Exosomes from Adipose-Derived Stem Cells Regulate M1/M2 Macrophage Phenotypic Polarization to Promote Bone Healing via miR-451a/MIF

Objectives: Bone defects caused by diseases and trauma are usually accompanied by inflammation, and the implantation of biomaterials as a common repair method has also been found to cause inflammatory reactions, which affect bone metabolism and new bone formation. This study investigated whether exosomes from adipose-derived stem cells (ADSC-Exos) plays an immunomodulatory role in traumatic bone defects and elucidated the underlying mechanisms.Methods:ADSC-Exos were loaded by a biomaterial named gelatine nanoparticles (GNPs), physical and chemical properties were analyzed by zeta potential, surface topography and rheology. A rat model of skull defect was used for our in vivo studies, micro-CT and histological staining were used to analyse histological changes in the bone defect area. RT-qPCR and western blotting were performed to verify that ADSC-Exos could regulate M1/M2 macrophage polarization. MicroRNA (miRNA) array analysis was conducted to determine the miRNA expression profiles of ADSC-Exos. After macrophages were treated with a miR-451a mimic, miR-451a inhibitor and ISO-1, the relative expression of genes and proteins was measured by RT-qPCR and western blotting.Results: In vivo, micro-CT and histological staining showed that exosome-loaded GNPs(GNP-Exos) hydrogel, with good biocompatibility and strong mechanical adaptability,exhibited immunomodulatory effect mainly by regulating macrophage immunity and promoting bone tissue healing. Immunofluorescence further indicated that ADSC-Exos reduced M1 marker (iNOS) expression and increased M2 marker (CD206) expression. Moreover, in vitro studies, western blotting and RT-qPCR showed that ADSC-Exos inhibited M1 macrophage marker expression and upregulated M2 macrophage marker expression. MiR-451a was enriched in ADSC-Exos and targeted macrophage migration inhibitory factor (MIF). Macrophages treated with the miR-451a mimic showed lower expression of M1 markers. In contrast, miR-451a inhibitor treatment upregulated the expression of M1 markers and downregulated the expression of M2 markers, while ISO-1 (a MIF inhibitor) treatment upregulated miR-451a expression and downregulated M1 macrophage marker expression.Conclusion: GNP-Exos can effectively regulate bone immune metabolism and futher promote bone healing partly through immune regulation of miR-451a,which may provide a therapeutic direction for bone repair.

Predictive potential of macrophage population phenotyping in malignization of H. pylori-associated chronic gastritis

Tumor-associated macrophages are able to regulate the tumor cell proliferation and to affect the tumor cell dissemination. The study was aimed to assess the predictive potential of the macrophage population immunohistochemical phenotyping in early malignization of H. pylori-associated chronic gastritis. Gastic biopsy samples of male and female patients aged 48 ± 7.2 infected with Helicobacter pylori were used as the research material. The patients were divided into three groups: non-atrophic chronic gastritis (NACG, n = 10), atrophic chronic gastritis (ACG, n = 10), G1/G2 gastric adenocarcinoma (GAC, n = 10). The macrophage population was visualized using the CD68 pan-macrophage marker and the type 2 monocyte/macrophage marker CD163. Intensity of neoangiogenesis was defined using the CD31 endothelial marker by assessing the total cross sectional area of blood vessels. It was found that chronic gastritis was accompanied by the dynamic increase in the size of the general macrophage population with the progression of atrophic and metaplastic processes. According to immunohistochemical study of biopsies obtained from patients with NCG, the CD163 : CD68 ratio was 0.67 ± 0.02, and the total cross sectional area of blood vessels was 3590.92 ± 356.27 µm2. Atrophic gastritis and adenocarcinoma were characterized by vector redistribution of monocytes/macrophages into the 2nd functional phenotype. The CD163 : CD68 expression index in the group with ACG was 0.81 ± 0.04, and in the group with GAC it was 0.88 ± 0.03. Microvascular area was significantly increased in the groups with ACG and GAC, which reflected tumor neoangiogenesis intensification under the influence of М2 monocytes/macrophages. The increased expression of CD163 can serve as a predictor of chronic gastritis malignization together with evaluation of the glandular epithelium atrophy and metaplasia degree.

