Role And Its Mechanism Of P2X₇ Receptor In The Enteral Stage Of Trichinella Spiralis Infection In Mice

Trichinellosis, caused by the parasitic nematode Trichinella spiralis, is a worldwide foodborne zoonosis. Transmission to humans involves the consumption of raw or undercooked meat and its derivatives from infected animals. Upon activity of digestive enzymes in the host’s stomach, larvae are released and migrate to the small intestine while they mature and then develop into adults(enteral phase). After mating, adults release new borne larvae(NBL) and NBL migrate into striated muscle where they encapsulate and form cysts(muscle phase). The intestinal mucosa is the first line of defense and important barrier in the body’s resistance to T. spiralis infection. The intestinal mucosal immune response determines clearance of parasites and interactions between T. spiralis and hosts. Therefore, the intestinal immune response of hosts plays a vital role in T. spiralis infection.P2X7 receptor(purinergic receptor, P2X7R) is one of the most studied receptors among the P2 receptor family. It has been identified that P2X7 R plays an important role in many immune mediated diseases. P2X7 R is expressed on immune cells especially macrophages. Adenosine triphosphate(ATP)is the natural ligand of P2X7 R. Activation of P2X7 R by ATP induces formation of pore channels of ionic membrane, resulting in activation of inflammasome, and leads to production and secretion of IL-1 beta(IL-1β) and IL-18. It has been demonstrated that many intracellular pathogens, such as Mycobacterium tuberculosis, chlamydia, Leishmania, and Toxoplasma gondii, can evade killing effects of infected hosts through interrupting the P2X7 R pathway, which indicates that P2X7 R acts as a trigger receptor of immune protection in infectious diseases.In the intestinal phase of T. spiralis infection, adults and larvae feed on intestinal villus and invade intestinal tissue, which results in damage of intestinal tissue. Abundant ATP is released from damaged cells and acts as an extracellular messenger to bind and activate P2X7 R. Activation of P2X7 R opens a cation-specific channel that alters the ionic environment inside cells, and as a result promoting inflammatory response. It’s reported that both P2X7 R expression and levels of IL-1β were increased in the jejunum of infected mice with T. spiralis. IL-1β is an important cytokine of intestinal innate immune response and one of the most important effector molecules to clear the pathogen in T. spiralis infection. However, the related immunological mechanism of how P2X7 R mediates immune repsonse in T. spiralis infection has not been further explored.There are lots of macrophages accumulated in intestinal mucosa lamina propria and abdominal cavity during the enteral phase in infected mice with T. spiralis, which indictates that macrophages serves as an important component in intestinal immune response. Macrophages are the main source cells of IL-1β in intestine. What is more, P2X7 R is highly expressed on macrophages. But it is not clear that whether P2X7 R mediates function of macrophages in T. spiralis infection.Based on these above findings, to explore the role and its related mechanism of P2X7 R in the enteral phase of T. spiralis infection, our study detected expression of P2X7 R in small intestine, spleen and mesenteric lymph node(MLN) at the enteral phase. Then we studied effects of blockade of P2X7 R on worm burden and macrophages function in infected mice. Finally effects of T. spiralis antigens on macrophage function and its related signaling pathways in vitro were investigated. This paper includes two following parts: PartⅠ Expression of P2X7 R and its Role in the enteral phase of T. spiralis infection in mice Objective: To observe P2X7 R expression level and its role in the enteral phase in infected mice with T. spiralis. Methods: Female BALB/c mice, aged 7 to 8 weeks old, were randomly divided into 3 groups(n=15): control group(0.2ml 0.9% saline administered by oral gavage), infected group(300 T. spiralis larvae per mouse in 0.2ml 0.9% saline administered by oral gavage); brilliant blue G(BBG) + infected group(infection plus intraperitoneal injections of BBG, each mouse was injected with BBG dissolved in distilled water, 50 mg/kg/day, began at 1 d before infection and lasting for 7 days). Behavior states, activities body weight of each mouse were recorded every day. On 7 d after infection, animals were sacrificed. Blood samples, intestinal tissues, spleen and MLN were collected, respectively. Abdominal fluid was aspirated with sterile PBS and peritoneal macrophages were collected. Western blot and immunohistochemical method were used to detect P2X7 R protein expression in small intestine tissue, spleen, MLN. Flow cytometry was used to detect