Effects And Its Mechanism Of AIM2 In Salmonella Infection

AIM2(Absent in melanoma 2), a member of the HIN200 protein family, is originally identified as a tumor suppressor. Recently, it is found as a DNA sensor. AIM2 gene has a microsatellite instability site that results in its inactivation in many tumors. Since its function as a cytosolic DNA sensor was disclosed, researches on AIM2 have predominantly focused its role in sensing of both intracellular bacterial and viral pathogens and triggering anti-microbial innate immunity when assembled as inflammasome with ASC and caspase-1. Evidences have showed that Salmonella can not active the AIM2 inflammasome in bone marrow-derived macrophage. The biological implications of AIM2 during Salmonella infection have not been characterized in vivo. To find the role of AIM2 played in Salmonella infection and make a better understand of the innate immunity to Salmonella infection, our study mainly focuses on the function of AIM2 in Salmonella infection, and tries to uncover the underlying mechanism. The study will provide theoretical guidance for Salmonella disease prevention.To explore the possible involvement of AIM2 in the host response to Salmonella infection, we initially examined the mortality rate and bacterial dissemination of wild-type(WT) and AIM2-deficient(AIM2-/-) mice following oral or systemic administration of Salmonella strain SL1344. Compared with WT mice, AIM2-/- mice were more susceptible to Salmonella mucosal infection and displayed significantly higher mortality and increased bacterial loads in spleen and liver indicating a function of AIM2 in protecting against Salmonella infeciton. Following Salmonella enter into the GI tract, it breaks the intestinal barrier and subsequently disseminates to the systemic site. The susceptibility might due to a defect either in intestinal barrier function or in bacteria clearance of AIM2-/- mice. However, both WT and AIM2-/- mice have similar mortality and bacterial loads in spleen and liver during Salmonella systemic infection. According to these data, the function of AIM2 was limited in GI tract, and whether AIM2 deficiency affects Salmonella induced colitis was determined.Following Salmonella mucosal infection, we found that AIM2-/- mice suffered from more body weight loss from day 2 on, severe splenomegaly and cecal weight loss and damage and a significant higher bacterial number in spleen, liver and MLN. Also there was an exacerbated intestinal inflammatory media secretion in AIM2-/-mice. At a later time point, AIM2-/- mice had a higher histological score of the cecum on days 3, 4 and 5 post infection compared with WT mice. These results indicated that AIM2 can restrict Salmonella induced colitis and facilate the maintenance of intestinal homeostasis.Intestinal epithelial cell turnover is important to the barrier integrity and intestinal homeostasis. Therefore, we subsequently examined whether there was a difference in cell proliferation and apoptosis. We first examined cell proliferation by immunofluoresence staining for PCNA. Our results revealed that the epithelial crypts presented with more PCNA-positive cells in naive AIM2-/- mice compared with WT mice; however, the level of epithelial cell proliferation decreased following oral administration of Salmonella in AIM2-/- mice. Subsequently, TUNEL staining of histological sections of cecum showed that AIM2-/- mice had greatly increased numbers of apoptotic cells in the epithelium and lamina propria(LP) on day 2 post infection, whereas WT mice showed only a mild increase in apoptotic cells in the epithelium. Apoptosis was further confirmed by immunoblotting for cleaved caspase-3 in cecal lysates, whereas AIM2-/- mice had a higher level of cleaved caspase-3 production. These results indicate AIM2-/- mice have a defect in maintaining enterocyte proliferative/apoptotic homeostasis.We can not rule out the possibility that the abnormal intestinal epithelial cell turnover was caused by exorbitant intestinal inflammation, however the abnormal intestinal epithelial cell proliferation of naive AIM2-/- mice may contribute to abnormal bacterial dissemination, consequently exacerbating damage and inflammation in the gastrointestinal tract. To explore this possibility, bacteria disseminate to cecal lamina propria and lymph nodes were examined at 3 h post infection. There were significantly more bacteria detected in both sites of AIM2-/-mice compared with WT mice indicating a defect in intestinal barrier in the absence of AIM2. In vitro, an AIM2-overexpressing Caco-2 cell monolayer was used to analyze the translocation of Salmonella. The bacterial translocation was significantly decreased in AIM2-overexpressing Caco-2 cells. These results indicated AIM2 can promote barrier function and restrict bacterial dissemination via paracellular pathway.Tight junction is an important structure in paracellular pathway which seals the gap between adjacent cells. To further evident there is a defect in intestinal barrier in the absence of AIM2, we set out to characterize the effects of AIM2 on tight junction by means of immunofluoresence, western blot and RT-PCR. Compared with WT mice, the basal levels of claudin3 and occludin in AIM2-/- mice were lower whereas there was no difference in the basal level of ZO-1. To further confirm our conduct, we performed in vitro study. We found that AIM2-overexpressing Caco-2 cells had a higher level of claudin3 expression and an increased TER compared with vector-transfected cells. Following Salmonella infection, there is a decrease in claudin3 expression; however, the level of claudin3 in AIM2-overexpressing Caco-2 cells was higher than vector transfected Caco-2 cells. These results showed that AIM2 can promote tight junction protein expression and intestinal barrier function to limit bacterial dissemination.Subsequently, we further analyzed how did AIM2 regulated tight junction protein expression. In vitro, AIM2 overexpressing cells had a higher level of Akt phosphorylation compared with vector-transfected cells. Following Salmonella challenge up to 60 min, inhibition of Akt phosphorylation accompanied by decreased claudin3 expression was observed in AIM2-overexpressing cells. Salmonella challenge promoted Akt phosphorylation and claudin3 expression at 120 min, the promotion of claudin3 expression can be restored by Akt inhibitor. Similarly, Akt inhibitor inhibit the increase of TER and the limitation of bacteria translocation which mediated by AIM2 over expression. However, this effect could not be inhibited with a caspase-1 inhibitor. These results indicated AIM2 can promote tight junction protein expression via Akt signaling.Collectively, these results indicated AIM2 absence results in intestinal barrier disruption and increased intestinal permeability which contribute to bacterial dissemination, ultimately, leading to AIM2-/- mice were more susceptible to Salmonella mucosal infection.