1. Conditional ablation of LYVE-1+ cells unveils defensive roles of lymphatic vessels in intestine and lymph nodes
by Jeon Yeob Jang, Young Jun Koh, Seung-Hun Lee, Junyeop Lee, Kyoo Hyun Kim, Daesoo Kim, Gou Young Koh, and Ook Joon Yoo
Blood
Volume 122(13):
September 26, 2013
©2013 by American Society of Hematology

2. Systemic analysis of structural changes in lymphatic vessels in LYVE-1-Cre/iDTR mice after DT administration.
Systemic analysis of structural changes in lymphatic vessels in LYVE-1-Cre/iDTR mice after DT administration. (A) Schematic diagram of a working model of the LYVE-1-Cre/iDTR double-transgenic mouse (LCiDT) and the iDTR (control) mouse. (B) Kaplan-Meier survival curves after DT administration into control and LCiDT mice. (C) CD31+ blood vessels and LYVE-1+ lymphatic vessels of the ear skin, trachea, diaphragm, intestine, and inguinal lymph node. 4,6 Diamidino-2-phenylindole (DAPI) staining for nuclei. Scale bars, 100 μm. (D) Quantification of LYVE-1+ areas in different organs. Each group, n = 4. *P < .05. (E) LYVE-1+/Prox1+ lymphatic vessels in the submedullary region of the inguinal lymph node. Scale bars, 50 μm.
Jeon Yeob Jang et al. Blood 2013;122:
©2013 by American Society of Hematology

3. Major phenotypes of LYVE-1-Cre/iDTR mice after DT administration.
Major phenotypes of LYVE-1-Cre/iDTR mice after DT administration. Comparisons between control and LCiDT for major findings at 24 hours after DT administration. Each group, n = 4 to 5. *P < .05. (A) Gross images of the abdominal contents. The arrow indicates a hemorrhagic and swollen duodenum, and the arrowhead indicates a hyperemic and enlarged Peyer patch. (B-C) Duodenums and Peyer patches in jejunums stained with hematoxylin and eosin. Scale bars, 200 μm. (D) Quantification of sizes of Peyer patch. (E) LYVE-1+ lymphatic vessels and CD31+ blood vessels in jejunums. Indicated areas (square with white dotted line) are magnified in the right panels. The arrows indicate mucosal lymphatic vessels, and the arrowheads indicate lacteal lymphatic vessels. Scale bars, 100 μm. (F) Quantification of LYVE-1+ area in the jejunum. (G) Images showing abundant distribution of LPS derived from E coli at the base of the villi (arrowheads) and within LYVE-1+ mucosal lymphatic vessels (arrow) of the LCiDT mice at 24 hours after DT administration, whereas LPS is rarely detected in the control mice. 4,6 Diamidino-2-phenylindole (DAPI) staining for nuclei. Scale bars, 50 µm. (H) Serum endotoxin levels. (I) Blood profiles of hemoglobin, hematocrit, neutrophils, lymphocytes, and platelets. NS, not statistically significant.
Jeon Yeob Jang et al. Blood 2013;122:
©2013 by American Society of Hematology

4. Damage of LYVE-1+ lacteals occurs before damage to the surrounding blood capillaries and to the whole architecture of the villi, and the lymphatic vessels in Peyer patches disappear in the LYVE-1-Cre/iDTR mice after DT administration.
Damage of LYVE-1+ lacteals occurs before damage to the surrounding blood capillaries and to the whole architecture of the villi, and the lymphatic vessels in Peyer patches disappear in the LYVE-1-Cre/iDTR mice after DT administration. Control and LCiDT mice were given DT (100 ng), and their intestines were harvested at indicated times and analyzed. (A) Images showing LYVE-1+ lacteals and CD31+ blood vessels in the villi of the jejunum at 6, 12, and 24 hours after DT administration. (B) Quantification of the LYVE-1+ area and the CD31+ area in the intestinal villi. Each group, n = 4. *P < .05. (C) Images showing LYVE-1+ lacteals and E-cadherin+ epithelial cells in the intestinal villi at 24 hours after DT administration. (D) Images showing LYVE-1+ lymphatic vessels and CD31+ blood vessels in the Peyer patches. Indicated areas (square with white dotted line) are magnified in the right panel. The arrowhead indicates lymphatic vessels located at the serosal side of the Peyer patch. (E) Quantification of the LYVE-1+ area in the Peyer patches. Each group, n = 4. *P < .05. All images were stained with 4,6 diamidino-2-phenylindole (DAPI) for nuclei imaging. Scale bars, 50 µm.
Jeon Yeob Jang et al. Blood 2013;122:
©2013 by American Society of Hematology

