Mast cells play no role in the pathogenesis of postoperative ileus induced by intestinal manipulation

Abstract

Introduction: Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3(Cre/+) , devoid of mast cells but with intact Kit signaling.

Design: The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. Kit(W-sh/W-sh) and Cpa3(Cre/+) mice, and by use of the mast cell stabilizer cromolyn.

Results: Kit(W-sh/W-sh) mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3(Cre/+) mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3(Cre/+) mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3(+/+) ). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI.

Conclusions: Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI.

Figure 1. Intestinal manipulation in Kit^W-sh/W-sh mice induces intestinal inflammation in the absence of mast cells.

WT and Kit^W-sh/W-sh mice were subjected to laparotomy alone (Lap) or to laparotomy plus intestinal manipulation (Lap + IM). (A) Mesenteric windows from WT and Kit^W-sh/W-sh mutant mice were collected 30 min after surgeries and stained with 0.1% toluidine blue. Scale bar 50 µm. (B) 30 minutes after surgery the levels of mouse mast cell protease-1 (mMCP-1) was determined by ELISA in the peritoneal lavage fluid of WT and Kit^W-sh/W-sh. (C) Geometric center (GC) values representing the dextran distribution through the GI tract 24 hours after surgery. (D) Infiltration of MPO-positive cells in the muscularis externa 24 hours after surgery. Data expressed as mean ± SEM. * P<0.01 (one-way ANOVA followed by Bonferroni post-hoc test). Dots represent individual mice.

Figure 2. Intestinal manipulation in Kit^W-sh/W-sh mice induces cytokines expression in the absence of mast cells.

WT and Kit^W-sh/W-sh mice were subjected to laparotomy alone (Lap) or to laparotomy plus IM (Lap + IM). Twenty four hours after surgery muscularis externa was collected and cytokines mRNA expression assessed by qPCR. Il6 (A), Il1a (B), Il1b (C), Tnfa (D), Cxcl1 (E) and Ccl2 (F) mRNA expression was evaluated in the jejunum muscularis externa after 24 h. Data expressed as mean ± SEM. * P<0.05, ** P<0.01 or *** P<0.001 (one-way ANOVA followed by Bonferroni post-hoc test). Dots represent individual mice.

Figure 3. Intestinal manipulation in Kit^W-sh/W-sh mice induces recruitment of immune cells in the muscularis externa in the absence of mast cells.

WT and Kit^W-sh/W-sh mice were subjected to laparotomy alone (Lap) or to laparotomy plus intestinal manipulation (Lap + IM) and immune cells recruitment in the muscle layer of the small intestine was assessed by flow cytometry. Typical dots plot showing different population of CD45-positive cells in WT and Kit^W-sh/W-sh mice after (A) laparotomy or (B) laparotomy plus intestinal manipulation. Absolute number of CD45 positive immune cells (C), monocytes (D) and neutrophils (E) were calculated from flow cytometry frequencies using viable cell counts. Data expressed as mean ± SEM. ** P<0.01 or *** P<0.001 (one-way ANOVA followed by Bonferroni post-hoc test). Dots represent individual mice.

Figure 4. Deficient ICC network and intestinal dysmotility in Kit^W-sh/W-sh mice.

ICC network in the intestinal wall and GI transit were assessed in WT and Kit^W-sh/W-sh mice. Jejunum sections from naïve WT mice (A) or Kit^W-sh/W-sh mice (B) were immunolabeled with anti-Kit (red) and anti-Ano1 (green) antibodies. Sections were counterstained with DAPI (blue) to identify nuclei. White arrows are pointing Kit-positive and Ano1-negative mast cells in the jejunum from a WT mouse. DMP, deep muscular plexus; MYP, myenteric plexus; CM, circular muscle layer and LM, longitudinal muscle layer. Scale bar 50 µm. Ninety min after oral gavage with dextran-FITC naïve (C) or animals subjected to laparotomy (D) WT and Kit^W-sh/W-sh were sacrificed and dextran-FITC distribution through the GI tract was determined as indicative of GI transit. Data expressed as mean. *** P<0.001 (two-way ANOVA).

