Identification And Functional Study Of Novel Effector Proteins Controlled By Inflammasome Activity

As a critical component of the innate immune system,inflammasomes play indis-pensable roles in fighting against invading pathogens and maintaining homeostasis.Inflammasomes are cytosolic multi-protein complexes and mainly reside in the innate immune cells.These complexes comprise cytosolic pattern recognition receptors,adaptor protein ASC and cysteinyl aspartate specific proteinase caspase-1.Upon in-sult challenge,aggregated receptors,ASC and caspase-1 complex leads to self-cleavage and auto-activation of caspase-1,which further cleaves its substrate gasdermin D,IL-1βand IL-18.Cleaved GSDMD results in pyroptosis of the cells,and secretion of mature IL-1βand IL-18.In addition to IL-1βand IL-18,many cyto-solic proteins are secreted from cells upon inflammasome activation noncanonically.Moreover,emerging evidence suggests that many functions of inflammasomes cannot be explained by the well-known effectors IL-1βand IL-18.We still don’t know what the other effectors are,and what their functions are.To address these questions,my PhD project started from mass spectrometry identi-fication of macrophage secretome.A set of proteins were identified,among them ga-lectin-3 is particularly interesting because it was secreted abundantly after inflam-masome activation.It has many known extracellular functions,and plays important roles in many diseases.No specificity of inflammasome is required for galectin-3 se-cretion,activation of NLRP3,NLRC4 or AIM2 all induced the secretion of galectin-3.However,in contrast to other studies,we found galectin-3 was secreted through nei-ther the exosome pathway,nor several well-known unconventional protein secretion pathways.The gene Capns1,which was reported to mediate galectin-3 secretion from mouse embryonic fibroblasts,was not required for inflammasome-induced galectin-3secretion.In addition,signals that induce IL-1βexpression did not affect the tran-scription of galectin-3,and caspase-1 did not cleave galectin-3 protein for its secretion.Further study found that galectin-3 passed through the GSDMD-formed membrane pores for secretion without any requirement for active regulatory signals,including calcium signaling.In addition,the plasma membrane penetrating reagent digitonin rescued the secretion of galectin-3 from Gsdmd^-/-BMDM.In vivo the serum level of galectin-3 in mice was tightly controlled by the activity of inflammasome.Galectin-3is well-known for its pro-inflammatory activity,and it was reported to induce the production of TNF-αin a TLR4-dependent manner.However,we found two recom-binant galectin-3 proteins were different in inducing TNF-αproduction.Moreover,deletion of galectin-3 in the NLRP3 gain-of-function mutation mice(Nlrp3^+/A350V)didn’t relieve the disease severity and inflammatory responses.In the diet-induced obese mouse model,NLRP3 inflammasome was activated and subsequently led to dramatical increase of serum galectin-3,which eventually resulted in decrease of in-sulin sensitivity.In the Cre-ER^T2-Nlrp3^+/A350V mouse,tamoxifen specifically induced the activation of NLRP3 inflammasome,elevation of serum galectin-3 level,and in-sulin resistance.Therefore,these data strongly support the hypothesis that galectin-3is a key effector protein that contributes to inflammasome-dependent insulin re-sistance.Our findings not only identified a novel and functionally important effector downstream of inflammasome,but also provided a new perspective to find out more inflammasome effectors.In addition,our findings suggest inflammasome effectors have particular functions under different conditions,and the combination of these in-dividual functions equals to the functions of inflammasome.Moreover,both inflam-masome and galectin-3 are deeply involved in many diseases,the inflam-masome-galectin-3 axis will deepen our understanding of these diseases,and may provide a new strategy to treat these diseases.