5. Abnormal lymphatic functions of LYVE-1-Cre/iDTR mice after DT administration.
Abnormal lymphatic functions of LYVE-1-Cre/iDTR mice after DT administration. Comparisons between control and LCiDT mice for major findings at 24 hours after DT administration. Each group, n = 4–5. *P < .05. (A) Popliteal lymph nodes after injection of fluorescein isothiocyanate–conjugated lectin into the footpad. Scale bars, 200 μm. (B) Quantification of the lectin-drained area density inside the popliteal lymph nodes. (C) Distributions of CD11c+ dendritic cells inside of the inguinal lymph nodes. Indicated areas (square with white dotted line) are magnified in the right panel. The white asterisk indicates abnormally distributed CD11c+ dendritic cells in the B-cell zone. Scale bars, 100 μm. (D) Distributions of CD169+ sinusoidal macrophages in the subscapular and medullary sinuses of the inguinal lymph nodes. The yellow asterisks indicate abnormally distributed CD169+ sinusoidal macrophages. Scale bars, 100 μm. (E) Lymphatic drainage of Evans blue dye in the ear skins. Inside dotted line, Evans blue injection site. The arrowhead indicates leakage areas of skin lymphatic vessels. (F) Quantification of the number of leakage points of Evans blue dye. DAPI, 4,6 diamidino-2-phenylindole.
Jeon Yeob Jang et al. Blood 2013;122:
©2013 by American Society of Hematology

6. No apparent phenotypes are detected in BM-LCiDT mice at 24 hours after DT administration.
No apparent phenotypes are detected in BM-LCiDT mice at 24 hours after DT administration. (A) Comparisons of flow cytometric profiles of LYVE-1+/CD45+ macrophages, CD3+/CD45+ T lymphocytes, B220+/CD45+ B lymphocytes, CD11b+/F4/80+ macrophages, and CD11b+/F4/80− monocytes in the peripheral bloods. Note that there are no differences in the population of each immune cell between BM-iDT and BM-LCiDT. (B) Gross images of the abdominal contents. The arrow indicates the proximal portion of the small intestine, and the arrowhead indicates the Peyer patch. (C) Intestinal villi in the duodenum stained with hematoxylin and eosin. Scale bars, 100 μm. (D) LYVE-1+ lymphatic vessels and CD31+ blood vessels in jejunums. Scale bars, 100 μm. Similar findings were observed in 3 to 4 different mice. (E) Blood profiles of hemoglobin, hematocrit, neutrophils, lymphocytes, and platelets. Each group, n = 4 to 5. DAPI, 4,6 diamidino-2-phenylindole; H&E, hematoxylin and eosin.
Jeon Yeob Jang et al. Blood 2013;122:
©2013 by American Society of Hematology

7. Local ablation of lymphatic vessels after intradermal injection of DT into the ear skin.
Local ablation of lymphatic vessels after intradermal injection of DT into the ear skin. (A) Diagram of experimental scheme for local lymphatic ablation by intradermal injection of DT (10 ng/mL) into the left ear skin of LCiDT mice. Samples were harvested at 7 days after triple injections of DT. (B) Images showing LYVE-1+ lymphatic vessels and CD31+ blood vessels in the left and right ear skins of LCiDT and control mice. Scale bars, 100 μm. (C) Quantifications for LYVE-1+ area in the ear skins. Each group, n = 4. *P < .05 vs control. (D) Images showing LYVE-1+ lymphatic vessels in draining cervical lymph node (cLN) and distal inguinal lymph node (iLN) of LCiDT and control mice. Scale bars, 100 μm. (E) Quantifications for LYVE-1+ area in the LNs. Each group, n = 4. *P < .05 vs cLN of control. (F) Lymphatic drainage of Evans blue dye in the ear skins. The dotted line shows the Evans blue injection site. The arrowhead indicates no lymph drainage. Similar findings were observed in 3 to 4 different mice. (G) Images showing LYVE-1+ lymphatic vessels in jejunums. Scale bars, 100 μm. (H) Quantifications for LYVE-1+ area in the jejunums. Each group, n = 4. NS, not statistically significant. DAPI, 4,6 diamidino-2-phenylindole.
Jeon Yeob Jang et al. Blood 2013;122:
©2013 by American Society of Hematology

8. Schematics depicting defensive roles of lymphatic vessels in the small intestine.
Schematics depicting defensive roles of lymphatic vessels in the small intestine. In the control mice (A), the intestinal lymphatic vessels play critical roles not only in draining absorbed lipids but also in the maintenance of villi structure and function. In the LYVE-1-Cre/iDTR mice (B), ablation of intestinal lacteals causes disruptions of blood vessels and villi architecture, which leads to invasion of intestinal pathogens into the circulatory system via the blood vessels.
Jeon Yeob Jang et al. Blood 2013;122:
©2013 by American Society of Hematology