Figure 6. Higher TLR4 expression on inactivated whole cell (IWC)-trained alveolar macrophages (AMs) mediates rapid protection.
(A) Heatmap of differentially expressed genes (DEGs) associated with myeloid leukocyte activation at day 7 after A. baumannii IWC (LAC-4) immunization in Rag1-/- mice. (B) Reads per kilobase per million reads (RPKM) of Tlr4 in lungs on days 2 and 7 after IWC (LAC-4) immunization. (C) Representative histogram of TLR4 expression and mean fluorescence index (MFI) of TLR4 on AMs in BALF on day 2 (red line) and day 7 (gray line) after IWC (LAC-4) immunization or control (blue line). n = 3. For (B) and (C), *p<0.05, **p<0.01, evaluated by ordinary one-way ANOVA. (D) Representative histogram of TLR4 expression and MFI of TLR4 on AMs after IWC stimulation in vitro (red line) for 7 days. n = 3. Data are mean ± SD. *p<0.05, determined by two-tailed unpaired t test. (E–H) Tlr4-/- and WT mice were immunized intranasally (i.n.) with IWC (LAC-4) and challenged with LAC-4 7 days later. (E) Survival curve (n = 5 for WT mice, n = 10 for Tlr4-/- mice). (F) Bacterial burdens at 24 hr post infection (hpi). (G) TNF-α in lungs at 2 hpi. (H) Neutrophil infiltration in lungs at 4 hpi. n = 4–5 mice for (F–H). (I) TNF-α levels in 2 hr culture supernatants of ex vivo IWC (LAC-4)-stimulated AMs from 7-day-vaccinated WT or Tlr4-/- mice. n = 3. For survival, p-value was calculated by log-rank test. From (F) to (I), *p<0.05, **p<0.01, ***p<0.001, ns, not significant, compared by ordinary two-way ANOVA. In (F), each dot represents one mice and the line means median. Data are representative of at least two independent experiments.
Figure 6—figure supplement 1. Upregulated differentially expressed genes (DEGs) at day 7 after inactivated whole cell (IWC) immunization in Rag1-/- mice.
