The field of innate immunity has been significantly rewritten since the discovery of mammalian TLRs, a family of pattern-recognition receptors (PRRs). TLRs have been implicated in immunoregulation in a wide range of disease states including atherosclerosis, allergies, autoimmunity, burn and sepsis [20
]. Because TLRs recognize pathogen associated conserved molecules shared among members of a particular class of microbes (e.g., LPS from Gram-negative pathogens and ssRNA from RNA viruses), a wide range of pathogens can be recognized by a small group of receptors [21
]. Studies also indicate that TLRs can regulate responses to endogenous stimuli, such as necrotic cells, heat-shock proteins and extracellular matrix breakdown products, these stimuli have come to be known as “alarmins” or damage associated molecular patterns (DAMPs). In this regard, necrotic cell death may be a primary cytotoxic mechanism following tissue injury. Necrotic cells can release their intracellular contents, which might contribute to inflammation following injury [22
]. Matzinger has proposed a concept involving danger signaling [23
]. In the Matzinger model, initiation of the immunoinflammatory response is the result of the recognition of molecular patterns by cellular receptors in response to molecular patterns that can be associated with either pathogens or specific normal cellular components released by damaged cells. This concept reconciles the paradox of the immunoinflammatory response in sterile and nonsterile conditions. Iwasaki and Medzhithov have discussed that exogenous TLR agonists (pathogen associated molecular patterns, PAMPS) activate genes involved in inflammation, tissue repair and adaptive immune response. In contrast, endogenous TLR ligands (DAMPS) only activate genes involved in inflammation and tissue repair [24
]. DAMPS associated with TLR activation include HMGB1, components of the extracellular matrix (hyaluronic acid and heparin sulfate), and heat shock proteins (HSP60 and HSP72) [25
]. All these DAMPs are associated with cellular injury or stress responses common to burn. Recent findings by Zhang et al. [26
] have also shown that injury releases mitochondrial DAMPs into the circulation that can activate neutrophils through TLRs and elicit neutrophil-mediated organ injury. Due to the nature of burns extensive tissue damage and tissue necrosis, rather than apoptosis, is commonplace. Thus, burn provides a fertile environment for the activation of the immunoinflammatory response by DAMPs via TLR activation.
Activation of an inflammatory cascade after burn injury is important in the development of subsequent immune dysfunction. In this regard, previous findings have shown that macrophage productive capacity for these mediators is markedly enhanced after burn and thereby contributing to immune dysfunction [27
]. Our study shows that the TLR2 and TLR4-mediated inflammatory responses by circulating leukocytes after burn occurs primarily at 3-7 days post-injury, as evidenced by the significantly increased production of IL-6, IL-10, KC and MIP-1β. IL-6, in general, is considered a marker of injury severity and levels correlate with burn severity in both animal models and humans [28
]. In addition, elevated levels of IL-6 early after sepsis (6 hrs) are predictive of death [30
]. In contrast, IL-10 is counter-inflammatory and has been shown to be important in the down regulation of inflammation after injury [31
]. While the current study and most of these referenced studies involve mice, rather than human subjects, studies by Finnerty et al, have validated the applicability of the mouse model of burn in terms of its similarity in magnitude and duration of the inflammatory response to that seen in humans [33
Cairns et al, have also shown that mouse splenocytes activated by TLR2 and TLR4 ligands 14 days after burn resulted in an increased inflammatory response [35
]. Similarly, Paterson et al have also shown increased splenocyte TLR responses up to 7 days post-burn [36
]. Recent findings from our laboratory have shown a marked increase in TLR expression on circulating γδ T-cells, but early (i.e., 24 hr) after burn [37
]. Our findings here extend those of other investigators by examining the impact of burn on TLR reactivity in the circulating immune cells, rather than in fixed tissues. Our previous findings have shown that the post-burn immune derangements differ between fixed tissues (ie, spleen) and circulating immune cells, indicating the importance of examining multiple tissues [38
]. Blood also represents the tissue that is predominately studied in humans, therefore understanding the responses in this tissue compartment is paramount in applying findings between animal models and the human condition. Moreover, our study examined a much wider array of cytokines and chemokines than the previously cited studies and showed that the burn-induced enhancements in TLR responses were specific for given cytokines rather than global in nature.
In general, the current study demonstrates a stronger shift towards enhanced TLR4 responses, rather than TLR2 responses after burn. Moreover, these responses were delayed till 3-7 days post-injury. Previous studies have shown, primarily via LPS-induced activation, that TLR4 responses are enhanced after burn, but are not immediate [39
]. The enhanced TLR4 reactivity after burn is mediated, at least in part, by enhanced activation of the p38 signaling pathway [41
]. The mechanism for the enhanced TLR2 responses is unknown, but may also be related to the p38 signaling pathway [43
The current study did not examine TLR expression on the circulating leukocytes, however; we have previously shown upregulation of TLR2 and TLR4 expression on circulating γδ T-cells, but not αβ T-cells early after burn [44
]. Nonetheless, while the regulation of TLR expression is not clearly understood [45
], under “normal conditions”, TLR expression is most likely limited to avoid excessive activation. In this regard, studies have shown that TLR expression is transiently upregulated on monocytes in response to LPS or TNF-α [46
]. Whether burn induces a dysregulated expression of TLRs by circulating leukocytes which contributes to the enhanced response to TLR agonists remains to be determined.
In conclusion, our findings show that at 3-7days post-burn TLR responses are enhanced in circulating leukocytes. This TLR hyperresponsiveness likely contributes to inflammatory complications after burn. An improved understanding of how injury modulates innate immune responses will reveal new insights into ways in which normal immune function could be restored after critical injury.