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Cited by 225Neuroinflammation and Neurodegeneration MechanismsGut microbiota and healthTryptophan and brain disorders

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Inhibition of inflammatory microglia by dietary fiber and short-chain fatty acids

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Caetano-Silva, Maria Elisa, Rund, Laurie, Hutchinson, Noah T., Woods, Jeffrey A., Steelman, Andrew J., Johnson, Rodney W.·2023·Scientific Reports
DOI 10.1038/s41598-022-27086-xPMC PMC9935636
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Abstract

Microglia play a vital role maintaining brain homeostasis but can also cause persistent neuroinflammation. Short-chain fatty acids (SCFAs) produced by the intestinal microbiota have been suggested to regulate microglia inflammation indirectly by signaling through the gut-brain axis or directly by reaching the brain. The present work evaluated the anti-inflammatory effects of SCFAs on lipopolysaccharide (LPS)-stimulated microglia from mice fed inulin, a soluble fiber that is fermented by intestinal microbiota to produce SCFAs in vivo, and SCFAs applied to primary microglia in vitro. Feeding mice inulin increased SCFAs in the cecum and in plasma collected from the hepatic portal vein. Microglia isolated from mice fed inulin and stimulated with LPS in vitro secreted less tumor necrosis factor α (TNF-α) compared to microglia from mice not given inulin. Additionally, when mice were fed inulin and injected i.p with LPS, the ex vivo secretion of TNF-α by isolated microglia was lower than that secreted by microglia from mice not fed inulin and injected with LPS. Similarly, in vitro treatment of primary microglia with acetate and butyrate either alone or in combination downregulated microglia cytokine production with the effects being additive. SCFAs reduced histone deacetylase activity and nuclear factor-κB nuclear translocation after LPS treatment in vitro. Whereas microglia expression of SCFA receptors Ffar2 or Ffar3 was not detected by single-cell RNA sequencing analysis, the SCFA transporters Mct1 and Mct4 were. Nevertheless, inhibiting monocarboxylate transporters on primary microglia did not interfere with the anti-inflammatory effects of SCFAs, suggesting that if SCFAs produced in the gut regulate microglia directly it is likely through an epigenetic mechanism following diffusion.

