The burn wound inflammatory response was assessed by measuring the cytokine content of skin lysates. As expected, a number of proinflammatory cytokines were elevated in the burn wound of WT mice (Fig. 7). In particular, a 50-fold increase in the levels of IL-6 and a 150-fold increase in the levels of MIP-1α were observed in the burn wound compared with sham skin. Interleukin 10 was the only cytokine measured that did not increase in the burn wound compared with sham skin. A number of the inflammatory cytokines were further increased in the burn wound of δTCR-/-, as the levels of MIP-1α, MIP-1β, and TNF-α in the burn wound of δTCR-/- mice were approximately 2- to 3-fold greater than that of wounds from WT mice.
Major burn is associated with immunoinflammatory and wound healing complications (4, 9, 10, 17–19). However, although inflammatory complications are deleterious, inflammation also plays an important role in the progression of the injury healing process, via the recruitment of immune cells to the injury site (9, 17). These immune cells, that include myeloid cells (i.e., neutrophils and macrophages) and T cells, release a wide range of factors, including cytokines, chemokines, and growth factors that are essential for proper wound healing (17, 20–22). Our group has previously shown that γδ T cells play a pivotal role after burn in the regulation of inflammation and wound healing (9, 16, 19). Interestingly, γδ T cells also have been shown to induce macrophage infiltration of the wound site (23), which are central in the immune complications associated with burn (2, 19). The current study was conducted to assess the role of γδ T cells in the regulation of myeloid cells at the burn wound site. Our findings herein demonstrate that the γδ T cells are critical in the regulation of myeloid cell trafficking at the burn wound site. In the absence of γδ T cells, (δTCR-/- mice) CD11b+, F4/80+, and Gr1+ myeloid cell numbers were markedly increased over that observed in WT burn mice. This increased influx of myeloid cells included both CD11b+F4/80+ macrophages and CD11b+Gr1+ MDSCs.
Wound healing after burn is an intricate process orchestrated by the complex interplay of myeloid cells, T cells, and other immune cells (10). Previous studies have shown a role for myeloid cells in the immune response to burn, trauma, and sepsis (17, 19, 24–26). Gr-1, CD11b, and F4/80 antigens have been shown to be expressed on the surface of immature myeloid cells and monocytes. In the present study, characterization of the wound infiltrating cells revealed that CD11b+, F4/80+, and Gr1+ myeloid cells were increased over that observed in uninjured skin. Further characterization of myeloid cells demonstrated that these cells were composed of both traditional macrophages (CD11b+F4/80+) and CD11b+Gr1+ myeloid cells. These myeloid cells have been shown to increase in animal models and patients with cancer, injury, and infection (5, 17, 26, 27). Cairns et al. (28) have also shown an accumulation in the periphery of CD11b+F4/80+ macrophages after burn injury. Further characterization of F4/80+ based on the CD11blow and CD11bhigh expression revealed that, after injury, both of these populations were increased significantly at the burn wound site; however, there was a profound shift toward a CD11bhigh-expressing population. Holt et al. (29) have also identified two distinct macrophage populations in mouse liver after acetaminophen challenge. Although they observed CD11blowF4/80high macrophages in PBS-treated control mice, CD11bhighF4/80low macrophages were present in the mice challenged with acetaminophen. In another study, Arnold et al. (30) demonstrated a change in the phenotype of recruited monocytes during the resolution of inflammation and tissue repair. They demonstrated that the recruited macrophages at the tissue injury site were changed from inflammatory to anti-inflammatory phenotype, which was tissue protective. The different subsets observed in our study may represent activated resident macrophages that have increased the expression of CD11b or, alternatively, they may be derived from circulating monocytes that are recruited at the wound site after burn.
The expansion of MDSCs was shown to be beneficial by increasing immune surveillance and innate immune responses in different injury models (17, 25). In addition to their suppressive effects on adaptive immune responses, MDSCs have also been reported to regulate innate immune responses by modulating macrophage cytokine production.
Our findings suggest that, early after burn (i.e., 3 days), there is a transition in the myeloid cell population at the injury site from a traditional macrophage phenotype (F4/80+) to a MDSC phenotype (i.e., Gr1+), as in WT mice, MDSC numbers increased and the numbers of F4/80+ cells decreased.
The lack of γδ T cells profoundly influenced the myeloid cell populations at the wound site. Relative to WT mice, the numbers of CD11b+, F480+, and Gr1+ myeloid cells markedly increased after burn. This supports the concept that γδ T cells at the burn wound site can act to suppress myeloid cell influx. In sharp contrast, Jameson et al. (23) have shown that γδ T cells are essential in the rapid migration of macrophages to the wound site in a murine punch wound model. These differences between our study and that of Jameson et al. may be, in part, related to the type of injury (burn versus punch wound) and the overall systemic inflammatory response associated with burn as opposed to an isolated punch wound injury that would induce a minimal systemic response. We have previously shown that punch wound closure rates are suppressed in burn mice (37), supporting the concept that the wound inflammatory response differs between these two models.
Although γδ T cells are the predominant dermal T cells in the mouse skin, in humans, the majority of the T cells are of αβ TCR lineage (19, 40). Nonetheless, it is clear from different clinical studies that γδ T cells, in human skin, play an important role in dermal pathologies, such as systemic lupus erythemoatosis, leprosy, leishmaniasis, and malignancies (41, 42). Thus, although the absolute numbers of γδ T cells in human skin may be less than that observed in rodents, they are an active cell population in humans and their role in human dermal pathology is clearly evident.
In the current study, in parallel to the infiltration of myeloid cells and MDSCs, we also observed an increase in the number of inflammatory cytokines and chemokines such as, IL-1β, IL-6, TNF-α, MIP-1α, MIP-1β, and monocyte chemoattractant protein 1 at the burn site. The levels of epidermal TNF-α, MIP-1α, and MIP-1β were further elevated in the injured skin of δ TCR-/- mice compared with WT mice. These data are consistent with our previous findings by Oppeltz et al. (39). In contrast, a study by Daniel et al. (9) showed a profound attenuation in cytokine/chemokine levels at the wound site in δ TCR-/- mice. This suggests that γδ T cell–mediated regulation of resident immune cells (in the current study) and that of infiltrating cells in the study by Daniel et al. (9) are markedly different.
In conclusion, γδ T cells play in important role in myeloid cell recruitment to the wound site early after burn and appear to act to transition the wound from an inflammatory stage to a proliferative stage of healing. Based on these findings and that wound healing after burn is a relevant clinical problem and clearer understanding of potential targets of therapeutic intervention (i.e., γδ T cells and MDSCs), data may provide improvements in burn care, leading to decreased morbidity and mortality.
These findings were presented in part at the Experimental Biology 2013 in Boston, Mass. MR was responsible for the animal experiments, cell isolation, FACs, data analysis, and drafting of the manuscript. QZ was responsible for the animal experiments and cell isolation. MGS was responsible for scientific conception, design, and interpretation and assisted in the final drafting of the manuscript. All authors read and approved the final version of the manuscript. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
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