MARCO+ Macrophage Dynamics in Regenerating Liver after 70% Liver Resection in Mice

Background: Macrophages play a key role in liver regeneration. The fates of resident macrophages after 70% resection are poorly investigated. In this work, using the MARCO macrophage marker (abbreviated from macrophage receptor with collagenous structure), we studied the dynamics of mouse liver resident macrophages after 70% resection. Methods: In BALB/c male mice, a model of liver regeneration after 70% resection was reproduced. The dynamics of markers CD68, TIM4, and MARCO were studied immunohistochemically and by using a Western blot. Results: The number of MARCO- and CD68-positive macrophages in the regenerating liver increased 1 day and 3 days after resection, respectively. At the same time, the content of the MARCO protein increased in the sorted macrophages of the regenerating liver on the third day. Conclusions: The data indicate that the number of MARCO-positive macrophages in the regenerating liver increases due to the activation of MARCO synthesis in the liver macrophages. The increased expression of MARCO by macrophages can be regarded as a sign of their activation. In the present study, stimulation with LPS led to an increase in the expression of the Marco gene in both Kupffer cells and macrophages of bone marrow origin.

Anti-Inflammatory Effect of Curcumin on the Mouse Model of Myocardial Infarction through Regulating Macrophage Polarization

Inflammation causes tissue damage and promotes ventricular remodeling after myocardial infarction (MI), and the infiltration and polarization of macrophages play an important role in regulating inflammation post-MI. Here, we investigated the anti-inflammatory function of curcumin after MI and studied its relationship with macrophage polarization. In vivo, curcumin not only attenuated ventricular remodeling 3 months after MI but also suppressed inflammation during the first 7 days post-MI. Importantly, the results of qPCR and immunochemistry showed that curcumin decreased M1 (iNOS, CCL2, and CD86) but increased M2 macrophage (Arg1, CD163, and CD206) marker expression in the myocardium of MI mice during the first 7 days post-MI. And flow cytometry analysis indicated that curcumin suppressed M1 (CD45+Gr-1-CD11b+iNOS+ cells) but enhanced M2 macrophage (CD45+Gr-1-CD11b+Arg+ cells) expansion in the myocardium of MI mice during the first 7 days post-MI. In vitro, curcumin decreased LPS/IFNγ-elevated M1 macrophage marker (iNOS and CD86) expression and the proportion of M1 macrophages (iNOS+F4/80+ cells) but increased LPS/IFNγ-suppressed M2 macrophage marker (Arg1 and CD206) expression and the proportion of M2 macrophages (Arg1+F4/80+ cells). In addition, curcumin modulates M1/M2 macrophage polarization partly via AMPK. In conclusion, curcumin suppressed the MI-induced inflammation by modulating macrophage polarization partly via the AMPK pathway.

Influence of Epstein–Barr virus and human papillomavirus infection on macrophage migration inhibitory factor and macrophage polarization in nasopharyngeal carcinoma

Background To assess the effects of Epstein–Barr virus (EBV) and human papillomavirus (HPV) infection on the tumor microenvironment, we examined the relationship between viral infection status, macrophage migration inhibitory factor (MIF), and tumor-associated macrophages in nasopharyngeal carcinoma (NPC). Methods A tissue microarray containing 150 cores from 90 patients with NPC and six with chronic inflammation was used. EBV and HPV status were detected using in situ hybridization with commercial EBER1 and HPV16/18 probes. Immunofluorescence double staining of MIF, pan-macrophage marker CD68, M1 macrophage marker CD11c, and M2 macrophage marker CD163 were analyzed using the same tissue microarray. The levels of these markers between NPC and inflammation cases and between tumor nests and stroma were compared. Correlations among these markers were analyzed. Results We found EBER1(+) cases in 90% of NPC patients, including 10% EBV/HPV co-infection. M1 macrophages mainly infiltrated the tumor nest, while M2 macrophages infiltrated the tumor stroma. We found a significant positive correlation between EBER1 levels and MIF levels in tumor nests and a significant positive correlation between HPV16/18 and CD11c(+) cell levels in NPC tissues. Conclusions It is suggested that MIF is associated with EBV, and M1 macrophage infiltration is affected by HPV status in NPC.

Developmental chronodisruption alters placental signaling in mice

Chronodisruption has been largely overlooked as a developmental exposure. The placenta, a conduit between the maternal and fetal environments, may relay circadian cues to the fetus. We have previously shown that developmental chronodisruption causes visual impairment and increased retinal microglial and macrophage marker expression. Here, we investigated the impacts of environmental chronodisruption on fetal and placental outcomes in a C57BL/6J mouse (Mus musculus) model. Developmental chronodisruption had no effect on embryo count, placental weight, or fetal sex ratio. When measured with RNAseq, mice exposed to developmental chronodisruption (CD) had differential placental expression of several transcripts including Serpinf1, which encodes pigment epithelium-derived factor (PEDF). Immunofluorescence of microglia/macrophage markers, Iba1 and CD11b, also revealed significant upregulation of immune cell markers in CD-exposed placenta. Our results suggest that in utero chronodisruption enhances placental immune cell expression, potentially programming a pro-inflammatory tissue environment.

