Severe injury causes a dramatic host response that disrupts immune homeostasis and predisposes the injured host to opportunistic infections. Because Toll-like receptors (TLRs) recognize conserved microbial Ags and endogenous danger signals that may be triggered by injury, we wanted to determine how injury influences TLR responses. Using an in vivo injury model, we demonstrate that injury significantly increased TLR2-and TLR4-induced IL-1␤, IL-6, and TNF-␣ production by spleen cells. This influence of injury on TLR reactivity was observed as early as 1 day after injury and persisted for at least 7 days. The outcome of similar studies performed using TLR4-mutant C57BL/10ScN/Cr mice revealed that TLR2 responses remained primed, thus suggesting that injury-induced priming can occur independently of endogenous TLR4 signaling. Increased TLR4 reactivity was also observed in vivo, because LPS-challenged injured mice demonstrated significantly higher cytokine expression levels in the lung, liver, spleen, and plasma. Macrophages and dendritic cells were the major source of these cytokines as judged by intracellular cytokine staining. Moreover, ex vivo studies using enriched macrophage and dendritic cell populations confirmed that T cells did not contribute to the enhanced TLR2 and TLR4 responses. The results of flow cytometry studies using TLR2-and TLR4-MD-2-specific Abs indicated that injury did not markedly alter cell surface TLR2 or TLR4-MD-2 expression. Taken together, these findings establish that injury primes the innate immune system for enhanced TLR2-and TLR4-mediated responses and provides evidence to suggest that augmented TLR reactivity might contribute to the development of heightened systemic inflammation following severe injury.

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Substantial clinical and laboratory research has revealed that major injury causes abnormalities in both the innate and adaptive immune systems. However, the relative importance of each of these systems in the immune dysfunction after injury is poorly understood and difficult to establish by clinical studies alone. Rag1 (-/-) C57BL/6 mice (Rag1), which lack an adoptive immune system, and immune-sufficient wild-type (WT) C57BL/6 mice underwent 25% total body surface area burn injury or sham injury under anesthesia and were subjected to cecal ligation and puncture (CLP) at day 10 postinjury, a time of high CLP mortality in this model. To test the effect of adaptive immune deficiency on inflammatory cytokine production after injury, adaptive cell-depleted splenocytes from sham and burn WT and Rag1 mice were stimulated with LPS, and TNF-alpha and IL-6 production were assayed at days 1 and 7 postinjury. Intracellular expression of TNFalpha and IL-6 by F4/80 macrophages was also assessed on day 7 by intracellular cytokine staining. Finally, Rag1 animals were reconstituted with WT splenocytes, and the effect of such reconstitution on CLP survival and cytokine production was determined. Survival of sham WT animals after CLP was significantly higher (P < 0.01) than survival of burn WT and Rag1 sham and burn animals, all of which had equivalently low survival. Reconstitution of Rag1 animals with WT splenocytes restored CLP survival to WT sham levels. Splenocytes from Rag1 burn mice showed significantly augmented cytokine production when compared with WT burn mice on day 7 (P < 0.05). Reconstitution of Rag1 mice with WT splenocytes at the time of injury returned cytokine production to WT levels. Intracellular cytokine expression in F4/80 macrophages was increased to a similar degree after burn, but not sham burn injury in Rag1, reconstituted Rag1 and WT animals. These studies demonstrate that the adaptive immune system is necessary for protection from polymicrobial sepsis and plays a significant role in regulating the inflammatory response to injury.

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