Fig 2: DARC expression on whole mount and frozen section stainings. Representative confocal micrographs of whole mount staining of (a) cremaster muscle (20× objective, scale bar = 200 µm), (b) bladder (10× objective, scale bar = 300 µm), and (c) conjunctival vessels (10× objective, scale bar = 300 µm) with MAbs against CD31 (green) and DARC (red). Representative confocal micrographs of frozen sections from (d) brain (20× objective, scale bar = 200 µm), (e) Peyer’s patches (10× objective, scale bar = 300 µm), (f) mesenteric lymph nodes (20× objective, scale bar = 200 µm), (g) thymus (10× objective, scale bar = 300 µm), (h) pancreas (20× objective, scale bar = 200 µm), (i) small intestine (20× objective, scale bar = 100 µm), and (j) colon (10× objective, scale bar = 300 µm) with MAbs against: CD31 (green), DARC (red), insulin (white), MAdCAM1 (white), or UEA-1 (white) as indicated. L indicates lumen of small intestine and colon. n = at least three experiments for each tissue

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: Lymphoma growth disrupts CCL21 migration cues on FRCs(A) Microscopic analysis of the FRC network (Ccl19RFP) in LN sections. Bottom: examples of FRC sphericity (Ccl19RFP segmentation).(B) Quantification of the FRC density in LN sections of Ccl19RFP mice. n = 7–10 mice per group.(C) Representative images of CCL21 expression in LNs sections of Ccl19RFP mice and gradients of the fluorescence intensity over the distance.(D) CCL21 distribution around and within HEVs (PNAd+) and FRCs (Ccl19RFP).(E) Microscopic analysis of CCL21 (MFI) within masked Ccl19RFP (FRCs) and PNAd+ (HEVs). Control n = 5, tumor n = 7.(F) Immunohistochemistry of CCL21 expression in controls and LNs from diseased tg. Eµ-Myc (n = 7) and Cd19-TAg mice (n = 11); box indicates magnified area. Bar graph depicts percentage of LNs with preserved (pos.) or lost (neg.) CCL21 expression.(G) Proportions of mesenchymal cell subsets (all Ccl19RFP), CD21/CD35+ follicular DCs (FDCs); MadCAM-1+ marginal reticular cells (MRCs); SCA-1+ pericytic reticular cells (PRCs); and T-zone reticular cells (TRCs). n = 4 mice per group.(H and I) FCM quantification of CCL21+ (n = 6–7 mice per group) (H) and PDPN+ expression (n = 7–8 mice per group) (I) in LNs of Ccl19RFP mice.(J and K) Exemplary images (J) and analysis (K) of aSMA (MFI) and CCL21 (integrated density) in a FRC line cultured under static conditions or with laminar flow.Scale bars, 20 µm (A, D, and J) and 100 µm (C and F). Mean and SEM are indicated. Data points represent individual cells (K) or mice (B, E, and G–I). A Mann-Whitney U-test was applied. *p < 0.05; **p < 0.01; ***p < 0.005.

Fig 5: 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.