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Cited by 263Gut microbiota and healthTryptophan and brain disordersGastrointestinal motility and disorders

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Gut microbiota from NLRP3-deficient mice ameliorates depressive-like behaviors by regulating astrocyte dysfunction via circHIPK2

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Zhang, Yuan, Huang, Rongrong, Cheng, Mengjing, Wang, Lirui, Chao, Jie, Li, Junxu, Zheng, Peng, Xie, Peng, Zhang, Zhijun, Yao, Honghong·2019·Microbiome
DOI 10.1186/s40168-019-0733-3PMC PMC6706943
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Abstract

BACKGROUND: Inflammasomes have been found to interact with the gut microbiota, and this effect is associated with depression, but the mechanisms underlying this interaction have not been elucidated in detail. RESULTS: The locomotor activity of NLRP3 KO mice was significantly greater than that of their WT littermates, while cohousing and transplantation of the NLRP3 KO gut microbiota avoid the effects of NLRP3 KO on the general locomotor activity at baseline. Meanwhile, transplantation of the NLRP3 KO microbiota alleviated the CUS-induced depressive-like behaviors. The compositions of the gut microbiota in NLRP3 KO mice and WT mice were significantly different in terms of the relative abundance of Firmicutes, Proteobacteria, and Bacteroidetes. Fecal microbiota transplantation (FMT) from NLRP3 KO mice significantly ameliorated the depressive-like behavior induced by chronic unpredictable stress (CUS) in recipient mice. Given the correlation between circular RNA HIPK2 (circHIPK2) and depression and the observation that the level of circHIPK2 expression was significantly increased in CUS-treated mice compared with that in the control group, further experiments were performed. FMT significantly ameliorated astrocyte dysfunction in recipient mice treated with CUS via inhibition of circHIPK2 expression. CONCLUSIONS: Our study illustrates the involvement of the gut microbiota-circHIPK2-astrocyte axis in depression, providing translational evidence that transplantation of the gut microbiota from NLRP3 KO mice may serve as a novel therapeutic strategy for depression.

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Fig. 1 NLRP3 KO gut microbiota affected depressive-like behavior. a Compared with WT mice, NLRP3 inflammasome deficiency exerted no significant effect on the sucrose preference induced by CUS. b–d Compared with WT mice, NLRP3 inflammasome deficiency inhibited the increased immobility time in the FST (b) and TST (c) induced by CUS. N = 6–13 mice/group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the WT control group. ##p < 0.01 and ###p < 0.001 vs. the CUS-treated WT group using one-way ANOVA followed by the Holm-Sidak test. d–f Cohousing reduced the significant differences in behavior between WT and NLRP3 KO littermates, as demonstrated by the FST (d) and TST (e). f Cohousing exerted no significant effect on sucrose preference. N = 11 mice/group. *p < 0.05 and ***p < 0.001 vs. the si-house-treated WT group using Student’s t test. si-house, only WT mice or KO mice were housed in a cage; cohouse, WT and KO mice were housed in a cage
Fig. 1 NLRP3 KO gut microbiota affected depressive-like behavior. a Compared with WT mice, NLRP3 inflammasome deficiency exerted no significant effect on the sucrose preference induced by CUS. b–d Compared with WT mice, NLRP3 inflammasome deficiency inhibited the increased immobility time in the FST (b) and TST (c) induced by CUS. N = 6–13 mice/group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the WT control group. ##p < 0.01 and ###p < 0.001 vs. the CUS-treated WT group using one-way ANOVA followed by the Holm-Sidak test. d–f Cohousing reduced the significant differences in behavior between WT and NLRP3 KO littermates, as demonstrated by the FST (d) and TST (e). f Cohousing exerted no significant effect on sucrose preference. N = 11 mice/group. *p < 0.05 and ***p < 0.001 vs. the si-house-treated WT group using Student’s t test. si-house, only WT mice or KO mice were housed in a cage; cohouse, WT and KO mice were housed in a cageCC BY · PubMed Central
Fig. 2 NLRP3 inflammasome deficiency affected the gut microbiota composition. a Three-dimensional PCoA of unweighted UniFrac distances showed obvious differences in the gut microbiota composition between WT and NLRP3 KO mice. Heatmap of the 120 discriminative OTUs between WT and NLRP3 KO mice. Each OTU ID and taxonomic assignment is provided to the right of the heatmap. b Relative abundances of genera significantly altered by NLRP3 inflammasome deficiency in the gut microbiota. N = 14 mice/group. **p < 0.01 and ***p < 0.001 vs. the WT group using the Mann-Whitney test
Fig. 2 NLRP3 inflammasome deficiency affected the gut microbiota composition. a Three-dimensional PCoA of unweighted UniFrac distances showed obvious differences in the gut microbiota composition between WT and NLRP3 KO mice. Heatmap of the 120 discriminative OTUs between WT and NLRP3 KO mice. Each OTU ID and taxonomic assignment is provided to the right of the heatmap. b Relative abundances of genera significantly altered by NLRP3 inflammasome deficiency in the gut microbiota. N = 14 mice/group. **p < 0.01 and ***p < 0.001 vs. the WT group using the Mann-Whitney testCC BY · PubMed Central
