Induced M2 Macrophage Research Articles

IL‐25 Supplementation Induces M2 Macrophage Polarization, Reduces Blood Pressure, and Improves Fetal Weight in Placental Ischemic Rats

Preeclampsia (PE) is characterized by maternal hypertension, intrauterine growth restriction (IUGR), and increased macrophage activation. Macrophages can be broadly categorized into pro‐inflammatory M1 macrophages (M1s) and anti‐inflammatory M2 macrophages (M2s). The M1:M2 ratio is significantly increased in PE women. However, the precise contribution of increased M1s to PE pathophysiology remains unclear. Using the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia, we tested the hypothesis that supplementation of RUPP rats with IL‐25, a cytokine that drives macrophage polarization to anti‐inflammatory M2s, will increase M2s, decrease mean arterial pressure (MAP), and improve fetal growth. On gestation day (GD) 14, timed pregnant 10‐12 week old Sprague Dawley rats underwent the RUPP or Sham procedure and a mini‐osmotic pump infusing IL‐25 (0.5µg/day) or vehicle was implanted i.p., in both groups. Carotid catheters were placed on GD18. On GD19, MAP was measured, animals were euthanized, and blood and tissues were collected for analysis. Flow cytometry analysis was performed on placental and renal tissues. M2 macrophages (CD3‐/CD68+/CD163+) were markedly reduced in placentas of RUPP rats compared to Sham (0.2% vs. 4.2% of total macrophages) and in kidneys of RUPP rats vs. Sham (0.19% vs. 1.445% total of macrophages). IL‐25 supplementation elevated M2 populations in placental (3.985%) and renal (5.985%) tissues of RUPP. Likewise, IL‐25 treatment increased M2 macrophages in Sham placental (18.56%) and renal (12.99%) tissues. Increased MAP was observed in RUPP rats compared with Sham (123.5 mmHg vs. 106.5 mmHg, respectively) and was reduced in RUPP + IL‐25 (107.5 mmHg). No changes were observed in MAP of Sham rats treated with IL‐25. Fetal weights were reduced in RUPP vs. Sham (2.035g vs. 2.405g, respectively) and were normalized in RUPP after IL‐25 supplementation (2.425g). IL‐25 supplementation had no effect on fetal weight of Sham rats. Additional analyses will include assessment of blood and tissue cytokines and reactive oxygen species, and vascular function in all groups. In conclusion, IL‐25 supplementation increased placental and renal anti‐inflammatory M2 macrophage populations, reduced MAP, and improved fetal weights in RUPP recipients. These data suggest that macrophage activation is a key contributor to PE pathophysiology and may represent a therapeutic target to improve maternal and fetal outcomes in PE.

Modeling macrophage response to periodontal infections in vitro

Periodontal disease (PD) is a chronic inflammatory disorder characterized by the destruction of connective tissue, tooth loss and systemic infections. Numerous virulence factors derived from chronic opportunistic pathogens such as Porphyromonas gingivalis are responsible for enhanced microbial colonization, stimulation of inflammatory responses, and suppression of host defense response. Individuals with PD present with a complex array of inflammatory mediators, and cellular infiltration by neutrophils, lymphocytes and monocytes/macrophages. Although macrophages comprise 5–30% of cells in the cellular infiltrate of human periodontal lesions, how macrophage skewing contributes to PD, and more specifically in the downstream development of a macrophage immune response to oral bacteria are unclear. We, including others have investigated the activation of monocytes/macrophages, and discovered the means of type I vs. type II inflammatory activation vs. deactivation of macrophages both in vitro and in vivo. We have shown previously that P. gingivalis infection induces M1 macrophage formation, whereas opsonized‐P. gingivalis challenge was able to switch to M2‐like phenotype demonstrating macrophage plasticity. However, the mechanism of modulation and kinetics of macrophage inflammatory reactions during the process is unclear. Prior research in the field has failed to appreciate that it is the same monocyte/macrophage cell population which is initially polarized towards an effector “inflammatory” program and subsequently re‐polarized to the deactivation program not just in PD but in any inflammation. The overarching goal of our research is to understand how macrophage skewing contributes to PD, and the downstream development of immune response to PD‐associated bacteria towards development of new macrophage‐based therapeutics. Since macrophage polarization dynamics cannot be studied in an animal model, a reliable in vitro system is needed to provide fundamental insights into macrophage biology. We have developed an in vitro model that recapitulates different phases of the inflammatory response (recruitment, initiation, development and resolution), based on purified human peripheral blood monocytes exposed to a battery of microenvironmental cues mimicking macrophage polarization/depolarization in vivo and we have created a kinetic profile of polarization by RNA analysis. Further we have applied our model to macrophage/gingival fibroblast coculture system to study kinetics of polarization. And by comparing gene clusters that are differentially expressed in M1 and M2, we have successfully recapitulated the macrophage polarization kinetics of acute inflammatory and resolution phases during periodontal inflammation and validated the key inflammatory and transcriptional factors using RT‐PCR at different phases. In conclusion, we developed an in vitro model which can be further employed to test novel macrophage‐based therapeutics to treat periodontal disease.

