The Study On The Function Of NLRP3 Inflammasome And Caspase-11 Inflammasome In Bone Associated Mesenchymal Stromal Cells

In spite of the protective effect of inflammation on tissues,excessive and persistent inflammation is associated with septic shock or autoimmune pathologies,which underlie a large group of inflammatory diseases,such as sepsis,allergic reactions,myopathies,colitis,and even cancer.In addition to the ability to differentiate into tissues and low immunogenicity,mesenchymal stromal cells(MSCs)have also been shown to possess broad immunoregulatory abilities.Therefore,MSCs have engaged great interest for clinical application in treating various infla mmatory diseases.Some studies have already demonstrated the beneficial effects of MSCs in reducing various inflammatory disorders,including sepsis,inflammatory bowel disease and the experimental allergy encephalitis.However,the therapeutic effects are not always achieved.Consequently,further studies on the molecular mechanisms by which MSCs respond to the inflammatory microenvironment may have dramatic impact on the clinical application of these unique cells.Most studies have reported that MSCs participate in inflammation mainly by direct cell-to-cell contact with immune cells or paracrine soluble cytokines.Some investigations reported that Toll-like receptors(TLRs)on MSCs are involved in their contribution to sense inflammation.MSCs are able to transduct inflammatory signals into immune cells to fight against inflammatory diseases.However,the intracellular pathway within MSCs responding to inflammatory stimuli is far from completely clarified.Inflammasomes are a set of intracellular protein co mplexes,which drive host and immune responses by releasing cytokines and inducing pyroptosis.Recently,NLRP3,NLRC4,NLRP1,AIM2,NLRP2,NLRP6,NLRP7,NLRP12 and IFI16 have been reported.NLRP3 is an essential inflammasome in immune responses and can be activated by diverse stimuli.NLRP3 inflammasome contains NLRP3,apoptotic speck protein(ASC)and pro-Caspase-1.The NLRP3 inflammasome cleaves pro-Caspase-1 into p20 and p10 subunits,inducing maturation and release of pro-inflammatory cytokines,such as IL-1β and IL-18.The NLRP3 inflammasome also induces pyroptotic cell death,which is characterized by the formation of membrane pores.Pyroptosis is not only induced by the activation of Caspase-1 but can also be led by the activation of murine Caspase-11 and human Caspase-4/5 key players of non-canonical inflammasomes.Non-canonical inflammasomes also release IL-1β with the help of the NLRP3 inflammasome and are crucial to innate immune defenses.The primary effectors of inflammasome-mediated inflammation are classically believed to be immune cells,such as monocytes,macrophages,dendritic cells,and neutrophils.Recently,Wang et al.reported that the expressions of NLRP3 and Caspase-1 were increased in human MSCs derived from the human umbilical cord in vitro under LPS treatment.Moreover,it was reported that IL-1β was expressed in MSCs upon shear stress,and our previous study found that the transcription level of IL-1a in bone-associated MSCs was very high.These results support the idea that MSCs may participate in inflammation through inflammasomes.MSCs can be expanded from various tissues including bone marrow,adipose tissue,umbilical cord blood,skin,tendon,muscle,dental pulp and endosteum.In the present study,we employed murine bone associated MSCs(BA-MSCs)investigate the NLRP3 and Caspase-11 inflammasomes activation by detecting pyroptosis,IL-1β/18 and key proteins level.The inflammasomes in BA-MSCs were further probed by NLRP3,Caspase-11 and Caspase-1/11 gene knockout mice respectively.Bacterial infected mice and inflammation bowel disease model were also established to confirm the observation.In the end we tried to use a novel synthesized small molecule to inhibit inflammasome activation for the sake of better utilization of MSCs.The main results were as follows: 1.Nigericin and LPS activated the NLRP3 inflammasome in BA-MSCs ex vivoUnder Nigericin and LPS stimulation,we observed that many membrane pores on BA-MSCs were observed by scanning electron microscopy and the percentage of GSDMDC1 foci significantly increased in BA-MSCs by immunofluorescence,suggesting that inflammatory stimulation induces pyroptosis of BA-MSCs in vitro.To explore the activation of the NLRP3 inflammasome,we employed qRT-PCR and western blot and found that the NLRP3,pro-IL-1β,and Caspase-1 gene transcripts and expression were up-regulated after stimulation.ELISA showed mature IL-1β/18 was released from BA-MSCs after stimulation.These results suggested that the NLRP3 inflammasome was activated in BA-MSCs in this system.To further confirm the function of the NLRP3 inflammsome in BA-MSCs,we employed NLRP3 gene knockout mice to analyze IL-1β/18 release and pyroptosis.According to the data of scanning electron microscopy,immunofluorescence,western blot and ELISA,we found NLRP3 gene knockout blocked IL-1β/18 release from BA-MSCs but unaltered pyroptosis.2.Caspase-11 inflammasome was