M2 Macrophage Polarization Research Articles

Vitamin D 1,25-Dihydroxyvitamin D3 reduces lipid accumulation in hepatocytes by inhibiting M1 macrophage polarization

BACKGROUND Non-alcoholic fatty liver disease (NAFLD), which is a significant liver condition associated with metabolic syndrome, is the leading cause of liver diseases globally and its prevalence is on the rise in most nations. The protective impact of vitamin D on NAFLD and its specific mechanism remains unclear. AIM To examine the role of vitamin D in NAFLD and how vitamin D affects the polarization of hepatic macrophages in NAFLD through the vitamin D receptor (VDR)-peroxisome proliferator activated receptor (PPAR)γ pathway. METHODS Wild-type C57BL/6 mice were provided with a high-fat diet to trigger NAFLD model and administered 1,25-dihydroxy-vitamin D [1,25(OH)2D3] supplementation. 1,25(OH)2D3 was given to RAW264.7 macrophages that had been treated with lipid, and a co-culture with AML12 hepatocytes was set up. Lipid accumulation, lipid metabolism enzymes, M1/M2 phenotype markers, proinflammatory cytokines and VDR-PPARγ pathway were determined. RESULTS Supplementation with 1,25(OH)2D3 relieved hepatic steatosis and decreased the proinflammatory M1 polarization of hepatic macrophages in NAFLD. Administration of 1,25(OH)2D3 suppressed the proinflammatory M1 polarization of macrophages induced by fatty acids, thereby directly relieving lipid accumulation and metabolism in hepatocytes. The VDR-PPARγ pathway had a notable impact on reversing lipid-induced proinflammatory M1 polarization of macrophages regulated by the administration of 1,25(OH)2D3. CONCLUSION Supplementation with 1,25(OH)2D3 improved hepatic steatosis and lipid metabolism in NAFLD, linked to its capacity to reverse the proinflammatory M1 polarization of hepatic macrophages, partially by regulating the VDR-PPARγ pathway. The involvement of 1,25(OH)2D3 in inhibiting fatty-acid-induced proinflammatory M1 polarization of macrophages played a direct role in relieving lipid accumulation and metabolism in hepatocytes.

The CCL5/CCR5/SHP2 axis sustains Stat1 phosphorylation and activates NF-κB signaling promoting M1 macrophage polarization and exacerbating chronic prostatic inflammation

Background and objectiveChronic prostatitis (CP) is a condition markered by persistent prostate inflammation, yet the specific cytokines driving its progression remain largely undefined. This study aims to identify key cytokines involved in CP and investigate their role in driving inflammatory responses through mechanistic and therapeutic exploration.MethodsA 48-cytokine panel test was conducted to compare the plasma cytokine profiles between participants with CP-like symptoms (CP-LS) and healthy controls. Experimental autoimmune prostatitis (EAP) models were used for functional validation, with further mechanistic studies performed through in vivo and in vitro assays. Pharmacological inhibition was applied using maraviroc, and pathway inhibitors to assess therapeutic potential.ResultsOur analysis identified CCL5 as one of the most prominently elevated cytokines in CP-LS patients. Further validation in the EAP model mice confirmed elevated CCL5 levels, highlighting its role in driving prostatic inflammation. Mechanistic studies revealed that CCL5 interacts with the CCR5 receptor, promoting M1 macrophage polarization and activating key inflammatory signaling pathways, including Stat1 and NF-κB, as indicated by increased phosphorylation of Stat1 and p65. In vitro, CCL5 combined with LPS stimulation amplified these effects, further promoting M1 polarization. CCL5 also sustained Stat1 activation by inhibiting its dephosphorylation through reduced interaction with SHP2, leading to prolonged inflammatory signaling. Single-cell transcriptomics confirmed high CCR5 expression in macrophages, correlating with inflammatory pathways. Pharmacological inhibition of CCR5, or its downstream signaling, significantly reduced macrophage-driven inflammation both in vivo and in vitro.ConclusionThese findings establish the CCL5/CCR5 axis as a critical driver of persistant prostatic inflammation and present it as a potential therapeutic target for CP.Graphic The graphic abstract illustrates the central role of the CCL5/CCR5 axis in promoting chronic prostatitis progression. CCL5 binds to the CCR5 receptor on macrophages, enhancing M1 polarization and activating key inflammatory pathways, including Stat1 and NF-κB, as shown by the increased phosphorylation of Stat1 and p65. The interaction also inhibits SHP2-mediated Stat1 dephosphorylation, sustaining prolonged inflammatory signaling. Pharmacological inhibition of CCR5 or its downstream pathways attenuates macrophage-driven inflammation, highlighting this axis as a critical driver and potential therapeutic target for chronic prostatitis.

