Macrophage-conditioned Medium Research Articles

M2 macrophage-derived exosomal miR-26b-5p regulates macrophage polarization and chondrocyte hypertrophy by targeting TLR3 and COL10A1 to alleviate osteoarthritis

Osteoarthritis (OA) is one of the most prevalent chronic musculoskeletal diseases among the elderly population. In this study, macrophage-derived exosomes were isolated and identified. Exosomes were subjected to microRNA (miRNA) sequencing and bioinformatic analysis, and differentially expressed miRNAs were verified. miR-26b-5p target genes were confirmed through target-site mutation combined with a dual-luciferase reporter assay. The effects of miR-26b-5p on macrophage polarization and chondrocyte hypertrophy were assessed in vitro. miR-26b-5p agomir was applied to mice with OA induced by anterior cruciate ligament transection (ACLT). The therapeutic effects of miR-26b-5p were evaluated via pain behavior experiments and histological observations. In vitro, miR-26b-5p repolarized M1 macrophages to an anti-inflammatory M2 type by targeting the TLR3 signaling pathway. miR-26b-5p could target COL10A1, further inhibiting chondrocyte hypertrophy induced by M1 macrophage-conditioned medium (M1-CM). In vivo, miR-26b-5p agomir ameliorated gait abnormalities and mechanical allodynia in OA mice. miR-26b-5p treatment attenuated synovitis and cartilage degeneration, thereby delaying OA progression. In conclusion, M2 macrophage-derived exosomal miR-26b-5p could protect articular cartilage and ameliorate gait abnormalities in OA mice by targeting TLR3 and COL10A1. miR-26b-5p further affected macrophage polarization and chondrocyte hypertrophy. Thus, this exosomal miR-26b-5p-based strategy might be a potential method for OA treatment.Graphical

Macrophages Promote Subtype Conversion and Endocrine Resistance in Breast Cancer.

The progression of tumors from less aggressive subtypes to more aggressive states during metastasis poses challenges for treatment strategies. Previous studies have revealed the molecular subtype conversion between primary and metastatic tumors in breast cancer (BC). However, the subtype conversion during lymph node metastasis (LNM) and the underlying mechanism remains unclear. We compared clinical subtypes in paired primary tumors and positive lymph nodes (PLNs) in BC patients and further validated them in the mouse model. Bioinformatics analysis and macrophage-conditioned medium treatment were performed to investigate the role of macrophages in subtype conversion. During LNM, hormone receptors (HRs) were down-regulated, while HER2 was up-regulated, leading to the transformation of luminal A tumors towards luminal B tumors and from luminal B subtype towards HER2-enriched (HER2-E) subtype. The mouse model demonstrated the elevated levels of HER2 in PLN while retaining luminal characteristics. Among the various cells in the tumor microenvironment (TME), macrophages were the most clinically relevant in terms of prognosis. The treatment of a macrophage-conditioned medium further confirmed the downregulation of HR expression and upregulation of HER2 expression, inducing tamoxifen resistance. Through bioinformatics analysis, MNX1 was identified as a potential transcription factor governing the expression of HR and HER2. Our study revealed the HER2-E subtype conversion during LNM in BC. Macrophages were the crucial cell type in TME, inducing the downregulation of HR and upregulation of HER2, probably via MNX1. Targeting macrophages or MNX1 may provide new avenues for endocrine therapy and targeted treatment of BC patients with LNM.

Degrees of macrophage-facilitated healing in aneurysm occlusion devices.