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Figure 1 Effect of dietary fiber intake on the SCFAs (acetate, butyrate, and propionate) levels in the cecum, portal vein plasma, and heart plasma from mice fed 0 or 2.5% inulin for 2 weeks. Samples (0 or 2.5% inulin) were compared by unpaired t-test (cecal content) or 2-way ANOVA (sample location × diet) *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Figure 1 Effect of dietary fiber intake on the SCFAs (acetate, butyrate, and propionate) levels in the cecum, portal vein plasma, and heart plasma from mice fed 0 or 2.5% inulin for 2 weeks. Samples (0 or 2.5% inulin) were compared by unpaired t-test (cecal content) or 2-way ANOVA (sample location × diet) *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.CC BY · PubMed Central
Figure 2 Effect of dietary fiber intake on the TNF-α protein secretion (measured by ELISA) by primary microglia from mice fed 0 or 2.5% inulin for 2 months. TNF-α secretion by microglia was measured (A) after isolating and stimulating in vitro with LPS (0 or 1 ng/mL) or (B) after injecting mice i.p. with LPS (0 or 500 µg/kg) 4 h before isolating and culturing microglia 4 h, 8 h, 12 h, or 24 h. In (A), samples (0 or 2.5% inulin) were compared by unpaired t-test. Statistical difference between inulin levels was considered for P < 0.05. In (B), samples were analyzed by 2-way ANOVA (2 × 2 factorial arrangement of LPS and diet), followed by Šidák’s multiple comparisons test (*P < 0.05).
Figure 2 Effect of dietary fiber intake on the TNF-α protein secretion (measured by ELISA) by primary microglia from mice fed 0 or 2.5% inulin for 2 months. TNF-α secretion by microglia was measured (A) after isolating and stimulating in vitro with LPS (0 or 1 ng/mL) or (B) after injecting mice i.p. with LPS (0 or 500 µg/kg) 4 h before isolating and culturing microglia 4 h, 8 h, 12 h, or 24 h. In (A), samples (0 or 2.5% inulin) were compared by unpaired t-test. Statistical difference between inulin levels was considered for P < 0.05. In (B), samples were analyzed by 2-way ANOVA (2 × 2 factorial arrangement of LPS and diet), followed by Šidák’s multiple comparisons test (*P < 0.05).CC BY · PubMed Central
Figure 3 Effect of short-chain fatty acids on (A) cytokine mRNA expression (measured by qPCR) and (B) TNF-α protein secretion (measured by ELISA) after LPS stimulation of primary microglia. Cells were pre-treated with acetate, butyrate, or combination of both (0 or 100 µM) for 4 h, and then stimulated with LPS (1 ng/mL) for 4 h. Control = cells stimulated with LPS and no pre-treatment with SCFA. Samples were analyzed by 2-way ANOVA (2 × 2 factorial arrangement of acetate x butyrate), followed by (A) Dunnet’s test (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) or (B) Tukey’s test. Different lowercase letters are statistically different (P < 0.05) among treatments.
Figure 3 Effect of short-chain fatty acids on (A) cytokine mRNA expression (measured by qPCR) and (B) TNF-α protein secretion (measured by ELISA) after LPS stimulation of primary microglia. Cells were pre-treated with acetate, butyrate, or combination of both (0 or 100 µM) for 4 h, and then stimulated with LPS (1 ng/mL) for 4 h. Control = cells stimulated with LPS and no pre-treatment with SCFA. Samples were analyzed by 2-way ANOVA (2 × 2 factorial arrangement of acetate x butyrate), followed by (A) Dunnet’s test (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) or (B) Tukey’s test. Different lowercase letters are statistically different (P < 0.05) among treatments.CC BY · PubMed Central
Figure 4 Effect of short-chain fatty acids on HDAC activity in primary microglia after LPS stimulation. Cells were pre-treated with SCFA mix (0 or 100 µM) for 4 h, and then stimulated with LPS (1 ng/mL) for 4 h. Control = cells stimulated with LPS and no pre-treatment with SCFA. Samples were analyzed by t-test with Welch's correction. ***P < 0.001.
Figure 4 Effect of short-chain fatty acids on HDAC activity in primary microglia after LPS stimulation. Cells were pre-treated with SCFA mix (0 or 100 µM) for 4 h, and then stimulated with LPS (1 ng/mL) for 4 h. Control = cells stimulated with LPS and no pre-treatment with SCFA. Samples were analyzed by t-test with Welch's correction. ***P < 0.001.CC BY · PubMed Central
Figure 5 Effect of short-chain fatty acids on nuclear translocation of p65 NF-κB in primary microglia from adult mice 30 min after stimulation with LPS. (Panel A) Fluorescence microscopy depicting immunocytochemical localization of NF-κB p65 (green) in primary microglia treated with no SCFA or SCFA mix (100 µM acetate + 100 µM butyrate) with no stimulation (two pictures on the left) and LPS 1 ng/mL stimulation (two pictures on the right). The white arrows point to the nucleus, indicating an example of the intensity of nuclear NF-κB in each treatment. (Panel B) Relative Nuclear p65 NF-kB of primary microglia, expressed as percentage of control (cells stimulated with LPS and no SCFA treatment). Primary microglia were plated and pre-treated with 0 or 100 µM acetate and butyrate for 4 h. After 30 min of LPS stimulation (0 or 1 ng/mL), cells were fixed and stained for p65 NF-κB. Values were expressed as the mean of four replicates, whose means resulted from 20 cells from three independent fields of view. Samples were analyzed by 2-way ANOVA (2 × 2 factorial arrangement of SCFA mix x LPS), followed by Šidák multiple comparisons test. Different lowercase letters are statistically different (P < 0.05) among treatments.
