Dendritic Cell-Like Cells Accumulate in Regenerating Murine Skeletal Muscle after Injury and Boost Adaptive Immune Responses Only upon a Microbial Challenge.

Skeletal muscle injury causes a local sterile inflammatory response. In parallel, a state of immunosuppression develops distal to the site of tissue damage. Granulocytes and monocytes that are rapidly recruited to the site of injury contribute to tissue regeneration. In this study we used a mouse model of traumatic skeletal muscle injury to investigate the previously unknown role of dendritic cells (DCs) that accumulate in injured tissue. We injected the model antigen ovalbumin (OVA) into the skeletal muscle of injured or sham-treated mice to address the ability of these DCs in antigen uptake, migration, and specific T cell activation in the draining popliteal lymph node (pLN). Immature DC-like cells appeared in the skeletal muscle by 4 days after injury and subsequently acquired a mature phenotype, as indicated by increased expression of the costimulatory molecules CD40 and CD86. After the injection of OVA into the muscle, OVA-loaded DCs migrated into the pLN. The migration of DC-like cells from the injured muscle was enhanced in the presence of the microbial stimulus lipopolysaccharide at the site of antigen uptake and triggered an increased OVA-specific T helper cell type 1 (Th1) response in the pLN. Naïve OVA-loaded DCs were superior in Th1-like priming in the pLN when adoptively transferred into the skeletal muscle of injured mice, a finding indicating the relevance of the microenvironment in the regenerating skeletal muscle for increased Th1-like priming. These findings suggest that DC-like cells that accumulate in the regenerating muscle initiate a protective immune response upon microbial challenge and thereby overcome injury-induced immunosuppression

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

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

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

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

Diversity of Interferon γ and Granulocyte-Macrophage Colony-Stimulating Factor in Restoring Immune Dysfunction of Dendritic Cells and Macrophages During Polymicrobial Sepsis

The development of immunosuppression during polymicrobial sepsis is associated with the failure of dendritic cells (DC) to promote the polarization of T helper (Th) cells toward a protective Th1 type. The aim of the study was to test potential immunomodulatory approaches to restore the capacity of splenic DC to secrete interleukin (IL) 12 that represents the key cytokine in Th1 cell polarization. Murine polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Splenic DC were isolated at different time points after CLP or sham operation, and stimulated with bacterial components in the presence or absence of neutralizing anti-IL-10 antibodies, murine interferon (IFN) γ, and/or granulocyte macrophage colony-stimulating factor (GM-CSF). DC from septic mice showed an impaired capacity to release the pro-inflammatory and Th1-promoting cytokines tumor necrosis factor α, IFN-γ, and IL-12 in response to bacterial stimuli, but secreted IL-10. Endogenous IL-10 was not responsible for the impaired IL-12 secretion. Up to 6 h after CLP, the combined treatment of DC from septic mice with IFN-γ and GM-CSF increased the secretion of IL-12. Later, DC from septic mice responded to IFN-γ and GM-CSF with increased expression of the co-stimulatory molecule CD86, while IL-12 secretion was no more enhanced. In contrast, splenic macrophages from septic mice during late sepsis responded to GM-CSF with increased cytokine release. Thus, therapy of sepsis with IFN-γ/GM-CSF might be sufficient to restore the activity of macrophages, but fails to restore DC function adequate for the development of a protective Th1-like immune response