The Role And Mechanism Of NAMPT-Mediated T-Cell Pyroptosis In The Development Of Immunosuppression In Traumatic Sepsis

Part 1 Clinical significance of serum NAMPT in patients with severemultiple trauma and traumatic sepsis Background: Sepsis is a common syndrome in patients with severe multiple trauma,and triggers a series of complicated inflammatory and immune responses.The initial “systemic out-of-control inflammatory response” during sepsis causes multiple organ failure(MOF),which subsequently transforms into a complex state of intertwined systemic inflammatory response syndrome(SIRS)and compensatory antiinflammatory response syndrome(CARS),and leads to immune disorder.It addresses a formidable challenge for clinicians.Nicotinamide phosphoribosyl transferase(NAMPT),plays an important role in cell megabolism,inflammation and immune modulation.In addition,studies have reported that serum NAMPT could be a potential circulating biomarker in sepsis and critically ill patients.Therefore,our research aimed to evaluate the changes of the serum NAMPT levels in patients with severe multiple trauma and traumatic sepsis,explore the relationship between the NAMPT levels and the severity of multiple trauma and traumatic infection,and analyze the clinical significance of the NAMPT levels in progression and prognosis of patients with severe multiple trauma and traumatic sepsis.Methods: Fifty patients with severe multiple trauma in trauma center of Tongji hospital(according to the diagnostic and exclusion criteria)and ten healthy volunteers(medical examination meets health standards)were enrolled in this study.According to the diagnostic criteria of sepsis 3.0,patients were divided into sepsis group(30 subjects)and non-sepsis group(20 subjects).Clinical data of all subjects were collected and analysed.Peripheral venous blood samples were aquired within 24 h after severe muitiple trauma or sepsis diagnosis.Serum were collected,and NAMPT levels were quantitatively measured by human NAMPT ELISA kits according to the manufacturers' instructions.All subjects were followed up for 60 days for prognosis analyze,and to confirm the relationship between plasma NAMPT levels and the patients with severe multiple trauma and traumatic sepsis.Results:(1)Levels of serum NAMPT in patients with severe multiple trauma [2.41(2.01-2.8)] were significantly increased compared with healthy controls [0.97(0.75-1.13)].Moreover,levels of serum NAMPT in traumatic sepsis group [2.98(2.23-3.78)] were further higher than the non-septic group(P<0.05).(2)Levels of serum NAMPT in patients with severe multiple trauma were positively correlated with acute physiology and chronic health score(APACHE II)(r=0.2815,P=0.0477).(3)Levels of serum NAMPT in patients with severe multiple trauma were positively correlated with indicators of infection,such as leukocytes(r=0.4043,P=0.0014),neutrophils(r=0.4703,P=0.0001),CRP(r=0.4392,P=0.0006),etc.(4)Levels of serum NAMPT in patients with severe multiple trauma were negatively correlated with albumin(r=-0.44,P=0.0004),and were positively correlated with INR(r=0.4601,P=0.0002)?BUN(r=0.3543,P=0.0055).(5)Counts of lymphocytes in patients with severe multiple trauma [0.77(0.49-1.09)] were significantly decreased compared with healthy controls [1.93(1.64-2.42)].Additionally,counts of lymphocytes in traumatic sepsis group [0.65(0.39-0.81)] were further lower than the non-septic group(P<0.05).Levels of serum NAMPT in patients with severe multiple trauma were negatively correlated with lymphocytes(r=-0.6704,P<0.0001).(6)Serum NAMPT was an independent risk factor of patients with traumatic sepsis(OR: 5.374,95% CI: 1.07-26.93,P=0.041),and high serum NAMPT levels could increase the death risk of patients with traumatic sepsis(OR: 20.66,P=0.01).Conclusions: Increased levels of serum NAMPT in patients with severe multiple trauma are associated with the severity of trauma,APACHE II,traumatic infection and prognosis of patients,suggesting that serum NAMPT may be a potential biomarker for diagnosis and prognosis of traumatic sepsis.In addition,the levels of serum NAMPT in patients with severe multiple trauma are negatively correlated with lymphocyte counts,suggesting that serum NAMPT may play an important role in the modulation of immunosuppression in traumatic sepsis.Part 2 The role and mechanism of NAMPT-mediated T-cell pyroptosis in immune disorder in a mouse