Intestine-Derived Matrix Metalloproteinase-8 Is a Critical Mediator of Polymicrobial Peritonitis

Abstract

Objective: Inhibition of matrix metalloproteinase-8 improves survival following cecal ligation and puncture in mice, making it a potential therapeutic target. In the current study, we expand our understanding of the role of matrix metalloproteinase-8 in sepsis by using an adoptive transfer approach and alternative sepsis models.

Design: We used three different sepsis models: cecal ligation and puncture, cecal slurry, and intestinal implantation. In our first model, adoptive transfer experiments were followed by cecal ligation and puncture to test the hypothesis that matrix metalloproteinase-8-containing myeloid cells are a critical factor in sepsis following cecal ligation and puncture. Our second model, cecal slurry, used intraperitoneal injections of cecal contents to induce polymicrobial peritonitis without tissue compromise in the recipient. Our third model, intestinal implantation, involved ligating and puncturing a cecum from a donor, and then removing the cecum and placing it into the recipient's peritoneal cavity. Clinically, blood samples were drawn from pediatric patients within 24 hours of meeting criteria for septic shock.

Setting: Basic science laboratory.

Subjects: Wild type and genetically modified mice.

Interventions: Experimental models of sepsis.

Measurements and main results: In our adoptive transfer experiments, matrix metalloproteinase-8 null mice receiving wild-type marrow had a survival advantage when compared with wild-type mice receiving matrix metalloproteinase-8 null marrow, suggesting that matrix metalloproteinase-8-containing myeloid cells are not a critical factor in sepsis following cecal ligation and puncture. In our cecal slurry model, no survival advantage was seen among matrix metalloproteinase-8 null mice. Our third model, intestinal implantation, found that mice receiving matrix metalloproteinase-8 null intestine had a survival advantage when compared with mice receiving wild-type intestine, regardless of recipient genotype. Clinically, median matrix metalloproteinase-8 serum concentrations were higher in patients with sepsis and primary intestinal pathology than in septic patients without primary intestinal pathology.

Conclusions: Intestine-derived matrix metalloproteinase-8 is a critical component of septic peritonitis secondary to intestinal compromise.

Figure 1. Adoptive transfer experiments followed by CLP indicate non-myeloid cells as the critical source of MMP8

(Figure 1A) WT (n=12) and MMP8 null (n=11) mice were transplanted with bone marrow from the same genotype. Following CLP, MMP8 null mice transplanted with MMP8 null marrow showed a trend toward increased survival, consistent with our previous experiments. (Figure 1B) Following CLP, MMP8 null mice transplanted with WT marrow (n=22) had a survival advantage over WT mice transplanted with MMP8 null marrow (n=21, *p< 0.05).

Figure 2. Survival of WT (C57BL/6) and MMP8 null mice following intraperitoneal injections with cecal slurry

The mean survival time of WT mice (n=20) was 53.2 hours and did not differ significantly from the mean of 59.3 hours for MMP8 null mice (n=16).

Figure 3. Implantation of cecum into WT and MMP8 null recipients

Figure 3A shows the survival of WT mice following intraperitoneal implantation of either a WT or MMP8 null cecum. WT mice that received a MMP8 null cecum (MMP8 null into WT, n=7) had a significant survival advantage over WT mice that received a WT cecum (WT into WT, n=9, *p<0.05). Figure 3B shows survival of MMP8 null mice following implantation of a WT or MMP8 null cecum. Mice that received a MMP8 null cecum (MMP8 null into MMP8 null, n=8) had a significant survival advantage compared to those that received a WT cecum (WT into MMP8 null, n=8, ***p<0.001).

Figure 4. Implantation of distal small bowel in WT and MMP8 null recipients

Figure 4 shows the survival of WT mice following intraperitoneal implantation of either WT or MMP8 distal small bowel. WT mice that received MMP8 null distal small bowel (n=9) had a significant survival advantage over MMP8 null mice that received WT distal small bowel (n=9, ***p<0.001).

Figure 5. Intraperitoneal decay of WT cecum causes greater increase in recipient serum cytokines

Serum levels of MIP 1α (4A), TNFα (4B), IL-1β (4C), IL-10 (4D), and IL-6 (not shown) were significantly higher in mice who received a WT cecum compared to those that received a MMP8 null cecum. Error bars are the standard deviation. * p<0.05 vs. MMP8 null mice implanted with WT cecum. ^#p<0.05 vs. WT mice implanted with WT cecum.

Figure 6. Ratio of MMP protein expression relative to t=0 hours

The expression of MMPs was measured using immuno-assays and expressed as a percentage of the respective baseline concentrations. Ex vivo experiments demonstrated that as the cecal tissue decays, MMP8 protein expression is sustained at a much higher rate than other metalloproteinases (n=12, ANOVA on ranks p=0.006).

Figure 7. Log transformed serum MMP8 protein concentrations in children with septic shock, with and without primary intestinal pathology

MMP8 serum protein concentrations were significantly higher in patients with primary intestinal pathology (n = 24), compared to age- and illness severity- matched patients without primary intestinal pathology (n=48, p<0.05).