Trichinella Spiralis Inhibits The NLRP3 Inflammasome By Inducing Autophagy To Regulate Immune Expulsion In The Intestinal Phase

Trichinellosis is a dangerous food-borne parasitic zoonosis spread through consuming raw or undercooked flesh that has been contaminated with Trichinella spiralis(T.spiralis,Ts)larvae cysts.The pathological process of trichinellosis is generally divided into an intestinal phase and a muscle phase.During the intestinal phase,type 2 immune response mediated parasite expulsion plays an important role in host defense against T.spiralis.The small intestine is the site of maturation and reproduction of T.spiralis,which can continue their parasitic life only if they evade the expulsion of hosts.Therefore,it is vital to conduct research to explore how T.spiralis escapes immune-mediated worm expulsion in the intestinal stage.Studies have shown that Nod-like receptor thermoprotein domain-related protein 3(NLRP3)plays an important role in promoting a Type 2 immune response and participates in the immune escape targeted at T.spiralis.Autophagy is a process of maintaining cell homeostasis that can alleviate the onset of inflammation by inhibiting NLRP3 inflammasome activation.The purpose of this research was to investigate the effect of T.spiralis on autophagy and the NLRP3 inflammasome in the intestinal phase and to determine whether the immune evasion effect of T.spiralis is related to the interaction between autophagy and the NLRP3 inflammasome.The main research content and results are as follows:1.The role of T.spiralis in autophagy and NLRP3 inflammasome expression during the intestinal phaseHealthy BALB/c mice were given 300 Trichinella muscle larvae.After 3,and 7 days post-infection(dpi)with T.spiralis,mice were sacrificed,and serum as well as tissue samples,including small intestine,spleen,and mesenteric lymph nodes,were collected from each mouse.The transcript levels of Beclin-1,p62,and LC3 B,as well as NLRP3,ASC,Caspase-1,and IL-1β in the small intestine,spleen,and mesenteric lymph nodes,were determined by qPCR.The results showed that the expression of each factor was significantly increased after T.spiralis infection.The expression levels of p62 and NLRP3 were also detected by immunohistochemistry(IHC)in the small intestine,and the results of IHC were consistent with the results of qPCR.ELISA tests on the expression of IL-1β in the small intestine and serum of mice showed that the expression level of IL-1β in the small intestine reached a peak at 7 dpi,and IL-1β expression peaked at 3 dpi in the serum.The above results suggested that T.spiralis can induce autophagy and activate the NLRP3 inflammasome during the intestinal stage.2.Effects of Ts-AES on the interaction of autophagy with the NLRP3 inflammasome in vitroThe excretory-secretory products of T.spiralis adult worms(Ts-AES)were prepared and collected.Ts-AES was co-cultured with mouse intestinal epithelial cells(MODE-K)to further explore the effect of T.spiralis on autophagy and NLRP3 inflammatory bodies in the intestinal phase.First,in order to determine whether Ts-AES could induce autophagy and NLRP3 inflammation as a signal and the optimal stimulation concentration,MODE-K cells were treated with different concentrations(0,5,10,and 20 ng/μL)for 12 h,and the expression of autophagy and the NLRP3 inflammasome was analyzed by qPCR,MDC staining,Western blot,and ELISA.The qPCR results showed that different concentrations of Ts-AES increased gene expression of Beclin-1,p62,and LC3 B as well as NLRP3,ASC,Caspase-1,and IL-1β in MODE-K cells,and the effects were significantly improved when the concentration of TsAES was 10 ng/μL.The results of experiments such as MDC staining,Western blot,and ELISA were consistent with the results of qPCR,that is,different concentrations of Ts-AES could enhance the expression of autophagy and the NLRP3 inflammasome,and were most notable when the Ts-AES concentration was 10 ng/μL.The above results indicated that TsAES was able to activate autophagy and induce the NLRP3 inflammasome and that the optimal stimulation concentration of Ts-AES was 10 ng/μL.After determining the concentration of Ts-AES,autophagy was intervened with the autophagy inhibitor 3-MA and the autophagy activator Rapamycin to explore the role of autophagy on the Ts-AES-induced NLRP3 inflammasome.MODE-K cells were grouped and processed as follows: the control group,the Ts-AES group,the Ts-AES + 3-MA group,and the Ts-AES + Rapamycin group.The transcript levels of NLRP3,ASC,and Caspase-1 were detected by qPCR,and the results showed that,compared with the Ts-AES group,the expressions of NLRP3,ASC,and Caspase-1 were significantly increased after 3-MA treatment,and the levels of NLRP3 and ASC were significantly decreased after the treatment of Rapamycin.The expression of autophagy and the NLRP3 inflammasome was detected by MDC staining,Western