T follicular helper cell is essential for M2 macrophage polarization and pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension

BackgroundHypoxia-induced pulmonary hypertension (HPH) is a subgroup of type 3 pulmonary hypertension that may cause early right ventricular failure and eventual cardiac failure, which lacks potential therapeutic targets. Our previous research demonstrated that T follicular helper (TFH) cells that produce IL-21 were involved in HPH. However, the molecular mechanisms of TFH/IL-21-mediated pathogenesis of HPH have been elusive. Here we investigate the role of TFH cells and IL-21 in HPH.MethodsStudies were performed in C57BL/6 mice or IL-21 knockout mice exposed to chronic hypoxia to induce PH, and examined by hemodynamics. Molecular and cellular studies were performed in mouse lung and pulmonary arterial smooth muscle cells (PASMCs). M2 signature gene (Fizz1), M1 signature genes (iNos, IL-12β and MMP9), GC B cell and its marker GL-7, caspase-1, M2 macrophages, TFH cells, Bcl-6 and IL-21 level were measured. Proliferation rate of PASMCs was measured by EdU. Pyroptosis was assessed using Hoechst 33,342/PI double fluorescent staining.ResultsIn response to chronic hypoxia exposure-induced pulmonary hypertension, IL-21−/− mice or downregulation of TFH cells in WT mice developed blunted pulmonary hypertension, attenuated pulmonary vascular remodelling. Furthermore, chronic hypoxia exposure significantly increased the germinal center (GC) B cell responses, which were not present in IL-21−/− mice or downregulation of TFH cells in WT mice. Importantly, IL-21 promoted the polarization of primary alveolar macrophages toward the M2 phenotype. Consistently, significantly enhanced expression of M2 macrophage marker Fizz1 were detected in the bronchoalveolar lavage fluid of HPH mice. Moreover, alveolar macrophages that had been cultivated with IL-21 promoted PASMCs proliferation and pyroptosis in vitro, while a selective CX3CR1 antagonist, AZD8797 (AZD), significantly attenuated the proliferation and pyroptosis of the PASMCs. Finally, ECM1 knockdown promoted IL-2–STAT5 signaling and inhibited Bcl-6 signaling to inhibit TFH differentiation in HPH.ConclusionsTFH/IL-21 axis amplified pulmonary vascular remodelling in HPH. This involved M2 macrophage polarization, PASMCs proliferation and pyroptosis. These data suggested that TFH/IL-21 axis may be a novel therapeutic target for the treatment of HPH.

ROS Responsive Cerium Oxide Biomimetic Nanoparticles Alleviates Calcium Oxalate Crystals Induced Kidney Injury via Suppressing Oxidative Stress and M1 Macrophage Polarization.

Emerging studies have demonstrated that M1 macrophage polarization and oxidative stress play important roles in calcium oxalate (CaOx) induced kidney injury, which leads to increased crystals deposition. ROS scavenging nanozymes and kidney-targeted nanoparticles for antioxidant drugs delivery have emerged as an arisen methodology for kidney injury therapy. However, cell membrane biomimetic-modified nanozymes as anti-inflammatory drug delivery systems for the treatment of kidney injury is rarely reported. Herein, the ROS responsive red blood cell-membrane-coated resatorvid-loaded cerium oxide nanoparticles (RBCM@CeO2/TAK-242) are constructed to suppress CaOx induced kidney injury and crystals deposition. In vitro, RBCM@CeO2/TAK-242 shows effective internalization by renal tubular epithelial cells, along with demonstrated antioxidative, anti-inflammatory, and macrophage reprogramming effects. Glyoxalate(Gly)-induced renal CaOx crystals mouse model is established, RBCM@CeO2/TAK-242 shows excellent injured kidney targeting and biosafety, and could effectively suppress CaOx induced kidney injury and crystals deposition. RBCM@CeO2/TAK-242 has a dual protective effect by both inhibiting oxidative stress and modulating macrophage polarization in vivo. In addition, RNA seq analysis reveals that RBCM@CeO2/TAK-242 protects against CaOx induced kidney injury via suppressing the TLR4/NF-κB pathway. This study provides an innovative strategy for RBCM@CeO2/TAK-242 as injured kidney targeting and dual protective effects for the treatment of CaOx induced kidney injury and crystals deposition.

