Targeting IL-17A Improves the Dysmotility of the Small Intestine and Alleviates the Injury of the Interstitial Cells of Cajal during Sepsis

Abstract

Intestinal dysmotility is a frequent complication during sepsis and plays an important role in the development of secondary infections and multiple organ failure. However, the central mechanisms underlying this process have not been well elucidated. Currently, effective therapies are still lacking for the treatment of sepsis-induced intestinal dysmotility. In this study, we found that the activation of IL-17 signaling within the muscularis propria might be associated with dysmotility of the small intestine during polymicrobial sepsis. Furthermore, we demonstrated that targeting IL-17A partially rescued the motility of the small intestine and alleviated interstitial cells of Cajal (ICC) injury during sepsis. The blockade of IL-17A suppressed the dominant sepsis-induced infiltration of M1-polarized macrophages into the muscularis. Additionally, impaired ICC survival may be associated with the oxidative stress injury induced by dominant infiltration of M1-polarized macrophages. Our findings reveal the important role of the IL-17 signaling pathway in the small intestine during sepsis and provide clues for developing a novel therapeutic strategy for treating gastrointestinal dysmotility during sepsis.

Figure 1

IL-17 signaling pathway in the small intestinal muscularis propria in normal mice and septic mice. Microarray-based genome-wide gene expression profiles were investigated 48 h after CLP. (a) The results showed the top ten enrichment score (-log10 (P value)) values of the upregulated and the downregulated genes and significant enrichment pathways. (b) The significantly upregulated genes related to the IL-17 signaling pathway (heat map) in septic mice. The higher the enrichment score is, the more significant the pathway, n = 3 in each group. (c) Real-time PCR analysis for the validation of selected gene expression patterns. (d) Comparison of the protein expression of IL-17A in the muscularis propria between normal mice and septic mice (data are shown as the mean ± SD, n = 3 to 4, ^∗P < 0.05).

Figure 2

Motility of the small intestine in normal mice, septic mice treated with PBS, and septic mice treated with IL-17A-Ab. (a) Analysis of intestinal myoelectrical activity. The amplitude and frequency were compared among the groups 24 h and 48 h after CLP. (b) Analysis of intraluminal pressure. The intraluminal pressure of the small intestine was compared among the groups 24 h and 48 h after CLP (data are shown as the mean ± SD, n = 5 to 6, ^∗P < 0.05).

Figure 3

Histological analyses of the small intestine and changes in ICCs in normal mice, septic mice treated with PBS, and septic mice treated with IL-17A-Ab. (a) Small intestine samples were obtained 48 h after CLP. The histologic scoring of hematoxylin and eosin (H&E) staining was assessed by intestinal injury grading. Scale bar: 0.2 mm. (b) Immunohistochemical staining for c-Kit was performed to analyze ICC networks. The immunofluorescence intensity of c-Kit was assessed. Scale bar: 50 μm. (c) RT-PCR analysis of the c-Kit and Ano-1 expression in the small intestinal muscularis propria. (d) Ultrastructural changes in ICC morphology were examined by TEM analysis (10,000x magnification). The damage to ICCs (white) and swelling of the nerve filament (black) are indicated by arrows (data are shown as the mean ± SD, n = 5 to 7, ^∗P < 0.05).

Figure 4

The inflammatory response within the small intestinal muscularis propria in septic mice treated with PBS and septic mice treated with IL-17A-Ab. Samples were obtained 48 h after CLP. (a) Representative pictures of the small intestine stained with CD68 for macrophages. Quantification of the macrophages infiltrating the muscularis propria was compared. Scale bar: 100 μm. (b) The quantification of MPO, a marker of neutrophil activation, is presented among each group. (c) The activation of NF-κB and MAPK signaling was examined by DNA-binding activity. (d) The expression of IL-6, IL-1β, and NO was measured by ELISA. NF-κB: nuclear factor kappa B; MAPK: mitogen-activated protein kinase; MPO: myeloperoxidase (data are shown as the mean ± SD, n = 5 to 7, ^∗P < 0.05).

Figure 5

ICC injury is associated with M1 macrophage-mediated oxidative stress. Samples were obtained 48 h after CLP. (a) Altered M1 and M2 macrophage infiltration in the muscularis propria. Representative images of M1 (CD163) staining and M2 (arginase 1) staining are shown. The ratio of M1/M2 macrophages was further analyzed in each group. Scale bar: 100 μm. (b) RT-PCR analysis of the expression of marker genes related to M1 and M2 macrophages in the muscularis propria. There was a significant reduction of M1 macrophage marker genes. (c) Double-immunostaining of c-Kit (green) and iNOS (red) in the muscularis propria was performed, and the c-Kit-positive and iNOS-positive areas were analyzed. Representative images of c-Kit and iNOS staining from septic mice are shown. Scale bar: 20 μm. (d) The pathological changes in ICCs after treatment with M1- or M2-derived medium for 24 h. ICCs were detected by c-Kit immunocytochemical staining (200x magnification), TUNEL (400x magnification), and TEM (10,000x magnification). The number of ICCs, the expression of c-Kit, and the percentage of apoptotic cells were also analyzed. (e) Expression levels of iNOS and NADPH in ICCs treated with M1- or M2-derived medium for 24 h. NT: no treatment; Arg1: arginase 1; NADPH: nicotinamide adenine dinucleotide phosphate (data are shown as the mean ± SD, n = 5 to 7, ^∗P < 0.05).