Fig. 3 Transplantation of the NLRP3 KO gut microbiota ameliorated CUS-induced depressive-like behaviors. a FMT experimental design and behavioral tests. Mice were treated with antibiotics for 7 weeks and gavaged with the fecal contents of either WT or NLRP3 KO donor mice for 3 days. After 1 week of recovery, the FMT recipient mice were subjected to CUS for 4 weeks, and behavioral tests were performed before the mice were sacrificed. b Compared to WT microbiota recipient mice, NLRP3 KO microbiota recipient mice displayed an inhibition of the CUS-induced decrease in sucrose preference. c, d Compared to WT microbiota recipient mice, NLRP3 KO microbiota recipient mice displayed an inhibition of the CUS-induced increase in immobility time in the FST (c) and TST (d). N = 20 mice/group. **p < 0.01 and ***p < 0.001 vs. the FMT-WT control group. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. the CUS-treated FMT-WT group using one-way ANOVA followed by the Holm-Sidak test. e Three-dimensional PCoA of unweighted UniFrac distances showed obvious differences in the gut microbiota composition between FMT-WT and FMT-NLRP3 KO mice with/without CUS treatment. f Heatmap of the 61 discriminative OTUs among FMT-WT and FMT-NLRP3 KO mice with/without CUS treatment. Each OTU ID and taxonomic assignment is provided to the right of the heatmap. Relative abundances of the phyla present in samples from the FMT-WT control group (pink bar), CUS-treated FMT-WT group (blue bar), FMT-NLRP3 KO control group (purple bar), and CUS-treated FMT-NLRP3 KO group (green bar). g Mice transplanted with the NLRP3 KO microbiota showed an inhibition of the CUS-induced alteration in the relative abundances of genera compared to WT microbiota recipient mice. N = 12-16 mice/group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the FMT-WT control group. #p < 0.05 and ##p < 0.01 vs. the CUS-treated FMT-WT group using the Mann-Whitney test
Fig. 3 Transplantation of the NLRP3 KO gut microbiota ameliorated CUS-induced depressive-like behaviors. a FMT experimental design and behavioral tests. Mice were treated with antibiotics for 7 weeks and gavaged with the fecal contents of either WT or NLRP3 KO donor mice for 3 days. After 1 week of recovery, the FMT recipient mice were subjected to CUS for 4 weeks, and behavioral tests were performed before the mice were sacrificed. b Compared to WT microbiota recipient mice, NLRP3 KO microbiota recipient mice displayed an inhibition of the CUS-induced decrease in sucrose preference. c, d Compared to WT microbiota recipient mice, NLRP3 KO microbiota recipient mice displayed an inhibition of the CUS-induced increase in immobility time in the FST (c) and TST (d). N = 20 mice/group. **p < 0.01 and ***p < 0.001 vs. the FMT-WT control group. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. the CUS-treated FMT-WT group using one-way ANOVA followed by the Holm-Sidak test. e Three-dimensional PCoA of unweighted UniFrac distances showed obvious differences in the gut microbiota composition between FMT-WT and FMT-NLRP3 KO mice with/without CUS treatment. f Heatmap of the 61 discriminative OTUs among FMT-WT and FMT-NLRP3 KO mice with/without CUS treatment. Each OTU ID and taxonomic assignment is provided to the right of the heatmap. Relative abundances of the phyla present in samples from the FMT-WT control group (pink bar), CUS-treated FMT-WT group (blue bar), FMT-NLRP3 KO control group (purple bar), and CUS-treated FMT-NLRP3 KO group (green bar). g Mice transplanted with the NLRP3 KO microbiota showed an inhibition of the CUS-induced alteration in the relative abundances of genera compared to WT microbiota recipient mice. N = 12-16 mice/group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the FMT-WT control group. #p < 0.05 and ##p < 0.01 vs. the CUS-treated FMT-WT group using the Mann-Whitney testCC BY · PubMed Central
Fig. 4 Transplantation of the gut microbiota from NLRP3 KO mice alleviated astrocyte dysfunction in CUS mice. a NLRP3 inflammasome deficiency rescued the decreased GFAP levels induced by CUS. N = 6 mice/group. ***p < 0.001 vs. the WT control group. #p < 0.05 vs. the CUS-treated WT group using one-way ANOVA followed by the Holm-Sidak test. b Colonization with the NLRP3 KO microbiota attenuated the decreased GFAP expression induced by CUS. N = 11 mice/group. c Effect of NLRP3 KO microbiota colonization on astrocyte dysfunction induced by CUS. Representative images of astrocyte immunostaining for GFAP in mouse hippocampi, followed by 3D reconstruction and Sholl analysis. Scale bars, 50 μm. d Quantification of GFAP-positive cells per square millimeter in mouse hippocampi. N = 4 mice/group. e–g Average branch number (e), total branch length (f), and total branch volume (g). n = 4 mice/group, 40 cells/group. *p < 0.05 and ***p < 0.001 vs. the FMT-WT control group. #p < 0.05 and ###p < 0.001 vs. the CUS-treated FMT-WT group using one-way ANOVA followed by the Holm-Sidak test.