Locally Injectable, ROS-Scavenging, and ROS-/pH-Responsive Polymeric-Micelles-Embedded Hydrogels for Precise Minimally Invasive and Long-Lasting Rheumatoid Therapy.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovitis, bone-erosion, and joint-destruction. Here, we developed a locally injectable, ROS-scavenging, and ROS-/pH-responsive drug-delivery platform (HC@PTM) by bio-compositing of aldolizing hyaluronic acid (HA) crosslinked with chitosan (CS), and ROS-triggered/eliminated micelles (PTM) coupled with the drug methotrexate(MTX). The PTM efficiently eradicate excessive ROS in RA-joints, precisely triggering drug-release within inflamed arthritic-sites and further confer exceptional antioxidant origins to HC@PTM. HC@PTM with outstanding shape-adaptability and self-repairing properties effectively conformed to irregular articular cartilage while resisting joint-induced deformations. Further, the platform's pH-responsive nature enables on-demand drug-release within acidic inflamed synovium, serving as a drug-reservoir for precise and sustained therapeutic effects. Extensive in vitro and in vivo investigations confirm HC@PTM's ability to induce M2 macrophage polarization, downregulate inflammatory factor expression, and ameliorate the RA-microenvironment, ultimately achieving synergistic therapeutic outcomes. This study represents significant advancements in precise and long-term RA-treatment through a minimally invasive approach, offering potential strategies for novel precision medicine.

Ionizable cationic lipid nanoparticles loaded with miRNA-125b/BLZ945 for pancreatic cancer treatment.

In prior research, both miRNA-125b and BLZ945 have shown potential in effectively inhibiting M2 macrophage polarization and producing antitumor effects. Nevertheless, their physicochemical characteristics present significant challenges for efficient in vivo delivery. Ionizable cationic lipid nanoparticles (LNPs), recognized for their superior biocompatibility and drug-loading capacity, serve as a novel carrier for nucleic acid-based therapeutics. In our study, we successfully encapsulated both agents within LNPs and conducted a thorough characterization. Subsequently, we investigated their potential to repolarize M2 macrophages in vitro and evaluated their in vivo distribution, biosafety, and antitumor efficacy. The findings revealed that the LNPs maintained excellent drug-loading efficiency, consistent particle size, and stable zeta potential. All formulations effectively inhibited M2 macrophage polarization in vitro. Upon administration in vivo, the LNPs not only demonstrated favorable biosafety profiles but also accumulated efficiently in tumor tissues, substantially reducing tumor burden, particularly notable in co-loaded LNPs. Our results affirm that LNPs are an effective carrier for miRNA-125b and BLZ945, highlighting this encapsulation approach as promising for the treatment of solid tumors and meriting further investigation. Practitioner points: (i) Ionizable cationic nanoparticles provide high and stable encapsulation rates to efficiently load nucleic acid polymers into the LNP, avoiding the rapid accumulation of circulating macrophages, which can lead to reduced penetration of the LNP into target tissues. Therefore, it can be used as a novel drug delivery method to benefit clinical patients. (ii) miRNA-125b LNP/BLZ945 LNP attenuated the depleting effect of BLZ945 on macrophages and significantly inhibited macrophage M2 polarization. It could be effectively distributed in tumors and showed good biosafety while exerting antitumor effects, bringing hope to clinical pancreatic tumor patients.

Human leukocyte antigen DR alpha inhibits renal cell carcinoma progression by promoting the polarization of M2 macrophages to M1 via the NF-κB pathway

Human leukocyte antigen DR alpha (HLA-DRA) is recognized for its inhibitory effect on the progression of clear cell renal cell carcinoma (ccRCC); high HLA-DRA expression levels are positively correlated with improved prognosis in patients with ccRCC. In this study, we evaluated HLA-DRA expression in ccRCCs, its effects on tumor-associated macrophage recruitment, and the influence of polarization. Clinical cohort analyses revealed that elevated HLA-DRA expression in ccRCC cells was correlated with enhanced tumor infiltration by M1-type macrophages. In addition, ccRCC prognosis was predicted by combining HLA-DRA expression level analysis and the M1/M2 macrophage ratio. In vitro studies demonstrated that ccRCC cells with increased HLA-DRA expression promoted THP-1 cell migration and induced macrophage polarization toward the M1 phenotype. The effect was further substantiated in a mouse xenograft model in which an increase in M1 macrophages was observed. In addition, co-culturing macrophages with the supernatant from cells overexpressing HLA-DRA induced the expression of proteins associated with both M1 and M2 macrophage polarization. HLA-DRA was intricately linked to the expression and secretion of chemokines, including CCL2, CCL5, MIP-1ɑ, and CXCL-10. Moreover, the NF-κB pathway activation promoted polarization to M1 macrophages. This study shows that HLA-DRA and the M1/M2 ratio are indicators of favorable prognosis in patients with ccRCC. HLA-DRA promotes M1-like polarization by regulating NF-κB, which can be used as a therapeutic target to enhance anti-tumor immunity.

