Fig 2: Staggered commitment of splenic white pulp organizer cells along the developing arterial tree. a, b Ccl19-iEYFP mice were treated with doxycycline from E19.5 (a, c) or P7 (b, d). Distribution and phenotype of EYFP+ cells in the white pulp of 6-week-old mice were determined by confocal microscopy using the indicated antibodies. Boxes show region of higher magnification in right images, arrows indicate EYFP+MAdCAM-1+ MRC c, d, arrowheads indicate EYFP+PDPN+ TRC (c, d), asterisks indicate EYFP+CD21/35+ FDC (c, d) (scale bars = 500/20 µm). e Determination of EYFP+ cell distances from the central artery at the hilum. Cross-sections from Ccl19-iEYFP spleens from 6-week-old mice were stained with antibody against TER119 and EYFP expression was pseudocolor-coded (lower image) according to concentric distance from the hilum (0–2400 µm). Scale bar = 250 µm. f Frequency of EYFP+ cells in white pulp areas of indicated concentric distances from the central artery (n = 5–7 mice per group from two independent experiments, mean ± SEM). Statistical analysis was performed using a Kolmogorov–Smirnov test. Source data are provided as Source Data file

Fig 3: Local sympathetic activation attenuates endothelial MAdCAM-1 levelsChR2/TH mice and their controls (transgene negative littermates exposed to light stimulation) underwent daily optogenetic activation of their colon (as described in Figure 1E) and were supplemented with 3% DSS for 7 days.(A) Relative mRNA expression levels of CCR9, CCR7, a4, ß7, CCL20, CCL25, E-selectin, and P-selectin in the colon tissue of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. Fold change between the ChR2/TH mice and their controls relative to the mean of the control group.(B) Expression of the markers CD31 (blue) and TH (red) in the colon of C57BL/6 mice. Scale bar, 200 µm.(C) Expression of the markers CD31 (blue) and the ChR2 fluorescent marker (green) in the colon of ChR2/TH mice. Scale bar, 20 µm.(D) Relative mRNA expression levels of VCAM-1, MAdCAM-1 and ICAM-1 in the colon tissue of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. Fold change between the ChR2/TH mice and their controls relative to the mean of the control group. VCAM-1: N = 9, 11; MAdCAM-1: N = 10, 9; ICAM-1: N = 10, 10.(E) Left: flow cytometry analysis of MAdCAM-1 and ICAM-1 levels on endothelial cells (CD31+; indicated by the fold change in MFI) in the colons of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. Fold change between the ChR2/TH mice and their controls relative to the mean of the control group. MAdCAM-1: N = 8, 8; ICAM-1: N = 9, 8. Right: representative flow cytometry histogram demonstrating the MAdCAM-1 and ICAM-1 levels on CD31+ cells.(F) Relative mRNA expression levels of MAdCAM-1 and ICAM-1 in the small intestine tissue of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. Fold change between the ChR2/TH mice and their controls relative to the mean of the control group. MAdCAM-1: N = 7, 9; ICAM-1: N = 5, 8.(G) Left: flow cytometry analysis of MAdCAM-1 and ICAM-1 levels on endothelial cells (CD31+; indicated by the fold change in MFI) in the colons of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. The mice received daily IP injections of adrenergic blockers 15 min before each optogenetic stimulation (Phentolamine 10 mg/kg and Nadolol 5 mg/kg). Fold change between the ChR2/TH mice and their controls relative to the mean of the control group. MAdCAM-1: N = 10, 10; ICAM-1: N = 7, 8. Right: representative flow cytometry histogram demonstrating MAdCAM-1 and ICAM-1 levels on CD31+ cells.(H) Left: flow cytometry analysis of MAdCAM-1 and ICAM-1 levels on endothelial cells (CD31+; indicated by the fold change in MFI) in the colons of ChR2/TH mice and their controls. The mice were injected IP with 6-OHDA, and 5 days afterward received 7 days of 3% DSS and daily optogenetic activation. Fold change between the ChR2/TH mice and their controls relative to the mean of the control group. MAdCAM-1: N = 16, 13; ICAM-1: N = 16, 13. Right: representative flow cytometry histogram demonstrating MAdCAM-1 and ICAM-1 levels on CD31+ cells.