Fig. 4 Transplantation of the gut microbiota from NLRP3 KO mice alleviated astrocyte dysfunction in CUS mice. a NLRP3 inflammasome deficiency rescued the decreased GFAP levels induced by CUS. N = 6 mice/group. ***p < 0.001 vs. the WT control group. #p < 0.05 vs. the CUS-treated WT group using one-way ANOVA followed by the Holm-Sidak test. b Colonization with the NLRP3 KO microbiota attenuated the decreased GFAP expression induced by CUS. N = 11 mice/group. c Effect of NLRP3 KO microbiota colonization on astrocyte dysfunction induced by CUS. Representative images of astrocyte immunostaining for GFAP in mouse hippocampi, followed by 3D reconstruction and Sholl analysis. Scale bars, 50 μm. d Quantification of GFAP-positive cells per square millimeter in mouse hippocampi. N = 4 mice/group. e–g Average branch number (e), total branch length (f), and total branch volume (g). n = 4 mice/group, 40 cells/group. *p < 0.05 and ***p < 0.001 vs. the FMT-WT control group. #p < 0.05 and ###p < 0.001 vs. the CUS-treated FMT-WT group using one-way ANOVA followed by the Holm-Sidak test.CC BY · PubMed Central
Fig. 5 Transplantation of the NLRP3 KO gut microbiota inhibited the increased expression of circHIPK2 in CUS mice. a–e The correlation of the relative abundances of phyla (Bacteroidetes and Firmicutes) and families (S24-7, Ruminococcaceae and Lachnospiraceae) with circHIPK2 levels in the plasma of CUS-treated mice. f circHIPK2 levels in plasma and hippocampi were significantly increased by CUS treatment. N = 9–12 mice/group. *p < 0.05 and ***p < 0.001 vs. the control group using Student’s t test. g, h circHIPK2 levels in the plasma (g) and hippocampi (h) of the WT microbiota recipient mice were significantly increased by CUS treatment, and these levels were significantly inhibited in the NLRP3 KO microbiota recipient mice. N = 6–8 mice/group. *p < 0.05 and **p < 0.01 vs. the FMT-WT control group. #p < 0.05, and ##p < 0.01 vs. the CUS-treated FMT-WT group using one-way ANOVA followed by the Holm-Sidak test. i Illustration of lentivirus microinjection and the experimental procedure. Mice were microinjected with the GFP-labeled circCon or circHIPK2 siRNA lentivirus for 2 weeks, followed by CUS treatment for another 4 weeks. Behavioral tests were performed before the mice were sacrificed. j circHIPK2 levels decreased in circHIPK2 siRNA-injected mice compared with those in circ siRNA control-injected mice in both the control and CUS-treated groups. k circHIPK2 siRNA microinjection significantly attenuated the CUS-induced decrease in sucrose preference. l, m circHIPK2 siRNA microinjection significantly inhibited the CUS-induced increase in immobility time in the FST (l) and TST (m). N = 7–15 mice/group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the circCon control group. #p < 0.05 and ##p < 0.01 vs. the CUS-treated circCon group using one-way ANOVA followed by the Holm-Sidak test.
