Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Aug 24;14(17):3467.
doi: 10.3390/nu14173467.

Gut Microbiota Dysbiosis after Traumatic Brain Injury Contributes to Persistent Microglial Activation Associated with Upregulated Lyz2 and Shifted Tryptophan Metabolic Phenotype

Zhipeng Zheng  1   2 , Shuai Wang  2 , Chenghao Wu  2 , Yang Cao  3 , Qiao Gu  2 , Ying Zhu  2 , Wei Zhang  4 , Wei Hu  2
Affiliations

Gut Microbiota Dysbiosis after Traumatic Brain Injury Contributes to Persistent Microglial Activation Associated with Upregulated Lyz2 and Shifted Tryptophan Metabolic Phenotype

Zhipeng Zheng et al. Nutrients. .

Abstract

Traumatic brain injury (TBI) is a common cause of disability and mortality, affecting millions of people every year. The neuroinflammation and immune response post-TBI initially have neuroprotective and reparative effects, but prolonged neuroinflammation leads to secondary injury and increases the risk of chronic neurodegenerative diseases. Persistent microglial activation plays a critical role in chronic neuroinflammation post-TBI. Given the bidirectional communication along the brain-gut axis, it is plausible to suppose that gut microbiota dysbiosis post-TBI influences microglial activation. In the present study, hippocampal microglial activation was observed at 7 days and 28 days post-TBI. However, in TBI mice with a depletion of gut microbiota, microglia were activated at 7 days post-TBI, but not at 28 days post-TBI, indicating that gut microbiota contributes to the long-term activation of microglia post-TBI. In addition, in conventional mice colonized by the gut microbiota of TBI mice using fecal microbiota transplant (FMT), microglial activation was observed at 28 days post-TBI, but not at 7 days post-TBI, supporting the role of gut microbiota dysbiosis in persistent microglial activation post-TBI. The RNA sequencing of the hippocampus identified a microglial activation gene, Lyz2, which kept upregulation post-TBI. This persistent upregulation was inhibited by oral antibiotics and partly induced by FMT. 16s rRNA gene sequencing showed that the composition and function of gut microbiota shifted over time post-TBI with progressive dysbiosis, and untargeted metabolomics profiling revealed that the tryptophan metabolic phenotype was differently reshaped at 7 days and 28 days post-TBI, which may play a role in the persistent upregulation of Lyz2 and the activation of microglia. This study implicates that gut microbiota and Lyz2 are potential targets for the development of novel strategies to address persistent microglial activation and chronic neuroinflammation post-TBI, and further investigations are warranted to elucidate the specific mechanism.

Keywords: Lyz2; chronic neuroinflammation; gut microbiota; microglia; traumatic brain injury; tryptophan metabolism.

