Sestrin2 Protects Dendrite Cells Against Ferroptosis Induced By Sepsis

Sepsis is a life-threatening organ dysfunction caused by the response to infection.Due to the high morbidity and mortality,sepsis is recognized as one of the urgent problems to be solved worldwide in the field of intensive care unit.Sepsis is currently considered to be caused by dysregulated host response to infection,with complex pathogenesis and pathogenesis.Recent studies suggest that immune dysfunction and immuno-suppression play a central role in the development of sepsis.Septic patients suffer from severe immunosuppression accompanied by alternative cycle of pro-inflammatory and anti-inflammatory state,which is closely related to poor prognosis.Excessive inflammatory response and oxidative stress lead to immune dysfunction and even immune paralysis,which is considered as another major factor in secondary infection of sepsis patients.It is suggested that the dysfunction of immune cell is the major cause of immunosuppression in sepsis.And the reduction and dysfunction of dendritic cells（DCs）,one of the most important antigen presenting cells,are critical characteristics in the immune disorder of sepsis,which play a vital role in regulating T lymphocyte proliferation and differentiation.Ferroptosis as a newly discovered non-apoptotic form of cell death,is characterized by excessive lipid pero-xidation and overload iron,which is triggered by the inactivation of glutathione peroxidase 4（GPX4）and a surplus of reactive oxygen species（ROS）induced in an imbalanced redox reaction.It is accompanied by the release of danger-associated molecular patterns（DAMPs）,which promotes cells to a pro-inflammatory state and plays an important role in the progression of sepsis.And it is recognized as the leading factor to decrease DCs and drive DCs to dysfunction.Sestrin2（Sesn2）is an evolutionarily highly conserved stress-induced protein family,which is significantly up-regulated when cells are onset of stress.In our previous study,over-expression of Sesn2 showed significantly protective effect on attenuating the endoplasmic reticulum stress（ERS）-related cell death induced by lipopoly-saccharied（LPS）in DCs derived from mouse bone marrow.In the present study,we focus on the ferroptosis of DC and try to explore the protective effects and mechanisms of Sesn2 on DC dysfunction in sepsis.The potential roles of Sesn2 on immune regulating of DC in sepsis and the underling mechanisms warrant further research to provide a new strategy for the prevention and treatment of immune dysfunction in sepsis.Part One The regularity of ferroptosis and immune function of dendr-itic cells stimulated by LPS.Objective:To observe the regularity of ferroptosis and function of dendritic cells stimulated by LPS.Methods:In vitro study,DCs were isolated from spleens of the wide-type（WT）male C57BL/6J mice.DCs were cultured in vitro and stimulated by LPS with different dose at times.Fe、GSH、ROS colorimetric kits were used to detect the contents in DCs.Western blot were used to analyze the expression level of ferroptotic proteins（x CT、GPX4、ACSL4、TFRC）.Flow cytometry was applied to detect the immune function and activity of DCs on the setting of sepsis.DCs pretreated with ferroptosis inducer Erastin（Era）and ferroptosis inhibitor Lip-1,were stimulated by LPS（1μg/ml）at 24h.The phenotypic maturations were detected by flow cytometry,and the proliferation and differentiation of T cells were analyzed.Results:The GSH level was significantly reduced while contents of Fe²⁺and ROS were increased in DCs stimulated by LPS at a dose of 1μg/ml at 24h.Stimulation with LPS decreased x CT and GPX4 expression,and inversely up-regulated ACSL4 and TFRC expression compared with the levels in the control group.The immune response of DCs was gradually enhanced with stimulation of time and dose,peaking at 12h and a concentration of 1μg/ml LPS.When DCs were pretreated with Era or Lip-1 before LPS stimulation,the immune function of DCs were decreased in the WT-Era group and were partly recovered in the WT-Lip-1 group.Moreover,T-cell proliferative activity were significantly increased in the WT-Lip-1 group.Conclusions:Ferroptosis occurs obviously on DCs stimulated by LPS（1μg/ml,24h）,and LPS（1μg/ml,12h）stimulated the peak of immune function of DCs.Ferroptosis can inhibit the activation of DCs immune function and involves in the LPS-induced suppression of DCs immune function.Part Two The protective effect of Sestrin2 on ferroptosis in dendritic cells stimulated by LPS.Objective:To observe the protective effect of Sestrin2 on ferroptosis in dendritic cells stimulated by LPS.Methods:We treated DCs extracted from splenocytes of mice with different genetic designs including WT mice and Sesn2 gene defect mice(Sesn2^-/-),with 1μg/ml LPS for 24h to investigate the correlations among Sesn2,ferroptosis,and the immune response of DCs.Fe、GSH、ROS colorimetric kits were used to detect the contents.Western blot was used to analyze the expression level of ferroptotic proteins.Moreover,confocal laser scanning microscopy was applied to reveal the impact of LPS-induced ferroptosis on oxidative and antioxidative responses of DCs.To further study the effect of ferroptosis induced by sepsis on DC function,we pretreated DCs form WT and Sesn2^-/-mouse with Lip-1（1μM）before LPS stimulation.The immune functions of DCs were detected by flow cytometry.Flow cytometry was used to analyze the proliferation and differentiation of T cells.Results:Cellular GSH contents in DCs were significantly reduced and Fe²⁺and ROS levels were increased in Sesn2^-/--LPS group.The expression levels of the ferroptosis-associated proteins x CT and GPX4 were decreased;however,ACSL4 and TFRC were upregulated in LPS-stimulated DCs of the Sesn2^-/--LPS group.DCs from