Fig. 5 Transplantation of the NLRP3 KO gut microbiota inhibited the increased expression of circHIPK2 in CUS mice. a–e The correlation of the relative abundances of phyla (Bacteroidetes and Firmicutes) and families (S24-7, Ruminococcaceae and Lachnospiraceae) with circHIPK2 levels in the plasma of CUS-treated mice. f circHIPK2 levels in plasma and hippocampi were significantly increased by CUS treatment. N = 9–12 mice/group. *p < 0.05 and ***p < 0.001 vs. the control group using Student’s t test. g, h circHIPK2 levels in the plasma (g) and hippocampi (h) of the WT microbiota recipient mice were significantly increased by CUS treatment, and these levels were significantly inhibited in the NLRP3 KO microbiota recipient mice. N = 6–8 mice/group. *p < 0.05 and **p < 0.01 vs. the FMT-WT control group. #p < 0.05, and ##p < 0.01 vs. the CUS-treated FMT-WT group using one-way ANOVA followed by the Holm-Sidak test. i Illustration of lentivirus microinjection and the experimental procedure. Mice were microinjected with the GFP-labeled circCon or circHIPK2 siRNA lentivirus for 2 weeks, followed by CUS treatment for another 4 weeks. Behavioral tests were performed before the mice were sacrificed. j circHIPK2 levels decreased in circHIPK2 siRNA-injected mice compared with those in circ siRNA control-injected mice in both the control and CUS-treated groups. k circHIPK2 siRNA microinjection significantly attenuated the CUS-induced decrease in sucrose preference. l, m circHIPK2 siRNA microinjection significantly inhibited the CUS-induced increase in immobility time in the FST (l) and TST (m). N = 7–15 mice/group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the circCon control group. #p < 0.05 and ##p < 0.01 vs. the CUS-treated circCon group using one-way ANOVA followed by the Holm-Sidak test.CC BY · PubMed Central
Fig. 6 Knockdown of circHIPK2 expression alleviated the astrocyte dysfunction induced by CUS. a Colocalization of circHIPK2 and GFAP-positive cells in mouse hippocampi. Green represents circHIPK2; red, GFAP; blue, DAPI. Scale bar, 10 μm. b circHIPK2 siRNA microinjection significantly attenuated CUS-induced decreased GFAP expression. Mice were microinjected with the circCon or circHIPK2 siRNA lentivirus followed by CUS treatment. N = 6 mice/group. c Effect of circHIPK2 knockdown on astrocyte dysfunction induced by CUS. Representative images of astrocyte immunostaining for GFAP in mouse hippocampi, followed by 3D reconstruction and Sholl analysis. Scale bars, 50 μm. d Quantification of GFAP-positive cells per square millimeter in mouse hippocampi. N = 4 mice/group. e–g Average branch number (e), total branch length (f), and total branch volume (g). n = 4 mice/group, 40 cells/group. ***p < 0.001 vs. the circCon control group. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. the CUS-treated circCon group using one-way ANOVA followed by the Holm-Sidak test.
Fig. 6 Knockdown of circHIPK2 expression alleviated the astrocyte dysfunction induced by CUS. a Colocalization of circHIPK2 and GFAP-positive cells in mouse hippocampi. Green represents circHIPK2; red, GFAP; blue, DAPI. Scale bar, 10 μm. b circHIPK2 siRNA microinjection significantly attenuated CUS-induced decreased GFAP expression. Mice were microinjected with the circCon or circHIPK2 siRNA lentivirus followed by CUS treatment. N = 6 mice/group. c Effect of circHIPK2 knockdown on astrocyte dysfunction induced by CUS. Representative images of astrocyte immunostaining for GFAP in mouse hippocampi, followed by 3D reconstruction and Sholl analysis. Scale bars, 50 μm. d Quantification of GFAP-positive cells per square millimeter in mouse hippocampi. N = 4 mice/group. e–g Average branch number (e), total branch length (f), and total branch volume (g). n = 4 mice/group, 40 cells/group. ***p < 0.001 vs. the circCon control group. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. the CUS-treated circCon group using one-way ANOVA followed by the Holm-Sidak test.CC BY · PubMed Central
Fig. 7 Knockdown of circHIPK2 expression in astrocytes ameliorated the depressive-like behavior induced by CUS. a Illustration of AAV microinjection and the experimental procedure. Mice were microinjected with the eGFP-labeled circCon or circHIPK2 shRNA AAV for 2 weeks, followed by CUS treatment for 4 weeks. b circHIPK2 levels decreased in circHIPK2 shRNA AAV-injected mice compared with those in circCon shRNA AAV-injected mice in both the control and CUS-treated groups. c Specific circHIPK2 expression knockdown in astrocytes attenuated the CUS-induced decrease in sucrose preference. d, e Specific circHIPK2 expression knockdown in astrocytes inhibited the CUS-induced increase in immobility time in the FST (d) and TST (e). N = 7–15 mice/group. f Specific circHIPK2 expression knockdown in astrocytes attenuated the CUS-induced decrease in GFAP expression. N = 6 mice/group. g Specific knockdown of circHIPK2 expression in astrocytes attenuated the astrocyte dysfunction induced by CUS. Representative images of astrocyte immunostaining for GFAP in mouse hippocampi, followed by 3D reconstruction and Sholl analysis. Scale bars, 50 μm. h Quantification of GFAP-positive cells per square millimeter2in mouse hippocampi. N = 4 mice/group. i–k Average branch number (i), total branch length (j), and total branch volume (k). n = 4 mice/group, 40 cells/group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the circCon control group. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. the CUS-treated circCon group using one-way ANOVA followed by the Holm-Sidak test.
