Intravital imaging of lymph node and small intestine

Abstract

For intravital real-time imaging of fast dynamic behaviors of various cells in lymph node and small intestine, a video-rate laser scanning confocal microscopy system was built based on previous reported platform. The custom-built confocal microscope system was capable of obtaining sub-micron resolution images at 30 frame/sec including three colors.

Lymph node (LN) is a major checkpoint for the circulating lymphocytes to recognize foreign antigens. High endothelial venule (HEV) in LN facilitates an effective recruitment of circulating lymphocytes (mainly T and B cells) from the blood. HEV has distinctive cuboidal-shaped endothelial cells and prominent perivascular sheaths consisting of fibro-reticular cells (FRCs). There have been many efforts to visualize lymphocytes trafficking across HEV to understand the underlying mechanism. However, due to insufficient spatiotem-poral resolution and the lack of an in vivo labeling method, clear visualization of dynamic behaviors of rap-idly flowing lymphocytes in HEV has been difficult. In addition, transendothelial, intra-perivascular channel and trans-FRCs migration of T and B cells in HEV has been poorly understood yet.

In this work, I clearly visualized rapidly flowing T cells in HEV real-time in vivo by utilizing the custom-built confocal microscopy system. Fast dynamics of T cells interacting with HEV-endothelial cells were analyzed compared with RBCs by generating velocity colormaps. I also demonstrated that the endothelial cells and the FRCs in HEV can be fluorescently labeled in vivo by injection of anti-CD31 antibody and anti-ER-TR7 anti-body conjugated with Alexa Flour, respectively. Actin-DsRed transgenic mouse can be also used to visualize HEV-endothelial cells that highly express DsRed fluorescent protein compared with stromal cells and lym-phocytes. The antibody based in vivo labeling methods and Actin-DsRed mice enable the clear visualization of whole migration of GFP-expressing T and B cells in HEV including transendothelial migration, crawling in perivascular channel and trans-FRC migration. Interestingly, compared with T cells, B cells spent longer time in passing the perivascular channel although their total moving distances in the perivascular channel were similar. By time-lapse imaging during 2 hours, I also found there were preferred exit sites (“exit ramp”) from the perivascular channel for both of T and B cell. Indeed, T and B cells followed each other through the same exit ramp from the perivascular channel. In addition to the exit ramp, there existed an “entrance ramp” to perivascular channel, a preferred site for transendothelial migration for both of T and B cells.

Small intestine is a major organ in which digestion and absorption of nutrients actively occur. The luminal surface of the small intestine is densely covered with villi, which provide an extensive absorptive surface area. At the surface of each villus, enterocytes absorb the majority of digested and processed nutrients and materi-als, including drugs, across the apical membrane and release them into the lamina propria. Lacteals are lym-phatic vessels located at the center of each villus and provide essential transport routes for lipids and other lipophilic molecules. However, the dynamic process for the absorption and transport of lipids from villus en-terocytes to lacteals has been poorly understood in vivo, mostly because of the lack of appropriate experi-mental tools.

Here, I used reporter mice that express GFP under the control of the lymphatic-specific promoter Prox1 and the custom-built confocal microscope and performed intravital real-time visualization of the absorption and transport dynamics of fluorescence-tagged fatty acids (FAs) and various exogenous molecules in the intesti-nal villi in vivo. These analyses clearly revealed transepithelial absorption of these molecules via enterocytes, diffusive distribution over the lamina propria, and subsequent transport through lacteals. Moreover, I ob-served active contraction of lacteals, which seemed to be directly involved in dietary lipid drainage. Quantita-tive analysis revealed that the smooth muscles that surround each lacteal are responsible for contractile dy-namics and that lacteal contraction is ultimately controlled by the autonomic nervous system. These results indicate that the lacteal is a unique organ-specific lymphatic system and does not merely serve as a passive conduit but as an active pump that transports lipids. Collectively, using this efficient imaging method, this study uncovered drainage of absorbed molecules in small intestinal villus lacteals and the involvement of lacteal contractibility.