the Hip (b) and PFC (c). CNS resident cell types included excitatory neuron, interneuron, microglia, astrocyte, oligodendrocyte, OPC, ependymal cell, endothelial cell, smooth muscular cell, and fibroblast (Endo&SMC&Fibro). d In the Hip, GF mice had significantly decreased microglial proportion than SPF mice, and microbial colonization failed to rescue this change. The downregulated hippocampal astrocyte and OPC proportion in GF group relative to the SPF group were reversed by microbial colonization in the CGF group (SPF, n = 3; GF, n = 3; CGF, n = 3; data are mean ± SEM; GF vs. SPF, microglia, *P = 0.013, Excitatory neuron, *P = 0.0444, astrocyte, **P = 0.005, oligodendrocyte, *P = 0.0394, OPC, *P = 0.015; GF vs. CGF, microglia, P = 0.662, astrocyte, ***P = 0.0003, Excitatory neuron, *P = 0.0342, OPC, *P = 0.034; P values are from ANOVA post hoc analysis-LSD test). e In the PFC, there was an increased trend of proportion of microglia in GF group relative to SPF groups, which could be reversed by microbial colonization (SPF, n = 3; GF, n = 3; CGF, n = 3; data are mean ± SEM; GF vs. CGF, microglia, *P = 0.021; P values are from ANOVA post hoc analysis-LSD test).
Fig. 1 Gut microbiota absence changed microglial proportion in the Hip and PFC. a Schematic graph shows the workflow of acquiring hippocampus (Hip) and prefrontal lobe cortex (PFC) samples, nuclei isolation and droplet-based capture (10X Genomics) to produce cell type–specific transcriptomic signatures. b, c UMAP graph-based clustering and visualization of all captured nuclei for the Hip (b) and PFC (c). CNS resident cell types included excitatory neuron, interneuron, microglia, astrocyte, oligodendrocyte, OPC, ependymal cell, endothelial cell, smooth muscular cell, and fibroblast (Endo&SMC&Fibro). d In the Hip, GF mice had significantly decreased microglial proportion than SPF mice, and microbial colonization failed to rescue this change. The downregulated hippocampal astrocyte and OPC proportion in GF group relative to the SPF group were reversed by microbial colonization in the CGF group (SPF, n = 3; GF, n = 3; CGF, n = 3; data are mean ± SEM; GF vs. SPF, microglia, *P = 0.013, Excitatory neuron, *P = 0.0444, astrocyte, **P = 0.005, oligodendrocyte, *P = 0.0394, OPC, *P = 0.015; GF vs. CGF, microglia, P = 0.662, astrocyte, ***P = 0.0003, Excitatory neuron, *P = 0.0342, OPC, *P = 0.034; P values are from ANOVA post hoc analysis-LSD test). e In the PFC, there was an increased trend of proportion of microglia in GF group relative to SPF groups, which could be reversed by microbial colonization (SPF, n = 3; GF, n = 3; CGF, n = 3; data are mean ± SEM; GF vs. CGF, microglia, *P = 0.021; P values are from ANOVA post hoc analysis-LSD test).CC BY · PubMed Central
Fig. 2 Gut microbiota mainly affected microglia in the two brain regions. a, b Venn diagrams describe 846 and 1333 cell-specific DEGs across six major cell types in the Hip (a) and PFC (b). c DEGs (GF vs. SPF) downsampling analysis showed greater microglial gene dysregulations in the Hip (10 times repetition; Microglia vs Astrocyte, ***P = 4.9471E–11, Microglia vs Oligodendrocyte, *P = 0.0395, Microglia vs OPC, ***P = 3.4168E–21, Microglia vs Excitatory neuron, ***P = 1.0866E–19, Microglia vs Interneuron, ***P = 6.7238E–24; P values are from two-tailed Student’s t-test). d DEGs (GF vs. SPF) downsampling analysis showed greater microglial gene dysregulations in PFC, Microglia vs Astrocyte, ***P = 2.5005E–14, Microglia vs Oligodendrocyte, ***P = 1.1953E–17, Microglia vs OPC, *P = 4.019E–20, Microglia vs Excitatory neuron, ***P = 1.1021E–13, Microglia vs Interneuron, ***P = 1.562E–13; 10 times repetition; P values are from two-tailed Student’s t-test).