Mincle Maintains M1 Polarization of Macrophages and Contributes to Renal Aging Through the Syk/NF-κB Pathway.

Kidney is a classic organ undergoing senescence, and chronic inflammation has an important effect in cellular senescence. Mincle has been shown to be vital for maintaining the M1 phenotype of macrophages, but its role in regulating renal aging has yet to be explored. Young (2 months of age) and old (24 months of age) mice were used to analyze the changes of kidney damage during natural aging. Mice were subcutaneously injected with D-galactose (D-gal) to establish a renal aging model, and miR-6948-3p mimic and Mincle siRNA were administered via the tail vein every 3 days. Aged kidney and experimental aging kidney were characterized by decreased renal function and structural damage, and upregulated expression of senescence-related proteins and SPAP components. The ratio of M1 macrophages was increased in the aged kidney, and Mincle accumulated in the aged kidney macrophages. Administration of miR-6948-3p mimic or Mincle siRNA alleviated D-gal-induced renal senescence. LPS was used to induce M1 polarization of bone marrow-derived macrophages, and a coculture system of M1 macrophages and mouse renal tubular epithelial cells (TCMK-1) was established. Mincle was upregulated in LPS-induced M1 macrophages in vitro, and silencing Mincle in M1 macrophages attenuated M1 macrophage-induced TCMK-1 cell senescence. Mechanistically, Mincle was regulated by miR-6948-3p and maintained the M1 phenotype of macrophages through the Syk/NF-κB pathway. In conclusion, Mincle, posttranscriptionally suppressed by miR-6948-3p, modulated renal senescence by maintaining the phenotype of M1 macrophages through the Syk/NF-κB pathway.

Simultaneously blocking ANGPTL3 and CD47 prevents the progression of atherosclerosis through regulating lipid metabolism, macrophagic efferocytosis and lipid peroxidation

Atherosclerosis (AS) ultimately cause major adverse cardiovascular events (MACEs). While traditional strategies by lipid-reducing have reduced MACEs, many patients continue to face significant risks. It might attribute to the upregulation of CD47 expression in AS lesions, that mediated anti-efferocytosis of macrophages. Therefore, we propose simultaneously blocking ANGPTL3, a vital regulator of lipid metabolism, and CD47 might be a potential approach for AS therapy. Firstly, we investigate the role of a novel anti-ANGPTL3 nanobody-Fc (FD03) in AS. We found that FD03 treatment significantly decreased circulating lipids, plaque size, and lipid deposition in apoE-/- mice compared to control Ab, but there was a twofold increase in plaque formation in comparison to baseline. However, immunofluorescence indicated the upregulation of CD47 expression in the plaques even after FD03 treatment compared to normal vascular tissue. Next, a bifunctional protein containing signal regulatory protein alpha (SIRPα) and FD03 (SIRPαD1-FD03) was constructed to block CD47 and ANGPTL3 concurrently, which had high purity, robust stability, and high affinity to CD47 and ANGPTL3 with biological activity in vitro. Furthermore, SIRPαD1-FD03 fusion protein exhibited the enhanced therapeutic effect on AS compared with SIRPαD1-Fc or FD03, regressing plaque contents and the necrotic core equal to baseline. Mechanistically, SIRPαD1-FD03 reduced serum lipids, augmented the efferocytosis rate and macrophage M2 polarization, and decreased the reactive oxygen species (ROS) and lipid peroxidation level in atherosclerotic plaques. Collectively, our project suggests an effective approach for AS by simultaneously blocking ANGPTL3 and CD47 to regulate lipid metabolism, macrophage activity and lipid peroxidation.