model of sepsisBackground: Emerging evidence implicates nicotinamide phosphoribosyl transferase(NAMPT)and programmed cell death of immune cells in the pathogenesis of sepsisinduced immune disorders.Although lymphocyte apoptosis was found to play an important role in sepsis-induced immunosuppression,pyroptosis of T lymphocyte has attracted increasing attention in the context of sepsis.Our previous study indicated that levels of serum NAMPT and percentage of pyroptotic T lymphocytes were significantly increased in patients with traumatic sepsis,which were correlated with their prognosis.Herein,a specific non-competitive inhibitor of NAMPT,FK866 was used to further demonstrated the regulatory effects of NAMPT on T-cell pyroptosis and to clarify the role and mechanism of NAMPT in immune disorders in a mouse model of sepsis.Methods:(1)Male C57BL/6 mice(8-12 weeks,20-25 g)were used to establish a model of polymicrobial sepsis,which was induced by cecum ligation and puncture(CLP).Animals were divided into three groups randomly(15 mice in each group),namely the vehicle-treated Sham group,the vehicle-treatd CLP group,and the FK866-treated CLP group.All mice were constantly monitored for seven days after the CLP procedure for a survival study,and analyzed by Log-Rank test.(2)Bacterial culture of peripheral blood and peritoneal lavage fluid was used to evaluate the effect of FK866 on bacteria clearance.(3)Levels of NAMPT and GSDMD in spleen were assessed by Western Blot and immunohistochemistry.Serum and spleen NAMPT were measured by ELISA.(4)Levels of pyroptotic-associated protein in spleen were detected by Western Blot.The percentages of T-cell pyroptosis,including CD4+ T and CD8+ T cells,were measured by flow cytometry.(5)The effect of FK866 on counts of T lymphocytes was assessed.The effect of FK866 on function of CD4+ T lymphocytes was measured by delayed-type hypersensitivity test(DTH).The effect of FK866 on ultrastructure of T lymphocytes in spleen was detected by transmission electron microscopy(TEM).(6)Mice serum cytokines,namely IL-6,IL-10,IL-12p70,MCP-1,IFN-?,and TNF-?,were measured by FACS Calibur flow cytometer using a cytometric bead array(CBA)mouse inflammation kit according to the manufacturer's instructions.(7)The effect of FK866 on NAD+,NADH and NAD+/NADH in spleen was detected by WST-8.(8)Expression of Sirt-6 and NF-?B protein in spleen was measured by Western Blot.Results:(1)Bacterial load of peritoneal lavage fluid and peripheral blood was reduced by FK866 treatment compared with the CLP group(CLP group vs.FK866 group: D1,7.43±0.44 vs.5.75±0.45;8.53±0.35 vs.6.78±0.31;D3,8.13±0.37 vs.5.53±0.46;8.37±0.32 vs.6.23±0.20;D7,5.72±0.78 vs.3.78±0.31;6.42±0.24 vs.4.53±0.23;P < 0.05).And FK866 improved the survival rate of septic mice(CLP group vs.FK866 group: 44% vs.67%;P < 0.05).(2)NAMPT levels in spleen were significantly increased after sepsis onset,while treatment with FK866 decreased levels of NAMPT.(3)Compared with the CLP grouop,FK866 downregulated the levels of pyroptoticassociated protein in spleen.In addition,FK866 significantly attenuated T-cell pyroptosis during CLP-induced sepsis,including CD4+ T cells(CLP group vs.FK866 group: D1,14.53±4.01% vs.7.45±1.82%;D3,18.99±4.30% vs.9.23±2.99%;D7,25.62±6.16% vs.11.53±4.97%;P < 0.05)and CD8+ T cells(CLP group vs.FK866 group: D1,13.15±3.62%;D3: 17.47±4.37%;P < 0.05).(4)Compared with the CLP group,FK866 increased the counts of T cells,improved the T-cell function,and reversed the ultrastructural changes of T cells.(5)FK866 alleviated the levels of inflammatory cytokine in the plasma of septic mice,such as IL-6?IFN-??TNF-??IL-12p70?MCP-1?IL-10.(6)Compared with the CLP group,FK866 treatment depleted the increased NAD+ and reduced the NAD+/NADH ratio(P < 0.05).(7)Sirt-6 waslowered and p-NF-?B was increased in spleen after sepsis onset,while FK866 treatment reversed the changes.Conclusion: T-cell pyroptosis plays an important role in sepsis-induced immune disorder.NAMPT inhibitor FK866 could mitigate systermic inflammatory cytokine storm,alleviate immunosuppression by suppressing T-cell pyroptosis and protecting Tcell function,and improve the survival of septic mice.The protective effects of FK866 on sepsis-induced T-cell pyroptosis may regulated by NAD+-dependent Sirt-6 pathway and NF-?B pathway.This finding may provide a promising targeted therapy for sepsisinduced immune disorders.