Sutureless neurorrhaphy system using a macrophage-polarizing in situ visible light-crosslinkable adhesive protein hydrogel for functional nerve regeneration

Surgical intervention after traumatic peripheral nerve injury relies heavily on sutures. While the suture method often burdens functional nerve regeneration by causing secondary traumatic damage and eliciting prolonged inflammatory immune responses, replacing sutures has rarely been considered because secure anastomosis between severed nerve ends has been the utmost priority in neurorrhaphy. In the present work, a macrophage-polarizing sutureless neurorrhaphy system was proposed by using an in situ visible light-crosslinkable protein-based bioadhesive hydrogel containing a functional neurotransmitter peptide. The efficacy of the proposed bioadhesive hydrogel as a successful sutureless neurorrhaphy system was intensively evaluated in vitro and in vivo. Using a rat sciatic nerve defect model, our findings suggested that this macrophage-polarizing bioadhesive hydrogel offered effective sutureless anastomosis and the capability to induce M2 macrophage polarization for effective tissue remodeling, significantly enhancing functional nerve regeneration compared with conventional sutures. Taken together, these results suggest that our macrophage-polarizing functional bioadhesive hydrogel system can be utilized as a promising alternative to surgical sutures for nerve regenerative medicine applications.

The Combination of LPS and Melatonin Induces M2 Macrophage Apoptosis to Prevent Lung Cancer

Background and objectivesLasting LPS stimulation changes macrophages toward the M2 phenotype therefore resulting in immunodepression. Melatonin can improve sleep, adjust the time difference, regulate immunity, and anti-tumor. This study is to observe whether melatonin can induce M2 macrophage apoptosis to reverse lasting LPS-induced immunodepression for lung cancer prevention and explore the possible mechanism.

Retraction Note: Exosomal DLX6-AS1 from hepatocellular carcinoma cells induces M2 macrophage polarization to promote migration and invasion in hepatocellular carcinoma through microRNA-15a-5p/CXCL17 axis

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s13046-021-01973-z.

Mesenchymal Stem Cells-derived Exosomes Ameliorate Lupus by Inducing M2 Macrophage Polarization and Regulatory T Cell Expansion in MRL/lpr Mice