Fig. 2 Gut microbiota mainly affected microglia in the two brain regions. a, b Venn diagrams describe 846 and 1333 cell-specific DEGs across six major cell types in the Hip (a) and PFC (b). c DEGs (GF vs. SPF) downsampling analysis showed greater microglial gene dysregulations in the Hip (10 times repetition; Microglia vs Astrocyte, ***P = 4.9471E–11, Microglia vs Oligodendrocyte, *P = 0.0395, Microglia vs OPC, ***P = 3.4168E–21, Microglia vs Excitatory neuron, ***P = 1.0866E–19, Microglia vs Interneuron, ***P = 6.7238E–24; P values are from two-tailed Student’s t-test). d DEGs (GF vs. SPF) downsampling analysis showed greater microglial gene dysregulations in PFC, Microglia vs Astrocyte, ***P = 2.5005E–14, Microglia vs Oligodendrocyte, ***P = 1.1953E–17, Microglia vs OPC, *P = 4.019E–20, Microglia vs Excitatory neuron, ***P = 1.1021E–13, Microglia vs Interneuron, ***P = 1.562E–13; 10 times repetition; P values are from two-tailed Student’s t-test).CC BY · PubMed Central
Fig. 3 The gut microbiome modulates the mutual transformation between microglial subpopulations. a UMAP plot depicting microglial subpopulations divided by groups in the Hip. Graph clustering shows that GF cells were located primarily in region #1, and SPF and CGF cells were located in region #2. b Bar chart showing that subcluster 0 (Hip_M0) was enriched in SPF and CGF groups and diminished in GF group, subclusters 1 (Hip_M1) and 4 (Hip_M4) were enriched in the GF group and reversed by microbiome colonization. c UMAP plot depicting Hip_M1 and Hip_M4 located mainly at region #1, Hip_M0 at region #2. Combined with Pseudotime analysis (Supplementary Fig. 8a, c, e), the arrow showed a shifting trend of transforming relationships between Hip_M0, Hip_M1 and Hip_M4. d UMAP plot depicts microglial subpopulations divided by groups in the PFC. Graph clustering shows that GF cells contributed more plots in region #1 than SPF and CGF cells. Furthermore, cells in region #2 primarily belonged to SPF and CGF groups. e The bar chart showed that subcluster 0 (PFC_M0) was enriched in SPF and CGF groups and decreased in the GF group; subcluster 2 (PFC_M2) was enriched in the GF group and reversed by colonization. f UMAP plot depicted PFC_M2 mostly located at region #1, PFC_M0 at region #2. Combined with Pseudotime analysis (Supplementary Fig. 8 b, d, f), the arrow showed a shifting trend of transforming the relationship between PFC_M0 and PFC_M2.
Fig. 3 The gut microbiome modulates the mutual transformation between microglial subpopulations. a UMAP plot depicting microglial subpopulations divided by groups in the Hip. Graph clustering shows that GF cells were located primarily in region #1, and SPF and CGF cells were located in region #2. b Bar chart showing that subcluster 0 (Hip_M0) was enriched in SPF and CGF groups and diminished in GF group, subclusters 1 (Hip_M1) and 4 (Hip_M4) were enriched in the GF group and reversed by microbiome colonization. c UMAP plot depicting Hip_M1 and Hip_M4 located mainly at region #1, Hip_M0 at region #2. Combined with Pseudotime analysis (Supplementary Fig. 8a, c, e), the arrow showed a shifting trend of transforming relationships between Hip_M0, Hip_M1 and Hip_M4. d UMAP plot depicts microglial subpopulations divided by groups in the PFC. Graph clustering shows that GF cells contributed more plots in region #1 than SPF and CGF cells. Furthermore, cells in region #2 primarily belonged to SPF and CGF groups. e The bar chart showed that subcluster 0 (PFC_M0) was enriched in SPF and CGF groups and decreased in the GF group; subcluster 2 (PFC_M2) was enriched in the GF group and reversed by colonization. f UMAP plot depicted PFC_M2 mostly located at region #1, PFC_M0 at region #2. Combined with Pseudotime analysis (Supplementary Fig. 8 b, d, f), the arrow showed a shifting trend of transforming the relationship between PFC_M0 and PFC_M2.CC BY · PubMed Central
Fig. 4 Microglial genes regulated by the gut microbiota are linked with AD and MDD. a, b In Hip (a) and PFC (b), reversed microglial genes were closed to neuropsychiatric diseases, such as AD and MDD. c 19 genes overlapped between plague-induced genes (PIGs), disease-associated microglia (DAM), and reversed genes, including Apoe, Cx3cr1, Trem2, Fcer1g, C1qa, Fcrls, C1qb, Itm2b, C1qc, Man2b1, Cd9, Olfml3, Cst3, Ctsb, Ctsl, Gusb, Ctss, Hexa, and Ctsz. d Ten genes overlapped between microglial DEGs in depression like macaque (unpublished data), MDD risk gene in the DisGeNet database and reversed genes in microglia (converted to HUGO Gene Symbol), including FKBP5, AUTS2, ERBB4, NEGR1, NRG3, RABGAP1L, SLC1A3, ANK3, CTTNBP2, and ITGB5.
