MMP-8 expression and activity are increased in human sepsis and septic shock
We mined our previously published mRNA expression database to determine the expression levels of MMP-8 mRNA in whole blood-derived RNA from children with sepsis and septic shock (20
). Blood samples were obtained within 24 hours of meeting criteria for either sepsis or septic shock (day 1) and 48 hours later (day 3). As shown in , children with sepsis and septic shock had significantly increased expression of MMP-8 mRNA as compared to healthy, control patients on day 1. Furthermore, patients with septic shock had increased MMP-8 mRNA expression as compared to patients with sepsis. This pattern of gene expression persisted into day 3. shows that MMP-8 mRNA expression was significantly higher in children with septic shock that did not survive the 28 day study period, as compared to survivors, suggesting that the degree of MMP-8 mRNA expression correlates with illness severity.
Figure 1 MMP-8 mRNA expression and activity are increased in children with sepsis or septic shock, and the degree of expression correlates with illness severity. (a) Relative expression of MMP-8 mRNA in whole blood of children with either sepsis (N = 32) or septic (more ...)
To further demonstrate a link between MMP-8 mRNA expression and illness severity, we measured MMP-8 mRNA expression in 180 children with septic shock and grouped these patients into quartiles of MMP-8 mRNA expression. We then queried the clinical database to determine the maximal number of organ failures, over the first 7 days of admission, for patients in each quartile. As shown in , patients in the 3rd and 4th quartiles of MMP-8 mRNA expression had a significantly higher number of organ failures, as compared to patients in the 1st quartile, further demonstrating a link between MMP-8 mRNA expression and illness severity.
To confirm that increased MMP-8 mRNA expression led to increased MMP-8 activity, MMP-8 activity was measured in plasma samples from separate cohorts of healthy control patients and children with septic shock. As shown in , MMP-8 activity was significantly increased in the plasma of children septic shock, as compared to healthy control patients.
Collectively, these data demonstrate that substantially increased MMP-8 mRNA expression and activity are early and persistent features of pediatric sepsis and septic shock, and that the degree of MMP-8 expression correlates with illness severity. Thus, it is possible that MMP-8 plays an integral role in the biological response to sepsis and septic shock.
Deletion of Mmp-8 improves survival and blunts the inflammatory response in a murine model of sepsis
To further explore the role of MMP-8 in sepsis, Mmp-8 deficient mice (Mmp8 −/−) and C57BL6-J wild type (WT) mice were subjected to CLP. As shown in , Mmp-8 −/− mice displayed a significant survival advantage over their WT counterparts over the 10 day observation period after CLP. As MMP-8 is an important component of neutrophil function, we next determined if absence of MMP-8 compromised bacterial clearance. We found no difference in plasma, pulmonary, peritoneal, or splenic bacterial colony counts 24 hours after CLP in Mmp8 −/− mice as compared to WT mice, thus indicating that absence of MMP-8 does not compromise bacterial clearance in mice (data not shown).
Figure 2 Mmp8 −/− mice show a survival advantage and decreased lung neutrophil activity after CLP as compared to WT mice. (a) Cumulative survival rates of WT (N=18) and Mmp8 −/− (N=20) mice up to 10 days after CLP (time depicted (more ...)
Morbidity and mortality from sepsis is thought to occur, in part, from an excessive host inflammatory response to bacterial challenge, and the lung is often a primary target of this robust inflammatory response. We assessed whole lung inflammation by measuring myeloperoxidase (MPO) activity as a surrogate of neutrophil infiltration. MPO levels were assessed in whole lung specimens from WT and Mmp8 −/− mice at 3, 6, and 24 hours after CLP. As shown in , Mmp8 −/− mice showed a reduction in lung MPO activity after CLP at 3 and 6 hours after CLP as compared to WT mice. These data demonstrate that the survival advantage seen in Mmp8 −/− mice correlates with decreased early lung neutrophil infiltration.
Following the observations that genetic ablation of MMP-8 improves survival and reduces early lung neutrophil infiltration, we hypothesized that MMP-8 possesses a regulatory function in the sepsis inflammatory cascade. To test this hypothesis, we analyzed the circulating concentrations of several cytokines and chemokines in the plasma of WT and Mmp8 −/− mice after CLP, at baseline and at several time points after CLP. As shown in , significant reductions in the circulating levels of the pro-inflammatory cytokines IL-6 and IL-1β were observed in Mmp8 −/− mice, compared to WT mice, at 6 hours after CLP. The reduction of IL-6 persisted at 24 hours. Additionally, plasma concentration of the anti-inflammatory cytokine, IL-10, was significantly increased in Mmp-8 −/− mice, as compared to their WT counterparts, at 24 hours after CLP. Cumulatively, these results suggest that MMP-8 plays a role in regulating the inflammatory response to sepsis, with genetic ablation of MMP-8 leading to a survival advantage and correlative reduction in early lung neutrophil infiltration and selected systemic cytokines.
Figure 3 Mmp8 −/− mice show an overall decreased systemic inflammatory profile of cytokines and chemokines after CLP. The circulating plasma concentrations of (a) IL-6 (ng/mL), (b) KC (ng/mL), (c) IL-1β (pg/mL), (d) MIP-1α (pg/mL) (more ...)