PubMed Disclaimer

Conflict of interest statement

The authors declared that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Design of the experiment and evaluation of brain injury and microglial activation at 7 days postTBI. (a) Design of the experiment (n = 6 per group). Mice of group C7 received sham surgery, mice of group T7 were subjected to TBI surgery without perioperative antibiotic treatment, the mice of group F7 underwent sham surgery and FMT using fecal microbiota from the mice of group T7, and mice of group A7 were subjected to TBI surgery with perioperative antibiotic treatment. All mice were sacrificed 7 days after TBI for sample collection. (b) Change of body weight before and after TBI (n = 6 per group). (c) Representative images of the brain H&E staining (n = 3 per group). (d) Representative images of brain IBA-1 immunofluorescence staining (red) (n = 3 per group). (e) Number of IBA-1 positive cells in hippocampus. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using unpaired Student’s t test. TBI, traumatic brain injury; H&E, hematoxylin–eosin; IBA-1, ionized calcium binding adapter molecule 1; ns, no significant.
Figure 2
Figure 2
Design of the experiment and evaluation of brain injury and microglial activation at 28 days post-TBI. (a) Design of the experiment (n = 6 per group). Mice of group C28 received sham surgery, mice of group T28 were subjected to TBI surgery without perioperative antibiotic treatment, mice of group F28 underwent sham surgery and FMT using fecal microbiota from the mice of group T28, and the mice of group A28 were subjected to TBI surgery with perioperative antibiotic treatment. All mice were sacrificed 28 days after TBI for sample collection. (b) Change of body weight before and after TBI (n = 6 per group). (c) Representative images of brain H&E staining (n = 3 per group). (d) Representative images of brain IBA-1 immunofluorescence staining (red) (n = 3 per group). (e) The number of IBA-1 positive cells in the hippocampus. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01 using unpaired Student’s t test. TBI, traumatic brain injury; H&E, hematoxylin–eosin; IBA-1, ionized calcium-binding adapter molecule 1; ns, no significant.
Figure 3
Figure 3
Transcriptome analysis of hippocampus post-TBI. PCA of the hippocampus RNA sequencing data at 7 days (a) and 28 days (b) post-TBI (n = 3 per group). Heatmap of DEGs in the hippocampus at 7 days (c) and 28 days (d) post-TBI (n = 3 per group). GO biological process enrichment analysis of DEGs in the hippocampus at 7 days (e) and 28 days (f) post-TBI (n = 3 per group). TBI, traumatic brain injury; PCA, principal component analysis; DEGs, differential expressed genes; GO, gene ontology.
Figure 4
Figure 4
Gut microbiota-dependent persistent upregulation of Lyz2. Advanced volcano plot analysis of gene expression levels of the hippocampus: between control mice and TBI mice at 7 days (a) and 28 days (b) post-TBI; between TBI mice and antibiotic-treated TBI mice at 7 days (c) 28 days (d) post-TBI; between control mice and TBI-colonized mice at 7 days (e) and 28 days (f) post-TBI (n = 3 per group). TBI, traumatic brain injury; Lyz2, lysozyme 2.
Figure 5
Figure 5
Untargeted metabolome analysis of serum post-TBI. PCA of the serum untargeted metabolic data at 7 days (a) and 28 days (b) post-TBI (n= 6 per group). KEGG pathway enrichment analysis of differential metabolites in serum at 7 days (c) and 28 days (d) post-TBI (n = 6 per group). TBI, traumatic brain injury; PCA, principal component analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 6
Figure 6
Alterations of the serum tryptophan metabolic pathway at 7 days post-TBI. Comparison of serum tryptophan (a) and major tryptophan metabolites in the indole pathway (b), serotonin pathway (c), and kynurenine pathway (d) at 7 days post-TBI. (e) Major tryptophan metabolism pathways are involved: (1) Indole pathway: Tryptophan can be directly metabolized by the gut microbiota into indole derivatives; (2) Serotonin pathway: Peripheral production of the neurotransmitter serotonin by enterochromaffin cells is influenced by the gut microbiota, and the brain can also synthesize serotonin; (3) Kynurenine pathway: kynurenine-producing pathway plays a critical role in immune response and neurobiological functions, which is also affected by gut microbiota. (f) The ratios of KYN/TRP and KYNA/KYN at 7 days post-TBI. ↑, increased; ↓, decreased in 7 days post-TBI mice. The tryptophan and its metabolite listed in the pathways were marked with black (unchanged), red (increased), and blue (decreased) solid boxes. Data are shown as median with range. * p < 0.05, ** p < 0.01 *** p < 0.001 using unpaired Student’s t test. TBI, traumatic brain injury; TRP, tryptophan; IPYA, indole-3-pyruvic acid; ILA, indole-3-lactic acid; IA, indole acrylic acid; IAAld, indole-3-acetaldehyde; IEt, indole-3-ethanol (tryptophol); IAM, indole-3-acetamide; IAA, indole acetic acid; 5-HT, 5-hydroxytryptamine (serotonin); KYN, kynurenine; KYNA, kynurenic acid; XA, xanthurenic acid; ns, no significant.
Figure 7
Figure 7
Alteration of serum tryptophan metabolic pathway at 28 days post-TBI. Comparison of serum tryptophan (a) and major tryptophan metabolites in indole pathway (b), serotonin pathway (c), and kynurenine pathway (d) at 28 days post-TBI. (e) Major tryptophan metabolism pathways are involved: (1) Indole pathway: Tryptophan can be directly metabolized by the gut microbiota into indole derivatives; (2) Serotonin pathway: Peripheral production of the neurotransmitter serotonin by enterochromaffin cells is influenced by the gut microbiota, and the brain can also synthesize serotonin; (3) Kynurenine pathway: Kynurenine-producing pathway plays a critical role in immune response and neurobiological functions, which is also affected by gut microbiota. (f) The ratios of KYN/TRP and KYNA/KYN at 28 days post-TBI. ↑, increased; ↓, decreased in 28 days post-TBI mice. The tryptophan and its metabolite listed in the pathways were marked with black (unchanged), red (increased), and blue (decreased) solid boxes. Data are shown as median with range. * p < 0.05, ** p < 0.01, *** p < 0.001 using unpaired Student’s t test. TBI, traumatic brain injury; TRP, tryptophan; IPYA, indole-3-pyruvic acid; ILA, indole-3-lactic acid; IA, indole acrylic acid; IAAld, indole-3-acetaldehyde; IEt, indole-3-ethanol (tryptophol); IAM, indole-3-acetamide; IAA, indole acetic acid; 5-HT, 5-hydroxytryptamine (serotonin); 5-HIAA, 5-hydroxyindole acetic acid; IAA, indole acetic acid; KYNA, kynurenic acid; XA, xanthurenic acid; ns, no significant.
Figure 8
Figure 8
Compositional change of gut microbiota post-TBI. The α-diversity of the gut microbiota at 7 days (a) and 28 days (b) post-TBI (n = 6 per group). PCoA of the gut microbiota composition with weighted UniFrac distance at 7 days (c) and 28 days (d) post-TBI (n = 6 per group). Relative abundance of gut microbiota at the phylum level at 7 days (e) and 28 days (f) post-TBI (n = 6 per group). Data are shown as median with range. * p< 0.05, ** p < 0.01 using Wilcoxon rank-sum test. TBI, traumatic brain injury; PCoA, principal coordinate analysis; ns, no significant.
Figure 9
Figure 9
Linear discriminant analysis effect size of gut microbiota. Cladogram of LEfSe at 7 days (a) and 28 days (c) post-TBI. LDA score of LEfSe at 7 days (b) and 28 days (d) post-TBI.
Figure 10
Figure 10
Functional difference analysis of gut microbiota. A comparison of the KEGG functional categories inferred from the 16S rRNA gene sequences using PICRUSt at 7 days (a) and 28 days (b) post-TBI. (n = 6 per group). KEGG, Kyoto Encyclopedia of Genes and Genomes; PICRUSt, phylogenetic investigation of communities by reconstruction of unobserved states.
See this image and copyright information in PMC

