Inflammasome-derived Exosomes Activate NF-?B Signaling In Macrophages

Inflammasome is a kind of protein complexes,which mainly participates in caspase's activation and processes pro-IL-1? and pro-IL-18 into the mature form.The inflammasome is the sensor of the innate immune system and plays an important role in the development of a few human diseases.At present,inflammasomes are mainly divided into two types,one is the canonical inflammasomes,NLR family;the other is a non-canonical inflammasomes,such as AIM2 inflammasomes and pyrin inflammasomes.With the in-depth research in recent years,the study on the mechanisms of activation of inflammasome are more and more clear and complete.When the cell is stimulated,the inflammasomes are assembled.Canonical inflammasomes serve as a scaffold to recruit the inactive pro-caspase-1.Oligomerization of pro-caspase-1 proteins induces their own proteolytic cleavage into active caspase-1.Active caspase-1 cleaves cytokines pro-IL-1? and pro-IL-18 into active cytokines IL-1? and IL-18.Active caspase-1 also induces an inflammatory form of cell death known as pyroptosis.The NLRP3 inflammasome is one of the most studied inflammasomes at present.However,whether exosome can be affected by this stimulation and if inflammasome-derived exosome has specific function remain to be investigated.Therefore,it is of great significance to identify the components of exosome and study its effect on the function of the cells under stimulation of NLRP3 inflammasome signaling.Exosomes are vesicles of 30-150 nm in diameter with biological activity.Exosomes are secreted by almost all types of cells.Exosomes transport various mRNAs,miRNAs,proteins and other active molecules into the recipient cells to participate in a variety of physiological processes including tumorigenesis,metabolism,immune response,etc.It has been reported that exosomes and inflammation are closely related.Exosomes derived from dendritic cells regulate endotoxin-induced inflammatory responses.The secretion of IL-1? is related to the release of exosomes.However,it remains unclear whether the inflammasome activator affects the secretion and the function of exosomes.This study mainly investigated the relationship of exosomes and NLRP3 inflammasome,including the change of proteins of different exosomes and the effects of different exosomes on bone-marrow-derived macrophages.We found that the released exosomes have specific shape and proteome when NLRP3 inflammasome pathway is activated.Firstly,different inflammatory signals have a large effect on the size of exosome secreted by cells.The exosomes derived from mock or LPS treatment were homogeneous rounded vesicles with diameters of30-150 nm which were observed under a transmission electron microscope.But the exosomes from LPS and nigericin treatment were mixed with small-sized vesicles.Secondly,different inflammatory signals have great influence on the number of proteins secreted by the cells.Compared with the control group and the LPS/Nig group,the number of exosomes in the LPS group significantly decreased.And compared with the other two kinds of exosomes,513 specific proteins were found in the exosomes of LPS/Nig group.These proteins are likely to affect the activation of inflammation and play an important role in the activation of inflammasome.Finally,different inflammatory activating signals have a profound effect on the type of proteins secreted by the cells.The proteins found in the control group are mainly involved in the redox and metabolic processes,but inflammasome-derived exosomes participate in the inflammatory response and innate immune system processes.These results show thatthe different inflammatory activating signals have great influences on the morphology,quantity and composition of exosomes.At the same time?,we found that NLRP3 inflammasome-derived exosomes directly activate the inflammatory response.Firstly,the bone-marrow-derived macrophages were treated with fluorescence-labeled exosomes and observed under a fluorescence confocal microscope.Macrophages differ in their sensitivity to different exosomes passing through the cellular membrane.Inflammasome-derived exosomes more easily penetrated the cellular membrane into the cell,whereas the exosomes from the control group and those stimulated by the first signal were mostly attached to the cellular membrane.Secondly,the protein expression was detected by Western blot.The NF-?B signaling pathway markers,NLRP3 and pro-IL-1?,were detected in the cell lysates which were treated with the inflammasome-derived exosomes.After treating with nigericin,pro-caspase-1 was detected in all three exosomes,but the processed fragment of p20 was detected only in cell lysate with the treatment of the inflammasome-derived exosomes.Thirdly,the cytotoxicity was measured by LDH to evaluate the cell pyroptosis.The LDH level in the cells with treatment of inflammasome-derived exosomes was higher than the other two kinds of exosomes.Lastly,we showed that the three kinds of exosomes did not affect the level of IL-1? by ELISA assay.However,the level of IL-1? was elevated after treating with nigericin in the exosomal group that treated with LPS and nigericin.The exosomes from LPS and nigericin treatment augmented the expression level of TNF-? and IL-6.In general,NLRP3 inflammasme-derived exosomes activated NF-?B signaling pathway and induced cell pyroptosis slightly,whereas the control and LPS-derived exosomes failed.All these results show that inflammasme-derived exosomes activate the inflammatory response in neighbor cells.Altogether,this study found that the inflammasome-derived exosomes have specific morphology,protein number and proteome.This exosomes also specifically activate the inflammatory response of the cells.The exosomes that stimulated by the inflammasome signaling are more easily engulfed by macrophages and directly activate the NF-?B signaling pathway to induce pyroptosis.