Extended Data Fig. 5: Lung dysbiosis affects neither T cell and endothelial cell interactions at the CNS borders nor the barrier integrity.
a, b, I.tr. neomycin treatment does not influence the expression of either chemokine receptors or adhesion molecules in TMBP cells. Lung EAE or transfer EAE were induced in PBS- or neomycin-pre-treated rats. a, Chemokine receptor and integrin expression in TMBP cells isolated from blood on D5 after immunization (lung EAE, n = 5 per condition) or D4 after transfer (transfer EAE, n = 4 per condition). Quantitative PCR. Housekeeping gene: β-actin. Mean ± s.e.m. Representative data of 2 independent experiments. b, Corresponding protein expression of LFA-1 and VLA-4. Flow cytometry. Representative data of 2 independent experiments. c, d, I.tr. neomycin treatment does not affect T cell motility at the CNS borders. TMBP (c) or TOVA (d) cells were i.v. transferred into rats pre-treated with PBS or neomycin. c, Intravascular TMBP cell motility was recorded in the leptomeninges by TPLSM on D2.5 post transfer. Depicted are representative time-projection images over a period of 30 min, percentage of crawling versus rolling cells (n = 8 videos per group) and quantification of the indicated motility parameters. Number of analysed T cells is indicated. Mean ± s.e.m. Representative data of 2 independent experiments. n = 2-4 (both groups). Turquoise, TMBP cells; Red, 70 kDa Texas Red Dextran labelled vessels; Blue, Collagen. d, Intravascular (upper panel) and extravascular (lower panel) TOVA cell motility was recorded in the leptomeninges by TPLSM on D3 and D4 post transfer, respectively. Time projection images over a period of 30 min, percentage of crawling versus rolling cells (n = 9 videos per group) and intravascular and extravascular motility parameters derived from the indicated number of T cells. Mean ± s.e.m. Turquoise, TOVA cells; Red, 70 kDa Texas Red Dextran labelled vessels; Blue, Collagen. e, I.tr. neomycin treatment does not affect TOVA cell diapedesis. Intravital TPLSM overviews and corresponding magnified pictures depicting the distribution of TOVA cells (turquoise) in the leptomeningeal milieu at the indicated time points post transfer in PBS- or neomycin- pre-treated rats. Red, 70 kDa dextran Texas-Red labelled vessels; Blue, Collagen. Arrows, Representative TOVA cells. Graph, Corresponding quantification of TOVA cells in the extravascular environment. Each dot represents a single 30 min video. Mean ± SEM. Representative data of two independent experiments. n = 8 (both groups). f, I.tr. neomycin treatment does not change the expression of tight junction molecules and integrin ligands. Expression of the indicated genes in endothelial cells isolated from spinal cord leptomeninges and parenchyma of rats pre-treated for 7 days with PBS or neomycin. Quantitative PCR. Housekeeping gene: β-actin. Mean ± s.e.m. Cumulative data of 4 independent experiments. n = 17-18 (all groups). g, I.tr. neomycin treatment does not alter the permeability of leptomeningeal vessels. Intravital TPLSM overviews and corresponding magnified pictures of the thoracic spinal cord recorded 7 days after i.tr. PBS or neomycin treatment. Images were acquired 0, 30, 60 and 90 min after i.v. injection of 3 kDa Texas Red Dextran. No leakage of the dye was observed at any time point. Representative images from two independent experiments. a, e, f, Statistical significance determined by unpaired two-tailed t-test (Gaussian distribution) and Mann–Whitney test (non-Gaussian distribution). c, d, Statistical significance of percentage crawling versus rolling was determined with a two-way ANOVA; for the other motility parameters unpaired two-tailed t-test (Gaussian distribution) and Mann–Whitney test (non-Gaussian distribution) were used.
