Study On The Mechanism Of TIM-3+NK Cells In Immune Disorder After Severe Multiple Trauma And Traumatic Sepsis

Trauma is the leading cause of death for people under the age of 45 worldwide.Although polytrauma accounts for only 10% of total trauma,requiring multidisciplinary comprehensive management and long hospital stay and rehabilitation period,imposing a huge economic burden on individuals and society.Chest trauma constitutes the third most common cause of death in patients with multiple injuries after abdominal injury and head trauma.Overall,approximately one-third of total trauma related deaths are attributed to chest trauma and the ensuing complications.Although the overall mortality of patients with multiple injuries has decreased significantly due to improved pre-hospital treatment and resuscitation strategies,the incidence of post-traumatic sepsis has not changed in the past decade.Sepsis is the leading cause of death in patients with multiple injuries in the middle and late stages.Survivors may die of immune-related complications such as immune overreaction,severe infection or recurrent infection.There is conclusive evidence that persistent lymphopenia after trauma is associated with a higher incidence of multiple organ failure(MOF)and higher mortality.Natural killer(NK)cells are the core cells of the innate immune system,which are mainly distributed in peripheral blood,liver and spleen.In addition to their powerful killing function,they can also rapidly activate and secrete cytokines,regulate macrophage activation and Th1/Th2 balance,and act as a bridge between innate immunity and acquired immunity.It plays an important role in a variety of diseases such as autoimmunity,cancer,infection and ischemia/reperfusionrelated tissue injury.TIM-3,an important immune checkpoint protein,is increased in expression in various chronic infections and cancers,and is one of the important regulatory mechanisms for the induction of immune disorders.As an important part of the innate immune system,it is not clear whether TIM-3+NK cells are involved in the regulation of immune disorders in severe multiple trauma and post-traumatic sepsis.Therefore,to understand the epidemiological characteristics of multiple injuries with severe chest trauma and monitor the changes of immune system in post-traumatic patients in order to find intervenable cellular and molecular targets,so as to reduce the incidence of post-traumatic infection and improve the poor prognosis.However,there are few studies on the epidemiological characteristics of multiple trauma patients with chest trauma and the immune changes of TIM-3+NK cells after trauma.Therefore,we intend to explore the potential factors affecting the prognosis of multiple trauma patients with chest trauma and the role of TIM-3+NK cells in immune disorders in multiple trauma and post-traumatic sepsis.The present study consists of three parts.The first part is the analysis of clinical characteristics of multiple injuries with chest trauma.The second part is the expression and significance of NK cell Tim-3 in multiple trauma patients with chest trauma.The third part is the study of the mechanism of TIM-3+NK cells in immune disorder of posttraumatic sepsis.Part 1: Analysis of Clinical Characteristics of Multiple Injuries with Chest TraumaObjective: To explore the potential factors affecting the prognosis of multiple trauma patients with chest trauma.Methods: The clinical data of patients with multiple traumas admitted to our trauma center with a diagnosis of chest trauma between January 2015 and December 2019 were retrospectively reviewed.Outcomes were determined,including hospitalization time of ICU,30-day post-injury mortality and 6-month post-injury mortality.Results:A total of 1196 patients met the inclusion criteria.According to the type of chest injury,the patients were divided into four groups.There were 851 cases of pulmonary contusion(Pulmonary contusion,PC),215 cases of pulmonary contusion with flail chest(Pulmonary contusion and flail chest,PC+FC),46 cases of pulmonary contusion with upper thoracic spinal cord injury(Pulmonary contusion and upper thoracic spinal cord injury,PC+ UT-SCI),and 84 cases of thoracic trauma triad(Thoracic trauma triad,TTT)complicated with flail chest,pulmonary contusion and upper thoracic spinal cord injury.Polytrauma patients with triad of thoracic trauma had a longer ICU stay,relatively higher mortality at 30 days after injury,and especially higher mortality at 6 months after injury.The leading causes of death for patients with triad of thoracic trauma were acute respiratory distress syndrome(ARDS)and pulmonary infection during first 30 days after injury.For those patients who died later than 30 days during the 6 months,the predominant underlying cause of death was sepsis/multiple organ failure.Conclusion : Lethal triad of thoracic trauma were described in this study,which consisting of pulmonary contusion、flail chest