Background: Severe traumatic injury has been associated with high susceptibility for the development of secondary complications caused by dysbalanced immune response. As the first line of the cellular immune response, neutrophils and monocytes recruited to the site of tissue damage and/or infection, are divided into three different subsets according to their CD16/CD62L and CD16/CD14 expression, respectively. Their differential functions have not yet been clearly understood. Thus, we evaluated the phenotypic changes of neutrophil and monocyte subsets among their functionality regarding oxidative burst and the phagocytic capacity in severely traumatized patients. Methods: Peripheral blood was withdrawn from severely injured trauma patients (TP; n = 15, ISS ≥ 16) within the first 12 h post-trauma and from healthy volunteers (HV; n = 15) and stimulated with fMLP and PMA. CD16^dimCD62L^bright (immature), CD16^brightCD62L^bright (mature) and CD16^brightCD62L^dim (CD62L^low) neutrophil subsets and CD14^brightCD16⁻ (classical), CD14^brightCD16⁺ (intermediate) and CD14^dimCD16⁺ (non-classical) monocyte subsets of HV and TP were either directly analyzed by flow cytometry or the examined subsets of HV were sorted first by fluorescence-activated cell sorting and subsequently analyzed. Subset-specific generation of reactive oxygen species (ROS) and of E. coli bioparticle phagocytosis were evaluated. Results: In TP, the counts of immature neutrophils were significantly increased vs. HV. The numbers of mature and CD62L^dim neutrophils remained unchanged but the production of ROS was significantly enhanced in TP vs. HV and the stimulation with fMLP significantly increased the generation of ROS in the mature and CD62L^dim neutrophils of HV. The counts of phagocyting neutrophils did not change but the mean phagocytic capacity showed an increasing trend in TP. In TP, the monocytes shifted toward the intermediate phenotype, whereas the classical and non-classical monocytes became less abundant. ROS generation was significantly increased in all monocyte subsets in TP vs. HV and PMA stimulation significantly increased those level in both, HV and TP. However, the PMA-induced mean ROS generation was significantly lower in intermediate monocytes of TP vs. HV. Sorting of monocyte and neutrophil subsets revealed a significant increase of ROS and decrease of phagocytic capacity vs. whole blood analysis. Conclusions: Neutrophils and monocytes display a phenotypic shift following severe injury. The increased functional abnormalities of certain subsets may contribute to the dysbalanced immune response and attenuate the antimicrobial function and thus, may represent a potential therapeutic target. Further studies on isolated subsets are necessary for evaluation of their physiological role after severe traumatic injury.

This paper discusses how the assembly of pro-caspase-1 and apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) in macromolecular protein complexes, inflammasomes, activates caspase-1. The present study investigates the molecular mechanisms of inflammasome activation in HepG2 cells and examines how short exposures to ethanol (EtOH) affect inflammasome activation. HepG2 cells were treated with lipopolysaccharide (LPS), ATP or nigericin (NIG) in a two-step model. After LPS priming, ATP or NIG were added. As inhibitors, sodium orthovanadate (general inhibitor of tyrosine phosphatases), AC-YVAD-CMK (caspase-1 inhibitor) or AZ10606120 (purinergic receptor P2X7R inhibitor) were applied after LPS priming. To monitor the inflammasome activation, the caspase-1 activity, ASC speck formation, reactive oxygen species (ROS) production and cell death were analyzed. To elucidate the mechanistical approach of EtOH to the inflammasome assembly, the cells were treated with EtOH either under simultaneous LPS administration or concurrently with ATP or NIG application. The co-stimulation with LPS and ATP induced a significant ASC speck formation, caspase-1 activation, cell death and ROS generation. The inhibition of the ATP-dependent purinoreceptor P2X7 decreased the caspase-1 activation, whereas sodium orthovanadate significantly induced caspase-1. Additional treatment with EtOH reversed the LPS and ATP-induced caspase-1 activation, ASC speck formation and ROS production. The ASC speck formation and caspase-1 induction require a two-step signaling with LPS and ATP in HepG2 cells. Inflammasome activation may depend on P2X7. The molecular pathway of an acute effect of EtOH on inflammasomes may involve a reduction in ROS generation, which in turn may increase the activity of tyrosine phosphatases.

Traumatic brain injury (TBI) may cause damage to distant organs. Acute ethanol intoxication (EI) induces complex local and systemic anti-inflammatory effects and influences the early outcomes of traumatized patients. Here, we evaluated its effects on the BI-induced expression of local inflammatory mediators in the trauma-remote organs the lungs and liver. Male mice were exposed to ethanol as a single oral dose (5g·kg^–1, 32%) before inducing a moderate blunt TBI. Sham groups underwent the same procedures without TBI. Ether 3 or 6h after the TBI, the lung and liver were collected. The gene expression of HMGB1, IL-6, MMP9, IL-1β, and TNF as well as the homogenate protein levels of receptor for advanced glycation end products (RAGE), IL-6, IL-1β, and IL-10 were analyzed. Liver samples were immunohistologically stained for HMGB1. EI decreased the gene expressions of the proinflammatory markers HMGB1, IL-6, and MMP9 in the liver upon TBI. In line with the reduced gene expression, the TBI-induced protein expression of IL-6 in liver tissue homogenates was significantly reduced by EI at 3h after TBI. While the histological HMGB1 expression was enhanced by TBI, the RAGE protein expression in the liver tissue homogenates was diminished after TBI. EI reduced the histological HMGB1 expression and enhanced the hepatic RAGE protein expression at 6h post TBI. With regard to the lungs, EI significantly reduced the gene expressions of HMGB1, IL-6, IL-1β, and TNF upon TBI, without significantly affecting the protein expression levels of inflammatory markers (RAGE, IL-6, IL-1β, and IL-10). At the early stage of TBI-induced inflammation, the gene expression of inflammatory mediators in both the lungs and liver is susceptible to ethanol-induced remote effects. Taken together, EI may alleviate the TBI-induced pro-inflammatory response in the trauma-distant organs, the lungs and liver, via the HMGB1-RAGE axis.