Remote thermal injury increases LPS-induced intestinal IL-6 production

Abstract

Background: Patients suffering from burn injury are at high risk for subsequent infection. Thermal injury followed by endotoxemia may result in a "second hit," causing an exaggerated inflammatory response with increased morbidity and mortality. The role of the intestine in this "second hit" response is unknown. We hypothesized that remote thermal injury increases the inflammatory response of intestinal mucosa to subsequent treatment with lipopolysaccharide (LPS).

Methods: Mice underwent sham or scald injury. Seven days after injury, mice were treated with LPS. Blood and bowel specimens were obtained. Serum and intestinal inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Changes in TLR-4 pathway components in intestine were measured by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and electrophoretic mobility shift assay (EMSA). Intestinal leukocyte infiltration was analyzed by myeloperoxidase assay.

Results: A "second hit" of injected LPS resulted in increased IL-6 in intestine of burned mice compared with sham. Similarly, jejunal IL-6 mRNA levels increased in mice with prior thermal injury, suggesting a transcriptional mechanism. Of transcription factors known to drive IL-6 expression, only AP-1 activation was significantly elevated by a "second hit" of LPS.

Conclusion: Prior thermal injury potentiates LPS-induced IL-6 cytokine production in intestine. These results indicate a heightened inflammatory response to a second hit by intestine after burn injury.

FIG. 1

IL-6 levels on PBD7 in jejunum (A), colon (B), and serum (C) after injury alone and 1 and 4 h after LPS injection in sham (gray bar) and burn (black bar) injured mice. IL-6 levels in jejunum and colon were significantly elevated 4 h after LPS injection in burn compared with sham injured mice (*P < 0.05).

FIG. 2

Jejunum IL-6 mRNA levels after treatment with sham or burn injury, recovery for 7 d, then injection with LPS. Data is expressed as fold difference in burn/sham mice 1 and 4 h after LPS injection (*P < 0.05).

FIG. 3

Representative jejunum (A) and colon (B) Western blots comparing sham to burn injured mice on PBD7 after injury alone and 1 and 4 h after LPS injection. Actin control demonstrates even protein loading. Paired comparisons were cut from the same Western blots.

FIG. 4

Representative EMSA for NF-κB (A and B), C/EBP (C and D), or AP-1 (E and F) of nuclear extract samples from jejunum or colon of mice treated with sham or burn injury, allowed to recover for 7 d, then injected with LPS for 1 or 4 h. Paired comparisons were cut from the same EMSAs.

FIG. 5

MPO assay of colon samples from sham (grey bars) or burn (black bars) injured mice on PBD7 after injury alone and 4 h after LPS injection (⁺P < 0.05 sham versus burn only); (*P < 0.05 burn versus burn + LPS groups).