and the upper thoracic spine cord injury.Like the classic “lethal triad”,there was a synergy between the factors when they coexist,resulting in especially high mortality rates.We should pay much more attention to the patients with this kind of injury for further minimizing complications and mortality.Part II: The Expression and Significance of NK Cell Tim-3 in Multiple Trauma Patients with Chest TraumObjective: To explore the effect of TIM-3 expression on NK cells on the prognosis of multiple trauma patients with thoracic trauma.Methods: Patients who were hospitalized in the trauma surgery of Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of science and technology from October 2019 to June 2021,diagnosed with severe chest injuries(AIS ≥ 3)and medical volunteers who passed the physical examination of trauma surgery in Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of science and technology were selected as the research objects.The relevant clinical data and laboratory finding were collected,and the peripheral venous blood of each subject was extracted for detection.Flow cytometry was used to detect the expression of TIM-3 on the surface of peripheral blood NK cells and its correlation with disease prognosis and other clinical indexes were analyzed.Results:A total of 41 patients and 18 healthy volunteers were included in the study.Compared with healthy volunteers,the expression of TIM-3 in NK cells in polytrauma patients with severe chest injuries was significantly higher than that in healthy controls.The percentage of TIM-3+NK cells is not only related to the severity of patients,but also valuable in evaluating the prognosis of patients.Conclusion:The number of NK cells changed significantly after severe trauma,and the expression of TIM-3 on NK cells increased.The correlation analysis between the expression of receptor and clinical data showed that the expression of TIM-3 on NK cells in multiple trauma patients with severe chest trauma was related to the poor prognosis of multiple trauma patients with severe chest trauma.The percentage of TIM-3+NK cells can be used as a marker to predict the prognosis of multiple trauma patients with severe chest trauma.Part III: Study on the Mechanism of TIM-3+NK Cells in Immune Disorder of Posttraumatic SepsisObjective:To further explore the role of TIM-3 expression on NK cells and the effect of interfering with TIM-3 signal pathway on the immune regulation of NK cells in post-traumatic sepsis,in order to provide some help for the in-depth research on the pathophysiological mechanism of NK cells involved in immune regulation of sepsis.Methods: Traumatic sepsis(TS)animal model was established in wild-type(WT)and TIM-3 mutation(TIM-3mut)mice with C57BL/6 background,and intervention conditions such as cervical subcutaneous injection of PBS or α-lactose were used.The expression of TIM-3 on the surface of NK cells,the apoptosis of NK cells and the expression of IFN-γ on NK cells were detected by flow cytometry.Real-time quantitative polymerase chain reaction(RT-PCR)was used to detect the expression of inflammatory factors IL-6,IL-12,TNF-α,IFN-γ,TGF-β and IL-10 in mice liver.The 28-day survival rates of TS-WT mice and TS-TIM-3mut mice were compared by Kaplan-Meier analysis.The liver tissues of Sham group,α-lactose treatment group and traumatic sepsis animal model group were sequenced by RNA-seq technique.Results:(1)8 hours after operation,the TIM-3 expression of NK cells in WT-TS mice was significantly higher than that in the control group,accompanied by an increase in the proportion of NK cells secreting INF-γ.Compared with TIM-3 negative NK cell subsets,TIM-3 positive NK cell subsets had a higher proportion of INF-γ secreting cells and apoptotic cells.In addition,the results also showed that the increased apoptosis of NK cells in the liver of traumatic sepsis mice was related to the increased expression of TIM-3.(2)The treatment of TS mice with α-Lactose could competitively block the apoptosis of TIM-3 positive NK cells induced by galectin-9,directly or indirectly reduce the expression of inflammatory cytokines in the liver of traumatic sepsis mice.Through RNA sequencing(RNA-seq)analysis,we found that 53.25%(2064/3876)genes which were abnormally expressed in the liver tissue of traumatic sepsis mice back to completely or partially normal level through α-Lactose treatment.(3)Blocking or damaging TIM-3 signaling can reduce the apoptosis of NK cells in liver of traumatic sepsis mice and improve the survival rate of traumatic sepsis mice.Conclusion: In general,these results suggest that TIM-3 signaling in NK cells plays a key role in the immune pathogenesis of traumatic sepsis.TIM-3 may be involved in the immune regulation of traumatic sepsis by regulating the apoptosis of NK cells.Using α-Lactose to interfere with TIM-3 signal may be a promising immunomodulatory strategy in the clinical practice of traumatic sepsis treatment in the future.