Adam Delisle

Increased CD4+ CD25+ T regulatory cell activity in trauma patients depresses protective Th1 immunity

Objectives:We recently reported increased CD4+ CD25+ T regulatory (Treg) activity after burn injury in mice. This study sought to determine if Tregs mediate the reduction in TH1-type immunity after serious injury in man and if Treg function is altered by injury. Methods:Peripheral blood was withdrawn from 19 consenting adult patients (35.1 ± 16.3 years of age) with Injury Severity Scores (ISS) 36.6 ± 13.9 on days 1 and 7 after trauma and from 5 healthy individuals. CD4+ T cells were purified and sorted into Treg (CD25high) and Treg-depleted populations. After activation of cells with anti-CD3/CD28 antibody, production of the TH1-type cytokine IFNγ, TH2-type cytokines (IL-4 and IL-5), and the inhibitory cytokine IL-10 was measured using cytometric bead arrays. Treg activity was measured by in vitro suppression of autologous CD4+ T cell proliferation. Results:All patients survived, 9 (47%) developed infection postinjury. IFNγ production by patient CD4+ T cells was decreased on day 1 and day 7, when compared with healthy controls. However, when Tregs were depleted from the CD4+ T cells, the IFNγ production increased to control levels. Tregs were the chief source of IL-4 and IL-5 as well as IL-10. Treg suppression of T cell proliferation increased significantly from day 1 to day 7 after injury. Conclusions:We demonstrate for the first time that human Tregs are increased in potency after severe injury. Most significantly, Tregs are important mediators of the suppression of T cell activation and the reduction in TH1 cytokine production found after injury.

Injury Enhances Resistance to Escherichia coli Infection by Boosting Innate Immune System Function

Major injury is widely thought to predispose the injured host to opportunistic infections. This idea is supported by animal studies showing that major injury causes reduced resistance to polymicrobial sepsis induced by cecal ligation and puncture. Although cecal ligation and puncture represents a clinically relevant sepsis model, we wanted to test whether injury might also lead to greater susceptibility to peritoneal infection caused by a single common pathogen, Escherichia coli. Contrary to our expectation, we show herein that the LD50 for sham-injured mice was 103 CFU of E. coli, whereas the LD50 for burn-injured mice was 50 × 103 CFU at 7 days postinjury. This injury-associated enhanced resistance was apparent as early as 1 day after injury, and maximal resistance was observed at days 7 and 14. We found that burn-injured mice had higher numbers of circulating neutrophils and monocytes than did sham mice before infection and that injured mice were able to recruit greater numbers of neutrophils to the site of infection. Moreover, the peritoneal neutrophils in burn-injured mice were more highly activated than neutrophils from sham mice as determined by Mac-1 expression, superoxide generation, and bactericidal activity. Our findings suggest that the enhanced innate immune response that develops following injury, although it is commonly accepted as the mediator of the detrimental systemic inflammatory response syndrome, may also, in some cases, benefit the injured host by boosting innate immune antimicrobial defenses.

Regulatory T cells suppress antigen-driven CD4 T cell reactivity following injury

Injury initiates local and systemic host responses and is known to increase CD4 Treg activity in mice and humans. This study uses a TCR transgenic T cell adoptive transfer approach and in vivo Treg depletion to determine specifically the in vivo influence of Tregs on antigen‐driven CD4 T cell reactivity following burn injury in mice. We report here that injury in the absence of recipient and donor Tregs promotes high antigen‐driven CD4 T cell expansion and increases the level of CD4 T cell reactivity. In contrast, CD4 T cell expansion and reactivity were suppressed significantly in injured Treg‐replete mice. In additional experiments, we found that APCs prepared from burn‐ or sham‐injured, Treg‐depleted mice displayed significantly higher antigen‐presenting activity than APCs prepared from normal mice, suggesting that Tregs may suppress injury responses by controlling the intensity of APC activity. Taken together, these findings demonstrate that Tregs can actively control the in vivo expansion and reactivity of antigen‐stimulated, naïve CD4 T cells following severe injury.

