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. 2023 Feb 21;13:1041552. doi: 10.3389/fimmu.2022.1041552

Figure 3.

Figure 3

ICAM-1 and VCAM-1 compartmentalization and differential usage by innate leukocytes entering influenza infected lungs. (A) Experimental design and timeline of pulmonary imaging by three-dimensional (3D) light sheet fluorescence microscopy (LSM). WT and ICAM-1/2-/- mice were I.N. infected with a sublethal dose of PR8 influenza virus or administered with PBS. 4 days post infection (dpi), mice were subjected to intravital staining by an intravenous co-injection of Alexa-Flour 568 labeled anti ICAM-1 mAb (red) and Alexa-Flour 647 labeled anti VCAM-1 mAb (cyan) (B, C). Scale bars represent 500 μm or 200 μm, respectively. (B) The lungs of resting and PR8 infected WT mice were compared by the intravital staining described in (A). (C) Enlarged views of the white squares shown in (B). (D) WT and ICAM-1/2-/- mice were compared by similar intravital staining as in (B, C) with Alexa-Flour 647 anti-VCAM-1 mAb (cyan). Scale bars represent 500 μm. (E) Experimental design and timeline (left panel) for determination of neutrophil (middle panel) and NK cell counts (right panel) in the BALF of either WT or ICAM-1/2-/- mice 4 days post I.T. infection with the indicated doses of PR8 influenza. (F) Flow cytometry analysis of α4 (top), β1 (middle), and β7 (bottom) integrin expression on neutrophils (left column) and NK cells (right column). (G) Experimental design and timeline protocol for α4 blocking in WT and ICAM-1/2-/- mice infected with PR8 influenza. Mice were I.V. administered with 60 ug of anti α4 mAb (α4 block) or α4 isotype control (iso) and 2 hours later were I.N. infected with 30 PFU of PR8 influenza. For the following 3 days, 30 ug anti-α4 blocking or isotype control mAb were I.V. administered once a day. On day 4 post infection, flow cytometry analysis was performed to determine the total number of neutrophils (H) and NK cells (I) recovered in the BALF of the infected WT or ICAM-1/2-/- mice. I.N. PBS administration was performed in control experiments. Statistical significance was determined by two-tailed, unpaired Student’s t tests. *P < 0.05, **P < 0.01, ns, not significant. The numbers of each experimental group are indicated in the graphs. The error bars indicate the SEM of each measurement.