Dendritic cell-like cells in the injured muscle tissue take up ovalbumin after intramuscular application <i>in vivo</i> and migrate into the popliteal lymph node.

<p>Seven days after injury or sham treatment, unlabeled ovalbumin (OVA; Sigma), OVA-fluorescein isothiocyanate (FITC), or OVA-Alexa Fluor 647 was injected into the gastrocnemius muscles. After 24 or 48 h, total popliteal lymph node cells from individual mice were stained for CD11c and CD11b. The FL-2 channel remained free. <b>(A)</b> Representative dot plots of popliteal lymph node cells showing the gating strategy of OVA-FITC⁺ cells among total lymph node cells 48 h after OVA application. <b>(B)</b> CD11c and CD11b expression of gated OVA-FITC⁺ cells. <b>(C)</b> Absolute number of OVA-FITC⁺ dendritic cells (DCs) in the lymph nodes per mouse 24 h (n = 4 per group) and 48 h (n = 9 per group) after injury or sham treatment. <b>(D)</b> OVA-Alexa Fluor 647 alone or in combination with lipopolysaccharide (LPS) was injected, and the absolute number of CD11c⁺OVA-Alexa⁺ DCs in the lymph node was determined 48 h later (n = 4 per group). Data are presented as mean±SD. Symbols indicate statistical differences that were detected with analysis of variance (ANOVA). *, p<0.05; ***, p<0.001 sham treatment vs. injury. ##, p<0.01; ###, p<0.001.</p

Infiltration of innate immune cells into the muscle after injury.

<p><b>(A)</b> At indicated time points after injury or sham treatment, digested tissues of the gastrocnemius muscles were stained with fluorescent antibodies against Gr-1, CD11b, and CD11c. Region I depicts CD11c⁺Gr-1^– cells, and region II depicts Gr-1⁺CD11c^– neutrophilic granulocytes. Monocytes/macrophages (mono/mac) were identified as CD11b⁺ cells (region IV) among gated CD11c^–Gr-1^– total muscle cells (region III). Numbers indicate the percentage of gated cells. The dot plots from sham-treated muscle are representative of dot plots from all sham-treated muscles at any time point. The dot plot for the isotype control is shown for time point 4d after injury and is representative for muscles from sham and injured mice at any time point. <b>(B)</b> The absolute number of granulocytes, monocytes/macrophages, and CD11c⁺ cells per muscle was determined. Median (horizontal lines), 25^th to 75^th percentile (extension of boxes), and range (error bars) of n = 6–8 mice per group are shown. Asterisks indicate statistically significant differences that were detected with the Kruskal-Wallis test followed by Dunn’s post hoc test. <b>(C)</b> Localization of granulocytes in the skeletal muscle 24 h after sham treatment or injury. Representative sections of skeletal muscle underwent immunofluorescent staining against Gr-1 (green) and laminin-2 (red) and were examined using laser scanning microscopy as described in Materials and Methods. Nuclei are visualized in blue. Scale bar = 50 μm **, p <0.01; ***, p <0.005 sham treatment vs. injury. iso, isotype.</p

The injection of endotoxin-containing but not endotoxin-free OVA into the injured muscle increases T helper cells type 1-like priming in the popliteal lymph node.

One, 4, or 7 days after injury or sham treatment, lipopolysaccharide (LPS)-containing unlabeled ovalbumin (OVA; Sigma) (A-G) or endotoxin-free OVA (Endograde) (H, I) was injected into the gastrocnemius muscles of mice that, 24 h earlier, had received OVA-specific T cells labeled with carboxyfluorescein succinimidyl ester (CFSE) from DO11.10 mice. Three days after OVA application, popliteal lymph node (pLN) cells were isolated and pooled by group. (A) Schematic overview of the experimental setup. (B) Gating of CD4+ OVA-specific (KJ1-26+) T helper (Th) cells among total lymph node cells. The number depicts the percentage of OVA-specific Th cells. (C) CFSE dilution in gated OVA-specific Th cells. Numbers indicate the percentage of cells that underwent at least one division. (D) Representative dot plots of the expression of CD69 and CD25 on gated OVA-specific Th cells. (E) Percentage of CD69+ and (F) CD25+ cells among gated OVA-specific Th cells on days 1, 4, and 7 after treatment. Data are presented as mean±SD of 3 experiments, each with n = 3 mice per group. (G) Lymph node cells were restimulated with OVA peptide (pOVA) in vitro, and the concentrations of interferon (IFN) γ, interleukin (IL) 2, and IL-10 in the supernatants were determined after 3 days. Data are presented as mean±SD of triplicate cultures and are representative of one of 3 experiments, each with n = 3 mice per group. (H) Content of IFN-γ in the supernatants from restimulated lymph node cells of mice that had received endotoxin-free OVA on day 7 after injury or sham treatment. Data are presented as mean±SD of triplicate cultures (n = 4 mice per group). (I) CFSE dilution in gated OVA-specific T helper cells among lymph node cells from mice that had received endotoxin-free OVA on day 7 after injury or sham treatment. Numbers indicate the percentage of cells that had undergone at least one division. Asterisks indicate statistically significant differences that were detected with two-way analysis of variance (ANOVA). i.m., intramuscular. *, p<0.05; **, p<0.01; ***, p<0.001 sham treatment vs. injury.</p

Natural killer cells do not contribute to enhanced T helper cell priming mediated by dendritic cells in the injured muscle.

