Abstract
As the tissue macrophages of the CNS, microglia are critically involved in diseases of the CNS. However, it remains unknown what controls their maturation and activation under homeostatic conditions. We observed substantial contributions of the host microbiota to microglia homeostasis, as germ-free (GF) mice displayed global defects in microglia with altered cell proportions and an immature phenotype, leading to impaired innate immune responses. Temporal eradication of host microbiota severely changed microglia properties. Limited microbiota complexity also resulted in defective microglia. In contrast, recolonization with a complex microbiota partially restored microglia features. We determined that short-chain fatty acids (SCFA), microbiota-derived bacterial fermentation products, regulated microglia homeostasis. Accordingly, mice deficient for the SCFA receptor FFAR2 mirrored microglia defects found under GF conditions. These findings suggest that host bacteria vitally regulate microglia maturation and function, whereas microglia impairment can be rectified to some extent by complex microbiota.
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Acknowledgements
This work is dedicated to our former friend Uwe-Karsten Hanisch, past Professor for Neuroscience at the Paul-Flechsig-Institute for Brain Research, Leipzig, who devoted his whole life to the exploration of microglia function. We thank M. Oberle, M. Ditter and T. el Gaz for excellent technical assistance. We are grateful to J. Bodinek-Wersing and G. Rappl for cell sorting and to A. Spies for providing cDNA from cell cultures. M.P. was supported by the BMBF-funded competence network of multiple sclerosis (KKNMS), the Sobek Foundation, the DFG (SFB 992, FOR1336, PR 577/8-1), the Fritz-Thyssen Foundation and the Gemeinnützige Hertie Foundation (GHST). B.S. was supported by the SPP1656 “Intestinal Microbiota” (STE 1971/4-1).
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D.E., A.L.H.d.A., P.W., D.J., K.J., T.M., B.S., O.U., I.A., H.K.-S., E.D. and K.D.M. conducted the experiments. I.A. and O.S. analyzed the RNA sequencing and microarray data, respectively. T.B., B.S., A.D., P.S., V.S., E.C. and W.S.G. provided mice and designed experiments. M.P. supervised the project and wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Gene profile from microglia in GF mice.
mRNA expression values (number of reads) of genes from microglia in GF animals (white bars) or SPF mice (black bars). (a) Prototypical microglia genes are presented and compared to the non-microglia genes such as Gfap, Mog, Tubb3 and Rbfox3. (b) Epigenetic signature of microglia was examined by investigating genes (number of reads) related to histone acetylation, deacetylation, methylation and demethylation, respectively. Bars represent means ± s.e.m. with seven samples in each group. Significant differences were determined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). P values: Csf1r: 0.0001, Sfpi1: 0.0075, Hexb: 0.0060. Myst3: 0.0121, Hdac1: 0.0191, Sirt2: 0.0113, Sirt7: 0.0441, Mll3: 0.0489, Mll5: 0.0105, Setd1a: 0.0360, Setd7: 0.0134, Kdm4a: 0.0476, Kdm6b: 0.0005, Jarid2: <0.0001.
Supplementary Figure 2 Absence of microbes does not alter the amount of neurons, astrocytes and oligodendrocytes.
CNS histology of the neuroectodermal compartment of 6-8 weeks old GF or control SPF mice. NeuN labels mature neurons, Nogo-A oligodendrocytes and glial fibrillary acidic protein (GFAP) astrocytes. Representative pictures are shown. Scale bars: NeuN: 100 µm, Nogo-A and GFAP: 50 µm.
Supplementary Figure 3 Absence of microbes does not alter the amount of neurons, astrocytes and oligodendrocytes.
(a,b) Quantitative assessment of the neuroectodermal compartment of 6-8 weeks old GF or control SPF mice. Hippocampal NeuN+ cells were counted in the gyrus dentatus. Cerebellar NeuN+ cells were counted in the granular cell layer. Each symbol represents one mouse, with five samples per group. Means ± s.e.m. are indicated. No significant differences could be determined by using an unpaired t test. Representative pictures are shown. n.d. = not detectable. Data are representative of two independent experiments. Scale bars: H&E: 200 µm.
