IL-37 Gene Modification Enhances the Protective Effects of Mesenchymal Stromal Cells on Intestinal Ischemia Reperfusion Injury

Abstract

Background: Ischemia reperfusion injury (IRI) is the major cause of intestinal damage in clinic. Although either mesenchymal stromal cells (MSCs) or interleukin 37 (IL-37) shows some beneficial roles to ameliorate IRI, their effects are limited. In this study, the preventative effects of IL-37 gene-modified MSCs (IL-37-MSCs) on intestinal IRI are investigated.

Methods: Intestinal IRI model was established by occluding the superior mesenteric artery for 30 minutes and then reperfused for 72 hours in rats. Forty adult male Sprague-Dawley rats were randomly divided into the sham control, IL-37-MSC-treated, MSC-treated, recombinant IL-37- (rIL-37-) treated, and untreated groups. Intestinal damage was assessed by H&E staining. The levels of gut barrier function factors (diamine oxidase and D-Lactate) and inflammation cytokine IL-1β were assayed using ELISA. The synthesis of tissue damage-related NLRP3 inflammasome and downstream cascade reactions including cleaved caspase-1, IL-1β, and IL-18 was detected by western blot. The mRNA levels of proinflammatory mediators IL-6 and TNF-α, which are downstream of IL-1β and IL-18, were determined by qPCR. Data were analyzed by one-way analysis of variance (ANOVA) after the normality test and followed by post hoc analysis with the least significant difference (LSD) test.

Results: IL-37-MSCs were able to migrate to the damaged tissue and significantly inhibit intestinal IRI. As compared with MSCs or the rIL-37 monotherapy group, IL-37-MSC treatment both improved gut barrier function and decreased local and systemic inflammation cytokine IL-1β level in IRI rats. In addition, tissue damage-related NLRP3 and downstream targets (cleaved caspase-1, IL-1β, and IL-18) were significantly decreased in IRI rats treated with IL-37-MSCs. Furthermore, IL-1β- and IL-18-related proinflammatory mediator IL-6 and TNF-α mRNA expressions were all significantly decreased in IRI rats treated with IL-37-MSCs.

Conclusion: The results suggest that IL-37 gene modification significantly enhances the protective effects of MSCs against intestinal IRI. In addition, NLRP3-related signaling pathways could be associated with IL-37-MSC-mediated protection.

Figure 1

IL-37-MSCs and MSCs could migrate to the injured tissue. The morphology of MSCs. (a) Passages 0, 1, 2, and 3 of MSCs. (b) FACS analysis of MSC surface marker, surface expressions of CD29, CD45, CD79a, and CD90 were detected. (c) The IL-37 and GFP proteins were detected in IL-37-MSCs and MSCs. (d) IL-37-MSC GFP-positive rate was calculated; positive rate was above 99.8% which met our needs. (e) IL-37-MSC-treated and MSC-treated intestine exhibited significant GFP fluorescence while the sham group did not, which suggested that IL-37-MSCs and MSCs could migrate to the injured tissue.

Figure 2

IL-37-MSCs significantly ameliorated pathological intestine damage following IRI. Microscopic findings illustrated the architecture of the ileum by 72 hours after reperfusion; the damage score was assessed according to Chiu's score. (a) Compared with the sham group, the untreated group demonstrated severe damage such as inflammatory cell infiltration, hemorrhage, and ulcer. However, as shown in (b), the IL-37-MSC-treated IRI group showed more significant therapeutic effects compared with the MSC-treated and rIL-37-treated groups. The data suggested that IL-37-MSCs provide a better protective role in intestinal IRI. Data shown were representative, and the p value was determined by one-way ANOVA followed by the LSD test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 3

IL-37-MSCs could effectively improve intestinal barrier function following IRI. DAO and D-Lac were used to assess the gut barrier function. Serum samples were collected from the sham, IL-37-MSC-treated, MSC-treated, rIL-37-treated, and untreated IRI groups. In comparison with the MSC-treated and rIL-37-treated IRI groups, IL-37-MSCs significantly reduced serum levels of DAO and D-Lac, which meant IL-37-MSCs remarkedly improved gut barrier function. Data shown were representative, and the p value was determined by one-way ANOVA followed by the LSD test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 4

IL-37-MSCs could significantly decrease local and systemic inflammation activity. Local and systemic inflammation activity was assessed by IL-1β level in the local tissue and serum. Compared with MSC and rIL-37 treatment, IL-37-MSCs significantly alleviate the local and systemic inflammation activity. The p value was determined by one-way ANOVA followed by the LSD test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 5

IL-37-MSC treatment decreased NLRP3 and downstream cascade protein synthesis. (a) Compared with MSC and IL-37 treatment, IL-37-MSCs significantly decreased the NLRP3 activation. In parallel with NLRP3 synthesis, cleaved caspase-1, IL-1β, and IL-18 proteins were significantly decreased following IL-37-MSC treatment compared with MSC and/or rIL-37 treatments (b–d), which suggested that IL-37-MSCs could inhibit the NLRP3-mediated signaling pathway. Data shown were representative, and the p value was determined by one-way ANOVA followed by the LSD test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 6

IL-37-MSC transplantation decreased IL-6 and TNF-α mRNA expression. IL-1β and IL-18 are key proinflammatory cytokines, and as their downstream proinflammatory molecules, IL-6 and TNF-α play a pivotal role in inflammatory reactivity. Intestine IL-6 and TNF-α mRNA expressions in the IL-37-MSC-treated group were significantly decreased compared with those in the MSC- and rIL-37-treated group. The p value was determined by one-way ANOVA followed by the LSD test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.