(I) Experimental design: cells were enzymatically dissociated from colons and incubated for one hour with LPS at 37°C. Afterward, NA was added (0-1000 µM) and the cells were incubated overnight at 37°C. Then MAdCAM-1 levels were analyzed on endothelial cells (CD31+) by flow cytometry.(J) Left: MAdCAM-1 levels on endothelial cells (CD31+; indicated by the fold change in MFI) following exposure to increasing concentrations of NA (as described in Figure 4I). N = 6. Right: representative flow cytometry histogram demonstrating MAdCAM-1 levels on CD31+ cells.(K) Left: MAdCAM-1 levels on colon-derived enriched endothelial cells (CD31+) following exposure to 100 µM NA overnight at 37°C. N = 6. Right: Representative flow cytometry histogram demonstrating MAdCAM-1 levels on CD31+ cells.(L) Left: MAdCAM-1 levels on endothelial cells (CD31+; indicated by the fold change in MFI) that were incubated at 37°C with a- or ß-adrenergic blockers (Phentolamine or Nadolol respectively) followed by exposure to 100 µM NA overnight. N = 5. Right: representative flow cytometry histogram demonstrating MAdCAM-1 levels on CD31+ cells.(M) Left: pCREB levels on endothelial cells (CD31+) following exposure to 100 µM NA for 30 min at 37°C. N = 10. Right: representative flow cytometry histogram demonstrating pCREB levels on CD31+ cells.(N) Left: MAdCAM-1 levels on endothelial cells (CD31+) following exposure to 100 µM NA overnight at 37°C. Prior to the administration of NA, the cells were incubated with a CREB inhibitor (666-15). N = 9. Right: representative flow cytometry histogram demonstrating MAdCAM-1 levels on CD31+ cells. Mean ± SEM, as well as data from individual mice, are presented for each group. Students paired t- test for figures 4K, 4M and 4N and Student’s unpaired t test for the rest of the figures. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Data represent at least two independent repeats.

Fig 4: MAdCAM-1 is necessary to mediate the beneficial effects of local sympathetic activation on DSS-induced colitis(A) Experimental design: ChR2/TH mice and their controls (transgene negative littermates exposed to light stimulation) underwent daily optogenetic activation of their colon (as described in Figure 1E) and were supplemented with 3% DSS for 7 days. Two sets of groups were used: ChR2/TH mice and their controls that received daily IP injection of anti-MAdCAM-1 antibody (anti-MAdCAM-1 group) or daily IP injection of isotype control antibody (isotype control group).(B) Change in body weight of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. The mice received daily injections of anti-MAdCAM-1 or isotype control. Isotype control: N = 10, 10; anti-MAdCAM-1: N = 10, 8.(C) Colon length of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. The mice received daily injections of anti-MAdCAM-1 or isotype control. Isotype control: N = 10, 7; anti-MAdCAM-1: N = 10, 8.(D and E) Representative histological image (D) and histological severity score (E) of colons obtained from ChR2/TH mice and their controls following 7 days of 3% DSS, daily optogenetic activation and daily injections of anti-MAdCAM-1. N = 5, 5.(F) Left: fold change in abundance of total immune cells (number of CD45+ cells/gr colon) in the LP and IEL layers of the colon from ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. The mice received daily injections of anti-MAdCAM-1 or isotype control. Fold change between the ChR2/TH mice and their controls relative to the mean of the control in each group (the isotype control group and anti-MAdCAM-1 group). LP tissue: N = 7, 5 isotype control, N = 10, 7 anti-MAdCAM-1. IEL tissue: N = 5, 6 isotype control, N = 10, 7 anti-MAdCAM-1. Right: representative flow cytometry plot demonstrating the percentage of CD45+ population in the LP and in the IEL. Mean ± SEM, as well as individual mice, are presented for each group. Student’s unpaired t test. * = p < 0.05, ** = p < 0.01. Data represent at least two independent repeats.