Extended Data Fig. 6: Lung dysbiosis induces quantitative but not qualitative changes in the immune infiltrates in the CNS.

a, I.tr. neomycin treatment reduces T_MBP cell-mediated CNS inflammation. Expression of iNOS (Nos2), MHC-II (Rt1ba), chemokines, proinflammatory cytokines and regulatory genes in total spinal cord at the initiation stage of the EAE (i.e. 24 h after the onset of the clinical symptoms). Quantitative PCR. Housekeeping gene: β-actin. Mean ± s.e.m. n = 11 (PBS); n = 9 (Neo). b, c, T cell responses in the CNS are not affected by lung dysbiosis in transfer EAE. EAE was induced in rats pre-treated i.tr. with neomycin or PBS by transfer of T_MBP cells. Immune cell characterization was performed at the initiation stage of the disease. b, Percentage of T_MBP cells, endogenous αβTCR⁺ CD4⁺ and CD8⁺ T cells expressing FoxP3 and proinflammatory cytokines (steady state and PMA/I-stimulated conditions) in the CNS and in the indicated peripheral compartments. Flow cytometry. Mean ± s.e.m. Representative data of 2 independent experiments. n = 5 (all groups). c, Corresponding expression of the specified T cell lineage signature cytokines in the indicated T cell subsets isolated from the spinal cord. Quantitative PCR. Housekeeping gene: β-actin. Mean ± s.e.m. n = 2–4 (all groups). d, Myeloid cells recruited to the CNS are not impaired by lung dysbiosis. Expression of iNOS (Nos2), MHC-II (Rt1ba), chemokines, M2 macrophage marker and IFNβ in spleen-derived CD45^high CD11b⁺ MΦ and in CNS-derived CD45^high CD11b⁺ MΦ at the initiation stage of EAE. Quantitative PCR. Housekeeping gene: β-actin. Mean ± s.e.m. Representative data of 2 independent experiments. n = 3-4 (PBS); n = 4 (Neo) per condition. e, T cell response in the CNS is not affected by lung dysbiosis also in lung active EAE. Lung EAE was induced in rats pre-treated i.tr. with neomycin or PBS. Immune cell analysis was performed at the initiation stage of the disease. e,  Percentage of T_MBP cells, endogenous αβTCR⁺ CD4⁺ and CD8⁺ T cells expressing FoxP3 and proinflammatory cytokines (steady state and PMA/I-stimulated conditions) in the CNS and in the indicated peripheral compartments. Flow cytometry. Mean ± s.e.m. Representative data of 2 independent experiments. n = 5 (all groups). f, Corresponding expression of the specified T cell lineage-signature cytokines measured as in c. Quantitative PCR. Mean ± s.e.m. n = 2–5 (all groups). g, Myeloid cells recruited to the CNS are not functionally impaired. Expression of the indicated genes measured as in d in spleen-derived CD45^high CD11b⁺ MΦ and in CNS-derived CD45^high CD11b⁺ MΦ. Quantitative PCR. Mean ± s.e.m. Representative data of 2 independent experiments. n = 2–5 (all groups). a–g, Statistical significance was determined by unpaired two-tailed t-test (Gaussian distribution) and Mann–Whitney test (non-Gaussian distribution). *P < 0.05, ***P < 0.001; ND, not detected.

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