Insufficient healing of aneurysms following treatment with vascular occlusion devices put patients at severe risk of fatal rupture. Therefore, promoting healing and not just occlusion is vital to enhance aneurysm healing. Following occlusion device implantation, healing is primarily orchestrated by macrophage immune cells, ending with fibroblasts depositing collagen to stabilize the aneurysm neck and dome, preventing rupture. Several modified occlusion devices are available currently on-market. Previous in vivo work demonstrated that modifications of occlusion devices with a shape memory polymer foam had enhanced aneurysm healing outcomes. To better understand cellular response to occlusion devices and improve aneurysm occlusion device design variables, we developed an in vitro assay to isolate prominent interactions between devices and key healing players: macrophages and fibroblasts. We used THP-1 monocyte derived macrophages and human dermal fibroblasts in our cell culture models. Macrophages were allowed device contact with on-market competitor aneurysm occlusion devices for up to 96 h, to allow for any spontaneous device-driven macrophage activation. Macrophage secreted factors were captured in the culture media, in response to device-specific activation. Fibroblasts were then exposed to device-conditioned macrophage media (with secreted factors alone), to determine if there were any device-induced changes in collagen secretion. Our in vitro studies were designed to test the direct effect of devices on macrophage activation, and the indirect effect of devices on collagen secretion by fibroblasts to promote aneurysm healing and stabilization. Over 96 h, macrophages displayed significant migration toward and interaction with all tested devices. As compared to other devices, shape memory polymer foams (SMM, Shape Memory Medical) induced significant changes in gene expression indicating a shift toward an anti-inflammatory pro-healing M2-like phenotype. Similarly, macrophages in contact with SMM devices secreted more vascular endothelial growth factor (VEGF) compared with other devices. Macrophage conditioned media from SMM-contacted macrophages actively promoted fibroblast secretion of collagen, comparable to amounts observed with exogenous stimulation via VEGF supplementation. Our data indicate that SMM devices may promote good aneurysm healing outcomes, because collagen production is an essential step to ultimately stabilize an aneurysm.

Pentagalloyl glucose induces anti-inflammatory macrophage polarization - suppressing macrophage mediated vascular cell dysfunction and TGF-β secretion.

Background: Pentagalloyl glucose (PGG) is a polyphenol with vasoprotective properties. Targeted delivery of PGG reversed aortic aneurysm growth in several rodent models associated with decreased number of macrophages and transforming growth factor-β (TGF-β) expression. Thus, we sought to determine cellular mechanisms by which PGG reduces macrophage-induced aortic pathogenicity and its relationship to TGF-β. Methods: Using THP-1 cells, primary human aortic cells, and explanted rat aortas, we assessed the anti-inflammatory effect of PGG. Expression of pro/anti-inflammatory macrophage markers was analyzed. Adhesion of monocytes as well as oxidative stress status, viability, and TGF-β expression after primary aortic cell exposure to macrophage-conditioned medium with and without PGG were assessed. The release of TGF-β was also examined in elastase-treated cultured rat aortas. Results: PGG pre-treatment of human aortic cell monolayers reduced the adhesion of THP-1 monocytes. PGG enhanced the expression of anti-inflammatory markers in THP-1-derived macrophages, and increased mitochondrial reactive oxygen species as well as mitochondrial polarization. Conditioned medium from THP-1-derived macrophages induced reactive oxygen species, cell death, and TGF-β release from human aortic cells, which was suppressed by PGG. In explanted rat aortas, PGG reduced elastase mediated TGF-β release. Conclusions: Combining anti-inflammatory, cytotoxic, and oxidative effects, PGG has high cardiovascular therapeutic potential. We confirmed previous in vivo observations whereby PGG suppressed TGF-β response associated with disease resolution.

Bufalin suppresses hepatocellular carcinogenesis by targeting M2 macrophage-governed Wnt1/β-catenin signaling

BackgroundThe interplay of tumor-associated macrophages (TAMs) and tumor cells plays a key role in the development of hepatocellular carcinoma (HCC) and provides an important target for HCC therapy. The communication between them is still on the investigation. Bufalin, the active component derived from the traditional Chinese medicine (TCM) Chansu, has been evidenced to possess anti-HCC activity by directly suppressing tumor cells, while its immunomodulatory effect on the tumor microenvironment (TME) is unclear. PurposeTo explore the mechanism of M2 TAM-governed tumor cell proliferation and the inhibitory effect of bufalin on HCC growth by targeting M2 macrophages. MethodsMorphology and marker proteins were detected to evaluate macrophage polarization via microscopy and flow cytometry. Cellular proliferation and malignant transformation of HCC cells cultured with macrophage conditioned medium (CM) or bufalin-primed M2-CM, were assessed by cell viability, colony formation and soft agar assays. Regulations of gene transcription and protein expression and release were determined by RT-qPCR, immunoblotting, immunoprecipitation, ELISA and immunofluorescence. Tumorigenicity upon bufalin treatment was verified in orthotopic and diethylnitrosamine-induced HCC mouse model. ResultsIn this study, we first verified that M2 macrophages secreted Wnt1, which acted as a mediator to trigger β-catenin activation in HCC cells, leading to cellular proliferation. Bufalin suppressed HCC cell proliferation and malignant transformation by inhibiting Wnt1 release in M2 macrophages, and dose-dependently inhibited HCC progression in mice. Mechanistically, bufalin specially targeted to block Wnt1 transcription, thus inactivating β-catenin signaling cascade in HCC cells and leading to tumor regression in HCC mouse model. ConclusionThese results clearly reveal a novel potential of bufalin to suppress HCC through immunomodulation, and shed light on a new M2 macrophage-based modality of HCC immunotherapy, which additively enhances direct tumor-inhibitory efficacy of bufalin.