Inhibition of the canonical Wnt signaling pathway by a β-catenin/CBP inhibitor prevents heart failure by ameliorating cardiac hypertrophy and fibrosis

In heart failure (HF) caused by hypertension, the myocyte size increases, and the cardiac wall thickens. A low-molecular-weight compound called ICG001 impedes β-catenin-mediated gene transcription, thereby protecting both the heart and kidney. However, the HF-preventive mechanisms of ICG001 remain unclear. Hence, we investigated how ICG001 can prevent cardiac hypertrophy and fibrosis induced by transverse aortic constriction (TAC). Four weeks after TAC, ICG001 attenuated cardiac hypertrophy and fibrosis in the left ventricular wall. The TAC mice treated with ICG001 showed a decrease in the following: mRNA expression of brain natriuretic peptide (Bnp), Klf5, fibronectin, β-MHC, and β-catenin, number of cells expressing the macrophage marker CD68 shown in immunohistochemistry, and macrophage accumulation shown in flow cytometry. Moreover, ICG001 may mediate the substrates in the glycolysis pathway and the distinct alteration of oxidative stress during cardiac hypertrophy and HF. In conclusion, ICG001 is a potential drug that may prevent cardiac hypertrophy and fibrosis by regulating KLF5, immune activation, and the Wnt/β-catenin signaling pathway and inhibiting the inflammatory response involving macrophages.

Lymphocyte-macrophage colonization in various molecular biological types of endometrial cancer: comparison with the receptor status and proliferative activity of tumor tissue

Recent years have been marked by a gradual shift from the previous division of endometrial cancer (EC) into two main types to modern molecular biological classifications of this disease, one of which (at present, most likely, the most popular: Talhouk et al., 2017, 2019) is based on the use of a combination of genetic and immunohistochemical analysis. The study involved material from untreated EC patients, the number of which varied depending on the method used. The average age of patients was close to 55-60 years, and over 80% of patients were postmenopausal. Deparaffinized blocks of EC tissue were analyzed for POLE (DNA polymerase epsilon) mutations, evaluated by immunohistochemistry (IHC) the expression of the oncoprotein p53 and MMR (mismatch-repair) proteins / MLH1, MSH2, MSH6 and PMS2 /, and also helped to identify the type of disease without a characteristic molecular profile (WCMP). In addition to studying the expression of p53 and MMR proteins, the IHC method was also used to study the expression of estrogen (ER) and progesterone (PR) receptors, the Ki-67 proliferative activity index, and the severity of macrophage-lymphocytic infiltration of the EC tissue based on the analysis of the macrophage marker (CD68) and markers of lymphocytic cells (cytotoxic CD8 and regulatory FoxP3) using reagents from Ventana and Dako.

P-420 Pre-selected for an award: Uncomplicated oocyte donation pregnancies display elevated CD163 positive type 2 macrophage load in the decidua, which is associated with fetal-maternal HLA class II mismatches

