Figure 1. Clec4d^−/− mice display increased mortality accompanied with overwhelming bacterial burden in systemic organs during Gram-negative pneumonia.

(A) Sixteen WT (C57BL/6) and 17 Clec4d^−/− mice were infected intranasally with 5 × 10⁴ CFUs of KPn in 20 μl sterile PBS and were assessed daily for disease severity. The increased susceptibility of Clec4d^−/− mice compared with WT mice is statistically significant, as determined by the Kaplan-Meier survival curve statistical analysis (P<0.001). (B) Total RNA was extracted by the Trizol method from lungs harvested at the indicated times after infection of WT mice with 5 × 10⁴ CFUs of KPn. The mRNA levels of Clec4d were analyzed by real-time PCR, as described in Materials and Methods, and are expressed as fold changes over the levels in mock control mice calculated by using the formula 2^−(ΔΔCt). Data shown are the averages of six to eight mice at each time point in two independent experiments. (C) WT and Clec4d^−/− mice were infected intranasally with 5 × 10⁴ CFUs of KPn. At indicated times p.i., the mice were sacrificed, and systemic organs were isolated, homogenized, and plated, as described in Materials and Methods. Bacterial burden was enumerated after incubating the plates overnight at 37°C. The data shown are from one representative experiment (five animals at each time-point) out of three performed with similar results. Significant differences in bacterial burden (using the nonparametric Mann-Whitney test) in WT and Clec4d^−/− mice are denoted by asterisks (**P<0.005; ***P<0.001).