Astragaloside IV alleviates macrophage senescence and d-galactose-induced bone loss in mice through STING/NF-κB pathway

BackgroundSenile osteoporosis (SOP) is an age-related metabolic bone disease that currently lacks specific therapeutic interventions. Thus, this study aimed to investigate the effect of Astragaloside IV (AS-IV) on macrophage senescence, bone marrow mesenchymal stem cell (BMSC) osteogenesis, and SOP progression. MethodsA senescent macrophage model was established and treated with varying concentrations of AS-IV. Cell activity was measured using the CCK8 assay. The senescence levels of macrophages were evaluated through β-galactosidase staining, PCR, and immunofluorescence. Macrophage mitochondrial function was assessed using ROS and JC-1 staining. Macrophage polarization was evaluated through PCR, Western blot, and immunofluorescence. The inhibitory effects of AS-IV on macrophage senescence were investigated using Western blot analysis. Furthermore, the effects of macrophage conditioned medium (CM) on BMSCs osteogenic were detected using ALP, alizarin red, and PCR. ResultsAS-IV inhibited macrophage senescence and M1 polarization, alleviated mitochondrial dysfunction, and promoted M2 polarization. Mechanistically, it suppressed the STING/NF-κB pathway in H2O2-activated macrophages. Conversely, the STING agonist c-di-GMP reversed the effects of AS-IV on macrophage senescence. Additionally, AS-IV-induced macrophage CM promoted BMSC osteogenic differentiation. In vivo, AS-IV treatment ameliorated aberrant bone microstructure and bone mass loss in the SOP mouse model, inhibited macrophage senescence, and promoted M2 polarization. ConclusionsBy modulating the STING/NF-κB signaling pathway, AS-IV potentially inhibited macrophage senescence and stimulated osteogenic differentiation of BMSCs, thus exerting an anti-osteoporotic effect. Consequently, AS-IV may serve as an effective therapeutic candidate for the treatment of osteoporosis.

Circular RNA FMN2 motivates colorectal cancer development by mediating tumor-associated macrophage polarization by controlling the microRNA-150-5p/PIK3R3 axis

BackgroundIt has been reported that circular RNA formin 2 (circFMN2) can motivate colorectal cancer (CRC) proliferation. However, the effect of circFMN2 on the polarization of tumor-associated macrophages (TAMs) in the CRC tumor microenvironment remains unclear. The study was to figure out the latent mechanism by which circFMN2 impacts TAM polarization to motivate the malignant behavior of CRC cells. ResultscircFMN2 and PIK3R3 levels were reduced in M1 macrophages but elevated in M2 macrophages, whereas miR-150-5p level was the opposite. circFMN2 knockdown downregulated M2 macrophage markers CD163, CCL22 and CD206 and upregulated M1 macrophage markers CD86, TNF-α and IL-1β in M2 macrophages. Co-culture with M2 macrophage-conditioned medium with circFMN2 knockdown reduced CRC proliferation, invasion, and migration, while knockdown of miR-150-5p had the opposite effect. CircFMN2 adsorbed miR-150-5p to mediate PIK3R3 in M2 macrophages. Overexpression of miR-150-5p can reverse the promoting effects of overexpression of circFMN2 on M2 polarization, CRC cell proliferation, invasion, and migration. Elevation of PIK3R3 could turn around the repressive effect of circFMN2 knockdown on M2 polarization and CRC cell proliferation, invasion, and migration. In an in vivo model, M2 macrophages expressing low or high circFMN2 were co-transplanted with CRC cells into nude mice, resulting in inhibition and promotion of tumor growth, respectively. ConclusionsAll in all, circFMN2 mediates TAM polarization to M2 type by controlling the miR-150-5p/PIK3R3 axis to motivate CRC development and may offer a latent molecular target for CRC treatment.How to cite: Cao Y, Cao D, Zhu T. Circular RNA FMN2 motivates colorectal cancer development by mediating tumor-associated macrophage polarization by controlling the microRNA-150-5p/PIK3R3 axis. Electron J Biotechnol 2024;68. https://doi.org/10.1016/j.ejbt.2023.12.002.

