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. 2018 Nov 13;9:2599.
doi: 10.3389/fimmu.2018.02599. eCollection 2018.

Leukocyte-Derived Interleukin-10 Aggravates Postoperative Ileus

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Free PMC article

Leukocyte-Derived Interleukin-10 Aggravates Postoperative Ileus

Kathy Stein et al. Front Immunol. .
Free PMC article

Abstract

Objective: Postoperative ileus (POI) is an inflammation-mediated complication of abdominal surgery, characterized by intestinal dysmotility and leukocyte infiltration into the muscularis externa (ME). Previous studies indicated that interleukin (IL)-10 is crucial for the resolution of a variety of inflammation-driven diseases. Herein, we investigated how IL-10 affects the postoperative ME inflammation and found an unforeseen role of IL-10 in POI. Design: POI was induced by a standardized intestinal manipulation (IM) in C57BL/6 and multiple transgenic mouse strain including C-C motif chemokine receptor 2-/-, IL-10-/-, and LysMcre/IL-10fl/fl mice. Leukocyte infiltration, gene and protein expression of cytokines, chemokines, and macrophage differentiation markers as well as intestinal motility were analyzed. IL-10 serum levels in surgical patients were determined by ELISA. Results: IL-10 serum levels were increased in patient after abdominal surgery. In mice, a complete or leucocyte-restricted IL-10 deficiency ameliorated POI and reduced the postoperative ME neutrophil infiltration. Infiltrating monocytes were identified as main IL-10 producers and undergo IL-10-dependent M2 polarization. Interestingly, M2 polarization is not crucial to POI development as abrogation of monocyte infiltration did not prevent POI due to a compensation of the IL-10 loss by resident macrophages and neutrophils. Organ culture studies demonstrated that IL-10 deficiency impeded neutrophil migration toward the surgically traumatized ME. This mechanism is mediated by reduction of neutrophil attracting chemokines. Conclusion: Monocyte-derived macrophages are the major IL-10 source during POI. An IL-10 deficiency decreases the postoperative expression of neutrophil-recruiting chemokines, consequently reduces the neutrophil extravasation into the postsurgical bowel wall, and finally protects mice from POI.

Keywords: interleukin-10; intestinal motility; macrophages; neutrophils; postoperative ileus.

