Regulatory T Cells Modulate CD4 Proliferation after Severe Trauma via IL-10.

OBJECTIVE: Severely injured patients frequently develop an immunological imbalance following the traumatic insult, which might result in infectious complications evoked by a persisting immunosuppression. Regulatory T cells (Tregs) maintain the immune homeostasis by suppressing proinflammatory responses, however, their functionality after trauma is unclear. Here, we characterized the role of Tregs in regulating the proliferation of CD4+ lymphocytes in traumatized patients (TP). METHODS: Peripheral blood was obtained daily from 29 severely injured TP (Injury Severity Score, ISS ≥16) for ten days following admission to the emergency department (ED). Ten healthy volunteers (HV) served as controls. The frequency and activity of Tregs were assessed by flow cytometry. Proliferation of CD4+ cells was analyzed either in presence or absence of Tregs, or after blocking of either IL-10 or IL-10R1. RESULTS: The frequencies of CD4+CD25high and CD4+CD25+CD127- Tregs were significantly decreased immediately upon admission of TP to the ED and during the following 10 post-injury days. Compared with HV CD4+ T cell proliferation in TP increased significantly upon their admission and on the following days. As expected, CD4+CD25+CD127- Tregs reduced the proliferation of CD4+ cells in HV, nevertheless, CD4+ proliferation in TP was increased by Tregs. Neutralization of IL-10 as well as blocking the IL-10R1 increased further CD4+ T cell proliferation in Tregs-depleted cultures, thereby confirming an IL-10-mediated mechanism of IL-10-regulated CD4+ T cell proliferation. Neutralization of IL-10 in TP decreased CD4+ T cell proliferation in Tregs-depleted cultures, whereas blocking of the IL-10R1 receptor had no significant effects. CONCLUSIONS: The frequency of Tregs in the CD4+ T lymphocyte population is reduced after trauma; however, their inductiveness is increased. The mechanisms of deregulated influence of Tregs on CD4+ T cell proliferation are mediated via IL-10 but not via the IL-10R1.

Comparative Analysis of the Regulatory T Cells Dynamics in Peripheral Blood in Human and Porcine Polytrauma.

Background: Severely injured patients experience substantial immunological stress in the aftermath of traumatic insult, which often results in systemic immune dysregulation. Regulatory T cells (Treg) play a key role in the suppression of the immune response and in the maintenance of immunological homeostasis. Little is known about their presence and dynamics in blood after trauma, and nothing is known about Treg in the porcine polytrauma model. Here, we assessed different subsets of Treg in trauma patients (TP) and compared those to either healthy volunteers (HV) or data from porcine polytrauma. Methods: Peripheral blood was withdrawn from 20 TP with injury severity score (ISS) ≥16 at the admittance to the emergency department (ED), and subsequently on day 1 and at day 3. Ten HV were included as controls (ctrl). The porcine polytrauma model consisted of a femur fracture, liver laceration, lung contusion, and hemorrhagic shock resulting in an ISS of 27. After polytrauma, the animals underwent resuscitation and surgical fracture fixation. Blood samples were withdrawn before and immediately after trauma, 24 and 72 h later. Different subsets of Treg, CD4+CD25+, CD4+CD25+FoxP3+, CD4+CD25+CD127-, and CD4+CD25+CD127-FoxP3+ were characterized by flow cytometry. Results: Absolute cell counts of leukocytes were significantly increasing after trauma, and again decreasing in the follow-up in human and porcine samples. The proportion of human Treg in the peripheral blood of TP admitted to the ED was lower when compared to HV. Their numbers did not recover until 72 h after trauma. Comparable data were found for all subsets. The situation in the porcine trauma model was comparable with the clinical data. In porcine peripheral blood before trauma, we could identify Treg with the typical immunophenotype (CD4+CD25+CD127-), which were virtually absent immediately after trauma. Similar to the human situation, most of these cells expressed FoxP3, as assessed by intracellular FACS stain. Conclusion: Despite minor percental differences in the recovery of Treg populations after trauma, our findings show a comparable decrease of Treg early after polytrauma, and strengthen the immunological significance of the porcine polytrauma model. Furthermore, the Treg subpopulation CD4+CD25+CD127- was characterized in porcine samples.