ABSTRACT Previous studies have implicated that the transplantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) effectively alleviates systemic lupus erythematosus (SLE) primarily due to immunomodulatory effects. However, little is known about the role of hUC-MSC-derived exosomes in SLE. This study is carried out to investigate the modifying effects of hUC-MSC-exosomes on the differentiation and function of immune cells in SLE. hUC-MSC-derived exosomes were extracted from the cultural supernatant of hUC-MSCs by ultrahigh speed centrifugation. Quantitative real-time polymerase chain reaction, western blot, enzyme-linked immunosorbent assay, and flow cytometry were performed to estimate the effect of hUC-MSC-derived exosomes on macrophage and regulatory T cell (Treg) polarization. In vivo, hUC-MSC-exosomes were injected intravenously into 28-week-old MRL/lpr mice. We had found that exosomes derived from hUC-MSC restrained the proliferation and inflammation of macrophages in vitro. Besides, MSC-exosomes inhibited CD68+M1 and HLA-DR+M1 but promoted CD206+M2 and CD163+M2 in vitro. Moreover, MRL/lpr mice administrated by intravenous injection of MSC-exosomes had less infiltration of CD14+CD11c+M1 cells but more CD14+CD163+M2 cells as well as Tregs in spleens compared with those in MRL/lpr mice treated by PBS. Additionally, MSC-exosomes could alleviate nephritis, liver and lung injuries of MRL/lpr mice. The survival of lupus mice could be improved after MSC-exosome treatment. This study has suggested that MSC-derived exosomes exert anti-inflammatory and immunomodulatory effects in SLE. MSC-exosomes ameliorate nephritis and other key organ injuries by inducing M2 macrophages and Tregs polarization. As natural nanocarriers, MSC-exosomes may serve as a promising cell-free therapeutic strategy for SLE. Abbreviations: SLE: Systemic lupus erythematosus; hUC-MSCs: Human umbilical cord mesenchymal stem cells; MSCs: Mesenchymal stem cells; qRT-PCR: Quantitative real-time polymerase chain reaction; ELISA: Enzyme-linked immunosorbent assay; Tregs: Regulatory cells; TNF-α: Tumor necrosis factor alfa; IL: Interleukin; COVID-19: Coronavirus disease 2019; pTHP-1: PMA-induced THP-1 macrophages; TEM: Transmission electron microscopy; LPS: Lipopolysaccharide; EVs: Extracellular vesicles; TRAF1: Tumor necrosis factor receptor-associated factor 1; IRAK1: Interferon-α-interleukin-1 receptor-associated kinase 1; NF-κB: Nuclear factor-κB; BLyS: B lymphocyte stimulator; APRIL: A proliferation-inducing ligand

Immunopathogenesis of endometriosis: An overview of the role of innate and adaptive immune cells and their mediators.

Endometriosis is a chronic inflammatory disease associated with the growth and proliferation of endometrial-like tissues outside the uterus. Although the exact etiology and mechanism of the pathogenesis of the disease have not been fully elucidated, the immune system cells and the mediators produced by them can be named as effective factors in the onset and progression of the disease. We aim to attempt to review studies on the role of the immune system in endometriosis to better understand the pathogenesis of endometriosis. Abundant production of inflammatory mediators by neutrophils and macrophages and reduced cytotoxicity of defined cells promote endometriosis at the early stages of the disease. Following an increase in the inflammation of the environment, the body takes compensatory mechanisms to reduce inflammation and establish homeostasis. For this purpose, the body produces remodeling and anti-inflammatory factors leading to slow conversion of the inflammatory environment into a non-inflammatory environment with proliferative and immunosuppressive properties. Environmental conditions induce M2 macrophages, TH2 cells, and Tregs differentiation, promoting disease progression by producing angiogenic and immunosuppressive factors. However, the exact molecular mechanism involved in changing inflammatory to non-inflammatory conditions is not yet fully understood. Due to the common characteristics of endometriotic cells and cancer cells, most potential treatment options for endometriosis have been suggested due to the results of these methods in the treatment of cancer. In this pathway, immune system cells and soluble mediators can be used as targets.

Synergistic effect of Abraxane that combines human IL15 fused with an albumin-binding domain on murine models of pancreatic ductal adenocarcinoma.

Nab‐paclitaxel (Abraxane), which is a nanoparticle form of albumin‐bound paclitaxel, is one of the standard chemotherapies for pancreatic ductal adenocarcinoma (PDAC). This study determined the effect of Abraxane in combination with a fusion protein, hIL15‐ABD, on subcutaneous Panc02 and orthotopic KPC C57BL/6 murine PDAC models. Abraxane combined with hIL15‐ABD best suppressed tumour growth and produced a 40%–60% reduction in the tumour size for Panc02 and KPC, compared to the vehicle group. In the combination group, the active form of interferon‐γ (IFN‐γ)‐secreting CD8+ T cells and CD11b+CD86+ M1 macrophages in tumour infiltrating lymphocytes (TILs) were increased. In the tumour drainage lymph nodes (TDLNs) of the combination group, there was a 18% reduction in CD8+IFN‐γ+ T cells and a 0.47% reduction in CD4+CD25+FOXP3+ regulatory T cells, as opposed to 5.0% and 5.1% reductions, respectively, for the control group. Superior suppression of CD11b+GR‐1+ myeloid‐derived suppressor cells (MDSCs) and the induction of M1 macrophages in the spleen and bone marrow of mice were found in the combination group. Abraxane and hIL15‐ABD effectively suppressed NF‐κB‐mediated immune suppressive markers, including indoleamine 2,3‐dioxygenase (IDO), Foxp3 and VEGF. In conclusion, Abraxane combined with hIL15‐ABD stimulates the anticancer activity of effector cells, inhibits immunosuppressive cells within the tumour microenvironment (TME) of PDAC, and produces a greater inhibitory effect than individual monotherapies.