M2a macrophages regulate fibrosis and affect the outcome after stroke via PU.1/mTOR pathway in fibroblasts

The moderate formation of the fibrotic scar plays an important role in functional recovery after stroke. M2a macrophages have been identified as an important source of early fibrosis after cerebral ischemia. However, the underlying mechanisms by which macrophages interact with fibroblasts in this context remain largely unknown. Therefore, our study aimed to further investigate the potential mechanisms underlying the effects of macrophages on fibroblasts following ischemic stroke. In vitro and in vivo, recombinant rat interleukin 4 (IL4) was used to induce macrophages to polarize into M2a macrophages. In vitro, primary Sprague–Dawley newborn rat meningeal-derived fibroblasts were treated with PU.1 knockdown, the PU.1 inhibitor DB1976 or the mTOR inhibitor rapamycin, which were then co-cultured with M2a macrophage conditioned medium (MCM). In vivo, Sprague–Dawley adult rats were infected with negative control adenoviruses or PU.1-shRNA adenoviruses. Ten days after infection, an injury model of middle cerebral artery occlusion/reperfusion (MCAO/R) was constructed. Subsequently, IL4 was injected intracerebroventricularly to induce M2a macrophages polarization. In vitro, M2a MCM upregulated PU.1 expression and promoted the differentiation, proliferation, migration and extracellular matrix generation of fibroblasts, which could be reversed by treatment with the PU.1 inhibitor DB1976 or PU.1 knockdown. In vivo, PU.1 expression in fibroblasts was increased within ischemic core following MCAO/R, and this upregulation was further enhanced by exposure to IL4. Treatment with IL4 promoted fibrosis, increased angiogenesis, reduced apoptosis and infarct volume, as well as mitigated neurological deficits after MCAO/R, and these effects could be reversed by PU.1 knockdown. Furthermore, both in vivo and in vitro studies showed that IL4 treatment increased the levels of phosphorylated Akt and mTOR proteins, which were markedly decreased by PU.1 knockdown. Additionally, the use of an mTOR inhibitor rapamycin obviously suppressed the migration and differentiation of fibroblasts, and Col1 synthesis. In conclusion, our findings suggest for the first time that M2a macrophages, at least in part, regulate fibrosis and affect the outcome after cerebral ischemic stroke via the PU.1/mTOR signaling pathway in fibroblasts.

HOW COULD MSC-POLARIZED MACROPHAGES PROMOTE CARTILAGE REPAIR?

In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription. The ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFβ) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90). Mo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair.

Less is more! Low amount of Fusobacterium nucleatum supports macrophage-mediated trophoblast functions in vitro.

F. nucleatum, involved in carcinogenesis of colon carcinomas, has been described as part of the commensal flora of the female upper reproductive tract. Although its contribution to destructive inflammatory processes is well described, its role as commensal uterine bacteria has not been thoroughly investigated. Since carcinogenesis shares similar mechanisms with early pregnancy development (including proliferation, invasion, blood supply and the induction of tolerance), these mechanisms induced by F. nucleatum could play a role in early pregnancy. Additionally, implantation and placentation require a well-balanced immune activation, which might be suitably managed by the presence of a limited amount of bacteria or bacterial residues. We assessed the effect of inactivated F. nucleatum on macrophage-trophoblast interactions. Monocytic cells (THP-1) were polarized into M1, M2a or M2c macrophages by IFN-γ, IL-4 or TGF-β, respectively, and subsequently treated with inactivated fusobacteria (bacteria:macrophage ratio of 0.1 and 1). Direct effects on macrophages were assessed by viability assay, flow cytometry (antigen presentation molecules and cytokines), qPCR (cytokine expression), in-cell Western (HIF and P-NF-κB) and ELISA (VEGF secretion). The function of first trimester extravillous trophoblast cells (HTR-8/SVneo) in response to macrophage-conditioned medium was microscopically assessed by migration (scratch assay), invasion (sprouting assay) and tube formation. Underlying molecular changes were investigated by ELISA (VEGF secretion) and qPCR (matrix-degrading factors and regulators). Inflammation-primed macrophages (M1) as well as high bacterial amounts increased pro-inflammatory NF-κB expression and inflammatory responses. Subsequently, trophoblast functions were impaired. In contrast, low bacterial stimulation caused an increased HIF activation and subsequent VEGF-A secretion in M2c macrophages. Accordingly, there was an increase of trophoblast tube formation. Our results suggest that a low-mass endometrial/decidual microbiome can be tolerated and while it supports implantation and further pregnancy processes.

