The Protective Effect And Mechanism Of Sestrin2 On The Pyroptosis Of Dendritic Cells In Sepsis

BACKGROUND AND OBJECTIVE:Sepsis is recognized as a global health priority yet a tough issue due to its high morbidity and mortality in the intensive care unit（ICU）worldwide.Sepsis is currently considered to be caused by dysregulated host response to infection,with both hyperinflammation and immunoparalysis occurring from the very early stage of sepsis,rather than a monophasic sustained hyperinflammation.Most patients with sepsis rapidly show severe immunosuppression,which is closely related to the poor prognosis.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.Pyroptosis,a lytic and proinflammatory programmed necrotic cell death mediated by caspase-1 or caspase-11/4/5,could instigate tissue damage and immune dysfunction,leading to the development of sepsis.Despite progresses in understanding of the alteration of DCs underlying sepsis,no specific treatment has been validated in clinical trials for restoring immune dysfunction in sepsis.In our previous study,over-expression of sestrin2（SESN2）,a stress-inducible protein,showed significantly protective effect on attenuating the ERS-related cell death induced by high mobility group box-1 protein（HMGB1）in DC2.4.The potential roles of SESN2 on immune regulating of DC in sepsis and the underling mechanisms warrant further research.In the present study,we focus on the pyroptosis of DC and try to explore the protective effects and mechanisms of SESN2 on DC dysfunction in sepsis.METHODS:The first part:In vivo study,DCs were isolated from spleens at 6 h,12 h,24 h,48 h,and 72 h post cecal ligation and puncture（CLP）surgery and sham operation.In vitro study,splenic DCs were primed with 1μg/ml LPS（6 h,24 h,and 72 h）and treated with 20μM Nigericin（Nig）treatment for 30 min before harvest.Then the pyroptotic rates of DCs were analyzed by flow cytometry.Transmission electron microscopy was used for observing morphological changes in cell membrane,ER,and mitochondria.Expressions of Cleaved-Casp-1,NLRP3,ASC,GSDMD and ERS related proteins（GRP78,ATF4,CHOP）were determined by Western blotting.Laser scanning confocal microscopy was applied to examine the activation of Casp-1/Tunel and NLRP3/ASC inflammasome.The concentrations of IL-1β,IL-18,HMGB1,TNF-α,IL-6,IL-25,and IL-10 were measured by ELISA.Furthermore,primary DCs of mice were treated with TM for 24 h in vitro and in vivo.Pyroptosis of DCs was assessed by flow cytometry.The expressions of Cleaved-Casp-1,GSDMD,NLRP3,ASC,GRP78,ATF4 and CHOP in DCs were determined by Western blotting.Meanwhile,the expressions of Cleaved-Casp-1,NLRP3 and ASC proteins were determined by Western blotting after pretreatment with Salubrinal.The second part:In vivo study,DCs were isolated from spleens at 6 h,12 h,24h,48 h,and 72 h post CLP surgery and sham operation.In vitro study,splenic DCs were primed with 1μg/ml LPS（6 h,24 h,and 72 h）followed by 20μM Nigericin（Nig）treatment for 30 min.Western blotting was used for examining the expression of SESN2.Laser scanning confocal microscopy was applied to examine the location of SESN2 in DC.Moreover,wile-type（WT）C57BL/6J and SESN2^-/-knockout mice were used for constructing septic model.Then pyroptosis of DCs was assessed by flow cytometry.Transmission electron microscopy was applied to observe the morphological changes of DCs.Expressions of Cleaved-Casp-1,NLRP3,ASC and GSDMD proteins were determined by Western blotting.Laser scanning confocal microscopy was used for examining the activation of NLRP3/ASC inflammasome.The concentrations of IL-1β,IL-18,HMGB1,TNF-α,IL-6,IL-25,and IL-10 were measured by ELISA.The third part:In vivo and vitro study,the co-location between ER and SESN2was examined by laser scanning confocal microscopy.After treated with TM for 24 h in vitro and in vivo,Western blotting was used for examining the expression of SESN2 in DCs.Meanwhile,the expression of SESN2 in DCs was determined by Western blotting after pretreatment with Salubrinal.Furthermore,pretreatment with or without Salubrinal,WT and SESN2^-/-knockout mice was used to examine the activation of ERS,the pyroptotic rates of DCs and the activation of NLRP3/ASC inflammasome based on the same methods as in Part2.RESULTS:1.The pyroptotic rates of DCs were increased,the expression of Cleaved-Casp-1,GSDMD-N,NLRP3,ASC were upregulated,and inflammatory cytokines（IL-1β,IL-18,HMGB1,TNF-α,IL-6,IL-25 and IL-10）showed significantly increased secretion at 24 h post CLP surgery or LPS treatment.Meanwhile,sepsis induced the activation of ERS response,evidenced by the increased expressions of GRP78,ATF4 and CHOP proteins.Moreover,tunicamycin（Tm）obviously increased the pyroptosis of DCs and the expressions of Cleaved-Casp-1,NLRP3,ASC and GSDMD proteins.After pretreated with Salubrinal for 30 min,then treated with 1 μg/ml LPS plus Nig for 24 h.These DCs showed remarkably decreased activation of NLRP3,cleaved-Casp-1,and ASC.2.As shown in vivo and vitro studies,sepsis increased the expression of SESN2.Treatment with Tm upregulated SESN2 expression in DCs,while it was inhibited by pretreated with Salubrinal.3.After the onset of septic challenge,DCs of SESN2^-/-mice showed obviously increased pyroptosis when compared with that of WT mice.Meanwhile,SESN2 knockout significantly aggravated the activation of Casp-1,GSDMD,NLRP3,and ASC.Likewise,SESN2 knockout obviously increased the secretion of IL-1β, IL-18,HMGB1,TNF-α,IL-6,IL-25and IL-10.Furthermore,the survival rates were significantly decreased after SESN2 knockout4.Sepsis obviously induced the activation of ERS response.The morphology of ER showed fragmented and lumped in SESN2^-/-DCs at 24 h after CLP.When compared with those in WT group,SESN2 deficiency significantly facilitated the transfer of ATF4 to the nucleus,and upregulated expressions of GRP78,ATF4, and CHOP during sepsis.5.Pretreatment with Salubrinal downregulating ERS by inhibiting PERK-ATF4-CHOP signaling pathway,and reversed the effect of SESN2 on NLRP3/ASC inflammasome activation.Meanwhile,pretreatment with Salubrinal showed significant protection against CLP-induced high mortality in both WT and SESN2^-/-mice.CONCLUSIONS:The results in our study confirmed that sepsis induced obvious pyroptosis in mice splenic DC and the pyroptosis was mediated by NLRP3/ASC/Casp-1 activation,which closely related to the excessive ERS response.The up-regulated expression of SESN2 could inhibit the over activation of NLRP3/ASC inflammasome by antagonizing PERK-ATF4-CHOP signaling pathway,further reducing the pyroptosis of DC,which was capable of protecting mice against immune-dysfunction in sepsis.