Murine dendritic cell antigen-presenting cell function is not altered by burn injury

Severe injury disrupts normal immune regulation causing a transient hyperinflammatory reaction and suppressed adaptive immune function. This report addresses the potential contribution of dendritic cells (DC) to changes in adaptive immune function after injury by specifically measuring injury‐induced changes in splenic DC numbers and subsets, cell‐surface markers, TLR responses, and APC function. Using a mouse burn injury model, we found that injury did not markedly alter the relative percentage of lymphoid, myeloid, or plasmacytoid DC in the spleens of burn‐injured mice. Moreover, we did not observe a significant reduction in cell‐surface expression of several major costimulatory molecules, CD40, CD80, CD86, programmed death 1 ligand, ICOS ligand, and B7‐H3, on DC. Instead, we observed increased cell‐surface expression of CD86 at 1 day after injury with no significant changes in costimulatory molecule expression at 7 days after injury, suggesting that burn injury causes an early activation of DC. In addition, injury did not suppress DC reactivity to TLR2, TLR4, or TLR9 agonists. Most important, DC prepared from injured mice were able to present peptide antigen to naïve OTII TCR transgenic CD4+ T cells as efficiently and effectively as DC from sham‐injured mice. We also found that CD4 T cells stimulated with antigen presented by DC from sham or burn mice showed similar levels of IL‐2, IFN‐γ, IL‐10, and IL‐13 production. Taken together, these findings support the conclusion that DC do not acquire a suppressive phenotype following severe injury in mice.

Injury-induced GR-1+ macrophage expansion and activation occurs independently of CD4 T-cell influence.

Burn injury initiates an enhanced inflammatory condition referred to as the systemic inflammatory response syndrome or the two-hit response phenotype. Prior reports indicated that macrophages respond to injury and demonstrate a heightened reactivity to Toll-like receptor stimulation. Since we and others observed a significant increase in splenic GR-1+ F4/80+ CD11b+ macrophages in burn-injured mice, we wished to test if these macrophages might be the primary macrophage subset that shows heightened LPS reactivity. We report here that burn injury promoted higher level TNF-&agr; expression in GR-1+, but not GR-1− macrophages, after LPS activation both in vivo and ex vivo. We next tested whether CD4+ T cells, which are known to suppress injury-induced inflammatory responses, might control the activation and expansion of GR-1+ macrophages. Interestingly, we found that GR-1+ macrophage expansion and LPS-induced TNF-&agr; expression were not significantly different between wild-type and CD4 T cell-deficient CD4(−/−) mice. However, further investigations showed that LPS-induced TNF-&agr; production was significantly influenced by CD4 T cells. Taken together, these data indicate that GR-1+ F4/80+ CD11b+ macrophages represent the primary macrophage subset that expands in response to burn injury and that CD4 T cells do not influence the GR-1+ macrophage expansion process, but do suppress LPS-induced TNF-&agr; production. These data suggest that modulating GR-1+ macrophage activation as well as CD4 T cell responses after severe injury may help control the development of systemic inflammatory response syndrome and the two-hit response phenotype.ABBREVIATIONS-SIRS-systemic inflammatory response syndrome; WT-wild type; MSCs-myeloid suppressor cells; TLR-Toll-like receptor; Tregs-CD4+ regulatory T cells; GR-1-granulocyte receptor 1; FACS-fluorescence-activated cell sorting

Immune System Phenotyping of Radiation and Radiation Combined Injury in Outbred Mice

The complexity of a radionuclear event would be immense due to varying levels of radiation exposures and injuries caused by blast-associated trauma. With this scenario in mind, we developed a mouse model to mimic as closely as possible the potential consequences of radiation injury and radiation combined injury (RCI) on survival, immune system phenotype, and immune function. Using a mouse burn injury model and a 137CsCl source irradiator to induce injuries, we report that the immunological response to radiation combined injury differs significantly from radiation or burn injury alone. Mice that underwent radiation combined injury showed lower injury survival and cecal ligation and puncture (CLP) induced polymicrobial sepsis survival rates than mice with single injuries. As anticipated, radiation exposure caused dose-dependent losses of immune cell subsets. We found B and T cells to be more radiation sensitive, while macrophages, dendritic cells and NK cells were relatively more resistant. However, radiation and radiation combined injury did induce significant increases in the percentages of CD4+ regulatory T cells (Tregs) and a subset of macrophages that express cell-surface GR-1 (GR-1+ macrophages). Immune system phenotyping analysis indicated that spleen cells from radiation combined injury mice produced higher levels of proinflammatory cytokines than cells from mice with radiation or burn injury alone, especially at lower dose radiation exposure levels. Interestingly, this enhanced proinflammatory phenotype induced by radiation combined injury persisted for at least 28 days after injury. In total, our data provide baseline information on differences in immune phenotype and function between radiation injury and radiation combined injury in mice. The establishment of this animal model will aid in future testing for therapeutic strategies to mitigate the immune and pathophysiological consequences of radionuclear events.