QiShenYiQi pills preserve endothelial barrier integrity to mitigate sepsis-induced acute lung injury by inhibiting ferroptosis

Ethnopharmacological relevanceThe QiShengYiQi pill (QSYQ) is a traditional Chinese medicinal formulation. The effectiveness and safety of QSYQ in treating respiratory system disorders have been confirmed. Its pharmacological actions include anti-inflammation, antioxidative stress, and improving energy metabolism. However, the mechanism of QSYQ in treating sepsis-induced acute lung injury (si-ALI) remains unclear. Aim of the studySi-ALI presents a clinical challenge with high incidence and mortality rates. This study aims to confirm the efficacy of QSYQ in si-ALI and to explore the potential mechanisms, providing a scientific foundation for its application and insights for optimizing treatment strategies and identifying potential active components. Materials and methodsThe impact of QSYQ on si-ALI was evaluated using the cecal ligation and puncture (CLP) experimental sepsis animal model. The effects of QSYQ on endothelial cells were observed through coculturing with LPS-stimulated macrophage-conditioned medium. Inflammatory cytokine levels, HE staining, Evans blue staining, lung wet/dry ratio, and cell count and protein content in bronchoalveolar lavage fluid were used to assess the degree of lung injury. Network pharmacology was utilized to investigate the potential mechanisms of QSYQ in treating si-ALI. Western blot and immunofluorescence analyses were used to evaluate barrier integrity and validate mechanistically relevant proteins. ResultsQSYQ reduced the inflammation and alleviated pulmonary vascular barrier damage in CLP mice (all P < 0.05). A total of 127 potential targets through which QSYQ regulates si-ALI were identified, predominantly enriched in the RAGE pathway. The results of protein-protein interaction analysis suggest that COX2, a well-established critical marker of ferroptosis, is among the key targets. In vitro and in vivo studies demonstrated that QSYQ mitigated ferroptosis and vascular barrier damage in sepsis (all P < 0.05), accompanied by a reduction in oxidative stress and the inhibition of the COX2 and RAGE (all P < 0.05). ConclusionsThis study demonstrated that QSYQ maintains pulmonary vascular barrier integrity by inhibiting ferroptosis in CLP mice. These findings partially elucidate the mechanism of QSYQ in si-ALI and further clarify the active components of QSYQ, thereby providing a scientific theoretical basis for treating si-ALI with QSYQ.

Abstract A122: Deciphering macrophage targeting cancer immunotherapies using a novel in vitro assay modelling the tumor microenvironment

Abstract Background: Macrophages are one of the major infiltrating immune cells in the tumor microenvironment and have been proposed as a primary driver of epithelial to mesenchymal transition (EMT) through interactions with tumour cells. These interactions play a key role in tumor progression, metastasis and fibrosis, processes which are common to all types of cancer. Understandably therefore, targeting macrophages is an attractive approach for treating wide range of tumors including those resistant to checkpoint inhibitors. Evaluation of cancer therapeutics is hampered by a lack of suitable in vitro models which closely recapitulate the complexity of tumor microenvironment. We addressed this challenge by developing a model system with potential to faithfully represent the key molecular interactions occurring between tumor associated macrophages and cancer cells, driving the latter to become invasive and metastatic. In order to demonstrate suitability of the assay, galunisertib, a TGFbRI/ALK5 inhibitor was used to block one of pathways that has been evaluated clinically in cancer patients. Methods: Human macrophages were differentiated using key growth factors prior to polarisation with various cytokines to generate different putative macrophage phenotypes as evidenced by differences in cytokine secretion, gene transcription and protein expression. Epithelial cells were stimulated with macrophage conditioned medium in a scratch wound assay (Incucyte) and analysed by immunocytochemistry. Results: Macrophages derived supernatants were shown to drive EMT, both in migratory capacity and EMT associated protein expression. Our results demonstrate that whilst galunisertib could potently inhibit TGF-b driven EMT it was only partly capable of reversing the effects associated with addition of macrophage conditioned media. Conclusions: The data show that macrophages can directly drive the EMT process in vitro via multiple mechanisms. One such mechanism is through the secretion of TGF-b. Indeed, blockade of TGF-b signalling appears to be a tangible approach in cancer immunotherapy, but to date this approach has delivered mixed results for clinical efficacy as a monotherapy. By way of example, development of galunisertib has recently been halted. As shown herein, macrophages effects on cancer migration and metastasis span beyond TGF-b only effects. We offer a potential explanation for why single therapies such as galunisertib alone are ineffective in cancer treatment. The described model provides a powerful tool to researchers to better understand and replicate in vitro the complex interactions occurring in the tumor microenvironment. Thus, enabling the identification of molecular targets and combination therapies with potential to block cancer cell migration and metastasis and to evaluate candidate drug efficacy. Citation Format: Darryl Turner, Ross Dobie, Justyna Rzepecka, Hayley Gooding, Matilda Bingham. Deciphering macrophage targeting cancer immunotherapies using a novel in vitro assay modelling the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A122.