BMP2 enhance the osteogenic effect of BMSCs-derived exosomes in skull defect of diabetic rats

Diabetic patients often face poor repair of bone defects, which may be related to the reduced osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs) and the polarization of M1/M2 macrophage imbalance leading to an inflammatory response. The ability of BMSC-derived exosomes (BMSC-Exos) and BMP2 to exert both bone tissue regeneration and immunomodulation is expected to resolve this clinical dilemma. In this study, we demonstrate that exosomes derived from BMP2-treated BMSCs (BMP2-Exos) in a diabetic microenvironment exhibit enhanced osteogenic effects. Additionally, compared to the blank control group, macrophages stimulated by BMP2-Exos were more polarized towards the M2 type. These results were verified in vivo by GelMA loaded exosomes in the diabetic rat skull defect model. Importantly, we found that BMP2-Exos can upregulate the PI3K/AKT signaling pathway in diabetic BMSCs and improved osteogenic function and M2 macrophage polarization in diabetes-impaired BMSC-Exos in vitro. In conclusion, the present study revealed that BMP2 pretreatment can enhance the ability of BMSC-Exos to promote osteogenesis in diabetic bone defect areas, provides a preliminary exploration of the underlying mechanisms. Our study provided important experimental basis and innovative ideas for the application of exosomes therapy in the field of bone tissue regeneration.

MSCs-derived ECM functionalized hydrogel regulates macrophage reprogramming for osteoarthritis treatment by improving mitochondrial function and energy metabolism

Osteoarthritis (OA) is a degenerative disease that affects the entire joint, with synovial inflammation being a major pathological feature. Macrophages, as the most abundant immune cells in the synovium, have an M1/M2 imbalance that is closely related to the occurrence and development of OA. Mesenchymal stem cells (MSCs) have been shown to effectively suppress inflammation in the treatment of OA, but they still pose issues such as immune rejection and tumorigenicity. The extracellular matrix (ECM), as a major mediator of MSCs' immunoregulatory effects, offers a cell-free therapy to circumvent these risks. In this study, we developed an ECM-functionalized hydrogel by combining MSC-derived ECM with gelatin methacryloyl (GelMA). To enhance the immunomodulatory potential of MSCs, we pre-stimulated MSCs with the inflammatory factor interleukin-6 (IL-6) present in OA. In vitro results showed that the ECM-functionalized hydrogel promoted M2 macrophage polarization and inhibited the expression of various inflammatory genes, strongly indicating the hydrogel's powerful immunoregulatory capabilities. In an in vivo rat OA model, the ECM-functionalized hydrogel significantly reduced synovial inflammation and cartilage matrix degradation, alleviating the progression of OA. Furthermore, we utilized proteomics and transcriptomics analysis to reveal that the hydrogel accomplished macrophage metabolic reprogramming by regulating mitochondrial function and energy metabolism, thereby reducing inflammation. These findings suggest that the ECM-functionalized hydrogel is a promising biomaterial-based strategy for treating OA by targeting key pathological mechanisms.

Study of the mechanism of fibroblast-like synoviocytes-derived exosomes inducing macrophages M1 polarization and CD8+T cells immune regulation ferroptosis and autophagy in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects the joints. The pathogenesis of RA is complex, involving membrane lipid antioxidant systems, oxidative stress, and lipid peroxidation. In this study, it was found that cysteine dioxygenase 1 (CDO1) is significantly upregulated in RA fibroblast-like synoviocytes (RA-FLS) and that exosomes derived from these RA-FLS deliver CDO1 to promote M1 polarization of macrophages, thus facilitating RA progression. In the immune microenvironment, CD8+T cells play a role in immune regulation by producing cytokines such as interferon gamma (IFNγ) in various diseases. The results of this study suggested that in RA-FLS, CD8+T cells deliver IFNγ, which not only inhibits the viability of RA-FLS but also affects glutathione (GSH) through CDO1, regulating the GPX4 antioxidant signaling pathway to promote ferroptosis and autophagy in cells. It was also discovered that IFNγ enhances the expression of TRI69, ubiquitinates and degrades FSP1, thereby forming a cooperative regulation process of GPX4 and FSP1 in ferroptosis. These findings provide a new direction for the treatment of RA.

Moxibustion inhibits the macrophage M1 polarization toll-like receptor 4/myeloid differentiation factor 88/nuclear factor kappa B signaling pathway by regulating T-cell immunoglobulin and mucin-containing protein-3 in rheumatoid arthritis.

