Myeloid differentiation protein-2-dependent and -independent neutrophil accumulation during Escherichia coli pneumonia - PubMed (original) (raw)

Myeloid differentiation protein-2-dependent and -independent neutrophil accumulation during Escherichia coli pneumonia

Shanshan Cai et al. Am J Respir Cell Mol Biol. 2009 Jun.

Erratum in

Abstract

Bacterial pneumonia remains a serious disease. Pattern recognition receptors play an integral role in neutrophil accumulation during pneumonia. Although myeloid differentiation protein (MD)-2 has been recognized as a key molecule for LPS signaling, the role of MD-2 in neutrophil accumulation in the lung during bacterial infection has not been explored. Here, we investigate the role of MD-2 in Escherichia coli LPS-induced lung inflammation and E. coli-induced pneumonia. LPS-induced CD14-independent neutrophil accumulation was abolished in CD14/MD-2(-/-) mice. MD-2(-/-) mice challenged with LPS displayed attenuated neutrophil influx, NF-kappaB activation, cytokine/chemokine expression, and lung histopathology. MD-2(-/-) mice transplanted with MD-2(+/+) bone marrow demonstrated decreased neutrophil influx and cytokine/chemokine expression in the lungs when challenged by LPS. MD-2(-/-) mice infected with E. coli demonstrated reduced neutrophil influx and cytokine/chemokine expression in the lungs, whereas heat-killed E. coli did not induce either neutrophil accumulation or cytokine/chemokine expression in MD-2(-/-) mice infected with E. coli. Furthermore, MD-2(-/-) mice displayed increased bacterial burden in the lungs and enhanced bacterial dissemination. Toll-like receptor (TLR)-5(-/-) mice infected with E. coli exhibited attenuated neutrophil accumulation, whereas MD-2/TLR5(-/-) mice inoculated with E. coli showed further attenuated neutrophil influx and impaired bacterial clearance. Taken together, these new findings demonstrate: (1) the important role of MD-2 in the CD14-independent LPS-mediated cascade of neutrophil influx; (2) the relative importance of bone marrow- and non-bone marrow cell-derived MD-2 in LPS-induced inflammation; and (3) the essential role of MD-2-dependent and MD-2-independent (TLR5) signaling in E. coli-induced neutrophil accumulation and pulmonary host defense.

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Figures

<b>Figure 1.</b>

**Figure 1.

Neutrophil accumulation in the lung after saline or LPS exposure. Animals were treated with aerosolized saline (control) (A) or 3,000 μg of LPS (B) for 20 minutes, and bronchoalveolar lavage fluid (BALF) was collected at 8 or 24 hours after challenge. BALF neutrophil counts were determined. A total of three to five mice were used in each group at each time point. Data are presented as mean (±SEM).

<b>Figure 2.</b>

**Figure 2.

Innate immune responses in the lung after LPS challenge. Animals were treated with aerosolized LPS (300 μg) or saline (control) for 20 minutes, and BALF and lungs were collected at various time points after LPS challenge. BAL total white blood cell (WBC) (A) and neutrophil (B) counts, myeloperoxidase activity in the lung homogenates (C), BALF cytokine/chemokine levels (D), lung NF-κB activation at 2 hours (E), lung histology at 24 hours (arrows indicate infiltrating inflammatory cells and edema; original magnification: ×400) (F), and actin filament assembly in bone marrow–derived neutrophils (G) were determined in MD-2−/− and MD-2+/+ mice. A total of five to seven animals were examined in each group at each time point. *Significant differences between MD-2−/− and MD-2+/+ mice (P < 0.05). Histopathology shown is representative of three lungs in each group. Data are presented as mean (±SEM).

<b>Figure 3.</b>

**Figure 3.

Role of MD-2 originating from bone marrow and non–bone marrow cells in LPS-induced inflammation (A and B). Bone marrow chimeras were prepared by lethal irradiation of MD-2−/− and MD-2+/+ mice and reconstituted with bone marrow cells via tail vein injection. BALF neutrophil counts (A) and cytokine/chemokine expression (B) in BALF were obtained at 8 h after LPS (300 μg aerosolized for 20 min) challenge.*Significant differences between groups (P < 0.05); a total of four to seven animals were used. (C) LIX expression by alveolar epithelial type II (AEII) cells after LPS exposure. Murine primary AEII cells were stimulated with LPS (30 ng/ml) for 18 hours at 37°C, and the protein levels were measured. These data are an average (±SEM) of four or five wells from two animals.