References

    1. Jiang J.-Y., Gao G.-Y., Feng J.-F., Mao Q., Chen L.-G., Yang X.-F., Liu J.-F., Wang Y.-H., Qiu B.-H., Huang X.-J. Traumatic brain injury in China. Lancet Neurol. 2019;18:286–295. doi: 10.1016/S1474-4422(18)30469-1. - DOI - PubMed
    1. Maas A.I.R., Menon D.K., Adelson P.D., Andelic N., Bell M.J., Belli A., Bragge P., Brazinova A., Büki A., Chesnut R.M., et al. Traumatic brain injury: Integrated approaches to improve prevention, clinical care, and research. Lancet Neurol. 2017;16:987–1048. doi: 10.1016/S1474-4422(17)30371-X. - DOI - PubMed
    1. Brett B.L., Gardner R.C., Godbout J., Dams-O’Connor K., Keene C.D. Traumatic Brain Injury and Risk of Neurodegenerative Disorder. Biol. Psychiatry. 2021;91:498–507. doi: 10.1016/j.biopsych.2021.05.025. - DOI - PMC - PubMed
    1. Morganti-Kossmann M.C., Semple B.D., Hellewell S.C., Bye N., Ziebell J.M. The complexity of neuroinflammation consequent to traumatic brain injury: From research evidence to potential treatments. Acta Neuropathol. 2019;137:731–755. doi: 10.1007/s00401-018-1944-6. - DOI - PubMed
    1. Loane D.J., Kumar A. Microglia in the TBI brain: The good, the bad, and the dysregulated. Pt 3Exp. Neurol. 2016;275:316–327. doi: 10.1016/j.expneurol.2015.08.018. - DOI - PMC - PubMed