blot,indirect immunofluorescence,and ELISA.Experimental results showed that Ts-AES increased the levels of autophagy and the NLRP3 inflammasome,and the secretion of IL-1β in the culture supernatant was also significantly raised.After autophagy suppression,the expression of the NLRP3 inflammasomes and IL-1β was enhanced,and after autophagy activation,the expression of the NLRP3 inflammasomes and IL-1β decreased with the increase in the autophagy level.The expression of Th2 cytokines IL-4 in cell supernatant was detected by ELISA,and it was found that the trend of IL-4 changes was consistent with IL-1β.Above we illustrated that Ts-AES was able to inhibit the expression of the NLRP3 inflammasome by autophagy and that the expression of IL-4 was positively associated with the activation of the NLRP3 inflammasome.3.The effect of the interaction between autophagy and NLRP3 inflammasome on host immune expulsion after T.spiralis infectionHealthy mice were randomly divided into four groups: the control group,the Ts group,the Ts + 3-MA group,and the Ts + Rapamycin group.One day before infection with T.spiralis,3-MA and Rapamycin were injected intraperitoneally once every two days,for a total of 7 days.At 3 and 7 days after infection,five mice were selected from each group to collect the serum,small intestine,and mesenteric lymph nodes of each mouse.Analysis of Beclin-1,p62,NLRP3,ASC,and IL-1β transcript expression in the small intestine and mesenteric lymph nodes by qPCR.The results showed that 3-MA treatment significantly reduced the level of Ts-induced autophagy while enhancing the expression of NLRP3 inflammasome;rapamycin enhanced the autophagy level induced by T.spiralis infection,but the expression of NLRP3 inflammasome has not seen any noticeable changes.The secretion of IL-1β in the small intestine and serum was analyzed by ELISA,and the results showed a significant improvement in the expression of IL-1β after 3-MA intervention compared with the Ts group at 3 and 7 days after the infection;rapamycin intervention did not have a noticeable effect on IL-1β expression in the small intestine and serum at 3 dpi,while the expression of IL-1β was significantly increased in the small intestine and serum at 7 dpi.Immunofluorescence was used to test the expression of p62,NLRP3,and IL-1β in the small intestine,and the results were consistent with the results of qPCR and ELISA.The above results showed that autophagy suppression further increased the levels of T.spiralis-induced NLRP3 inflammasome and promoted the expression and release of the inflammation cell factor IL-1β,indicating that T.spiralis can inhibit excessive inflammation caused by NLRP3 inflammasome activation through autophagy.In order to investigate the influence of the inhibition of autophagy on the NLRP3 inflammasome in the intestinal phase of T.spiralis infection on Th2 immune expulsion,ELISA was used to detect IL-4 levels in the small intestine and serum,HE staining was used to evaluate the pathological damage of the small intestine,tongue muscle,and masseter muscle,and the number of adults and muscle larvae was counted.The results of IL-4 secretion assays using ELISA were consistent with the expression results of IL-1β.HE staining showed that,compared with the Ts group,the pathological changes of the small intestine in the Ts + 3-MA group were significantly aggravated,and the number of muscle larvae in the tongue muscle and masseter muscle was significantly reduced;at 3 dpi,the intestinal injury in the Ts + Rapamycin group significantly improved,but at 7 dpi,the degree of the pathological damage to the small intestine significantly worsened compared to other groups.The statistical results of the number of adults and muscle larvae showed a significant decrease in Ts + 3-MA group adult worms and muscle larvae compared to the Ts group;the number of adult worms in the Ts + Rapamycin group was significantly higher than that in the Ts group,but the number of muscle larvae was significantly lower than that in the Ts group and higher than that in the Ts + 3-MA group.All of the above illustrated that T.spiralis could induce autophagy to inhibit NLRP3 inflammasome activation,thus suppressing the host Th2 response in the intestinal phase and guaranteeing their long and stable parasitic lives.In summary,our study demonstrated that the intestinal phase of T.spiralis can induce autophagy to inhibit NLRP3 inflammasome activation.The Th2 response-mediated expulsion of T.spiralis was dependent on NLRP3 inflammasome activation during the intestinal phase.Therefore,we concluded that T.spiralis-mediated autophagy induction escaped the Th2-related expulsion reaction in the intestinal phase by inhibiting NLRP3 inflammasome activation.This experiment provides a new idea to study T.spiralis escaping the intestinal expulsion effect to establish a long-term infection.