Fig. 4 Microglial genes regulated by the gut microbiota are linked with AD and MDD. a, b In Hip (a) and PFC (b), reversed microglial genes were closed to neuropsychiatric diseases, such as AD and MDD. c 19 genes overlapped between plague-induced genes (PIGs), disease-associated microglia (DAM), and reversed genes, including Apoe, Cx3cr1, Trem2, Fcer1g, C1qa, Fcrls, C1qb, Itm2b, C1qc, Man2b1, Cd9, Olfml3, Cst3, Ctsb, Ctsl, Gusb, Ctss, Hexa, and Ctsz. d Ten genes overlapped between microglial DEGs in depression like macaque (unpublished data), MDD risk gene in the DisGeNet database and reversed genes in microglia (converted to HUGO Gene Symbol), including FKBP5, AUTS2, ERBB4, NEGR1, NRG3, RABGAP1L, SLC1A3, ANK3, CTTNBP2, and ITGB5.CC BY · PubMed Central
Fig. 5 Cross-species analysis described that microglia subpopulations regulated by the gut microbiota were associated with AD and MDD. a Circos graph shows microglial subpopulations highly associated with AD and MDD cross humans, mice and macaca (proportion of segment means –logFDR, Fisher’s exact tests, FDR-BH corrected). b The OFT showed no differences in locomotion activity in the three groups (SPF, n = 17; GF, n = 16; CGF, n = 16; data are mean ± SEM; NA means P = 0.109; P values are from ANOVA test). c GF mice displayed decreased immobility time in FST and restored partially in CGF mice (n = 6 in GF and CGF, n = 7 in SPF; data are mean ± SEM; GF vs. SPF, ***P = 3.00E–06; GF vs. CGF, **P = 0.0011, SPF vs. CGF, ***P = 6.10E–05; P values are from ANOVA post hoc analysis-Tamhane T2 test). d The decrease of spontaneous alternation rate of GF mice in the Y-maze test compared to SPF mice was restored in CGF mice (n = 10/group; data are mean ± SEM; GF vs. SPF, *P = 0.022; GF vs. CGF, *P = 0.012; P values are from ANOVA post hoc analysis-LSD test).
Fig. 5 Cross-species analysis described that microglia subpopulations regulated by the gut microbiota were associated with AD and MDD. a Circos graph shows microglial subpopulations highly associated with AD and MDD cross humans, mice and macaca (proportion of segment means –logFDR, Fisher’s exact tests, FDR-BH corrected). b The OFT showed no differences in locomotion activity in the three groups (SPF, n = 17; GF, n = 16; CGF, n = 16; data are mean ± SEM; NA means P = 0.109; P values are from ANOVA test). c GF mice displayed decreased immobility time in FST and restored partially in CGF mice (n = 6 in GF and CGF, n = 7 in SPF; data are mean ± SEM; GF vs. SPF, ***P = 3.00E–06; GF vs. CGF, **P = 0.0011, SPF vs. CGF, ***P = 6.10E–05; P values are from ANOVA post hoc analysis-Tamhane T2 test). d The decrease of spontaneous alternation rate of GF mice in the Y-maze test compared to SPF mice was restored in CGF mice (n = 10/group; data are mean ± SEM; GF vs. SPF, *P = 0.022; GF vs. CGF, *P = 0.012; P values are from ANOVA post hoc analysis-LSD test).CC BY · PubMed Central

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Authors
Huang, Y., Wu, J., Zhang, H., Li, Y., Wen, L., Tan, X., Cheng, K., Liu, Y., Pu, J., Liu, L., Wang, H., Li, W., Perry, S. W., Wong, M.-L., Licinio, J., Zheng, P., & Xie, P.
Affiliations
  • The Affiliated Yongchuan Hospital of Chongqing Medical University (CN)
  • Stomatological Hospital of Chongqing Medical University (CN)
  • SUNY Upstate Medical University (US)
Corresponding author
  • Zheng, Peng
  • Xie, Peng
ORCID
Venue
Molecular Psychiatry
Publisher
Springer Science and Business Media LLC
Published
2023-03-13
Volume
28
Issue
4
Pages
1611-1621
ISSN
  • 1359-4184 (ISSN-L)
  • 1476-5578
DOI
10.1038/s41380-023-02017-y
PMC
PMC10208978
OpenAlex
W4324129695
Cited by
111
References
65
Citations by year
2026: 33 · 2025: 37 · 2024: 31 · 2023: 10
FWCI
11.36
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Psychiatry (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • State University of New York
  • National Natural Science Foundation of China (82101596, 81820108015, 82171523, 81971296)
  • China Postdoctoral Science Foundation (2021MD703928, 2021M693926, 2021TQ0396, 2020TQ0393)
  • Chinese Academy of Medical Sciences (2019PT320002)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Life Sciences › Neuroscience › Biological Psychiatry › Tryptophan and brain disorders
Keywords
gut microbiota, microbiota-gut-brain axis, microglial subtypes, germ-free mice, microbial colonization, prefrontal cortex, hippocampus, cell type-specific transcriptomics, Alzheimer's disease, major depressive disorder, neuroinflammation
MeSH terms
Gastrointestinal Microbiome, Alzheimer Disease, Animals, Depression, Major Depressive Disorder, Prefrontal Cortex, Microglia, Mice

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