Surfactant Protein D Influences Mortality During Abdominal Sepsis by Facilitating Escherichia coli Colonization in the Gut

Abstract

Determine the role of surfactant protein D (SPD) in sepsis.

Design: Murine in vivo study.

Setting: Research laboratory at an academic medical center.

Patients: SPD knockout (SPD^-/-) and wild-type (SPD^+/+) mice.

Interventions: SPD^-/- and SPD^+/+ mice were subjected to cecal ligation and puncture (CLP). After CLP, Escherichia coli bacteremia was assessed in both groups. Cecal contents from both groups were cultured to assess for colonization by E. coli. To control for parental effects on the microbiome, SPD^-/- and SPD^+/+ mice were bred from heterozygous parents, and levels of E. coli in their ceca were measured. Gut segments were harvested from mice, and SPD protein expression was measured by Western blot. SPD^-/- mice were gavaged with green fluorescent protein, expressing E. coli and recombinant SPD (rSPD).

Measurements and main results: SPD^-/- mice had decreased mortality and decreased E. coli bacteremia compared with SPD^+/+ mice following CLP. At baseline, SPD^-/- mice had decreased E. coli in their cecal flora. When SPD^-/- and SPD^+/+ mice were bred from heterozygous parents and then separated after weaning, less E. coli was cultured from the ceca of SPD^-/- mice. E. coli gut colonization was increased by gavage of rSPD in SPD^-/- mice. The source of enteric SPD in SPD^+/+ mice was the gallbladder.

Conclusions: Enteral SPD exacerbates mortality after CLP by facilitating colonization of the mouse gut with E. coli.

Keywords: Escherichia coli; cecal ligation and puncture; critical illness; sepsis; surfactant protein D.

Figure 1.

Surfactant protein D (SPD) deficiency reduces Escherichia coli bacteremia and mortality after cecal ligation and puncture (CLP) by reducing cecal colonization by E. coli. A, CLP (100 % ligation, 19-G puncture, two holes) was performed on SPD^+/+ and SPD^−/− mice (n = 16/group) (Gehan-Breslow-Wilcoxon, *p < 0.05). B, Blood E. coli load following CLP. E. coli colony forming units (CFU) in blood 24 hr after CLP (100% ligation, 19-G puncture, one hole) in SPD^+/+ (n = 9) and SPD^−/− (n = 18) mice (Mann-Whitney, ****p < 0.0001). C, Ex vivo phagocytosis. Neutrophils were isolated from SPD^+/+ and SPD^−/− mice (n = 3/group) and incubated with green fluorescent protein (GFP) expressing E. coli. The percentage of GFP+ cells was determined by flow cytometry (p > 0.05, Mann-Whitney). D, In vivo phagocytosis. SPD^+/+ mice (n = 5–6) and SPD^−/− mice (n = 6) were injected intraperitoneally with GFP expressing E. coli. Neutrophils and macrophages were identified by cell surface markers, and the percentage of GFP+ cells was determined by flow cytometry (p > 0.05, Mann-Whitney). E, Post E. Coli fibrin clot survival of SPD^+/+ and SPD^−/− mice (n = 9/group) (Gehan-Breslow-Wilcoxon, p > 0.05), where both SPD^+/+ and SPD^−/− mice were exposed to exogenous E. coli. E. coli for fibrin clot was isolated from SPD^+/+ cecum.

Figure 2.

Surfactant protein D (SPD) is synthesized by the gallbladder and promotes colonization of both the cecum and colon with Escherichia coli. A, SPD^+/+ gut organs were harvested at baseline or post-cecal ligation and puncture (CLP) with Western blots performed for SPD or β-actin (loading control). Blots represent pooled samples, n = 2/group. Representative gel shown from three experiments. Lungs from SPD^−/− and SPD^+/+ mice were used as negative and positive controls, respectively. B, Gallbladder was isolated from SPD^+/+ mice after CLP or sham surgery and from SPD^−/− mice after CLP with Western blots performed for SPD or glyceraldehyde 3-phosphate dehydrogenase antibody (GAPDH; loading control). Blots represent pooled samples, n = 5–7/group. Lungs from SPD^−/− mice and SPD^+/+ were used as negative and positive controls, respectively. C, SPD^−/− mice (n = 9) were gavaged with recombinant surfactant protein D (rSPD), followed by gavage with green fluorescent protein (GFP)-labeled E. coli, and compared with SPD^−/− mice gavaged only with GFP-labeled E. coli (n = 9). After 24 hr, cecum and colon were harvested. GFP-labeled E. coli were then detected by culture (Mann-Whitney *p < 0.05, **p < 0.01). CFU = colony forming units.

Figure 3.

Surfactant protein D (SPD)^−/− mice have diminished cecal Escherichia coli colonization. A, E. coli cultured from cecal culture of homozygote bred SPD^+/+ mice (n = 24) and SPD^−/− mice (n = 15) (**p < 0.0001, Mann-Whitney). B, E. coli cultured from cecal culture of heterozygote bred, cohoused SPD^+/+ (n = 5) mice and SPD^−/− mice (n = 12) (p > 0.05, Mann-Whitney). C, E. coli cultured from cecal culture of heterozygote bred, SPD^+/+ mice (n = 5) and SPD^−/− mice (n = 10) after 7 wk of separation, by genotype (*p < 0.05, Mann-Whitney). D, Post-cecal ligation and puncture (CLP) survival in heterozygote bred, cohoused SPD^+/+ mice (n = 12) and SPD^−/− mice (n = 14), where SPD^−/− mice had exposure to E. coli from the feces of SPD^+/+ mice (p = ns, Gehan-Breslow-Wilcoxon). E, Post-CLP survival of homozygote bred SPD^+/+ (n = 6) and SPD^−/− (n = 9) mice (Gehan-Breslow-Wilcoxon, **p < 0.01) for concurrent recapitulation of the findings in Figure 1A. CFU = colony forming units.