To explore whether moxibustion exerts therapeutic effects on rheumatoid arthritis (RA) by regulating the expression of T-cell immunoglobulin and mucin-containing protein-3 (TIM-3) and subsequently modulating the macrophage M1 polarization toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-nuclear factor kappa B (NF-κB) signaling pathway. We utilized moxibustion treatment in RA rat models using the Zusanli (ST36) and Shenshu (BL23) acupoints. Hematoxylin and eosin (HE) staining was used to observe the pathological changes of the synovial tissue under a section light microscope, and pathological scoring was performed according to the grading standard of the degree of synovial tissue disease. Enzyme-linked immunosorbent assay (ELISA) was applied to verify the efficacy of moxibustion in reducing inflammation. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of the TIM-3/TLR4-MyD88-NF-κB signaling pathway-related molecules, and Western blot was used to detect the contents of synovial NF-κB. We established the Freund's complete adjuvant (FCA)-induced RA model in rats. The expression level of M1 polarization signaling pathway TLR4-MyD88-NF-κB and the inflammatory factors interleukin-12(IL-12), tumor necrosis factor alpha (TNF-α), and tumor necrosis factor beta (TNF-β) were significantly increased in the RA model. After moxibustion treatment, the expression level of TLR4-MyD88-NF-κB was significantly decreased, and the inflammatory factors IL-12, TNF-α, and TNF-β were decreased, but the expression level was significantly increased in the RA model. When TIM-3 expression was inhibited, the expression level of TLR4-MyD88-NF-κB, and the inflammatory factors IL-12, TNF-α, and TNF-β were not suppressed, even after moxibustion treatment. Moxibustion regulates the key target TIM-3 by acting on the Zusanli (ST36) and Shenshu (BL23) points, thereby inhibiting the M1 polarization of macrophages; that is, it inhibits the TLR4-MyD88-NF-κB signaling pathway, and finally achieves alleviation of pathological changes and anti-inflammatory effects.

STING agonists and PI3Kγ inhibitor co-loaded ferric ion-punicalagin networks for comprehensive cancer therapy

Nanoparticles-based drug delivery system has been a promising approach for the treatment of colorectal cancer (CRC), which can be combined with chemotherapy, targeted therapy and immunotherapy to improve the treatment of CRC. 2′3’ cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) is an agonist of the STING signaling pathway activating antitumor immunity. IPI-549 is a small-molecule inhibitor for phosphatidylinositol 3-kinase γ (PI3Kγ), which can induce M1 macrophages polarization to provide pro-inflammatory microenvironment to suppress tumors. Here, we developed a ferric ion-punicalagin network (Fe-PU), which can be not only used as an inducer of ferroptosis, but also serve as a carrier to load cGAMP and IPI-549 to obtain nanohybrid (Fe-PU/CD-IPI). In order to improve the delivery effect and targeted ability to CRC, a cyclic arginine-glycine-aspartic acid peptide linked-bovine serum albumin were utilized to modify Fe-PU/CD-IPI to prepare nanohybrid Fe-PU/CD-IPI@cBSA. The therapeutic effect of various nanohybrids were validated in the mice with spontaneous tumor in the colorectal area and tumor-bearing mice, which lead to the increase of ferroptosis, the activation of STING signaling pathway, and the repolarization of macrophages. Collectively, the cGAMP and IPI-549 co-loaded nanohybrids effectively reshaped the tumor immune microenvironment, and exhibited prominent treatment effect of anti-colorectal cancer in vitro, patient-derived organoids, and in vivo.

Cooperative tumor inhibition by CpG-oligodeoxynucleotide and cyclic dinucleotide in head and neck cancer involves T helper cytokine and macrophage phenotype reprogramming

Head and neck cancer ranks as the sixth most common cancer worldwide, highlighting the critical need for the development of new therapies to enhance treatment efficacy. The activation of innate immune receptors given their potent immune stimulatory properties aid in the eradication of cancer cells. In this study, we investigated the immune mechanism and anti-tumor function of a Toll-like receptor 9 (TLR9) agonist, CpG-oligodeoxynucleotide-2722 (CpG-2722), in combination with cyclic dinucleotides, which are agonists of stimulator of interferon genes (STING). Our results revealed that CpG-2722 stimulation increased the expression of Th1 pro-inflammatory cytokines. Stimulation by STING agonists exhibited lower expression of Th1 cytokines but higher expression of Th2 cytokines compared to CpG-2722. However, the combination of these two agonists significantly enhanced Th1 cytokines while reducing Th2 cytokines. Moreover, in vivo experiment showed that both CpG-2722 and 2'3'-c-di-AMP suppressed head and neck tumor growth, with their combination proving more effective than the use of these agonists alone. The combined treatment cooperatively promoted the production of Th1 cytokines and type I interferons, while suppressing Th2 cytokines in the tumors as observed in vitro. Additionally, it led to the accumulation of M1 macrophages, dendritic cells, and T cells, shaping a favorable tumor microenvironment for T cell-mediated tumor killing. The anti-tumor activity of the CpG-2722 and 2'3'-c-di-AMP combination depends on the macrophage presence but does not directly activate M1 macrophage polarization, instead working through a reprogrammed cytokine profile. Furthermore, this combination shows a cooperative anti-tumor activity with anti-PD-1 in treating head and neck tumors. Overall, these findings highlight a Th response and macrophage phenotype reprograming involved functional mechanism underlying the cooperative activity of the combination of TLR9 and STING agonists in the immunotherapy of head and neck cancer.

