Damage-associated molecular patterns (DAMPs) released after burn are associated with inflammation and monocyte activation

Abstract

Burns are associated with activation of the innate immunity that can contribute to complications. Damage-associated molecular patterns (DAMPs) released after tissue injury play a critical role in the activation of the innate immunity, which appears to be mediated via toll-like receptors (TLRs). Previous findings have shown that TLRs and TLR-mediated responses are up-regulated after burn. Nonetheless, it is unclear what impact burn has on circulating levels of DAMPs. To study this, male C57BL/6 mice were subjected to a major burn or sham procedure. Three hours to 7days thereafter, plasma was collected and assayed for the representative DAMPs (i.e., HMGB1, cytochrome C, DNA and S100A) and extracellular cleavage products (fibronectin and hyaluronan). HMGB1, cytochrome C, fibronectin and hyaluronan levels were elevated in a time-dependent manner after burn as compared to sham levels. A significant elevation in TNF-α, IL-6 and IL-10 cytokine plasma levels was also found after burn. All cytokine levels were increased as early as 3h and remained elevated up to 24h. Circulating CD11b⁺ monocytes were increased at 24h after burn and showed increased expression of TLR-2. In conclusion, these findings support the concept that burn-induced elevations in circulating DAMPs are in part responsible for monocyte activation and the development of inflammatory complications under such conditions and warrants further investigation.

Keywords: Alarmins; CD11b; Danger Theory; HMGB-1; Toll-like receptors; Trauma.

Figure 1. Plasma DAMP levels

Plasma samples were collected from sham and burn mice and assessed for HMGB1, cytochrome C, DNA, and S100A8 (which was not measurable) as described in the Materials & Methods. Data are expressed as the mean ± SEM for 5–7 mice/group. *p<0.05 vs. respective sham group.

Figure 2. Plasma levels of extracellular matrix cleavage products

Plasma samples were collected from sham and burn mice and assessed for fibronectin and hyaluronan levels as described in the Materials & Methods. Data are expressed as the mean ± SEM for 4–5 mice/group. *p<0.05 vs. respective sham group.

Figure 3. Plasma cytokine levels

Plasma samples from sham and burn mice were assessed for cytokine levels as described in the Materials & Methods. Data are expressed as the mean ± SEM pg/ml for 4 mice/group. nd = not detectable. *p<0.05 vs. respective sham group.

Figure 4. Analysis of CD11b and TLR expression on monocytes

Blood samples were collected after 24 hr of sham or burn procedure and the blood cells were stained with CD11b alone or in combination with TLR-2 and TLR-4 antibodies as described in the Materials & Methods. The monocyte population was gated as determined by forward and side scatter. Panel A shows the entire cell population with the lymphocyte/monocytes gate (Lymph/mono, as determined by forward, FSC and side scatter, SSC) used for monocytes analysis. In panel B the upper right quadrant of the graphs represents the monocytes positive for CD11b and TLR-2 or TLR-4. The numbers are the mean percentage of the gated population. Panel C shows the CD11b⁺ monocytes based on the forward scatter (FSC) and CD11b expression. The numbers are the percentages of the CD11b^low and CD11b^high populations. Data shown are representative of 4 independent experiments.