IL-10-induced modulation of macrophage polarization suppresses outer-blood-retinal barrier disruption in the streptozotocin-induced early diabetic retinopathy mouse model.

Diabetic retinopathy (DR) is associated with ocular inflammation leading to retinal barrier breakdown, vascular leakage, macular edema, and vision loss. DR is not only a microvascular disease but also involves retinal neurodegeneration, demonstrating that pathological changes associated with neuroinflammation precede microvascular injury in early DR. Macrophage activation plays a central role in neuroinflammation. During DR, the inflammatory response depends on the polarization of retinal macrophages, triggering pro-inflammatory (M1) or anti-inflammatory (M2) activity. This study aimed to determine the role of macrophages in vascular leakage through the tight junction complexes of retinal pigment epithelium, which is the outer blood-retinal barrier (BRB). Furthermore, we aimed to assess whether interleukin-10 (IL-10), a representative M2-inducer, can decrease inflammatory macrophages and alleviate outer-BRB disruption. We found that modulation of macrophage polarization affects the structural and functional integrity of ARPE-19 cells in a co-culture system under high-glucose conditions. Furthermore, we demonstrated that intravitreal IL-10 injection induces an increase in the ratio of anti-inflammatory macrophages and effectively suppresses outer-BRB disruption and vascular leakage in a mouse model of early-stage streptozotocin-induced diabetes. Our results suggest that modulation of macrophage polarization by IL-10 administration during early-stage DR has a promising protective effect against outer-BRB disruption and vascular leakage. This finding provides valuable insights for early intervention in DR.

Cigarette smoke sustains immunosuppressive microenvironment inducing M2 macrophage polarization and viability in lung cancer settings.

BACKGROUND: It is amply demonstrated that cigarette smoke (CS) has a high impact on lung tumor progression worsening lung cancer patient prognosis and response to therapies. Alteration of immune cell types and functions in smokers' lungs have been strictly related with smoke detrimental effects. However, the role of CS in dictating an inflammatory or immunosuppressive lung microenvironment still needs to be elucidated. Here, we investigated the effect of in vitro exposure to cigarette smoke extract (CSE) focusing on macrophages. METHODS: Immortalized murine macrophages RAW 264.7 cells were cultured in the presence of CS extract and their polarization has been assessed by Real-time PCR and cytofluorimetric analysis, viability has been assessed by SRB assay and 3D-cultures and activation by exposure to Poly(I:C). Moreover, interaction with Lewis lung carcinoma (LLC1) murine cell models in the presence of CS extract were analyzed by confocal microscopy. RESULTS: Obtained results indicate that CS induces macrophages polarization towards the M2 phenotype and M2-phenotype macrophages are resistant to the CS toxic activity. Moreover, CS impairs TLR3-mediated M2-M1 phenotype shift thus contributing to the M2 enrichment in lung smokers. CONCLUSIONS: These findings indicate that, in lung cancer microenvironment of smokers, CS can contribute to the M2-phenotype macrophages prevalence by different mechanisms, ultimately, driving an anti-inflammatory, likely immunosuppressive, microenvironment in lung cancer smokers.

Codonopsis pilosula-derived glycopeptide dCP1 promotes the polarization of tumor-associated macrophage from M2-like to M1 phenotype.

