Extended Data Fig. 8: Effects of lung dysbiosis on microglia and the lung milieu.
a, I.tr. neomycin treatment reduces microglial reactivity towards inflammatory cytokines in the CNS. Rats were i.tr. treated daily with neomycin or PBS. After 7 days, PBS or TNF & IFNγ were administered intrathecally (i.th.). Percentage of MHC-II+ CD45low CD11b+ microglia and number of CD45high CD11b+ MΦ in spinal cord and brain 4 h and 18 h after i.th. injection. Flow cytometry. Mean ± s.e.m. Representative data from 3 independent experiments. For each CNS compartment, n = 4 (PBS and PBS); n = 5 (PBS and TNF & IFNγ); n = 3 (Neo and PBS); n = 5 (Neo and TNF & IFNγ). b, Lung dysbiosis impairs the capacity of microglia to respond to proinflammatory stimuli in vitro. Microglial cells, isolated from rats treated for 7 days with PBS or neomycin, were stimulated in vitro with increasing doses of IFNγ. Expression of chemokines, cytokines β2MG (B2m), MHC-II (Rt1ba) and iNOS (Nos2) 4h after stimulation. Quantitative PCR. Representative data of 2 independent experiments. Each value represents the pooled microglia of at least 6 rats per group. c, Oral administration of inactivated P. melaninogenica does not affect EAE. EAE was induced by transfer of TMBP cells. Clinical parameters after daily oral treatment started 7 days before TMBP cell transfer and continued throughout the entire disease course. Mean ± s.e.m. Cumulative data of 2 independent experiments. n = 5 (PBS); n = 4 (P. melaninogenica). d, Neomycin treatment increases pulmonary LPS. Concentration of LPS in BALF of rats treated for 7 days with PBS, neomycin or vancomycin (Vanco). ELISA. Mean ± s.e.m. Cumulative data of 2 independent experiments. n = 9 (PBS); n = 6 (Vanco); n = 8 (Neo). e, Neomycin treatment induces a shift to a type I IFN phenotype in pulmonary immune cells. Expression of type I IFN-stimulated genes in pulmonary stromal cells (CD45−) and immune cell subsets in PBS- or neomycin-pre-treated rats. Quantitative PCR. Housekeeping gene: β-actin. Mean ± s.e.m. Cumulative data of 3 independent experiments. n = 4–13 (PBS); n = 4–11 (Neo) per condition. a, c, e, Statistical significance determined by unpaired two-tailed t-test (Gaussian distribution) and Mann–Whitney test (non-Gaussian distribution). d, Statistical significance determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.