Impaired Surface Expression of HLA-DR, TLR2, TLR4, and TLR9 in Ex Vivo-In Vitro Stimulated Monocytes from Severely Injured Trauma Patients.

Objective. Trauma patients (TP) frequently develop an imbalanced immune response that often causes infectious postinjury complications. Monocytes show a diminished capability of both producing proinflammatory cytokines and antigen presentation after trauma. TLR2, TLR4, and TLR9 recognize pathogens and subsequently activate monocytes. While there are conflictive data about TLR2 and TLR4 expression after trauma, no studies about the expression of TLR2, TLR4, TLR9, and HLA-DR on monocytes from TP after their secondary ex vivo-in vitro "hit" have been reported. Methods/Results. Ex vivo-in vitro lipopolysaccharide- (LPS-) stimulated blood from TP showed diminished interleukin- (IL-) 1ß-release in TP for five postinjury days compared to healthy volunteers (HV). The recovery was observed at day 5. In parallel, monocytes from TP showed an impaired capability of TLR2, TLR4, and TLR9 expression after secondary stimulation compared to HV, while the measurement of unstimulated samples showed significant reduction of TLR4 and TLR9 at ED. Furthermore, HLA-DR decreased after trauma and was even more profound by stimulation of monocytes. Ratio of monocytes to leukocytes was significantly increased at days 6 and 7 after trauma compared to HV. Conclusion. Impaired expression of TLRs and HLA-DR in acute inflammatory conditions may be responsible for the well-described monocyte paralysis after severe trauma.

Phagocytizing activity of PMN from severe trauma patients in different post-traumatic phases during the 10-days post-injury course.

OBJECTIVE: Phagocytizing leukocytes (granulocytes and monocytes) play a fundamental role in immunological defense against pathogens and clearance of cellular debris after tissue injury due to trauma. According to the "two-hit hypothesis", phagocytes become primed due to/after trauma. Subsequently, a secondary stimulus may lead to their exaggerated response. This immune dysfunction can result in serious infectious complications, also depending on trauma injury pattern. Here, we investigated the phagocytizing capacity of leukocytes, and its correlation to trauma injury pattern. MATERIAL/METHODS: Peripheral whole blood was taken daily from 29 severely injured trauma patients (TP, Injury Severity Score, ISS≥28) for ten days (1-10) following admission to the emergency department (ED). Sixteen healthy volunteers served as controls (HV). Samples were incubated with opsonized Staphylococcus aureus labelled with pHrodo fluorescent reagent and the percentage of phagocytizing activity was assessed by flow cytometry. Abbreviated Injury Scales (AIS)≥3 of head, chest and extremities were used for injury pattern analysis. RESULTS: Overall distribution of active phagocytes (out of 100% phagocytizing leukocytes) in TP included granulocytes with 28.6±1.5% and monocytes with 59.3±1.9% at ED, and was comparable to HV (31.5±1.6% granulocytes and 60.1±1.6% monocytes). The percentage of phagocytizing granulocytes increased significantly after D2 (39.1±1.2%), while the percentage of phagocytizing monocytes (52.0±1.2%, p<0.05) decreased after D2. These changes persisted during the whole time course. Phagocytizing activity of granulocytes (27.9±2.8%) and monocytes (55.2±3.3%) was significantly decreased at ED compared to HV (42.4±4.1% and 78.1±3.1%, respectively). After D2 up to D10, phagocytizing activity was significantly enhanced in granulocytes. Phagocytizing activity of monocytes remained decreased on D1 and has risen continuously during the ten days time course to values comparable to HV. No significant differences in phagocytosis could be associated to certain injury pattern. CONCLUSIONS: Our data demonstrate that the increasing percentage of phagocytizing granulocytes may indicate their enhanced mobilization out of bone marrow persisting until post-injury day 10. Furthermore, an initially decreased phagocytizing activity of granulocytes is strongly increased in the 10-days post-injury course. The altered activity of phagocytes due to injury could not be linked to any trauma injury pattern, and emerged rather as a general characteristic of phagocytes after severe trauma.