Sesn2^-/-mice exposed to LPS exhibited more severe oxidation.DCs stimulated in the WT-LPS and Sesn2^-/--LPS groups exhibited cytoplasmic dilation instead of perinuclear dilated in addition to increased mitochondrial membrane and destroyed mitochondrial cristae.The immune functions of DCs were decreased in Sesn2^-/-mice.DCs co-cultured with T cells tended to exhibit increases in CD4+T-cell proliferation.Conclusions:Sestrin2 plays a protective role in LPS stimulated ferroptosis of DCs,which improves the immune function by providing protection against ferroptosis.Part Three The protective effect of Sestrin2 on ferroptosis in dendritic cells of septic miceObjective:To observe protective effect of Sestrin2 on ferroptosis in dendritic cells of septic mice.Methods:We adopted CLP to construct a sepsis model with WT and Sesn2^-/-mouse.DCs were isolated from spleens at 24h post CLP surgery.Fe、GSH、ROS colorimetric kits were used to detect the contents.Western blot was used to analyze the expression level of ferroptotic proteins.Moreover,confocal laser scanning microscopy was applied to reveal the impact of LPS-induced ferroptosis on oxidative and antioxidative responses of DCs.TEM was applied to observe alterations in the cytomembrane,cytoplasm,and mitochondria.Spleen tissues of WT and Sesn2^-/-mice were isolated for tissue staining to observe the iron deposition in spleen tissues by Prussian blue staining.Mice were treated by intraperitoneal injection of Lip-1（10 mg/kg）1h before CLP operation.Flow cytometry was used to analyze the proliferation and differentiation of T cells.Then the 7d survival of two types mice were also analyzed.Results:In comparison to the sham group,decreased GSH and overload of Fe²⁺as well as ROS in the CLP of the WT and Sesn2^-/-mouse groups were obvious.After the CLP,procedure at 24h,x CT and GPX4 levels were decreased,ACSL4 and TFRC expression was significantly enhanced in comparison to the levels in the sham groups.Sesn2^-/--CLP group revealed that more oxidative production resulted in brighter green than that observed in the WT group.Histomorphological alterations of ferroptosis were observed in spleens following septic challenge,which presented as brilliant blue staining and reunion distribution.Mice were intraperitoneally injected with ferroptotic inhibitor Lip-1 24h after CLP surgery.The proliferation of DCs on T cells in the Sesn2^-/--CLP+Lip-1 group was obviously lower than that in the WT-CLP+Lip-1 group.The survival rate of Sesn2^-/-mice with CLP surgery of septic model was significantly lower than that of WT mice.Conclusions:Sestrin2 plays a protective role in ferroptosis of DCs in septic mice,which improves the immune function by providing protection against ferroptosis.Part Four The signaling mechanism of sepsis-induced ferroptosis in dendritic cells regulated by Sestrin2.Objective:To observe signaling mechanism of sepsis-induced ferroptosis in DCs regulated by Sestrin2.Methods:DCs were pretreated with Salubrinal（Sal）（20Μm/L）,an inhibitor of an upstream signaling pathway in endoplasmic reticulum stress（ERS）response for 1h,which could effectively block the action of ATF4-CHOP.Western blot were used to analyze the expression level of ferroptotic proteins and the signaling pathway-related proteins.The immune functions of DCs were detected by flow cytometry.Flow cytometry was used to analyze the proliferation and differentiation of T cells.Results:The expression of signaling related proteins including ATF4,CHOP,and CHAC1 was obviously up-regulated upon LPS stimulation（1μg/ml,24h）.When DCs were pretreated with Sal,an inhibitor of upstream pathway of ERS,the ferroptosis-related proteins x CT and GPX4 were accordingly increased whereas ACSL4 and TFRC were decreased.DCs pretreated with Sal exhibited an increased capacity to stimulate the differentiation of CD4+T cells,and the proliferation of DCs with T cells in the Sesn2^-/--LPS+Sal group was markedly lower.Conclusions:ERS-related pathway ATF4-CHOP-CHAC1 promoted ferrotptosis in DC stimulated by LPS.Sestrin2 protects LPS-stimulated DCs by down-regulation of ATF4-CHOP-CHAC1 signaling pathway.Part Five Study on ferroptosis-associated index of septic patients.Objective:To investigate the ferroptosis-associated index of septic patients.Methods:This study was administrated in severe patients who were admitted to the emergency intensive care unit（EICU）of The Second Hospital of Hebei Medical University between January 2020 and June 2021.The patients were divided into non-sepsis group（n=32）and sepsis group（n=41）.Demographic data,clinical and laboratory parameters of enrolled patients were collected and recorded on admission.The records of age,sex,and body mass index（BMI）were included in patients’demographic characteristics.Complications and vital signs such as body temperature,systolic blood pressure（SBP）,and heart rate（HR）should be collected.Of note,each participant should be evaluated the severity degree by means of Sequential Organ Failure Assessment（SOFA）scores and Acute Physiology and Chronic Health Evaluation II（APACHE II）scores.Blood of patients admitted to hospital were collected and supernatant were taken after centrifugation.Fe、GSH、ROS colorimetric kits were used to detect the contents of Fe²⁺、GSH、ROS in the supernatant.All statistical processes were performed by using SPSS software.Results:The scores of APACHE II and SOFA in patients with sepsis were significantly higher than those in patients without sepsis.The GSH of blood supernatant in patients with sepsis was lower than that in patients without sepsis,and the content of Fe and ROS was significantly higher than that in patients with sepsis.Conclusions:Septic patients presented oxidative stress response in the early course,and there was the possibility and risk of ferroptosis.