The majority of the immune cell population in the tumor microenvironment (TME) consists of tumor-associated macrophages (TAM), which are the main players in coordinating tumor-associated inflammation. TAM has a high plasticity and is divided into two main phenotypes, pro-inflammatory M1 type and anti-inflammatory M2 type, with tumor-suppressive and tumor-promoting functions, respectively. Considering the beneficial effects of M1 macrophages for anti-tumor and the high plasticity of macrophages, the conversion of M2 TAM to M1 TAM is feasible and positive for tumor treatment. This study sought to evaluate whether the glycopeptide derived from simulated digested Codonopsis pilosula extracts could regulate the polarization of M2-like TAM toward the M1 phenotype and the potential regulatory mechanisms. The results showed that after glycopeptide dCP1 treatment, the mRNA relative expression levels of some M2 phenotype marker genes in M2-like TAM in simulated TME were reduced, and the relative expression levels of M1 phenotype marker genes and inflammatory factor genes were increased. Analysis of RNA-Seq of M2-like TAM after glycopeptide dCP1 intervention showed that the gene sets such as glycolysis, which is associated with macrophage polarization in the M1 phenotype, were significantly up-regulated, whereas those of gene sets such as IL-6-JAK-STAT3 pathway, which is associated with polarization in the M2 phenotype, were significantly down-regulated. Moreover, PCA analysis and Pearson's correlation also indicated that M2-like TAM polarized toward the M1 phenotype at the transcriptional level after treatment with the glycopeptide dCP1. Lipid metabolomics was used to further explore the efficacy of the glycopeptide dCP1 in regulating the polarization of M2-like TAM to the M1 phenotype. It was found that the lipid metabolite profiles in dCP1-treated M2-like TAM showed M1 phenotype macrophage lipid metabolism profiles compared with blank M2-like TAM. Analysis of the key differential lipid metabolites revealed that the interconversion between phosphatidylcholine (PC) and diacylglycerol (DG) metabolites may be the central reaction of the glycopeptide dCP1 in regulating the conversion of M2-like TAM to the M1 phenotype. The above results suggest that the glycopeptide dCP1 has the efficacy to regulate the polarization of M2-like TAM to M1 phenotype in simulated TME.

Baricitinib protects ICIs-related myocarditis by targeting JAK1/STAT3 to regulate Macrophage polarization.

PURPOSE: The emergence of immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment, but these drugs can also cause severe immune-related adverse effects (irAEs), including myocarditis. Researchers have become interested in exploring ways to mitigate this side effect, and one promising avenue is the use of baricitinib, a Janus kinase inhibitor known to have anti-inflammatory properties. This study aimed to examine the potential mechanism by which baricitinib in ICIs-related myocarditis. METHODS: To establish an ICIs-related myocarditis model, BALB/c mice were administered murine cardiac troponin I (cTnI) peptide and anti-mouse programmed death 1 (PD-1) antibodies. Subsequently, baricitinib was administered to the mice via intragastric administration. Echocardiography, HE staining, and Masson staining were performed to evaluate myocardial functions, inflammation, and fibrosis. Immunofluorescence was used to detect macrophages in the cardiac tissue of the mice.In vitro experiments utilized raw264.7 cells to induce macrophage polarization using anti-PD-1 antibodies. Different concentrations of baricitinib were applied to assess cell viability, and the release of pro-inflammatory cytokines was measured. The activation of the JAK1/STAT3 signaling pathway was evaluated through western blot analysis. RESULTS: Baricitinib demonstrated its ability to improve cardiac function and reduce cardiac inflammation, as well as fibrosis induced by ICIs. Mechanistically, baricitinib treatment promoted the polarization of macrophages towards the M2 phenotype. In vitro and in vivo experiments showed that anti-PD-1 promoted the release of inflammatory factors. However, treatment with baricitinib significantly inhibited the phosphorylation of JAK1 and STAT3. Additionally, the use of RO8191 reversed the effects of baricitinib, further confirming our findings. CONCLUSION: Baricitinib demonstrated its potential as a protective agent against ICIs-related myocarditis by modulating macrophage polarization. These findings provide a solid theoretical foundation for the development of future treatments for ICIs-related myocarditis.

Ficolin-A induces macrophage polarization to a novel pro-inflammatory phenotype distinct from classical M1.