Exosomes Derived from Epidermal Stem Cells Improve Diabetic Wound Healing

Diabetic foot ulceration is a major diabetic complication with unmet needs. We investigated the efficacy of epidermal stem cells and epidermal stem cells-derived exosomes (ESCs-Exo) in improving impaired diabetic wound healing and their mechanisms of action. Invitro experiments showed that ESCs-Exo enhanced the proliferation and migration of diabetic fibroblasts and macrophages and promoted alternative or M2 macrophage polarization. In wounds of db/db mice, treatment with both epidermal stem cells and ESCs-Exo, when compared with fibroblast exosomes and PBS control, accelerated wound healing by decreasing inflammation, augmenting wound cell proliferation, stimulating angiogenesis, and inducing M2 macrophage polarization. Multiplex protein quantification of wound lysates revealed TGFβ signaling influenced by ESCs-Exo. High-throughput sequencing of small RNAs contained in the ESCs-Exo showed higher proportions of microRNAs than those contained in fibroblast exosomes. In silico functional analysis showed that the ESCs-Exo microRNAs‒target genes were primarily involved in homeostatic processes and cell differentiation and highlighted regulatory control of phosphatidylinositol-3 kinase/protein kinase B and TGFβ signaling pathways. This was also validated invitro. Collectively, our results indicate that epidermal stem cells and ESCs-Exo are equally effective in promoting impaired diabetic wound healing and that ESCs-Exo treatment may be a promising and technically advantageous alternative to stem cell therapies.

Hexapeptide induces M2 macrophage polarization via the JAK1/STAT6 pathway to promote angiogenesis in bone repair

Angiogenesis is essential for successful bone defect repair. In normal tissue repair, the physiological inflammatory response is the main regulator of angiogenesis through the activity of macrophages and the cytokines secreted by them. In particular, M2 macrophages which secrete high levels of PDGF-BB are typically considered to promote angiogenesis. A hexapeptide [WKYMVm, (Trp-Lys-Tyr-Met-Val-D-Met-NH2)] has been reported to modulate inflammatory activities. However, the underlying mechanisms by which WKYMVm regulates macrophages remain unclear.In this study, the possible involvement by which WKYMVm induces the polarization of macrophages and affects their behaviors was evaluated. In vitro results showed that macrophages were induced to an M2 rather than M1 phenotype and the M2 phenotype was enhanced by WKYMVm through activation of the JAK1/STAT6 signaling pathway. It was also found that WKYMVm played an important role in the PDGF-BB production increase and proangiogenic abilities in M2 macrophages. Consistent with the results in vitro, the elevated M2/M0 ratio induced by WKYMVm enhanced the formation of new blood vessels in a femoral defect mouse model. These findings suggest that WKYMVm could be a promising alternative strategy for angiogenesis in bone repair by inducing M2 macrophage polarization.