Figure 5 Effect of short-chain fatty acids on nuclear translocation of p65 NF-κB in primary microglia from adult mice 30 min after stimulation with LPS. (Panel A) Fluorescence microscopy depicting immunocytochemical localization of NF-κB p65 (green) in primary microglia treated with no SCFA or SCFA mix (100 µM acetate + 100 µM butyrate) with no stimulation (two pictures on the left) and LPS 1 ng/mL stimulation (two pictures on the right). The white arrows point to the nucleus, indicating an example of the intensity of nuclear NF-κB in each treatment. (Panel B) Relative Nuclear p65 NF-kB of primary microglia, expressed as percentage of control (cells stimulated with LPS and no SCFA treatment). Primary microglia were plated and pre-treated with 0 or 100 µM acetate and butyrate for 4 h. After 30 min of LPS stimulation (0 or 1 ng/mL), cells were fixed and stained for p65 NF-κB. Values were expressed as the mean of four replicates, whose means resulted from 20 cells from three independent fields of view. Samples were analyzed by 2-way ANOVA (2 × 2 factorial arrangement of SCFA mix x LPS), followed by Šidák multiple comparisons test. Different lowercase letters are statistically different (P < 0.05) among treatments.CC BY · PubMed Central
Figure 6 Uniform Manifold Approximation and Projection (UMAP) visualization of cell clusters identified during scRNA-seq analysis of mouse whole brain tissue and SCFA receptors and transporters expression. (Panel A) identifies each cell type by name and color. (Panel B) illustrates the expression of microglia markers (C1qa, C1qb, C1qc, Ctss, and Tmem 119) used to identify microglial cells. (Panels C and D) illustrate the expression of SCFA receptors, Ffar2, Ffar3, and Olfr78, and transporters, Mct1 (Slc16a1) and Mct4 (Slc16a3), respectively. Microglia lack the expression of all receptors but express both transporters.
Figure 6 Uniform Manifold Approximation and Projection (UMAP) visualization of cell clusters identified during scRNA-seq analysis of mouse whole brain tissue and SCFA receptors and transporters expression. (Panel A) identifies each cell type by name and color. (Panel B) illustrates the expression of microglia markers (C1qa, C1qb, C1qc, Ctss, and Tmem 119) used to identify microglial cells. (Panels C and D) illustrate the expression of SCFA receptors, Ffar2, Ffar3, and Olfr78, and transporters, Mct1 (Slc16a1) and Mct4 (Slc16a3), respectively. Microglia lack the expression of all receptors but express both transporters.CC BY · PubMed Central
Figure 7 Effect of monocarboxylate transporters (MCT) blockade on short-chain fatty acids activity in primary microglia after LPS stimulation. Cells were pre-treated with SCFA mix (0 or 100 µM) for 4 h, and then stimulated with LPS (1 ng/mL) for 4 h. Samples were analyzed 2-way ANOVA (2 × 2 factorial arrangement of MCT inhibitor x SCFA mix), followed by Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Figure 7 Effect of monocarboxylate transporters (MCT) blockade on short-chain fatty acids activity in primary microglia after LPS stimulation. Cells were pre-treated with SCFA mix (0 or 100 µM) for 4 h, and then stimulated with LPS (1 ng/mL) for 4 h. Samples were analyzed 2-way ANOVA (2 × 2 factorial arrangement of MCT inhibitor x SCFA mix), followed by Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.CC BY · PubMed Central
Figure 8 Experimental design. Specific conditions such as time and concentrations are described in the Methods section and in the respective Figure legend.
Figure 8 Experimental design. Specific conditions such as time and concentrations are described in the Methods section and in the respective Figure legend.CC BY · PubMed Central

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Metadata

Authors
Caetano-Silva, M. E., Rund, L., Hutchinson, N. T., Woods, J. A., Steelman, A. J., & Johnson, R. W.
Affiliations
University of Illinois Urbana-Champaign (US)
Corresponding author
Johnson, Rodney W.
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2023-02-16
Volume
13
Issue
1
Pages
2819-2819
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-022-27086-x
PMC
PMC9935636
OpenAlex
W4321018263
Cited by
225
References
32
Citations by year
2026: 80 · 2025: 77 · 2024: 51 · 2023: 17
FWCI
23.02
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Institutes of Health (R01 AG059622)
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
microglia, neuroinflammation, short-chain fatty acids, dietary fiber, inulin, gut microbiota, lipopolysaccharide, TNF-α, acetate, butyrate, histone deacetylase, NF-κB, FFAR2, FFAR3, MCT1, MCT4, monocarboxylate transporters, epigenetic regulation, gut-brain axis
MeSH terms
Animals, Anti-Inflammatory Agents, Dietary Fiber, Fatty Acids, Volatile, Inulin, Lipopolysaccharides, Tumor Necrosis Factor-alpha, Microglia, Membrane Transport Proteins, Mice

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