<b>Figure 4.</b>

**Figure 4.

(A_–_D) Effect of viable Escherichia coli (106 CFU/mouse) on total WBC (A), neutrophil accumulation (B), and KC and macrophage inflammatory protein (MIP)-2 expression in the airspaces (C and D) in MD-2−/− and MD-2+/+ mice at 6 and 24 hours after infection (n = 4–7 mice in each group; *Significant differences between MD-2−/− and MD-2+/+ [P < 0.05]). (E_–_G) Bacterial clearance of E. coli after intratracheal inoculation. The MD-2−/− and MD-2+/+ mice were administered E. coli intratracheally on Day 0 at a concentration of 106 CFU/mouse, and bacterial burden in the lungs (E) and spleens (F) were determined at 6 and 24 hours after infection (n = 4–6 mice in each group; *Significant differences between MD-2−/− and MD-2+/+ mice [P < 0.05]). (G) Neutrophil killing assay. A total of 106 CFU/ml of E. coli was cultured alone or cocultured with 106 bone marrow–derived polymorphonuclear cells from MD-2−/− or MD-2+/+ mice. Bacteria were then quantified after 2 hours of incubation (n = 3/group). (H_–_K) Effect of heat-killed E. coli (106 CFU/mouse) on neutrophil accumulation, and KC and MIP-2 expression in the lungs in MD-2−/− and MD-2+/+ mice at 6 and 24 h after infection (n = 4–6 mice in each group at 6 and 24 h; *Significant differences between MD-2−/− and MD-2+/+ [P < 0.05]). Data are presented as mean (±SEM).

<b>Figure 5.</b>

**Figure 5.

(A and B) Effect of viable E. coli (106 CFU/mouse) on neutrophil accumulation in the lungs in Toll-like receptor (TLR)-5−/− and MD-2−/−/TLR5 mice (n = 4–5 mice in each group at 6 and 24 h; *significant differences between knockout [KO] mice and their control littermates [P < 0.05]). (C) KC and MIP-2 expression in the lungs in MD-2−/−/TLR5 and wild-type (WT) mice at 6 and 24 hours after infection (n = 4–5). *Significant differences between MD-2−/−/TLR5−/− and their littermate controls (P < 0.05). (D and E) Bacterial clearance of E. coli after intratracheal inoculation. The MD-2/TLR5−/− and MD-2+/+/TLR5−/− mice were administered E. coli at a concentration of 106 CFU/ml on Day 0, and bacterial burden in the lungs (D) and bacterial dissemination (E) were determined. *Significant differences between MD-2/TLR5−/−−/− and WT mice. (F) Survival in MD-2−/−/TLR5 mice and control animals (C57Bl/6) after intratracheal E. coli infection. Mice were inoculated intratracheally with a dose of E. coli (108 CFU/mouse) on Day 0, and for survival (n = 16 mice from 2 separate experiments in each group; *significant differences between KO and WT mice [P < 0.05] determined by Wilcoxon rank sign test between groups). (G) Survival in MD-2−/−, TLR5−/− mice, and control animals (C57Bl/6) after intratracheal E. coli (108 CFU/mouse) infection (n = 18 mice from 2 separate experiments in each group; *P < 0.05 determined by Wilcoxon rank sign test between groups). Data are presented as mean (±SEM).

References

    1. Mizgerd JP. Lung infection: a public health priority. PLoS Med 2006;3:e76. - PMC - PubMed
    1. Fein AM. Pneumonia in the elderly: overview of diagnostic and therapeutic approaches. Clin Infect Dis 1999;28:726–729. - PubMed
    1. Lynch JP, Martinez FJ. Community-acquired pneumonia. Curr Opin Pulm Med 1998;4:162–172. - PubMed
    1. Mizgerd JP, Skerrett SJ. Animal models of human pneumonia. Am J Physiol Lung Cell Mol Physiol 2008;294:L387–L398. - PubMed
    1. Mizgerd JP. Acute lower respiratory tract infection. N Engl J Med 2008;358:716–727. - PMC - PubMed

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