Extended Data Fig. 4: TRC network tension increases upon lymph node swelling.
From: Multitier mechanics control stromal adaptations in the swelling lymph node
(a) Schematic of in vivo laser cutting in inguinal lymph nodes (iLNs) of FRC-mGFP mice. (b) Subcapsular T-zone reticular cell (TRC) network is imaged at interfollicular (IF) regions (blue arrow). Homeostatic (day 0) and inflammation (day 14) are shown. mGFP positive lymphatic endothelial cells (LECs), marginal reticular cells (MRCs) and TRCs are encountered while gradually focusing into the LN, while follicular dendritic cell (FDC) and CXCL12+ reticular cells (CRCs) regions are avoided. The T-zone at IF sites is identified by regular network morphology and bright fluorescent reporter intensity (1), compared to the more irregular and dimmer CRC (2) and dense and bright FDC (3) stromal networks found in the largely ECM-free (ER-TR7 negative) follicles. White dashed lines indicate the outline of follicles, and white dense spaced lines indicate the subcapsular region where MRCs are found (annotated in homeostasis). The area enclosed by the blue dashed line shows a typical area targeted for TRC laser cutting in the orthogonal plane. Scale bars: top = 50 µm, bottom = 20 µm. (c) Averaged and smoothed distribution of the subcapsular TRC network as analyzed by circle fitting gap analysis of homeostatic (day 0) and inflamed (day 2, 4, 8, 14) LNs from FRC-mGFP mice, plotted as the weighted area fraction as a function of the fitted circle diameter (n = 5, 5, 5, 5, 5). (d) Quantification of the mean fitted circle diameter as in c (n = 5, 5, 5, 5, 5). (e) Example of force propagation in the TRC network following a UV laser cut. Time point directly after cutting (t = 0 s) and late after cutting (t = 22.2 s) overlapped (left). Scissor and white line indicate the laser cut location. Scale bar = 20 µm. Overlay of movement vectors (red arrows) derived from particle imaging velocimetry analysis (right). Data from c shown as mean and d shown as mean ± s.e.m. Datapoints in d represent independently analyzed TRC network images from LNs used in laser cutting experiments. Statistical analysis was performed using one-way ANOVA (d). All experiments were repeated independently (3 mice and ≥2 experiments). For statistical details see Supplementary Information, table 1. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001.