BACKGROUND: Macrophages are key inflammatory immune cells that orchestrate the initiation and progression of autoimmune diseases. The characters of macrophage in diseases are determined by its phenotype in response to the local microenvironment. Ficolins have been confirmed as crucial contributors to autoimmune diseases, with Ficolin-2 being particularly elevated in patients with autoimmune diseases. However, whether Ficolin-A stimulates macrophage polarization is still poorly understood. METHODS: We investigated the transcriptomic expression profile of murine bone marrow-derived macrophages (BMDMs) stimulated with Ficolin-A using RNA-sequencing. To further confirm a distinct phenotype activated by Ficolin-A, quantitative RT-PCR and Luminex assay were performed in this study. Additionally, we assessed the activation of underlying cell signaling pathways triggered by Ficolin-A. Finally, the impact of Ficolin-A on macrophages were investigated in vivo through building Collagen-induced arthritis (CIA) and Dextran Sulfate Sodium Salt (DSS)-induced colitis mouse models with Fcna-/- mice. RESULTS: Ficolin-A activated macrophages into a pro-inflammatory phenotype distinct to LPS-, IFN-γ- and IFN-γ + LPS-induced phenotypes. The transcriptomic profile induced by Ficolin-A was primarily characterized by upregulation of interleukins, chemokines, iNOS, and Arginase 1, along with downregulation of CD86 and CD206, setting it apart from the M1 and M2 phenotypes. The activation effect of Ficolin-A on macrophages deteriorated the symptoms of CIA and DSS mouse models, and the deletion of Fcna significantly alleviated the severity of diseases in mice. CONCLUSION: Our work used transcriptomic analysis by RNA-Seq to investigate the impact of Ficolin-A on macrophage polarization. Our findings demonstrate that Ficolin-A induces a novel pro-inflammatory phenotype distinct to the phenotypes activated by LPS, IFN-γ and IFN-γ + LPS on macrophages.

TSG6-Exo@CS/GP Attenuates Endometrium Fibrosis by Inhibiting Macrophage Activation in a Murine IUA Model.

Intrauterine adhesion (IUA) is characterized by the formation of fibrous scar tissue within the uterine cavity, which significantly impacts female reproductive health and even leads to infertility. Unfortunately, severe cases of IUA currently lack effective treatments. This study presents a novel approach that utilizes tumor necrosis factor-(TNF) stimulated gene 6 (TSG6)-modified exosomes (Exos) in conjunction with an injectable thermosensitive hydrogel (CS/GP) to mitigate the occurrence of IUA by reducing endometrium fibrosis in a mouse IUA model. This study demonstrate that TSG6-modified Exos effectively inhibits the activation of inflammatory M1-like macrophages during the initial stages of inflammation and maintains the balance of macrophage phenotypes (M1/M2) during the repair phase. Moreover, TSG6 inhibits the interaction between macrophages and endometrial stromal fibroblasts, thereby preventing the activation of stromal fibroblasts into myofibroblasts. Furthermore, this research indicates that CS/GP facilitates the sustained release of TSG6-modified Exos, leading to a significant reduction in both the manifestations of IUA and the extent of endometrium fibrosis. Collectively, through the successful construction of CS/GP loaded with TSG6-modified Exos, a reduction in the occurrence and progression of IUA is achieved by mitigating endometrium fibrosis. Consequently, this approach holds promise for the treatment of IUA.

M2 Macrophage-Derived Extracellular Vesicles Encapsulated in Hyaluronic Acid Alleviate Osteoarthritis by Modulating Macrophage Polarization.

An imbalance between M1 and M2 macrophage polarization is critical in osteoarthritis (OA) development. We investigated the effect of M2 macrophage-derived extracellular vesicles (M2-EVs) to reprogramme macrophages from the M1 to M2 phenotype for OA treatment. M1 macrophages and mouse OA models were treated with M2-EVs. Proteomic analysis was performed to evaluate macrophage polarization in vitro. The OA models were as follows: destabilization of the medial meniscus (DMM) surgery-induced OA and collagenase-induced OA (CIOA). Hyaluronic acid (HA) was used to deliver M2-EVs. M2-EVs decreased macrophage accumulation, repolarized macrophages from the M1 to M2 phenotype, mitigated synovitis, reduced cartilage degradation, alleviated subchondral bone damage, and improved gait abnormalities in the CIOA and DMM models. Moreover, HA increased the retention time of M2-EVs and enhanced the efficiency of M2-EVs in OA treatment. Furthermore, proteomic analysis demonstrated that M2-EVs exhibited a macrophage reprogramming ability similar to IL-4, and the pathways might be the NOD-like receptor (NLR), TNF, NF-κB, and Toll-like receptor (TLR) signaling pathways. M2-EVs reprogrammed macrophages from the M1 to M2 phenotype, which resulted in beneficial effects on cartilage and attenuation of OA severity. In summary, our study indicated that M2-EV-guided reprogramming of macrophages is a promising treatment strategy for OA.

