Mechanism Of Antimicrobial Peptide Cathelicidin-2 Inducing NLRP3 Inflammasome Activation In Mouse Macrophages

Host defense peptides（antimicrobial peptides）are small peptides produced by organisms to control infection by directly killing pathogens or modulating host immune responses.Host defense peptides are mainly divided into two families,namely,the cathelicidin family and the defensin family.Cathelicidin-2（CATH-2）,a cathelicidin family antimicrobial peptide,has a broad spectrum of antibacterial and immunomodulatory activities.Innate immunity is the host’s first line of defense against pathogenic microbial infection,which relies on pattern recognition receptors on the cell surface and in the cytoplasm.NLRP3 protein is a pattern recognition receptor located in the cytoplasm,recognizes pathogens and stress factors,and regulates the secretion of the cytokine IL-1βby forming inflammasomes.Studies have shown that CATH-2 has the ability to regulate inflammatory response,and the activation of NLRP3 inflammasome is the key to inflammation.However,the mechanism by which CATH-2 regulates the activation of NLRP3 inflammasome is still unclear.In this study,lipopolysaccharide（LPS）-stimulated mouse peritoneal macrophages were used as a stimulation model.Firstly,the role of CATH-2 in regulating NLRP3 inflammasome was investigated,and then the mechanism of CATH-2-induced NLRP3 inflammasome activation in macrophages was elucidated by investigating potassium efflux,NEK7 and lysosomal leakage.1.The role of CATH-2 in NLRP3 inflammasome activationTo investigate whether CATH-2 acts on NLRP3 inflammasome,peritoneal macrophages from WT and NLRP3 inflammasome related component knockout mice(NLRP3^-/-,caspase-1^-/-,ASC^-/-)were stimulated with LPS,and then CATH-2 was added to the cells.ELISA,Western blot and q PCR were used to detect the expression of related proteins.The results showed that in the presence of CATH-2,lipopolysaccharide（LPS）induced macrophages produced IL-1βand IL-1α,indicating that CATH-2 acted on IL-1βsignaling pathway.In addition,CATH-2-induced IL-1βsecretion was significantly decreased in LPS-stimulated NLRP3^-/-,ASC^-/-,and caspase-1^-/-macrophages,indicating that CATH-2 induces IL-1βsecretion through the NLRP3 inflammasome pathway.In addition,the active forms of caspase-1 and IL-1βand the oligomerization of ASC could be detected after CATH-2 was added,indicating that CATH-2 could activate caspase-1 and induce ASC oligomerization,leading to IL-1βsecretion.Interestingly,there was no significant change in NLRP3 and IL-1βm RNA after the addition of CATH-2,indicating that CATH-2 did not affect the first signaling pathway.Taken together,these results suggest that CATH-2 acts as a second signal to activate the NLRP3 inflammasome,leading to ASC oligomerization and caspase-1 activation,thereby promoting IL-1βmaturation and secretion.2.The role of potassium efflux in CATH-2-induced NLRP3 inflammasome activationStudies have shown that NLRP3 inflammasome activation requires a second signal,including ATP,release of mitochondrial oxidized DNA,and potassium efflux.Among them,potassium efflux as an upstream event in the activation of NLRP3inflammasome has been widely studied.However,whether CATH-2 activates the NLRP3 inflammasome via potassium efflux is not known.To explore whether CATH-2could cause potassium ion efflux,KCl solution was used to inhibit potassium ion efflux,and ATP receptor（P2X7）inhibitor was used as a control.ELISA and Western blot were used to detect the expression of related proteins.The results showed that the cells in the LPS+CATH-2 group had higher mature secretion of IL-1βand caspase-1,and the cells in the LPS+CATH-2 group still had higher mature secretion of IL-1βand caspase-1 after inhibiting P2X7 receptor.This indicates that CATH-2 is independent of P2X7 to induce NLRP3 inflammasome activation.However,with the increase of potassium chloride concentration,the secretion of IL-1βand caspase-1 decreased in the LPS+CATH-2 group,indicating that CATH-2 induced the activation of NLRP3inflammasome through potassium efflux,thereby promoting IL-1βsecretion.3.The role of NEK7 in CATH-2-induced NLRP3 inflammasome activationNEK7 plays a crucial role in the activation of NLRP3 inflammasome by acting downstream of potassium efflux and can interact with NLRP3 protein to regulate the oligomerization of NLRP3 protein,which is essential for ASC spot formation and caspase-1 activation.However,whether CATH-2 activates the NLRP3 inflammasome by regulating NEK7 remains unclear.In this chapter,we used gene silencing technology to knock down NEK7 protein expression to explore whether CATH-2activates NLRP3 inflammasome through NEK7.ELISA and Western blot were used to detect the expression of related proteins.The results showed that after NEK7knockdown,the secretion of IL-1βand caspase-1 in the supernatant of LPS+CATH-2group was significantly decreased,indicating that CATH-2 activated NLRP3inflammasome through NEK7 pathway to promote IL-1βsecretion.4.The role of lysosomes in CATH-2-induced NLRP3 inflammasome activationThe phagocytosis of self-derived particles and foreign particles will lead to the rupture of lysosomes and the release of cathepsins into the cytoplasm,which is essential for the activation of NLRP3 inflammasome.However,whether CATH-2activates the NLRP3 inflammasome is dependent on the lysosomal pathway is not known.In this chapter,lysosomes were labeled and cathepsin inhibitor was used to investigate whether CATH-2 activates the NLRP3 inflammasome through lysosomes and cathepsin by fluorescence visualization of lysosomes.The results showed that the LPS+CATH-2 group had weaker green fluorescence and fewer green spots,indicating that CATH-2 could cause lysosomal leakage.In addition,inhibition of cathepsin B（CTSB）completely abolished the secretion of IL-1βand caspase-1,suggesting that CATH-2 activates NLRP3 inflammasome through the cathepsin B pathway.Similarly,a large number of ASC spots were detected in the LPS+CATH-2 group,while ASC spots were significantly reduced when the CTSB inhibitor was added,indicating that CATH-2 induced ASC spot formation through the CTSB pathway.In conclusion,our study shows that CATH-2 releases CTSB by disrupting lysosomes and subsequently promotes ASC spot formation and caspase-1 activation.In this study,we investigated the mechanism by which CATH-2 regulates NLRP3activation induced by LPS in primary mouse peritoneal macrophages.Our results suggest that CATH-2 promotes IL-1βmaturation and secretion through the NLRP3pathway.Our study reveals a novel immunomodulatory role for CATH-2 and provides a basis for the development of novel therapeutic immunomodulatory strategies from a host perspective to combat antibiotic-resistant pathogens.