Extended Data Fig. 7: Lung dysbiosis induces changes in microglial morphology and expression profile.
a, I.tr. neomycin treatment dampens microglial response in EAE. EAE was induced in rats pre-treated i.tr. with neomycin or PBS by transfer of TMBP cells. Confocal images acquired in spinal cord white matter and grey matter depicting the morphology of Iba1+ microglia (red) at the peak of EAE. b, c, PLX3397 ameliorates EAE but does not add to the disease-ameliorating effects of i.tr. neomycin treatment. b, Quantification of CD45low CD11b+ microglia in the brain after 7 days of oral treatment with PLX3397 or vehicle. Flow cytometry. Mean ± s.e.m. Cumulative data of 3 independent experiments. n = 9 (both groups). c, Rats were treated orally with PLX3397 or vehicle and i.tr. with neomycin or PBS. After 7 days EAE was induced by transfer of TMBP cells. The treatments were continued throughout the entire disease course. Clinical parameters. Mean ± s.e.m. Cumulative data of 2 independent experiments. n = 10 (vehicle and PBS, vehicle and Neo); n = 9 (PLX and PBS); n = 11 (PLX and Neo). d, I.tr. neomycin treatment does not induce quantitative microglia changes. Rats were treated i.tr. with PBS or neomycin for 7 days. Histological quantification of Iba1+ microglia in the grey matter of the spinal cord (n = 5 (PBS); n = 6 (Neo)), and cytofluorometric quantification of CD45low CD11b+ microglia in the spinal cord (n = 10 (PBS); n = 9 (Neo)). Mean ± s.e.m. e, f, I.tr. neomycin treatment changes microglia morphology in spinal cord and brain cortex without EAE induction. Rats were treated i.tr. with PBS or neomycin for 7 days. e, Quantification of the indicated morphological parameters extracted from confocal images of microglia in the spinal cord of PBS- or neomycin-pre-treated rats. Mean ± s.e.m. 16 cells from 3 different rats per group were analysed. f, Iba1+ microglia in cortical grey matter after 7 days of i.tr. treatment with PBS or neomycin. Representative confocal 3D-reconstructions and corresponding morphological parameters derived from 13 cells from 3 different rats per group. Mean ± s.e.m. g, I.tr. neomycin treatment induces a type I IFN signature in spinal cord microglia. Significantly enriched (P < 0.05) GO terms belonging to biological processes (BP) in genes upregulated in microglia of rats treated with neomycin compared to PBS. h, I.tr. neomycin treatment induces type I IFN-stimulated gene expression in brain-derived microglia. Differential expression of the indicated genes in microglial cells sorted from the brain of rats pre-treated with PBS or neomycin. Representative data of 2 independent experiments. Quantitative PCR. Housekeeping gene: β-actin. Mean ± s.e.m. n = 5 (both groups). i, I.tr. neomycin treatment induces upregulation of type I IFN-stimulated genes in the total spinal cord. Comparison of differential gene expression between neomycin- and PBS-treated rats. Light red dots, genes significantly upregulated (P < 0.05) but below the 0.5-fold change cut-off. Type I IFN-regulated genes are indicated. Bold, genes upregulated in both spinal cord and sorted microglia (Fig. 4b). j, Lung dysbiosis does not induce a shift to a type I IFN profile in astrocytes. Expression of type I IFN-regulated genes, β2MG (B2m), MHC-II (Rt1ba), and TNF. Note that no signal was detectable in most of the samples. Cumulative data of 2 independent experiments. n = 6 (PBS); n = 7 (Neo) per condition. b, d, e, h, j, Statistical significance determined by unpaired two-tailed t-test (Gaussian distribution) and Mann–Whitney test (non-Gaussian distribution). c, Statistical significance determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001; ND, not detected.