Bone marrow mesenchymal stem cells enhance angiogenesis and promote fat retention in fat grafting via polarized macrophages

BackgroundFat grafting is one of the most common soft tissue filling methods in plastic surgery. Bone marrow mesenchymal stem cell (BM-MSC) transplantation is an effective method for improving graft retention. However, the role of BM-MSCs in fat transplantation is not completely clear.MethodsHuman fat particles, together with BM-MSCs or PBS as a control, were subcutaneously transplanted into the backs of nude mice. Samples were taken on days 14, 30 and 90 post-grafting to calculate the fat graft retention rate, and tissue staining was evaluated. Furthermore, macrophages were treated with BM-MSC conditioned medium (BM-MSC-CM) to identify the beneficial component secreted by these stem cells.ResultsIn this study, we found that BM-MSCs improved retention by enhancing angiogenesis in fat grafting. Further analysis revealed that BM-MSCs could significantly inhibit the expression of the proinflammatory M1 macrophage markers interleukin (IL)-1β, tumor necrosis factor-α (TNF-α) and IL-6 in the early stages of fat grafting and promote the expression of the anti-inflammatory M2 macrophage markers Arg1, IL-10 and VEGF. Furthermore, our results showed that IL-10 secreted by BM-MSCs induced M2 macrophage polarization in vitro.ConclusionsBM-MSC transplantation can improve the fat retention rate and promote angiogenesis, which may be related to M2 macrophages. These results help elucidate the role of BM-MSCs in fat grafting.

NEAT1 in bone marrow mesenchymal stem cell-derived extracellular vesicles promotes melanoma by inducing M2 macrophage polarization.

Bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles (EVs) reportedly play an important role in melanoma pathogenesis. This study aimed to explore the mechanisms of EVs-carried long non-coding RNA (lncRNA) NEAT1 involvement in melanoma. Gain- and loss-of-function experiments were performed to determine biological characteristics of A-375 melanoma cells. Bioinfomatic prediction, RNA immunoprecipitation (RIP), and dual luciferase reporter gene experiments were applied to investigate the roles of NEAT1 and microRNA-374a-5p (miR-374a-5p), and leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4). A subcutaneous tumor model was constructed using nude mice, and in vivo fluorescence imaging was used to observe the effect of NEAT1 on the growth and metastasis of melanoma cells in vivo. The results indicated that BMSC-EVs could be internalized by macrophages to promote the expression of macrophages M2 markers. M2 type macrophages promoted malignancy of melanoma cells. NEAT1 derived from BMSC-EVs promoted the progression of melanoma by promoting M2 polarization of macrophages. NEAT1 inhibits miR-374 expression, while miR-374 could upregulate LGR4-dependent IQGAP1 expression. The tumor-inhibiting effect of NEAT1 silencing was validated in the nude mouse xenograft model. Collectively, the results demonstrated that BMSC-EVs carrying NEAT1 can promote the progression of melanoma by inducing M2 polarization of macrophages, and thus may be considered as a potential target for melanoma therapeutics.

The role of NFATc1/c-myc/PKM2/IL-10 axis in activating cervical cancer tumor-associated M2 macrophage polarization to promote cervical cancer progression

Nuclear factor of activated T cells 1 (NFATc1) is mainly expressed in tumor microenvironment, especially in macrophages. However, whether NFATc1 is involved in the polarization of tumor associated macrophages (TAMs) and tumor progression in cervical cancer (CC) remains unclear. Immunofluorescence staining was used to detect the expression of CD68 and NFATc1 in CC tissues or adjacent normal tissues of patients. RT-qPCR, flow cytometry, ELISA, and inhibitors treatment were used to observe the effect of NFATc1 on TAMs polarization. Clonal formation, scratch, and transwell assays were used to examine the effects of NFATc1-transfected macrophages or NFATc1-transfected TAM on tumor proliferation, migration, and invasion. Further, a xenograft model was established to confirm the roles of NFATc1+ TAM in CC tumorigenesis. NFATc1+CD68+/CD68+ TAMs ratio was significantly increased in CC tissues compared with the normal tissue, and NFATc1+ TAM showed an M2-like TAM subtype. NFATc1 induced macrophages to secrete IL-10, which further induced M2 polarization of macrophages. Mechanically, the c-myc-PKM2 pathway mediated the expression of IL-10 in NFATc1-induced macrophages. Functionally, NFATc1 induced M2 macrophages promoted the proliferation, migration, and invasion of CC cells, and the knockout of NFATc1 in TAMs significantly inhibited the tumor-promoting function of TAMs. Further, the tumorigenesis test in nude mice confirmed that NFATc1+ TAM promoted the tumorigenicity of CC cells in vivo. In conclusion, NFATc1 mediated IL-10 secretion by regulating the c-myc/PKM2 pathway, thereby inducing M2 polarization of TAMs and promoting the progression of CC.