Figures

Figure 1
Figure 1
The role of IL-10 in POI. WT and IL-10−/− mice underwent IM or left untreated (CTL). (A) Gene expression of IL-10 was analyzed in WT mice after indicated time points and compared to naïve controls (CTL). n = 5 mice per group. Statistical analysis was done by one-way ANOVA followed by Bonferroni's post-hoc test (***p < 0.001). (B) IL-10 release into the media of 24 h organ cultured ME harvested from intestinal manipulated (IM24h) WT or IL-10−/− mice. Groups were compared by one-way ANOVA followed by Bonferroni's post-hoc test (**p < 0.01 and ***p < 0.001 vs. control or indicated groups; n.d. not detected). Bar graphs demonstrate means ± SEM, n = 4 for both groups. (C) IL-10 human serum concentrations were measured in surgical patients who underwent abdominal (AS, n = 17) and extraabdominal (EAS, n = 7) surgery before and 24 h after surgery. Data sets are shown as means ± SEM and were compared via a paired t-test. (D) GI-transit was plotted as mean ± SEM calculated by the geometric center of distribution of a fluid meal. st, stomach, c, cecum. Bar graphs show means ± SEM and are representative for five independent experiments. (E) MPO+ cells were quantified in naïve and 24, 72, and 168 h after IM in WT and IL-10−/− mice. Cells were microscopically counted in five randomly chosen areas in ME whole mount specimens. Statistical analysis was performed by one-way ANOVA followed by Bonferroni's post-hoc test (***p < 0.001 vs. CTL or indicated groups).
Figure 2
Figure 2
Leukocyte restricted IL-10 deficiency limits inflammation and neutrophil extravasation in POI. WT and LysMcre/IL10fl/fl mice underwent IM. (A) GI-transit was plotted as mean ± SEM calculated by the geometric centers of distribution of a fluorescent marker. st, stomach, c, cecum. Bar graphs show means ± SEM and are representative for five independent experiments. (B) Quantification of MPO+ cells within the same animals used in (A). Statistical analysis was performed with a one-way ANOVA followed by Bonferroni's post-hoc test (***p < 0.001 vs. CTL or indicated groups). (C) Gene expression analysis of IL-10 was performed 24 h after IM and compared to naïve controls (CTL). Data are shown as mean fold change ± SEM in mRNA levels vs. CTL. n = 5 for all groups. ***p < 0.001 vs. strain-specific CTL or between indicated samples. Statistical analysis was done by one-way ANOVA followed by Bonferroni's post-hoc test. (D) Flow cytometry analysis of ME specimen was performed to identify Ly6GLy6C+ monocytes and Ly6G+Ly6C+ neutrophils 24 h after IM in WT and LysMcre/IL10fl/fl mice. Data sets are shown as mean fold change ± SEM, n = 5 for all groups. Statistical significance between mouse strains was determined by unpaired t-test (**p < 0.01 vs. indicated samples). (E–G) LysMcre/IL10fl/fl mice were treated with rmIL-10 or vehicle after IM. GI-transit (D), MPO+ cells (E), and flow cytometry analysis (F) of ME specimen was performed as described above. Data sets (n = 4) were compared by unpaired t-test (*p < 0.05, **p < 0.01 vs. vehicle treated group).
Figure 3
Figure 3
IL-10 is mainly produced by monocytes and macrophages during POI. (A) ITIB+/− mice underwent IM or left untreated (CTL). After 24 and 72 h, IL-10 reporter activity was detected by a CCF4 administration resulting in blue fluorescence. Gating on living IL-10+ cells (i) was followed by CD45+ as well as identification of F4/80+/Ly6C+/− (ii) and Ly6C+Ly6G+/− (iii) cell populations during flow cytometry analysis. Data are representative plots from three independent experiments. (B) Ratios of IL-10+CD45+ cells within the ME of CTL and intestinally manipulated WT mice. Data are shown as means ± SEM, n = 3 for all groups. (C) Resident F4/80+Ly6C macrophages, Ly6C+Ly6G monocytes, and Ly6C+Ly6G+ neutrophils were sorted by flow cytometry from CTL and IM24 h animals, respectively. Indicated cell populations underwent gene expression analysis of IL10. n = 3 for all groups. Statistical analysis was done by one-way ANOVA followed by Bonferroni's post-hoc test. **p < 0.01 vs. CTL.
Figure 4
Figure 4
IL-10 is mainly produced by monocytes and macrophages during POI. (A) Arginase 1 (Arg1) and Ym1 mRNA levels were analyzed in WT mice after indicated time points. n = 5 mice per group. (B) Arginase activity was determined by urea formation within the same ME specimens used in (A). (C) Confocal microscopy of F4/80 and Arg1 expressing cells 24 h after IM in the ME of WT mice. Asterisks, resident F4/80+/Arg1 muscularis macrophages; arrows, infiltrated F4/80+Arg1+ monocytes. Note the presence of numerous Arg1+ monocyte-shaped cells not yet expressing F4/80. Shown are representative images from three independent experiments. Scale = 50 μm. (D) Three different cell populations (1–3) were sorted by flow cytometry from CTL and IM24h animals. Populations were defined as F4/80+Ly6C resident macrophages (1, CTL green; 2, IM24h red) and F4/80+Ly6C+ monocytes (3, IM24h blue). Indicated cell populations underwent gene expression analysis of Arg1 and Ym1. n = 3 for all groups. Statistical analysis was done by one-way ANOVA followed by Bonferroni's post-hoc test. *p < 0.05 and **p < 0.01 vs. group 1. (E) Gene expression of Arg1 and Ym1 was analyzed 24 h postoperatively in WT and CCR2−/− mice. Data are shown as changes in mRNA levels (means ± SEM) compared to non-manipulated strain specific controls. Groups (n = 5) were compared via unpaired t-test (***p < 0.001).
Figure 5
Figure 5
Neutrophil transmigration is impaired by IL-10 deficiency. (A) Resident F4/80+Ly6C macrophages, Ly6C+Ly6G monocytes and Ly6C+Ly6G+ neutrophils were sorted by flow cytometry from CTL and IM24h animals, respectively. Indicated cell populations underwent gene expression analysis of IL-10Rα. n = 3 for all groups. Statistical analysis was done by one-way ANOVA followed by Bonferroni's post-hoc test. ***p < 0.001 vs. CTL or indicated populations. (B) Neutrophil transmigration was determined toward WT ME or IL-10−/− ME conditioned media (SN, supernatant). Data are shown as mean fold change ± SEM. n = 4 for both groups. Statistical significance was determined by unpaired t-test (**p < 0.01 vs. WT SN). (C) Analysis of neutrophil transmigration through a microporous membrane. Bone marrow derived WT neutrophils were cultured in triplicates in a transwell culture system and where were allowed to transmigrate toward IL-10 supplemented media (10, 100 ng/mL) or medium (Veh). No significance was determined between vehicle and IL-10 supplemented groups. (D) WT and LysMcre/IL10fl/fl mice underwent IM and qPCR analysis of neutrophil chemokines CXCL1/CXCL2 as well as monocyte chemokine CCL2 was performed 24 h after IM and compared to naïve controls (CTL). Data are shown as mean fold change ± SEM in mRNA levels vs. CTL. n = 5 for all groups. *p < 0.05; **p < 0.01; ***p < 0.001 vs. strain-specific CTL or between indicated samples. Statistical analysis was done by one-way ANOVA followed by Bonferroni's post-hoc test.

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