activated in BA-MSCs ex vivoNon-canonical inflammasomes can also trigger pyroptosis,which is dependent on Caspase-11 rather than caspase-1.We observed the Caspase-11 gene transcript level and the active Caspase-11 protein in BA-MSCs were significant increased by LPS and Nigericin,indicating that the Caspase-11 inflammasome could be activated in BA-MSCs.We further affirmed the existence and detected the function of the Caspase-11 inflammasome in BA-MSCs,using Caspase-11 gene knockout mice.The observation of scanning electron microscopy showed the membrane pore numbers were significantly decreased in Caspase-11 gene knockout BA-MSCs,compared with those in wild type BA-MSCs,Moreover,we observed consistent results that the GSDMDC1 foci in Caspase-11 gene deficient BA-MSCs were significantly fewer than those in normal BA-MSCs.Taken together,these observations indicated that the Caspase-11 inflammasome in BA-MSCs played a key role in pyroptosis.To further clarify the function of these two inflammasomes in BA-MSCs,we employed Caspase-1/11 knockout mice to assess the secretion of cytokines and pyroptosis.We found Caspase-1/11 genes knockout in BA-MSCs inhibited the secretion of IL-1β/18 and pyroptosis,and further affirmed the excitence of the Caspase-11 inflammasome.3.NLRP3 and Caspase-11 inflammasomes of BA-MSCs were activated in vivoIn view of the activation of NLRP3 and Caspase-11 inflammasomes in BA-MSCs in vitro,we further assessed whether they were activated in vivo.We challenged mice with Pseudomonas aeruginosa and Escherichia coli to induce the infected model.With the help of western blot and immunofluorescence,we found the infected mice had increased expression of NLRP3,pro-IL-1β,Caspase-1/11 and GSDMDC1.These results affirmed the canonical NLRP3 inflammsome and Caspase-11 inflammsome of BA-MSCs could be activated by bacterial infection-related inflammation in vivo.To determine whether the activation of inflammasomes in BA-MSCs is universal,we investigated inflammasomes in other inflammatory settings.We used dextran sodium sulfate to establish a murine inflammation bowel disease model.We found the canonical NLRP3 inflammsome was activated to participate in the inflammatory response in BA-MSCs from inflammatory bowel disease mice,whereas the Caspase-11 inflammasome might not be involved.4.A small-molecular compound inhibited the activation of inflammasomes in BA-MSCsGiven that the activation of canonical NLRP3 and Caspase-11 inflammasomes in BA-MSCs was observed in inflammation,an inhibitor of inflammasomes in BA-MSCs is highly desired not only for further validating our observations but also for therapeutic purposes.Western blot analysis showed that NLRP3 expression and activated Caspase-1 were significantly decreased after treatment with 66 PR.The cleavage of Caspase-11 protein was also obviously decreased by 66 PR.The release of IL-1β from BA-MSCs was also inhibited by 66 PR.Consistently,66 PR inhibited pyroptosis of BA-MSCs.Therefore,we found that 66 PR inhibited the activation of NLRP3 and Caspase-11 inflammasomes in BA-MSCs in vitro.To further validate the 66 PR inhibition on inflammasome in vivo,we injected 66 PR into IBD mice.As expected,66 PR blocked Caspase-1 protein cleavage in the BA-MSCs,and also inhibited BA-MSCs pyroptosis in vivo.Considering its potential clinical application in inflammation control,we transplantated BA-MSCs into IBD mice,and found 66 PR can improve BA-MSCs survival by restraining pyroptosis.【Conclusion】Under in vitro stimulation and animal inflammation model,we found the canonical NLRP3 and Caspase-11 inflammasomes were activated in BA-MSCs.The NLRP3 inflammasome in BA-MSCs dominated IL-1β and IL-18 release,whereas the Caspase-11 inflammasome managed pyroptosis.The current study uncovered an inflammasome-dependent pathway of MSCs directly participating in inflammation in response to inflammatory stimuli.We found one novel small-molecule compound 66 PR could inhibit the activation of inflammasomes in BA-MSCs in vitro and in vivo.Pretreatment of BA-MSCs with our small-molecule inhibitor might improve their potential therapeutic effect on inflammatory disorders.These findings indicated that BA-MSCs themselves could directly promote the inflammatory response without the help of immune cells.The collected data extended our understanding of the subtle functions of BA-MSCs other than hematopoiesis or tissue repair,which is important for BA-MSCs to be used in clinical settings.Our study was helpful for understanding the molecular mechanisms of MSCs respond to the inflammatory microenvironment,which may have dramatic impact on the clinical application of these unique cells.The present investigation supplemented the knowledge on inflammasome activation in non-immune cells.The findings remind us that the maintenance of a proper inflammation balance should not only target immune cells but also non-immune cells.Moreover,this inhibitory effect of 66 PR on inflammasomes may also be applied to other cells containing inflammasomes,to control inflammasome-associated diseases.