Dental pulp stem cell-derived exosomes suppress M1 macrophage polarization through the ROS-MAPK-NF\u03baB P65 signaling pathway after spinal cord injury

Stem cell-derived exosomes have recently been regarded as potential drugs for treating spinal cord injury (SCI) by reducing reactive oxygen species (ROS) and suppressing M1 macrophage polarization. However, the roles of ROS and exosomes in the process of M1 macrophage polarization are not known. Herein, we demonstrated that ROS can induce M1 macrophage polarization and have a concentration-dependent effect. ROS can induce M1 macrophage polarization through the MAPK-NFκB P65 signaling pathway. Dental pulp stem cell (DPSC)-derived exosomes can reduce macrophage M1 polarization through the ROS-MAPK-NFκB P65 signaling pathway in treating SCI. This study suggested that DPSC-derived exosomes might be a potential drug for treating SCI. Disruption of the cycle between ROS and M1 macrophage polarization might also be a potential effective treatment by reducing secondary damage.Graphical

Daeshiho-tang Attenuates Atherosclerosis by Regulating Cholesterol Metabolism and Inducing M2 Macrophage Polarization.

Dyslipidemia, the commonest cause of cardiovascular disease, leads to lipid deposits on the arterial wall, thereby aggravating atherosclerosis. DSHT (Daeshiho-tang) has long been used as an anti-dyslipidemia agent in oriental medicine. However, the anti-atherosclerotic effects of DSHT have not been fully investigated. Therefore, this study was designed to evaluate whether DSHT could exert beneficial anti-atherosclerotic effects. We fed apolipoprotein E-deficient (ApoE-/-) mice on a high-fat diet and treated them with atorvastatin (AT) or DSHT, or the combination of DSHT and AT for 12 weeks. To determine the role of DSHT, atherosclerotic lesions in the aorta, aortic root, and aortic arch; lipids and apolipoprotein levels in serum; and macrophage polarization markers in aorta tissues were examined. We show here that the DSHT decreased the atherosclerotic plaque ratio in the aortic arch, aorta, and aortic root. DSHT also regulated lipid levels by decreasing the ApoB level and increasing the ApoA1 level. Moreover, DSHT effectively regulated cholesterol metabolism by increasing the levels of PPARγ, ABCA1 and ABCG1, and the LDL receptor genes. We further found that DSHT promoted polarization to the M2 phenotype by increasing the levels of M2 macrophage (ARG1, CD163, and PPARγ) markers. Our data suggested that DSHT enhances the anti-atherosclerotic effect by regulating cholesterol metabolism through the activation of the PPARγ signaling pathway and by promoting anti-inflammatory M2 macrophage polarization.

Inflammatory level under different p53 mutation status and the regulation role of curcumin in tumor microenvironment

The inflammation is tightly associated with tumor development, promoting or inhibiting tumorigenesis. And mutant p53 is speculated to promote inflammation and tumorigenesis. The tumor associated macrophages are usually educated to present the anti-inflammatory profile to tune down antitumor immunity. However, the impact of p53 mutants on macrophages is not clear. Here, we compared the basal inflammatory level and macrophage profiles in tumor cells and tumor samples with different p53 mutations. Data revealed that a lower inflammatory level was maintained in immune organs and tumor cells with p53 point mutations than those with p53 null mutation. Using the tumor cell-derived conditional media to culture macrophages, we found that the media from cells with p53 mutations, especially the point mutations, could decrease M1 markers and inhibit phagocytosis, suggesting the p53 mutation promoted M2 profile polarization.To target the p53 mutation induced M2 macrophage polarization, we applied low-concentration curcumin to the tumor cells with different p53 mutations. The data showed that curcumin could inhibit STAT3 signal and decrease PPARγ and CSF1 in tumor cells and tumor samples. In vitro, the co-culture assays showed that the curcumin treatment shifted p53 mutation educated macrophages back towards M1 profile. In vivo, the curcumin-treated MEFs showed obvious tumor inhibition, and the tumor samples displayed inhibited M2 markers. Results suggested that curcumin could inhibit p53 mutation educated macrophage induction and suppress M2-promoted tumorigenesis.Our study illustrated the inflammatory level under different p53 status and the inflammatory regulated role of curcumin in tumor environment. This study might provide a potential method in tumor personalized treatment aiming immune therapy in different p53 status.