Nanoscaled biphasic calcium phosphate modulates osteogenesis and attenuates LPS-induced inflammation

Micron-scale structure biphasic calcium phosphate (BCP) materials have demonstrated promising clinical outcomes in the field of bone tissue repair. However, research on biphasic calcium phosphate materials at the nanoscale level remains limited. In this study, we synthesize granular-shaped biphasic calcium phosphate nanomaterials with multiple desirable characteristics, including negatively charged surfaces, non-cytotoxicity, and the capability to penetrate cells, using a nanogrinding dispersion process with a polymeric carboxylic acid as the dispersant. Our results reveal that treating human osteoblasts with 0.5 μg/mL biphasic calcium phosphate nanomaterials results in a marked increase in alkaline phosphatase (ALP) activity and the upregulation of osteogenesis-related genes. Furthermore, these biphasic calcium phosphate nanomaterials exhibit immunomodulatory properties. Treatment of THP-1-derived macrophages with BCP nanomaterials decreases the expression of various inflammatory genes. Biphasic calcium phosphate nanomaterials also mitigate the elevated inflammatory gene expression and protein production triggered by lipopolysaccharide (LPS) exposure in THP-1-derived macrophages. Notably, we observe that biphasic calcium phosphate nanomaterials have the capacity to reverse the detrimental effects of LPS-stimulated macrophage-conditioned medium on osteoblastic activity and mineralization. These findings underscore the potential utility of biphasic calcium phosphate nanomaterials in clinical settings for the repair and regeneration of bone tissue. In conclusion, this study highlights the material properties and positive effects of biphasic calcium phosphate nanomaterials on osteogenesis and immune regulation, opening a promising avenue for further research on inflammatory osteolysis in patients undergoing clinical surgery.

3D printed strontium-doped calcium phosphate ceramic scaffold enhances early angiogenesis and promotes bone repair through the regulation of macrophage polarization

The vascularization of bone repair materials is one of the key issues that urgently need to be addressed in the process of bone repair. The changes in macrophage phenotype and function play an important role in the process of vascularization, and endowing bone repair materials with immune regulatory characteristics to enhance angiogenesis is undoubtedly a new strategy to improve the effectiveness of bone repair. In order to improve the effect of tricalcium phosphate (TCP) on vascularization and bone repair, we doped strontium ions (Sr) into TCP (SrTCP) and prepared porous 3D printed SrTCP scaffolds using 3D printing technology, and studied the scaffold mediated macrophage polarization and subsequent vascularization and bone regeneration. The results of the interaction between the scaffold and macrophages showed that the SrTCP scaffold can promote the polarization of macrophages from M1 to M2 and secrete high concentrations of VEGF and PDGF-bb cytokines, which shows excellent angiogenic potential. When human umbilical vein endothelial cells (HUVECs) were co-cultured with macrophage-conditioned medium of SrTCP scaffold, HUVECs exhibited excellent early angiogenesis-promoting effects in terms of scratch healing, angiogenic gene expression, and in vitro tube formation performance. The results of in vivo bone repair experiments showed that the SrTCP scaffold formed a vascular network with high density and quantity in the bone defect area, which could increase the rate of new bone formation and advance the period of bone formation, and finally achieved a better bone repair effect. We observed a cascade effect in which Sr-doped SrTCP scaffold regulate macrophage polarization to enhance angiogenesis and promote bone repair, which may provide a new strategy for the repair of clinical bone defects.

HOW COULD MSC-POLARIZED MACROPHAGES PROMOTE CARTILAGE REPAIR?