Supplementary Figure 4 Lymphocytic cells are indistinguishable in the brains of SPF and GF mice.
Immunohistochemistry for T cells (CD3) and B cells (B220) in different brain regions of SPF and GF mice, respectively. Insets depict spleen sections as positive controls. Scale bars: overviews: cortex, corpus callosum and cerebellum (CD3): 50 µm; olfactory bulb (CD3): 20 µm; hippocampus (CD3), cortex, corpus callosum, hippocampus, olfactory bulb and cerebellum (B220): 100 µm. Inset: 50 µm (CD3) and 100 µm (B220).
Supplementary Figure 5 Increased microglia number in cerebellar grey and white matter of GF mice.
(a) Quantification of Iba-1+ parenchymal microglia from either SPF (black symbols) or GF (white symbols) mice was performed on cerebellar brain slices. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are shown. Significant differences were determined by an unpaired t test and marked with asterisks (*P < 0.05, ***P < 0.001). P values: granular cell layer: 0.0008, white matter: 0.0287. Representative pictures of nine examined mice per group are displayed.
(b) Imaris-based 3-D reconstruction of the cortical microglia networks under SPF (left) or GF (right) conditions. GF microglia have no distances to neighbouring microglia and show partial physical contacts to adjacent cells. Inset exhibits such an interaction. Scale bars: 20 µm (overview), 3 µm (inset).
(c) Quantitative RT-PCR for the indicated mRNAs. Data are expressed as ratio of mRNA expression versus endogenous Actb relative to SPF conditions and exhibited as mean ± s.e.m. Bars (black: SPF, white: GF) represent means ± s.e.m. with at least four samples in each group. Data are representative of two independent experiments. Significant differences were determined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). n.d. = not detectable. P values: Il-34: hippocampus: 0.0031. Csf1: cortex: 0.0071, cerebellum: 0.0009. Tgfß1: cortex: 0.0253, cerebellum: 0.031. Sfpi1: cortex: 0.0302, corpus callosum: 0.0290, cerebellum: 0.0345.
Supplementary Figure 6 Decreased inflammatory repertoire of microglia from GF mice upon intracerebral LPS challenge.
Gene ontology enrichment network on differentially expressed genes in microglia from SPF (a) or GF (b) mice on the basis of an Affymetrix DNA microarray analysis. Diagram depicts functional results of GO clustering through GORilla. Only very highly significantly overrepresented GO terms are included with P values ranging from P < 10-9 (yellow) to P < 10-24 (red). White framed terms depict not significantly overrepresented parent terms of the colored terms.
Supplementary Figure 7 Diminished inflammatory response of microglia from GF mice upon peripheral LPS challenge.
Quantitative RT-PCR of LPS-induced factors in microglia 6 hrs after intraperitoneal LPS challenge. Data are expressed as ratio of the mRNA expression compared to endogenous Actb relative to control and show mean ± s.e.m. At least three mice per group were analyzed. Data are representative of two independent experiments. Significant differences were determined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). ns: not significant. P values: PBS: Il1- b:0.3720, Il-6: 0.5625, Il12 b: 0.2442, Tnfα p=0.5955, Marco: 0.0642, Ccl2: 0.6608, Ccl7: 0.3316, Cxcl10: 0.0867, Csf1: 0.1194, cyclin D2: 0.1135, S100a4: 0.5357, S100a6: 0.4281, S100a8: 0.0642, S100a9: 0.4583, S100a10: 0.1771. LPS: Il-1 b: 0.0023, Il-6: 0.1329, Il-12 b: <0.0001, Tnfα: 0.0008, Marco: 0.0001, Ccl2: 0.0673, Ccl7: 0.2708, Cxcl10:.1522, Csf1: 0.1828, cyclin D2: 0.0344, S100a4: 0.0064, S100a6: 0.0341, S100a8: 0.0009, S100a9: 0.5782, S100a10: 0.0352.
Supplementary Figure 8 Determination of intestinal bacterial loads and complexity.