Fig. 7 Knockdown of circHIPK2 expression in astrocytes ameliorated the depressive-like behavior induced by CUS. a Illustration of AAV microinjection and the experimental procedure. Mice were microinjected with the eGFP-labeled circCon or circHIPK2 shRNA AAV for 2 weeks, followed by CUS treatment for 4 weeks. b circHIPK2 levels decreased in circHIPK2 shRNA AAV-injected mice compared with those in circCon shRNA AAV-injected mice in both the control and CUS-treated groups. c Specific circHIPK2 expression knockdown in astrocytes attenuated the CUS-induced decrease in sucrose preference. d, e Specific circHIPK2 expression knockdown in astrocytes inhibited the CUS-induced increase in immobility time in the FST (d) and TST (e). N = 7–15 mice/group. f Specific circHIPK2 expression knockdown in astrocytes attenuated the CUS-induced decrease in GFAP expression. N = 6 mice/group. g Specific knockdown of circHIPK2 expression in astrocytes attenuated the astrocyte dysfunction induced by CUS. Representative images of astrocyte immunostaining for GFAP in mouse hippocampi, followed by 3D reconstruction and Sholl analysis. Scale bars, 50 μm. h Quantification of GFAP-positive cells per square millimeter2in mouse hippocampi. N = 4 mice/group. i–k Average branch number (i), total branch length (j), and total branch volume (k). n = 4 mice/group, 40 cells/group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the circCon control group. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. the CUS-treated circCon group using one-way ANOVA followed by the Holm-Sidak test.CC BY · PubMed Central

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Authors
Zhang, Y., Huang, R., Cheng, M., Wang, L., Chao, J., Li, J., Zheng, P., Xie, P., Zhang, Z., & Yao, H.
Affiliations
  • Southeast University (CN)
  • China Pharmaceutical University (CN)
  • The Affiliated Yongchuan Hospital of Chongqing Medical University (CN)
  • Zhongda Hospital Southeast University (CN)
  • Nantong University (CN)
Corresponding author
Yao, Honghong
ORCID
Venue
Microbiome
Publisher
Springer Science and Business Media LLC
Published
2019-08-22
Volume
7
Issue
1
Pages
116-116
ISSN
2049-2618 (ISSN-L)
DOI
10.1186/s40168-019-0733-3
PMC
PMC6706943
OpenAlex
W2969822493
Cited by
263
References
48
Citations by year
2026: 17 · 2025: 36 · 2024: 46 · 2023: 47 · 2022: 55 · 2021: 38 · 2020: 24
FWCI
9.67
Type
journal article
Language
en
Open access
Licence
Locations
  • Microbiome (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (2017YFA0104303, 81603090, 81761138048, 81673410)
  • Government of Jiangsu Province
  • National Key Research and Development Program of China (2017YFA0104303)
  • Fundamental Research Funds for the Central Universities (2242019, 2017YFA0104303)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Life Sciences › Neuroscience › Biological Psychiatry › Tryptophan and brain disorders
  • Health Sciences › Medicine › Gastroenterology › Gastrointestinal motility and disorders
Keywords
gut microbiota, NLRP3 inflammasome, depression, depression-like behavior, astrocyte dysfunction, circular RNA, fecal microbiota transplantation, chronic unpredictable mild stress, locomotor activity, depression treatment
MeSH terms
Gastrointestinal Microbiome, Fecal Microbiota Transplantation, NLR Family, Pyrin Domain-Containing 3 Protein, Animals, Astrocytes, Behavior, Animal, Depression, Male, Mice, Inbred C57BL, Protein Serine-Threonine Kinases, Mice

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