Bone marrow mesenchymal stem cell-derived exosomal miR-181a-5p promotes M2 macrophage polarization to alleviate acute pancreatitis through ZEB2-mediated RACK1 ubiquitination.

As a common digestive disease, acute pancreatitis (AP) often threatens the life of patients. Bone marrow mesenchymal stem cells (BMSCs) derived exosomes have exhibited some benefits for AP. However, the mechanism remains unclear and deserves to be further investigated. The characteristics of BMSCs-exosomes (BMSCs-Exos) were identified. The abundance of genes and proteins was evaluated using quantitative real-time PCR (RT-qPCR), western blot, enzyme-linked immunosorbent assay (ELISA) and IF assay. Cell apoptosis and CD206-positive cells were measured by flow cytometry. The interactions among miR-181a-5p, Zinc finger E-box binding homeobox2 (ZEB2) and Receptor for Activated C Kinase 1 (RACK1) were verified using dual luciferase reporter assay, RNA immunoprecipitation (RIP), coimmunoprecipitation (Co-IP). BMSCs-Exos effectively improved AP injury through restraining AR42J cell apoptosis and promoting M2 macrophage polarization, which was realized due to BMSCs-Exos harboring an abundance of miR-181a-5p. Further experiments validated miR-181a-5p silenced ZEB2 and ZEB2 reduced RACK1 expression through mediating RACK1 ubiquitination. ZEB2 knockdown decreased AR42J cell apoptosis and induced M2 macrophage polarization to alleviate AP injury, whereas RACK1 downregulation abolished these phenomena. BMSCs-Exos harboring miR-181a-5p suppressed AR42J cell apoptosis and promoted M2 macrophage polarization to delay AP progression through ZEB2-mediated RACK1 ubiquitination.

Influence of Chitosan Purification on the Immunomodulator Potential of Chitosan Nanoparticles on Human Monocytes

The deproteinization of chitosan is a necessary purification process for materials with biomedical purposes; however, chitosan sourcing and purification methods can modify its molecular weight, deacetylation degree, and residual proteins. These factors affect the reactive groups that affect the immunomodulatory activities of cells, particularly macrophages and monocytes; considering this activity is key when developing successful and functional biomaterials. Here, two brands of chitosan were purified and used to synthesize nanoparticles to evaluate their immunomodulatory effect on monocyte and macrophage differentiation. Chitosan FT-IR showed bands related to its purification process, with increased OH group intensity. Nanoparticles (CtsNps) synthesized with purified chitosan were of a smaller size compared to those using unpurified chitosan due to the alkaline purification process’s shortening of the polymeric chain. At low concentrations (50 μg/mL), CtsNps showed a lower expression of CD80 and CD14, corroborating the differentiation effect of chitosan. Inducible nitric oxide synthase (iNOS) is related to a pro-inflammatory response and M1 macrophage polarization was detected in monocytes treated with purified and unpurified nanoparticles. Sigma-purified chitosan nanoparticles (CtsNps SigmaP), at 300 μg/mL, showed arginase production related to an anti-inflammatory response and M2 macrophage polarization. The chitosan purification process induces a shift in the polarization of macrophages to an anti-inflammatory M2 profile. This effect is concentration-dependent and should be further studied in each use case to favor the suitable biological response.

