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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-) 1β-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.
In their post-traumatic course, trauma patients suffering from multiple injuries have a high risk for immune dysregulation, which may contribute to post-injury complications and late mortality. Monocytes as specific effector cells of the innate immunity play a crucial role in inflammation. Using their Pattern Recognition Receptors (PRRs), notably Toll-Like Receptors (TLR), the monocytes recognize pathogens and/or pathogen-associated molecular patterns (PAMPs) and organize their clearance. TLR2 is the major receptor for particles of gram-positive bacteria, and initiates their phagocytosis. Here, we investigated the phagocytizing capability of monocytes in a long-term porcine severe trauma model (polytrauma, PT) with regard to their TLR2 expression. Polytrauma consisted of femur fracture, unilateral lung contusion, liver laceration, hemorrhagic shock with subsequent resuscitation and surgical fracture fixation. After induction of PT, peripheral blood was withdrawn before (-1 h) and directly after trauma (0 h), as well as 3.5 h, 5.5 h, 24 h and 72 h later. CD14+ monocytes were identified and the expression levels of H(S)LA-DR and TLR2 were investigated by flow cytometry. Additionally, the phagocytizing activity of monocytes by applying S. aureus particles labelled with pHrodo fluorescent reagent was also assessed by flow cytometry. Furthermore, blood samples from 10 healthy pigs were exposed to a TLR2-neutralizing antibody and subsequently to S. aureus particles. Using flow cytometry, phagocytizing activity was determined. P below 0.05 was considered significant. The number of CD14+ monocytes of all circulating leukocytes remained constant during the observational time period, while the percentage of CD14+H(S)LA-DR+ monocytes significantly decreased directly, 3.5 h and 5.5 h after trauma. The percentage of TLR2+ expressing cells out of all monocytes significantly decreased directly, 3.5 h and 5.5 h after trauma. The percentage of phagocytizing monocytes decreased immediately and remained lower during the first 3.5 h after trauma, but increased after 24 h. Antagonizing TLR2 significantly decreased the phagocytizing activity of monocytes. Both, decreased percentage of activated as well as TLR2 expressing monocytes persisted as long as the reduced phagocytosis was observed. Moreover, neutralizing TLR2 led to a reduced capability of phagocytosis as well. Therefore, we assume that reduced TLR2 expression may be responsible for the decreased phagocytizing capacity of circulating monocytes in the early post-traumatic phase.
Background/Aims: Alcohol (ethanol, EtOH) as significant contributor to traumatic injury is linked to suppressed inflammatory response, thereby influencing clinical outcomes. Alcohol-induced immune-suppression during acute inflammation (trauma) was linked to nuclear factor-kappaB (NF-ĸB). Here, we analyzed alcohol`s effects and mechanisms underlying its influence on NF-ĸB-signaling during acute inflammation in human lung epithelial cells. Methods: A549-cells were stimulated with interleukin (IL)-1β, or sera from trauma patients (TP) or healthy volunteers, with positive/negative blood alcohol concentrations (BAC), and subsequently exposed to EtOH (170 Mm, 1h). IL-6-release and neutrophil adhesion to A549 were analyzed. Specific siRNA-NIK mediated downregulation of non-canonical, and IKK-NBD-inhibition of canonical NF-ĸB signaling were performed. Nuclear levels of activated p50 and p52 NF-ĸB-subunits were detected using TransAm ELISA. Results: Both stimuli significantly induced IL-6-release (39.79±4.70 vs. 0.58±0.8 pg/ml) and neutrophil adhesion (132.30±8.80 vs. 100% control, p<0.05) to A549-cells. EtOH significantly decreased IL-6-release (22.90±5.40, p<0.05) and neutrophil adherence vs. controls (105.40±14.5%, p<0.05). IL-1β-induced significant activation of canonical/p50 and non-canonical/p52 pathways. EtOH significantly reduced p50 (34.90±23.70 vs. 197.70±36.43, p<0.05) not p52 activation. Inhibition of canonical pathway was further increased by EtOH (less p50-activation), while p52 remained unaltered. Inhibition of non-canonical pathway was unchanged by EtOH. Conclusion: Here, alcohol`s anti-inflammatory effects are mediated via decreasing nuclear levels of activated p50-subunit and canonical NF-ĸB signaling pathway.