Pharmacologic inhibition of MMP-8 activity replicates the Mmp8 −/− phenotype
While the experiments involving Mmp8 −/− mice implicate a role for MMP-8 in sepsis, there remains the possibility that chronic deletion of MMP-8 can lead to unexpected developmental adaptations that confer a survival advantage in the context of sepsis. Therefore, we attempted to replicate the phenotype observed in Mmp8 −/− mice via pharmacologic inhibition of MMP-8 in WT mice subjected to CLP. To directly measure the efficacy of our MMP-8 inhibitor, we measured MMP-8 activity in the plasma of WT mice treated with the MMP-8 inhibitor and subjected to CLP. shows that intraperitoneal (i.p.) administration of the MMP-8 inhibitor, after CLP, effectively decreased plasma MMP-8 activity in WT mice 6 hours after CLP. This trend continued at 24 hours, but did not reach statistical significance ().
Figure 4 WT mice treated with the MMP-8 inhibitor have a survival advantage after CLP. (a, b) Plasma MMP-8 activity (pM) at 6 hours (a) and 24 hours (b) after CLP in WT mice treated with vehicle (N=9) as compared to WT mice treated with MMP-8 inhibitor (N=9). (more ...)
To show that this reduced activity correlated to improved outcomes, we observed mice for survival during a 10 day observation period after CLP. Pharmacologic inhibition of MMP-8 activity correlated with a significant survival advantage as compared to vehicle-treated WT mice (). Thus, pharmacologic inhibition of MMP-8 reduces mortality in a rodent model of sepsis, successfully replicating the phenotype observed in Mmp8 −/− mice.
To examine if this replicated phenotype was also due to regulation of the inflammatory cascade by MMP-8, we again measured whole lung MPO as a marker of neutrophil infiltration. While whole lung MPO tended to be lower in mice treated with the MMP-8 inhibitor, compared to vehicle-treated animals, this trend did not reach statistical significance ().
To further assess the role of pharmacologic inhibition on the inflammatory cascade, we measured the plasma concentrations of several cytokines and chemokines 6 hours after CLP in WT mice treated with vehicle or the MMP-8 inhibitor. As shown in , pharmacologic inhibition of MMP-8 activity in WT mice significantly reduced plasma concentrations of IL-6, IL-1β, MIP-1α and TNFα at 6 hours after CLP, as compared to vehicle-treated WT mice.
Figure 5 WT mice treated with the MMP-8 inhibitor show an overall decreased systemic inflammatory profile of cytokines and chemokines 6 hours after CLP. The figure depicts the circulating plasma concentrations of (a) IL-6 (ng/mL), (b) KC (ng/mL), (c) IL-1β (more ...)
To test the specificity of the MMP-8 inhibitor, we treated Mmp8 −/− mice with the MMP-8 inhibitor or vehicle after CLP. We found no significant difference in lung MPO activity or plasma cytokine/chemokine concentrations between Mmp8 −/− mice treated with vehicle or inhibitor (data not shown).
Cumulatively, these data demonstrate that we can reduce mortality and systemic inflammation with pharmacologic inhibition of MMP-8, thus generally reproducing the phenotype observed in Mmp8 −/− mice.
MMP-8 activates the pro-inflammatory transcription factor NF-κB
To begin assessing a potential mechanism by which genetic ablation or inhibition of MMP-8 blunts inflammation following sepsis, we evaluated the effect of MMP-8 on activation of the pro-inflammatory transcription factor, NF-κB. RAW 264.7 murine macrophages were transfected with an NF-κB-dependent luciferase reporter and subsequently treated with active MMP-8 (1μg/ml) isolated from human neutrophils. Macrophages treated with MMP-8 showed a significant increase in luciferase activity (). LPS (1 μg/ml) was used as a positive control, and showed a vigorous activation of NF-κB.
Figure 6 MMP-8 activates the pro-inflammatory nuclear transcription factor NF-κB. (a) Luciferase activity from RAW 264.7 murine macrophages transfected with an NF-κB driven reporter plasmid. RAW 246.7 murine macrophages were treated for 4 hours (more ...)
To assess the potential role of LPS contamination of the MMP-8 preparation, we carried out a series of control experiments. We did not observe increased luciferase activity when macrophages were exposed to the same concentration of LPS (80 pg/mL) as that found in MMP-8 preparation, nor when we denatured the MMP-8 protein by boiling (). However, the increase in luciferase activity was maintained when active MMP-8 was delivered in conjunction with the endotoxin binder, polymyxin-B. Thus, MMP-8 can directly activate NF-κB in vitro, and this affect does not appear to be an artifact of LPS contamination of the MMP-8 preparation.
To confirm that MMP-8 led to a pro-inflammatory phenotype in macrophages, we measured the production of several pro-inflammatory cytokines by primary murine peritoneal macrophages treated with MMP-8 ex vivo. Peritoneal macrophages from WT mice were exposed to media alone, media with LPS (1 μg/ml) as a positive control, or media with MMP-8 (1 μg/ml) for 24 hours. shows that treatment with MMP-8 increased supernatant concentrations of IL-6, KC, TNFα, and MIP-1α, as compared to cells treated with media alone. This confirms that MMP-8 induces a pro-inflammatory phenotype in cultured macrophages.
We next evaluated the effect of MMP-8 on in vivo NF-κB activation. The concentration of the p65 active subunit of NF-κB was measured in murine whole lung nuclear protein extracts at 6 hours after CLP. We found that Mmp8 −/− mice showed a significant reduction of NF-κB activation in lung nuclear extracts as compared to WT mice (). WT mice treated with the MMP-8 inhibitor had a trend toward decreased NF-κB activation in lung nuclear extracts as compared to WT mice treated with vehicle, but this did not reach statistical significance (). Collectively, these data demonstrate that MMP-8 has the potential to directly activate NF-κB, thus suggesting a potential mechanism by which MMP-8 regulates the inflammatory state in experimental sepsis.