P2X7R+, CD11b+ and CD11b+P2X7R+ cells in spleen and MLN. Levels of IL-1β and IL-18 in serum, peritoneal lavage fluids and supernatants of cultured cells from spleen and MLN were investigated by ELISA. Furthermore, to investigate the role of P2X7 R in the enteral immune response, effects of blockade P2X7 R with BBG on worm burden(number of adult worms at 7 d post-infection and muscle larvae per gram at 42 d post-infection) and on these above immunological paramenters, were demonstrated too. Results: Compared with the control group, the infection group had significantly increased levels of P2X7 R protein expression in small intestine tissue, spleen and MLN. The ratio of P2X7 R expression in cells from spleen and MLN was as high as 95% in the infection group. The infection group had increased number of CD11b+ cells and mean fluorescence intensity of P2X7 R in spleen and MLN. Number of CD11b+P2X7R+ cells in MLN increased nearly 10 times of that of the control. In addition, the infection group had increased CD68+ macrophages and higher P2X7 R expression in the intestinal lamina propria. Levels of IL-1β and IL-18 increased significantly in the infection group. Upon blocking P2X7 R, infected mice showed decreased activity, back arched, towering hair, and more serious pathological damage in the intestinal mucosa. Compared with the infection control, blockade of P2X7 R resulted in increased adults and larvae per gram muscle, accompanied with decreased CD68+ macrophages in small intestinal tissue and lower levels of IL-1β and IL-18. Conclusion: P2X7 R expression was up-regulated in the enteral phase of T. spiralis infection. Activation of P2X7 R could promote macrophages to produce and release cytokine IL-1β and IL-18, participating in worm clearance in the intestinal immune response. PartⅡ Mechanism of worm clearance of macrophages mediated by P2X7 R in the enteral stage of T. spiralis infection Objective: To explore the mechanism of worm clearance of macrophages mediated by P2X7 R Methods: Peritoneal macrophages from uninfected and infected mice were collected and cultured. The protein levels of P2X7 R, NLRP3, Pro-caspase-1, IL-1β and IL-18 were tested by Western blot. U937(a human monocyte cell line) and PMA-U937(differentiated into an adherent macrophage phenotype using PMA in vitro), were treated for 24 h with 100 ng/ml LPS or T. spiralis adult or larva antigens, respectively, and then stimulated with 1mM ATP for 30 min. In all experiments, cells incubated with cell culture media were used as control groups. The activation of inflammasome NLRP3 was detected by Western blot. Effect of blockade of P2X7R(10 μM A740003 was used at 2 h before ATP stimulation) on activation of NLRP3 was detected, too. Supernatant of all groups were collected and incubated with 20 female adult worms per well for 30 h in the CO2 incubator. The mRNA levels of P2X7 R, NLRP3, Pro-caspase-1 and Pro-IL-1β in the cells were detected by quantitative Real-time PCR. The activity of female worm and number of newborn larvae were recorded under invert microscope. To understand whether the worm clearance of macrophages mediated by ATP-P2X7 R signaling pathway were associated with LPS/TLR4 pathway, total RNA of small intestinal tissue in the enteral phase was extracted and its transcription level was tested by high-throughput sequencing and information analysis. And differentially expressed genes were screened and differences in gene expression pattern clustering analysis were investigated. Results: Compared with those from the control group, the peritoneal macrophages from the infection group had higher protein levels of P2X7 R, NLRP3, Pro-caspase-1 and IL-18. The activation of inflammasome NLRP3 in monocytes-macrophages was dependent on ATP stimulation and could be inhibited by blockade of P2X7 R. The mRNA levels of NLRP3 and Pro-IL-1β, expressed by PMA-U937, were increased after LPS stimulation. Compared with those from other groups, supernatant from cultured cells with LPS and ATP had more obvious killing effects on both adults and larvae, shown as lower activity of female adult and fewer newborn larvae. Results of differences in gene expression pattern clustering analysis showed that there were higher expression levels of TLR4, NF-κB, Pro-IL-1β, IL-6 and IL-10 in the small intestine during the enteral phase in infected mice. Conclusion: P2X7 R on monocyte-macrophages, dependent on ATP stimulation, could mediate activation of inflammasome NLPR3, and thus promoted these cells to produce and secret IL-1β and IL-18. As a result, macrophages activated by P2X7 R had killing effect on parasites after the double stimulation of ATP and LPS, which was associated with higher gene expression levels of TLR4/NF-κB signaling pathway in the small intestine during the enteral phase of T. sprialis infection.