(a) qPCR analysis for eubacterial 16S rRNA could not detect significant amounts of bacteria in the caeca of GF and antibiotic-treated SPF animals. Representative data from fifteen SPF, five GF, six SPF (ABX), five ASF and five ASF (recol.) mice are depicted. Gene copy numbers were normalized to DNA content. Each symbol represents one mouse. Means ± s.e.m. are indicated. Significant differences were determined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01). P values: SPF vs. GF: 0.0151, SPF vs. SPF (ABX): 0.0046, SPF vs. ASF (recol.): 0.0280.
(b) Recolonized ASF mice (ASF recol.) harbour a greater microbial diversity as detected by qPCR using group specific primers. Besides Parabacteroides (ASF357), Lactobacillus (ASF 361) and Clostridium (ASF356), mouse intenstinal Bacteroides (mib) and Enterobacteriaceae groups are detectable in the caecum of SPF and re-colonised ASF animals using group-specific primers, respectively.
Supplementary Figure 9 Microglia maturation is independent from the TLRs2,3,4,7,9.
(a) Left: View of caecum from a SPF-TLRwt (control, left) and SPF-TLR2,3,4,7,9-deficient (SPF-TLRko) mouse (right). Ruler scale is shown. Representative pictures are shown. Right: Relative caecum weight of control SPF-TLRwt and SPF-TLRko mice. Symbols represent individual mice, with six animals per group. Means ± s.e.m. are shown. No statistically significant differences were found by an unpaired t test.
(b) Iba-1 immunohistochemistry in the cortex of SPF-TLRwt and SPF-TLRko animals (left) and quantification thereof (right). Every symbol represents one mouse. Six animals per group were analyzed and three to four sections per mouse were examined. Data are representative of two independent experiments. Means ± s.e.m. are indicated. No significant differences could be determined by an unpaired t test.
(c) Ddit4 mRNA measured by qRT-PCR in microglia from SPF-TLRwt (black bar) or SPF-TLRko (white bar) mice. Means ± s.e.m. with six samples in each group are displayed. No significant differences could be determined by an unpaired t test. Data are representative of two independent experiments.
(d) Three-dimensional reconstruction (Scale bars: 15µm) and Imaris-based automatic quantification of cell morphometry (e) of Iba-1+ microglia. Individual symbols represent one mouse per group with at least three measured cells per mouse. Six mice per group were analyzed. Means ± s.e.m. are indicated. No significant differences were detected by an unpaired t test.
(f) Flow cytometry of isolated microglia from control six SPF-TLRwt and six SPF-TLRko individuals. Percentages and mean fluorescence intensities (MFI) of the indicated surface molecules on microglia are shown. Each symbol represents one mouse. Means ± s.e.m. are indicated. Significant differences were evaluated by an unpaired t test and labelled with asterisks (*P < 0.05). P value: CD31 (MFI): 0.0419. Data are representative of two independent experiments.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–9 (PDF 1810 kb)
Supplementary Table 1: Altered microglial gene profile and immaturity in germ-free animals.
Significantly down- (upper table) and up- (lower table) regulated genes subjected to functional GO cluster analysis. The significance of genes is indicated by the given P values. (XLS 32 kb)
Supplementary Table 2: Reduced gene induction in microglia from non-colonized GF animals.
Affymetrix Genome 430A 2.0 array-based gene induction of microglia from SPF mice compared to GF individuals. 78 genes significantly differentially expressed in GF vs. SPF microglia 6 hr after i.c. LPS treatment are shown. Only genes also significantly up- or downregulated by LPS treatment compared to PBS treated controls of the same housing conditions (GF and SPF respectively) are included to account for differences in basal gene regulation. (P < 0.01). (XLS 41 kb)
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Erny, D., Hrabě de Angelis, A., Jaitin, D. et al. Host microbiota constantly control maturation and function of microglia in the CNS. Nat Neurosci 18, 965–977 (2015). https://doi.org/10.1038/nn.4030
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DOI: https://doi.org/10.1038/nn.4030