Figure 8. Epithelial cell protection in macrophage-depleted mice and in Let1 and LA-4 cells.

(A) Mice (n = 5/group) were inoculated with 1 nmol of INNA-X or diluent and 1 day later received clodronate liposomes via the i.v. (200 μL) and i.n. route (50 μL) or were left untreated. All mice were challenged the next day with 500 PFU of Udorn IAV and 1 day after viral challenge the numbers of (B) macrophages and (C) NP⁺ CD45^–CD31^–EpCAM⁺ epithelial cells in nasal turbinates were analyzed. (D and E) 5 × 10⁵ Let1 or 2 × 10⁵ LA-4 cells were incubated in the presence or absence of INNA-X for 24 hours (D) or 18 hours (E) (n = 3–6/group). Cell monolayers were washed prior to challenge with Udorn IAV (MOI of 0.01). Culture supernatants were harvested 24 hours later and assayed to quantitate viral titers. (F) LA-4 cells were treated with 1 nmol of INNA-X prior to infection. Cells were harvested 8 hours after infection and stained for surface expression of HA, neuraminidase (NA), matrix-2 (M2) influenza viral proteins, and intracellular expression of NP. (G) INNA-X–treated LA-4 cells were lysed and proteins analyzed by reverse-phase liquid chromatography with tandem mass spectrometry (LC-MS/MS) after proteolytic digestion. Sample comparisons were performed using the MaxQuant proteomics platform to determine differentially expressed proteins (red) in INNA-X–treated cells relative to those treated with diluent. Statistical analysis was performed using (B–D and E) 1-way ANOVA with Tukey’s post hoc test and (F) Welch t test. *P < 0.05, **P < 0.01, ***P < 0.001.