Intestine-specific Mttp deletion decreases mortality and prevents sepsis-induced intestinal injury in a murine model of Pseudomonas aeruginosa pneumonia

Abstract

Background: The small intestine plays a crucial role in the pathophysiology of sepsis and has been referred to as the "motor" of the systemic inflammatory response. One proposed mechanism is that toxic gut-derived lipid factors, transported in mesenteric lymph, induce systemic injury and distant organ failure. However, the pathways involved are yet to be defined and the role of intestinal chylomicron assembly and secretion in transporting these lipid factors is unknown. Here we studied the outcome of sepsis in mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO), which exhibit a block in chylomicron assembly together with lipid malabsorption.

Methodology/principal findings: Mttp-IKO mice and controls underwent intratracheal injection with either Pseudomonas aeruginosa or sterile saline. Mttp-IKO mice exhibited decreased seven-day mortality, with 0/20 (0%) dying compared to 5/17 (29%) control mice (p<0.05). This survival advantage in Mttp-IKO mice, however, was not associated with improvements in pulmonary bacterial clearance or neutrophil infiltration. Rather, Mttp-IKO mice exhibited protection against sepsis-associated decreases in villus length and intestinal proliferation and were also protected against increased intestinal apoptosis, both central features in control septic mice. Serum IL-6 levels, a major predictor of mortality in human and mouse models of sepsis, were elevated 8-fold in septic control mice but remained unaltered in septic Mttp-IKO mice. Serum high density lipoprotein (HDL) levels were reduced in septic control mice but were increased in septic Mttp-IKO mice. The decreased levels of HDL were associated with decreased hepatic expression of apolipoprotein A1 in septic control mice.

Conclusions/significance: These studies suggest that strategies directed at blocking intestinal chylomicron secretion may attenuate the progression and improve the outcome of sepsis through effects mediated by metabolic and physiological adaptations in both intestinal and hepatic lipid flux.

Figure 1. Effect of impaired intestinal lipid transport on mortality in P. aeruginosa pneumonia.

Mice with intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO) and control mice were subjected to P. aeruginosa pneumonia. Sham mice received intratracheal injections of saline. All mice were followed for survival for 7 days. Mice with impaired intestinal lipid transport exhibited significantly improved survival compared to control mice (p<0.05). All sham mice survived.

Figure 2. Effect of impaired intestinal lipid transport on intestinal morphology and proliferation.

Intestinal morphology (A) was evaluated in H&E-stained sections. Magnification 20×. Villus length (B) and crypt depth (C) were quantified in jejunal sections. Control mice subjected to P. aeruginosa pneumonia had significantly shorter villi and smaller crypts than sham mice, while both villus length and crypt depth was restored or nearly restored to sham levels in septic Mttp-IKO mice. n = 9 shams/genotype, n = 17 septics/genotype. (D) S-phase cells were quantified in 100 crypts. Control mice subjected to P. aeruginosa pneumonia had significantly decreased intestinal proliferation compared to control sham mice, while septic Mttp-IKO mice exhibited increased proliferative capacity. n = 6–7 shams/genotype, n = 9–11 septics/genotype. (E) Intestinal tissue was stained with osmium to detect intracellular lipid droplets, which appear as dark black staining material. (F) Mucosal concentrations of triglycerides and (G) cholesterol were measured in jejunum, the data expressed as µg/mg protein. n = 5/group.

Figure 3. Effect of impaired intestinal lipid transport on intestinal epithelial apoptosis.

Intestinal epithelial apoptosis was evaluated by active caspase-3 staining (A) and H&E staining (B) in 100 crypts. Control mice subjected to P. aeruginosa pneumonia exhibited increased intestinal apoptosis by both methods. In contrast, Mttp-IKO mice with pneumonia had similar levels of intestinal apoptosis as sham mice.n = 6–7 shams/genotype, n = 16–18 septics/genotype. The gene expression ratios of pro-apoptotic Bax to anti-apoptotic Bcl-2 (C) and Bcl-xL (D) were evaluated. Septic Mttp-IKO mice had significantly decreased ratios compared to septic control mice. n = 11/group.

Figure 4. Effect of impaired intestinal lipid transport on pulmonary bacterial clearance and neutrophil infiltration.

Control mice with pneumonia had increased bacterial burden in the lungs compared to shams (A). There was less bacteria in the BAL fluid of Mttp-IKO mice with pneumonia, but the difference was not statistically significant compared to control septic mice. Myeloperoxidase (MPO) activity was evaluated as an index of neutrophil infiltration and degranulation in BAL fluid (B). Mice subjected to P. aeruginosa pneumonia exhibited elevated MPO activity compared to shams; however, the lack of intestinal lipid absorption did not significantly alter neutrophil activation. n = 3–5 shams/genotype, n = 8–10 septics/genotype.

Figure 5. Effect of impaired intestinal lipid transport on lung cytokines.

Cytokines were measured in bronchoalveolar lavage (BAL) fluid 24 hr after induction of pneumonia. Although several cytokines were elevated during pneumonia, there were no differences between septic Mttp-IKO and control mice. n = 8–10/group.

Figure 6. Effect of impaired intestinal lipid transport on systemic cytokines.

Cytokines were measured in serum 24 hr after induction of pneumonia. The proinflammatory cytokines IL-6 and G-CSF were increased in septic control mice; however, these cytokines were reduced in septic Mttp-IKO mice. n = 4–5 shams/genotype, n = 13–15 septics/genotype.

Figure 7. Effect of impaired intestinal lipid transport on serum lipopolysaccharide (LPS).

Serum LPS was increased in septic control mice, but not in septic Mttp-IKO mice. Serum LPS concentration was measured using the LAL chromogenic endotoxin kit (Methods). N = 5–8 per group. *p<0.05.

Figure 8. Effect of impaired intestinal lipid transport on serum lipoproteins.

Lipoprotein distribution measured by fast protein liquid chromatography in control (A) and Mttp-IKO (B) mice. Pooled samples of serum from n = 5–10 mice per genotype were analyzed in sham animals and both before and 24 hr after induction of pneumonia in the experimental groups. Cholesterol was assayed enzymatically and peaks corresponding to fractions 10–16 indicate particles in the low density lipoprotein (LDL) range while fractions 19–26 correspond to high density lipoprotein (HDL). (C) Expression of genes implicated in hepatic cholesterol efflux were analyzed by qRT-PCR on samples of RNA from the indicated groups (n = 4 mice per genotype and treatment) (D) and (E). Expression of hepatic Abca1, apoA1 and apoA4 protein by SDS-PAGE and western blot. Gapdh was used as loading control. Panel D shows representative Western blotting results. Panel E shows densitometric scanning from groups of 4 mice per genotype and treatment. (F) Expression of genes implicated in intestinal lipid metabolism were analyzed by qRT-PCR on samples of small intestinal RNA from the indicated groups (n = 4 mice per genotype and treatment). *p<0.05.