<p><b>(A)</b> Schematic overview on the experimental setup. Natural killer (NK) cells were depleted through intraperitoneal application of anti–asialo ganglioside-monosialic acid (αGM-1) serum 24 h before and 48 and 96 h after injury or sham treatment. Normal rabbit serum (NRS) served as a control. All mice received ovalbumin (OVA)-specific T cells from DO11.10 mice intravenously (i.v.) 1 day before OVA application. Unlabeled lipopolysaccharide-containing OVA (Sigma) was injected intramuscularly (i.m.) into the gastrocnemius muscles 7 days after injury or sham treatment. After 3 days, the popliteal lymph node (pLN) cells were pooled by group and were stained with antibodies against CD49b or were restimulated with OVA peptide (pOVA). The content of interferon (IFN) γ in the supernatants was determined 3 days later. <b>(B)</b> Representative dot plots with numbers indicating the percentage of CD49b⁺ NK cells among popliteal lymph node cells. <b>(C)</b> Release of IFN-γ from restimulated lymph node cells. Data are presented as mean±SD of triplicate cultures and are representative of 2 experiments with n = 3 mice per group. Statistically significant differences were detected with two-way analysis of variance (ANOVA). *, p<0.05; **, p<0.01.</p

Dendritic cells exposed to the microenvironment in the injured muscle possess enhanced ability to prime T helper cells type 1 upon migration into the popliteal lymph node.

<p>Bone marrow-derived dendritic cells (BMDCs) labeled with carboxyfluorescein succinimidyl ester (CFSE) <b>(A)</b> or unlabeled <b>(B, C)</b> were loaded with lipopolysaccharide (LPS)-containing unlabeled ovalbumin (OVA) (Sigma) and were subsequently injected into the gastrocnemius muscles 4 days after injury or sham treatment. <b>(A)</b> Twenty-four or 48 h later, popliteal lymph node (pLN) cells were prepared and stained for CD11c. CFSE⁺ BMDCs were gated. Numbers indicate the percentage of CFSE⁺ BMDCs among total lymph node cells. <b>(B, C)</b> Before the injection of OVA-loaded BMDCs, the mice received CFSE-labeled OVA-specific T cells. After 3 d, pLN cells were isolated and pooled by group. <b>(B)</b> Representative histogram of CFSE dilution in gated OVA-specific CD4⁺KJ1-26⁺ T helper cells. <b>(C)</b> Lymph node cells were restimulated with 1 μg/ml OVA peptide (pOVA), and the concentration of interferon (IFN) γ in the supernatants was determined after 3 days. Data are presented as mean±SD of triplicate cultures and are representative of one of 3 experiments with n = 3 mice per group. Asterisks indicate statistically significant differences that were detected with Student’s <i>t</i>-test. **, p<0.01 sham treatment vs. injury.</p

Phenotype of CD11c⁺ cells in the muscle after injury.

<p><b>(A)</b> Four or 7 days after injury or sham treatment, cells from the gastrocnemius muscles were prepared, pooled per group, and stained for CD11c, CD11b, and MHC class II. Expression of MHC class II on gated CD11c⁺ cells and monocytes/macrophages (Mono/Mac). Representative dot plots are shown from n = 3 experiments each with n = 3 mice per group. <b>(B)</b> Expression of CD11b and Ly6C on gated CD11c⁺MHC class II⁺ cells 7 d after injury. The median (interquartile range) of the percentage of CD11c⁺MHC class II⁺ cells among total leukocytes was 11(8–15). <b>(C)</b> Expression of CD40 and CD86 on MHC class II⁺ cells among diverse subpopulations 7 d after injury. Among total leukocytes the percentage of CD11c⁺CD11b⁺, CD11c⁺CD11b^-, and CD11c^-CD11b⁺ cells was 14 (3–34), 0.9 (0.7–1.4), and 1.9 (1.2–2.9), respectively. Dot plots are representative for separate analyses of n = 6 muscles. <b>(D)</b> Pooled muscle cells were stained against CD11c, CD40, and CD86. The expression of CD40 and CD86 on gated total CD11c⁺ cells 4 and 7 d after injury or sham treatment was determined. Representative dot plots are shown from n = 3 experiments with n = 3 mice per group. Numbers in the dot plots indicate the mean±SD of the percentage of gated or double-positive cells.</p

Table_1_Leukocyte-Derived Interleukin-10 Aggravates Postoperative Ileus.docx

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.</p

Data_Sheet_1_Leukocyte-Derived Interleukin-10 Aggravates Postoperative Ileus.PDF

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.</p

Data_Sheet_4_Leukocyte-Derived Interleukin-10 Aggravates Postoperative Ileus.PDF

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.</p

Тагильский рабочий. 2017. № 037

<p>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.</p><p>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 LysM^cre/IL-10^fl/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.</p><p>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.</p><p>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.</p