Inhibition of lncEPS by TLR4/NF-κB pathway induces ventilator-induced lung injury by decreasing its binding to and upregulating Hspa5

Whether LincRNA erythroid prosurvival (LncEPS) reduces VILI remains unclear. A GSE200932 microarray was used to screen differentially expressed genes (DEGs). A VILI mouse model was constructed by mechanical ventilation (MV), with or without TAK242 or SN50 pretreatment. Airway transfection with adeno-associated virus (AAV) was used to overexpress lncEPS in alveolar macrophages (AMs). Lung tissues were collected to assess pathological injury and macrophage polarization. NLRP3 inflammasome, TLR4/ NF-κB pathway activation and heat shock protein family A member 5 (Hspa5) in lung tissue and AMs wre evaluated. LncEPS localization and regulatory changes were assessed using in situ hybridization and RT–PCR. Lung tissues after lncEPS overexpression were subjected to transcriptomics. Chromatin isolation and mass spectrometry (MS) were performed to identify proteins interacting with lncEPS. GSE200932 microarray showed that the DEGs were related to NF-κB pathway, Toll-like receptor pathway and NOD-like receptor pathway. TAK242 or SN50 treatment increased polarization of M2 macrophages and decreased NLRP3 inflammasome activation by inhibiting TLR4/NF-κB pathway in VILI. Inhibition of TLR4/NF-κB pathway upregulated lncEPS expression in AMs. Overexpression of lncEPS increased polarization of M2 macrophages and decreased NLRP3 inflammasome activation, eventually alleviating VILI in mice. Mechanistically, lncEPS bound to Hspa5 and downregulated its expression to inhibit inflammatory response.

BMSCs Downregulate CXCL12 by Secreting Exosomal miR-20a-5p to Promote Macrophage M2 Polarization and Alleviate the Development of Sepsis

ABSTRACT Objective Sepsis is a syndrome of the systemic inflammatory response caused by infection that can endanger a patient’s life. The aim of this study was to explore the molecular mechanism by which bone marrow mesenchymal stem cells-derived exosomes (BMSCs-exo) carrying miR-20a-5p regulate the progression of sepsis. Methods Clinical samples from sepsis patients were collected. Mouse and cell models of sepsis were induced by lipopolysaccharide (LPS). The levels of related genes and proteins were determined by RT‒qPCR, Western blotting and ELISA. CCK-8 and flow cytometry assays were used to assess cell viability, apoptosis, and markers of macrophage polarization. Results In septic patients, miR-20a-5p levels were significantly lower and CXCL12 expression was significantly increased. After LPS induction, M2 polarization of macrophages was significantly reduced, the level of inflammatory factors was increased, and apoptosis was increased. The addition of BMSCs-exo increased the miR-20a-5p level and decreased the expression of CXCL12 in macrophages, thereby promoting macrophage M2 polarization and reducing the levels of inflammatory factors. Conclusion This study demonstrated for the first time that BMSCs-exo promoted the polarization of M2 macrophages through the miR-20a-5p/CXCL12 axis, thus alleviating the development of sepsis. These findings provide a new theoretical basis for the targeted treatment of sepsis with exosomes or miR-20a-5p.

M2 macrophage-derived exosomes promote cell proliferation, migration and EMT of non-small cell lung cancer by secreting miR-155-5p.

Tumor-associated macrophages (TAMs) are a type of highly plastic immune cells in the tumor microenvironment (TME), which can be classified into two main phenotypes: classical activated M1 macrophages and alternatively activated M2 macrophages. As previously reported, M2-polarized TAMs play critical role in promoting the progression of non-small cell lung cancer (NSCLC) via secreting exosomes, but the detailed mechanisms are still largely unknown. In the present study, the THP-1 monocytes were sequentially induced into M0 and M2-polarized macrophages, and the exosomes were obtained from M0 (M0-exos) and M2 (M2-exos) polarized macrophages, respectively, and co-cultured with NSCLC cells (H1299 and A549) to establish the exosomes-cell co-culture system in vitro. As it was determined by MTT assay, RT-qPCR and Transwell assay, in contrast with the M0-exos, M2-exos significantly promoted cell proliferation, migration and epithelial-mesenchymal transition (EMT) process in NSCLC cells. Next, through screening the contents in the exosomes, it was verified that miR-155-5p was especially enriched in the M2-exos, and M2-exos enhanced cancer aggressiveness and tumorigenesis in in vitro NSCLC cells and in vivo xenograft tumor-bearing mice models via delivering miR-155-5p. The detailed molecular mechanisms were subsequently elucidated, and it was found that miR-155-5p bound with HuR to increase the stability and expression levels of VEGFR2, which further activated the tumor-promoting PI3K/Akt/mTOR signal pathway, and M2-exos-enhanced cancer progression in NSCLC cells were apparently suppressed by downregulating VEGFR2 and PI3K inhibitor LY294002 co-treatment. Taken together, M2-polarized TAMs secreted miR-155-5p-containing exosomes to enhanced cancer aggressiveness of NSCLC by activating the VEGFR2/PI3K/Akt/mTOR pathway in a HuR-dependent manner.