Up-regulation of cell surface Toll-like receptors on circulating gammadelta T-cells following burn injury

Abstract

Burn injury is associated with profound inflammation and activation of the innate immune system involving gammadelta T-cells. Similarly, Toll-like receptors (TLR) are associated with activation of the innate immune response; however, it is unclear whether TLR expression is altered in gammadelta T-cells after major burn injury. To study this, male C57BL/6 mice were subjected to burn injury (25% TBSA) and 1 or 7 days thereafter, blood and spleen cells were isolated and subjected to FACs analysis for TLRs and other phenotypic markers (gammadelta TCR, alphabeta TCR, CD69, CD120b). A marked increase in the number of circulating gammadelta T-cells was observed at 24h post-burn (14% vs. 4%) and a higher percentage of these cells expressed TLR-2. TLR-4 expression was also increased post-burn, but to a lesser degree. These changes in TLR expression were not associated with altered CD69 or CD120b expression in gammadelta T-cells. The mobilization of, and increased TLR expression in, gammadelta T-cells was transient, as phenotypic changes were not evident at 7 days post-burn or in gammadelta T-cells from the circulation or spleen. The increases in TLR expression were not observed in alphabeta T-cells after burn injury. In conclusion, 24h after burn injury mobilization of gammadelta T-cells with increased TLR expression was observed. This finding suggests that this unique T-cell population is critical in the innate immune response to injury, possibly through the recognition of danger signals by TLRs.

Figure 1. Effect of burn injury on circulating immune cell populations

Blood was collected from mice at 24 hr after sham or burn injury. The lymphocyte/monocyte and granulocyte populations were identified by forward (FSC) and side scatter (SSC) and analyzed as a percentage of the total cell population. Data are mean ± SEM for n=6/group. * p<0.05 vs. respective sham group.

Figure 2. Effect of burn injury on circulating γδ T-cells

Blood was collected from mice at 24 hr after sham or burn injury and stained for expression of γδ TCR as described in the materials and methods. The lymphocyte/monocyte population was identified by forward and side scatter and analyzed FITC γδ TCR. Bar indicates γδ TCR+ population. Data are representative of 6 experiments.

Figure 3. TLR expression on circulating γδ T-cells

Blood was collected from mice at 24 hr after sham or burn injury and stained for expression of γδ TCR and TLR-2 [A] or TLR-4 [B] as described in the materials and methods. The lymphocyte/monocyte population was identified by forward and side scatter and analyzed FITC γδ TCR and PE TLR-2 or TLR-4. Data are mean ± SEM for n=4/group. * p<0.05 vs. respective sham group. † vs. respective gd TCR⁺ TLR-2⁻ group.

Figure 4. TLR expression on circulating αβ T-cells

Blood was collected from mice at 24 hr after sham or burn injury and stained for expression of αβ TCR and TLR-2 [A] or TLR-4 [B] as described in the materials and methods. The lymphocyte/monocyte population was identified by forward and side scatter and analyzed FITC αβ TCR and PE TLR-2 or TLR-4. Data are mean ± SEM for n=4/group.

Figure 5. The impact of burn injury on systemic cytokine and chemokine levels

Plasma was collected from mice at 24 hr after sham or burn injury and assayed for the cytokines TNF-α, IL-6 and IL-10 [A] and the chemokines KC and MCP-1 [B] as described in the materials and methods. Data are mean ± SEM for n=5/group. * p<0.05 vs. respective sham group.

Figure 6. TLR expression on circulating T-cells late post-injury

Blood was collected from mice at 7 days after sham or burn injury and stained for expression of γδ TCR and TLR-2 [A], γδ TCR and TLR-4 [B], αβ TCR and TLR-2 [C] and γδ TCR and TLR-4 [D] as described in the materials and methods. The lymphocyte/monocyte population was identified by forward and side scatter and analyzed FITC TCR and PE TLR-2 or TLR-4. Data are mean ± SEM for n=4/group.