AbstractObjectivesIn relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome.MethodsHuman monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription.ResultsThe ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFa) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90).ConclusionsMo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

A Biomimetic Se-nHA/PC Composite Microsphere with Synergistic Immunomodulatory and Osteogenic Ability to Activate Bone Regeneration in Periodontitis.

Accumulation of reactive oxygen species (ROS) in periodontitis exacerbates the destruction of alveolar bone. Therefore, scavenging ROS to reshape the periodontal microenvironment, alleviate the inflammatory response and promote endogenous stem cell osteogenic differentiation may be an effective strategy for treating bone resorption in periodontitis. In this study, sericin-hydroxyapatite nanoparticles (Se-nHA NPs) are synthesized using a biomimetic mineralization method. Se-nHA NPs and proanthocyanidins (PC) are then encapsulated in sericin/sodium alginate (Se/SA) using an electrostatic injection technique to prepare Se-nHA/PC microspheres. Microspheres are effective in scavenging ROS, inhibiting the polarization of macrophages toward the M1 type, and inducing the polarization of macrophages toward the M2 type. In normal or macrophage-conditioned media, the Se-nHA/PC microspheres effectively promoted the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). Furthermore, the Se-nHA/PC microspheres demonstrated anti-inflammatory effects in a periodontitis rat model by scavenging ROS and suppressing pro-inflammatory cytokines. The Se-nHA/PC microspheres are also distinguished by their capacity to decrease alveolar bone loss, reduce osteoclast activity, and boost osteogenic factor expression. Therefore, the biomimetic Se-nHA/PC composite microspheres have efficient ROS-scavenging, anti-inflammatory, and osteogenic abilities and can be used as a multifunctional filling material for inflammatory periodontal tissue regeneration.

Dapagliflozin promotes angiogenesis in hindlimb ischemia mice by inducing M2 macrophage polarization.

Critical limb ischemia (CLI) is associated with a higher risk of limb amputation and cardiovascular death. Dapagliflozin has shown great potential in the treatment of cardiovascular disease. However, the effects of dapagliflozin on CLI and the underlying mechanisms have not been fully elucidated. We evaluated the effect of dapagliflozin on recovery from limb ischemia using a mouse model of hindlimb ischemia. The flow of perfusion was evaluated using a laser Doppler system. Tissue response was assessed by analyzing capillary density, arterial density, and the degree of fibrosis in the gastrocnemius muscle. Immunofluorescence and Western blot were used to detect the expression of macrophage polarization markers and inflammatory factors. Our findings demonstrate the significant impact of dapagliflozin on the acceleration of blood flow recovery in a hindlimb ischemia mouse model, concomitant with a notable reduction in limb necrosis. Histological analysis revealed that dapagliflozin administration augmented the expression of key angiogenic markers, specifically CD31 and α-SMA, while concurrently mitigating muscle fibrosis. Furthermore, our investigation unveiled dapagliflozin's ability to induce a phenotypic shift of macrophages from M1 to M2, thereby diminishing the expression of inflammatory factors, including IL-1β, IL-6, and TNF-α. These effects were partially mediated through modulation of the NF-κB signaling pathway. Lastly, we observed that endothelial cell proliferation, migration, and tube-forming function are enhanced in vitro by utilizing a macrophage-conditioned medium derived from dapagliflozin treatment. Taken together, our study provides evidence that dapagliflozin holds potential as an efficacious therapeutic intervention in managing CLI by stimulating angiogenesis, thereby offering a novel option for clinical CLI treatment.

Mechanism of M2 type macrophage-derived extracellular vesicles regulating PD-L1 expression via the MISP/IQGAP1 axis in hepatocellular carcinoma immunotherapy resistance