Study question Do quantity and composition of decidual macrophages differ between uncomplicated oocyte donation (OD) pregnancies and non-OD in vitro fertilization (IVF) pregnancies? Summary answer OD placentas show higher decidual CD163 positive fraction within the total macrophage population compared to non-OD IVF placentas. What is known already The embryo of an OD pregnancy is completely allogeneic to the mother, which may lead to a bigger challenge for the maternal immune system to tolerize the fetus compared to autologous pregnancies. Placental macrophages may be essential in maintaining a healthy pregnancy. Macrophages can be classified into different categories based on phenotype and characteristics, in which type 2 macrophages are thought to exhibit immune suppressive activity. Study design, size, duration This retrospective case-control study included patients who delivered in the Leiden University Medical Center between January 1st 2006 and July 1st 2016. A total of 42 pregnancies were enrolled in this study, conceived by uncomplicated singleton OD pregnancies (n = 25) or non-OD IVF pregnancies (n = 17). Medical records were reviewed and clinical data were collected. Placental tissue samples were collected for immunohistochemical staining and blood samples were collected for HLA typing. Participants/materials, setting, methods Placentas were collected and immunohistochemically stained for CD14 (pan-macrophage marker) and CD163 (type 2 macrophage marker). The extent of staining was quantitated by digital image analysis software. To assess mismatching, maternal and fetal DNA was typed for HLA-A, -B, C, -DRB1, and -DQB1. Main results and the role of chance A significantly lower percentage of CD14 positive staining was observed in the decidua basalis of OD pregnancies compared to non-OD IVF pregnancies (p = 0.030). Consequently, the CD163/CD14 ratio in OD group was higher than in non-OD IVF group (p = 0.243). In the parietalis, OD pregnancies demonstrated a significantly higher percentage of CD163+ staining (p = 0.040) and a significantly higher CD163/CD14 ratio (p = 0.032) compared to non-OD IVF group. The reproducibility of this quantitative analysis was found to be high. OD group was separated into a syngeneic group (number of mismatches lower than half of the antigens per HLA locus) and an allogeneic group (number of mismatches higher than half of the antigens per HLA locus). Significant differences of CD163+ and CD163/CD14 ratio were found in the decidua parietalis when comparing the HLA-classII-allogeneic OD group with the non-OD IVF group (p = 0.047). This difference was not found for the HLA-class-II-syngeneic OD group. Limitations, reasons for caution Our study only focused on decidua basalis and parietalis, no other locations in the placentas. Larger sample size might be needed to verify the association between macrophages and HLA mismatches. Wider implications of the findings To our knowledge, this study is the first to quantify a higher CD163 positive M2 macrophages load within the total decidual macrophages of uncomplicated OD pregnancy compared to non-OD IVF pregnancies. Trial registration number not applicable

P–420 uncomplicated oocyte donation pregnancies display elevated CD163 positive type 2 macrophage load in the decidua, which is associated with fetal-maternal HLA class II mismatches

Study question Do quantity and composition of decidual macrophages differ between uncomplicated oocyte donation (OD) pregnancies and non-OD in vitro fertilization (IVF) pregnancies? Summary answer OD placentas show higher decidual CD163 positive fraction within the total macrophage population compared to non-OD IVF placentas. What is known already The embryo of an OD pregnancy is completely allogeneic to the mother, which may lead to a bigger challenge for the maternal immune system to tolerize the fetus compared to autologous pregnancies. Placental macrophages may be essential in maintaining a healthy pregnancy. Macrophages can be classified into different categories based on phenotype and characteristics, in which type 2 macrophages are thought to exhibit immune suppressive activity. Study design, size, duration This retrospective case-control study included patients who delivered in the Leiden University Medical Center between January 1st 2006 and July 1st 2016. A total of 42 pregnancies were enrolled in this study, conceived by uncomplicated singleton OD pregnancies (n = 25) or non-OD IVF pregnancies (n = 17). Medical records were reviewed and clinical data were collected. Placental tissue samples were collected for immunohistochemical staining and blood samples were collected for HLA typing. Participants/materials, setting, methods Placentas were collected and immunohistochemically stained for CD14 (pan-macrophage marker) and CD163 (type 2 macrophage marker). The extent of staining was quantitated by digital image analysis software. To assess mismatching, maternal and fetal DNA was typed for HLA-A, -B, C, -DRB1, and -DQB1. Main results and the role of chance A significantly lower percentage of CD14 positive staining was observed in the decidua basalis of OD pregnancies compared to non-OD IVF pregnancies (p = 0.030). Consequently, the CD163/CD14 ratio in OD group was higher than in non-OD IVF group (p = 0.243). In the parietalis, OD pregnancies demonstrated a significantly higher percentage of CD163+ staining (p = 0.040) and a significantly higher CD163/CD14 ratio (p = 0.032) compared to non-OD IVF group. The reproducibility of this quantitative analysis was found to be high. OD group was separated into a syngeneic group (number of mismatches lower than half of the antigens per HLA locus) and an allogeneic group (number of mismatches higher than half of the antigens per HLA locus). Significant differences of CD163+ and CD163/CD14 ratio were found in the decidua parietalis when comparing the HLA-classII-allogeneic OD group with the non-OD IVF group (p = 0.047). This difference was not found for the HLA-class-II-syngeneic OD group. Limitations, reasons for caution Our study only focused on decidua basalis and parietalis, no other locations in the placentas. Larger sample size might be needed to verify the association between macrophages and HLA mismatches. Wider implications of the findings: To our knowledge, this study is the first to quantify a higher CD163 positive M2 macrophages load within the total decidual macrophages of uncomplicated OD pregnancy compared to non-OD IVF pregnancies. Trial registration number Not applicable