Long-Term Recruitment of Endogenous M2 Macrophages by Platelet Lysate-Rich Plasma Macroporous Hydrogel Scaffold for Articular Cartilage Defect Repair.

After cartilage damage, a large number of monocytes/macrophages infiltrate into adjacent synovium and the resident macrophages in synovial tissue transform to activated macrophages (M1), which secrete pro-inflammatory cytokines to induce sustained inflammation and chondrocyte apoptotic. However, current clinical therapies for cartilage repair can rarely achieve long-term anti-inflammatory regulation and satisfactory outcomes. Herein, a platelet lysate-rich plasma macroporous hydrogel (PLPMH) scaffold with around 100µm pore size and 1.25MPa Young's modulus is developed to sustainedly recruit and polarize endogenous anti-inflammatory macrophages (M2) for improving cartilage defect repair. PLPMH scaffold can steadily release sphingosine1-phosphate and proteins via gradual degradation, thus inducing M2 macrophages migration or resting (M0) macrophages migration and then polarization to M2 phenotype, and improving the secretion of anti-inflammatory cytokines. Furthermore, PLPMH scaffold exhibits negligible inflammatory responses in vivo and promotes endogenous M2 macrophage infiltration in large numbers and long-time duration to provide a local anti-inflammatory microenvironment, which even lasts for 42 d. In a rabbit model of cartilage defect, PLPMH scaffold increases the ratio of M2 macrophages and improves cartilage tissue regeneration. These studies support that PLPMH scaffold may have a great potential in articular cartilage tissue engineering by providing an anti-inflammatory and pro-regenerative microenvironment.

Periodontal ligament stem cells promote polarization of M2 macrophages.

Macrophages are widely distributed in a variety of tissues, and the different state of macrophages polarization is closely related to the occurrence, development, and prognosis of inflammation, including periodontitis, a chronic inflammatory disease leading to tooth loss worldwide. Periodontal ligament stem cells (PDLSCs) play a key role in immune regulation and periodontal tissues regeneration, contributing to cell-based therapy of periodontitis. However, the interactions between PDLSCs and macrophages are still elusive. The purpose of present study is to investigate the effect of PDLSCs conditioned medium (PDLSCs-CM) on the macrophage polarization and the possible mechanism. PDLSCs were isolated using tissue explant methods and characterized via multipotent differentiation test and examination of expression profiles of mesenchymal stem cells (MSCs) markers. The supernatant of PDLSCs was collected, centrifuged, filtered, and used as PDLSCs-CM. Then, PDLSCs-CM was cocultured with M0 macrophages or IL-4- and IL-13-induced M2 macrophages. The level of surface markers of M1/M2 macrophages and production of several proinflammatory or anti-inflammatory factors were evaluated by flow cytometric analysis and enzyme-linked immunosorbent assay, respectively. The associated genes and proteins involved in the JNK pathway were investigated to explore the potential mechanism that may regulate PDLSCs-CM-mediated macrophage polarization. PDLSCs expressed MSCs markers, including STRO-1, CD146, CD90, and CD73, and were negative for CD34 and CD45, could undergo osteogenic and adipogenic differentiation when cultured in defined medium. After incubation with PDLSCs-CM, no significant increase of CD80+ and HLA-DR+ M1 macrophages was shown while evaluated CD209+ and CD206+ M2 macrophages were observed. In addition, the levels of anti-inflammatory factors such as IL-10, TGF-β, and CCL18 were increased instead of proinflammatory factors such as IL-1β, TNF-α with PDLSC-CM treatment. There was a decrease of JNK expression on M0 macrophages by qRT-PCR analysis and an increase of protein phosphorylation on M0 macrophages after incubation with PDLSCs-CM. Furthermore, as for the enhancement of IL-4- and IL-13-mediated M2 polarization by PDLSCs-CM, the mRNA level of JNK decreased, and the protein phosphorylation level of JNK increased. In addition, the treatment of JNK pathway inhibitor, SP600125, could inhibit the expression and secretion level of anti-inflammatory factor such as IL-10 in M2 polarization induced by PDLSCs-CM. Collectively, PDLSCs were able to induce M2 macrophage polarization instead of M1 polarization, and capable of enhancing M2 macrophage polarization induced by IL-4 and IL-13. The JNK pathway was involved in the promotion of M2 macrophage polarization.