Figure 5. Cpa3^Cre/+ mice lack mesenteric and mucosal mast cells but have normal ICC network and gut motility.

Naïve Cpa3^Cre/+ and littermates control Cpa3^+/+ mice were used to analyze intestinal mast cells, ICCs and GI transit. (A) Mesenteric windows from Cpa3^+/+ and Cpa3^Cre/+ mice were stained with 0.1% toluidine blue. Scale bar 50 µm. (B) Jejunum mucosa sections from naïve Cpa3^+/+ and Cpa3^Cre/+ mice were immunolabeled with Kit antibody (red) and counterstained with DAPI (blue). Scale bar 25 µm. White arrows are pointing to mast cells in Cpa3^+/+ mice. To reveal ICCs, jejunum sections from Cpa3^+/+ (C) and Cpa3^Cre/+ (D) mice were immunolabeled with Kit (red) and counterstained with DAPI (blue). DMP, deep muscular plexus; MYP, myenteric plexus; CM, circular muscle layer and LM, longitudinal muscle layer. Scale bar 50 µm. GI transit was evaluated in naïve (E) or animal subjected to laparotomy (F) WT, Cpa3^+/+ and Cpa3^Cre/+ mice by assessing dextran-FITC distribution through the GI tract during 90 min after oral gavage. Data are expressed as means. No significant differences were found between the groups of animals (two-way ANOVA).

Figure 6. Intestinal manipulation induces postoperative ileus and recruitment of MPO-positive cells in the muscularis externa independently of mast cells.

Cpa3^Cre/+ and littermates control Cpa3^+/+ mice were subjected to laparotomy alone (lap) or to laparotomy plus IM (lap + IM). GI transit was evaluated 24 h after surgery by assessing dextran-FITC distribution through the gastrointestinal tract 90 min after oral gavage. (A) Graph represents GC values. (B) Peritoneal levels of mMCP-1 were determined by ELISA in Cpa3^+/+ and Cpa3^Cre/+ mice. (C) Representative images of MPO-positive cells in the muscularis externa 24 h after surgery in Cpa3^Cre/+ and littermates control Cpa3^+/+ mice. (D) Histogram represents numbers of MPO-positive cells in the muscularis externa 24 h after surgery in Cpa3^Cre/+ and littermates control Cpa3^+/+. Data expressed as mean ± SEM. * P<0.01 (one-way ANOVA followed by Bonferroni post-hoc test). Dots represent individual mice.

Figure 7. Intestinal manipulation induces muscularis externa inflammation independently of mast cells.

Cpa3^Cre/+ and littermate control Cpa3^+/+ mice were subjected to laparotomy alone (Lap) or to laparotomy plus IM (Lap + IM). Twenty four hours after surgery muscularis externa was collected and cytokines mRNA expression assessed by qPCR. Il6 (A), Il1a (B), Il1b (C), Tnfa (D), Cxcl1 (E) and Ccl2 (F) mRNA expression was evaluated in the jejunum muscularis externa after 24 h. Data expressed as mean ± SEM. * P<0.01 (one-way ANOVA followed by Bonferroni post-hoc test). Dots represent individual mice.

Figure 8. Intestinal manipulation induces recruitment of immune cells in the muscularis externa in the absence of mast cells.

Cpa3^Cre/+ and littermate control Cpa3^+/+ mice were subjected to laparotomy alone (Lap) or to laparotomy plus intestinal manipulation (Lap + IM) and immune cells recruitment in the muscle layer of the small intestine was assessed by flow cytometry. Typical dots plot showing different population of CD45-positive cells in Cpa3^Cre/+ and littermate control Cpa3^+/+ mice after (A) laparotomy or (B) laparotomy plus intestinal manipulation. Absolute number of CD45 positive immune cells (C), monocytes (D) and neutrophils (E) were calculated from flow cytometry frequencies using viable cell counts. Data expressed as mean ± SEM. *** P<0.001 (one-way ANOVA followed by Bonferroni post-hoc test). Dots represent individual mice.