BackgroundHepatocellular carcinoma (HCC) is a prevailing cancer affecting human health. M2 macrophages are essential in mediating immune responses in tumors. This study investigated the action of M2 macrophages in immune escape of HCC. MethodsMitotic spindle positioning (MISP), IQ motif containing GTPase activating protein 1 (IQGAP1) and programmed cell death-1 (PD-L1) levels in primary HCC/tumor-adjacent tissues were determined by Western blot, followed by correlation analysis. M2 macrophage and CD3+CD8+T cell percentages were estimated by flow cytometry. Hep3B and HepG2 cells were treated with M2 macrophage conditioned medium (M2-CM) and M2 macrophage-derived extracellular vesicles (M2-EVs) and/or co-cultured with CD8+T cells, followed by assessment of cell viability and apoptosis. TNF-α and INF-γ levels were measured by ELISA. MISP and IQGAP1 overexpression plasmids were transfected into HCC cells to explore their role in immune escape. The interactions among MISP, IQGAP1, STAT3, and PD-L1 were analyzed by co-immunoprecipitation. The mechanism of M2-EVs in HCC immune escape was verified in nude mice. ResultsMISP/IQGAP1/PD-L1 were upregulated in HCC tissues. MISP negatively-correlated with IQGAP1/PD-L1 and IQGAP1 positively-correlated with PD-L1. M2 macrophages were reduced but CD8+T cells were increased in HCC tissues with high MISP expression. M2-CM or M2-EVs inhibited the killing ability of CD8+T cells, increased HCC cell viability, impeded HCC cell apoptosis, induced CD8+T cell apoptosis, downregulated TNF-α and INF-γ, and upregulated PD-L1. M2-EVs facilitated HCC cell immune escape by potentiating IQGAP1 nuclear translocation and activating STAT3 phosphorylation through MISP downregulation. In vivo experiments further verified the action of M2-EVs through MISP. ConclusionM2-EVs promote HCC cell immune escape by upregulating PD-L1 through the MISP/IQGAP1/STAT3 axis.

A combination of 5/6-nephrectomy and unilateral ureteral obstruction model accelerates progression of remote organ fibrosis in chronic kidney disease.

Chronic kidney disease (CKD) involves progressive renal fibrosis, which gradually reduces kidney function and often causes various complications in extrarenal tissues. Therefore, we investigated fibrogenesis in extrarenal tissues (heart, liver, and lungs) in different experimental CKD models, such as the 5/6-nephrectomy (5/6 Nx), unilateral ureteral obstruction (UUO), and a combination (2/3 Nx + UUO). We evaluated the degree of fibrogenesis in kidneys and extrarenal tissues by histological analysis and quantification of fibrosis-related gene and protein expression. To elucidate the fibrosis mechanisms observed in 2/3 Nx + UUO mice, we evaluated the effect of indoxyl sulfate (IS), a typical uremic toxin accumulated in CKD, and transforming growth factor-β (TGF-β), a fibrosis-related factor, on fibrosis using human hepatoma (HepG2) and RAW264.7 cells. A significant decline in renal function was observed in the 5/6 Nx and 2/3 Nx + UUO models, whereas a significant increase in renal fibrosis was observed only in the obstructed kidneys. Notable amount of fibrosis was induced in the liver and heart in the 2/3 Nx + UUO model, with the induction of macrophage infiltration and increased tissue IS and TGF-β levels. In agreement with the results of in vivo experiments, co-stimulation with IS, TGF-β, and macrophage-conditioned medium increased the expression of fibrogenic genes in HepG2 cells. We demonstrated that the 2/3 Nx + UUO model induced both loss of renal function and renal fibrosis in the earlier stages, providing a novel CKD model that induces remote organ fibrosis in a shorter time.

MED1 induces M2 polarization of tumor-associated macrophages to aggravate breast cancer.

Breast cancer is a common malignant tumor in female, and its 5-year survival rate remains low. The correlation between mediator subunit 1 (MED1) gene and macrophage phenotypic transformation may be a key factor affecting the therapeutic effect on cancer. The present study intended to explore the role of MED1 in macrophage polarization and its further influence on the malignant behaviors of breast cancer. Bioinformatics analysis was carried out to predict the expression pattern of MED1 in breast cancer. Flow cytometry was conducted to detect the effect of MED1 overexpression or silencing on macrophage polarization. ELISA was applied to analyze the effect of abnormal MED1 expression on cytokine secretion of macrophages. CCK-8, colony formation, Transwell and scratch healing assays were applied to investigate the effects of macrophage conditioned medium on the malignant behaviors of breast cancer cells. MED1 expression was prominently increased in M2 macrophages, and overexpression of MED1 significantly increased M2 polarization of tumor-associated macrophages (TAMs) and IL-10 cytokine level. Meanwhile, M2 macrophages with MED1 overexpression could significantly promote the malignant behaviors of breast cancer cells. Dasatinib rescue experiment further confirmed that MED1-induced M2 macrophage polarization could facilitate the malignant progression of breast cancer cells. In summary, MED1 could induce M2 macrophage polarization and thus regulate the malignant behaviors of breast cancer cells.