Hypoxic Tumor-Derived Exosomes Induce M2 Macrophage Polarization via PKM2/AMPK to Promote Lung Cancer Progression

Hypoxia is a major regulator of tumor aggressiveness and metastasis in cancer progression. Exosomes (exos) play an important role in the communication between lung cancer and hypoxic microenvironment. However, the underlying mechanisms are largely undefined. Exos were isolated from A549 cells under hypoxia conditions. Transmission electron microscopy and nanoparticle tracking analysis were carried out to characterize exos. CCK-8 assay, flow cytometry, Western blot, wound healing, and transwell assays were performed to assess the proliferation, apoptosis, migration, and invasion of A549 cells, respectively. The M2 polarization of macrophages was evaluated by RT-qPCR and Western blot analysis. In vivo nude mice model was established to determine the regulatory effect of hypoxia/exos on the progression of lung cancer. Hypoxic A549 cell-derived exos (hypoxia/exos) promoted the proliferation and migration, and inhibited the apoptosis in A549 cells. The expression of PKM2 was significantly upregulated in hypoxia/exos. Hypoxic exosomal PKM2 induced M2 polarization of macrophages by activating AMPK pathway. Co-culture with hypoxia/exos-treated macrophages enhanced the migration, invasion, and epithelial-mesenchymal transition (EMT) in A549 cells. Moreover, treatment with hypoxia/exos facilitated the tumor growth and lung metastasis of A549 cells. Our findings reveal that hypoxic exosomal PKM2 induces M2 macrophage polarization via AMPK pathway, and thus exerts a simulative effect on the growth and metastasis of lung carcinoma.

Loss of myeloid cell-specific SIRPα, but not CD47, attenuates inflammation and suppresses atherosclerosis.

Inhibitors of the anti-phagocytic CD47-SIRPα immune checkpoint are currently in clinical development for a variety of haematological and solid tumours. Application of immune checkpoint inhibitors to the cardiovascular field is limited by the lack of preclinical studies using genetic models of CD47 and SIRPα inhibition. In this study, we comprehensively analysed the effects of global and cell-specific SIRPα and CD47 deletion on atherosclerosis development. Here, we show that both SIRPα and CD47 expression are increased in human atherosclerotic arteries and primarily co-localize to CD68+ areas in the plaque region. Hypercholesterolaemic mice homozygous for a Sirpa mutant lacking the signalling cytoplasmic region (Sirpamut/mut) and myeloid cell-specific Sirpa-knockout mice are protected from atherosclerosis. Further, global Cd47-/- mice are protected from atherosclerosis but myeloid cell-specific deletion of Cd47 increased atherosclerosis development. Using a combination of techniques, we show that loss of SIRPα signalling in macrophages stimulates efferocytosis, reduces cholesterol accumulation, promotes lipid efflux, and attenuates oxidized LDL-induced inflammation in vitro and induces M2 macrophage phenotype and inhibits necrotic core formation in the arterial wall in vivo. Conversely, loss of myeloid cell CD47 inhibited efferocytosis, impaired cholesterol efflux, augmented cellular inflammation, stimulated M1 polarization, and failed to decrease necrotic core area in atherosclerotic vessels. Finally, comprehensive blood cell analysis demonstrated lower haemoglobin and erythrocyte levels in Cd47-/- mice compared with wild-type and Sirpamut/mut mice. Taken together, these findings identify SIRPα as a potential target in atherosclerosis and suggest the importance of cell-specific CD47 inhibition as a future therapeutic strategy.