Selenium maintains intestinal epithelial cells to activate M2 macrophages against deoxynivalenol injury.

Selenium (Se) is indispensable in alleviating various types of intestinal injuries. Here, we thoroughly investigated the protective effect of Se on the regulation of the epithelial cell-M2 macrophages pathway in deoxynivalenol (DON)-induced intestinal damage. In the present study, Se has positive impacts on gut health by improving gut barrier function and reducing the levels of serum DON in vivo. Furthermore, our study revealed that Se supplementation increased the abundances of GPX4, p-PI3K, and AKT, decreased the levels of 4-HNE and inhibited ferroptosis. Moreover, when mice were treated with DON and Fer-1(ferroptosis inhibitor), ferroptosis was suppressed and PI3K/AKT pathway was activated. These results indicated that GPX4-PI3K/AKT-ferroptosis was a predominant pathway in DON-induced intestinal inflammation. Interestingly, we discovered that both the number of M2 anti-inflammatory macrophages and the levels of CSF-1 decreased while the pro-inflammatory cytokine IL-6 increased in the intestine and MODE-K cells supernatant. Therefore, Se supplementation activated the CSF-1-M2 macrophages axis, resulting in a decrease in IL-6 expression and an enhancement of the intestinal anti-inflammatory capacity. This study provides novel insights into how intestinal epithelial cells regulate the CSF-1-M2 macrophage pathway, which is essential in maintaining intestinal homeostasis confer to environmental hazardous stimuli.

2-Substituted-4,7-dihydro-4-ethylpyrazolo[1,5-a]pyrimidin-7-ones alleviate LPS-induced inflammation by modulating cell metabolism via CD73 upon macrophage polarization.

Macrophage polarization towards the M1 phenotype under bacterial product-related exposure (LPS) requires a rapid change in gene expression patterns and cytokine production along with a metabolic rewiring. Metabolic pathways and redox reactions are such tightly connected, giving rise to an area of research referred to as immunometabolism. A role in this context has been paid to the master redox-sensitive regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and to the 5'-ectonucleotidase CD73, a marker related to macrophage metabolism rearrangement under pro-inflammatory conditions. In this light, a cell model of LPS-stimulated macrophages has been established and nine 4,7-dihydro-4-ethylpyrazolo[l,5-a]pyrimidin-7-ones with a potential anti-inflammatory effect have been administered. Our data highlight that two selected compounds (namely, 5 and 8) inhibit the LPS-induced Nrf2 nuclear translocation and ameliorate the activity rate of the antioxidant enzyme catalase. Additionally, the pyridine-containing compound (8) promotes the shift from the pro-inflammatory immunophenotype M1 to the pro-resolving M2 one, by downregulating CD80 and iNOS and by enhancing CD163 and TGFß1 expression. Most importantly, CD73 is modulated by these compounds as well as the lactate production. Our data demonstrate that pyrazolo[l,5-a]pyrimidine derivatives are effective as anti-inflammatory compounds. Furthermore, these pyrazolo[l,5-a]pyrimidines exert their action via CD73-related signaling and modulation of cell metabolism of activated macrophages.

Puerarin Alleviates Experimental Autoimmune Thyroiditis by Regulating Macrophages.

Hashimoto's thyroiditis (HT) is the most common organ-specific autoimmune disease, predominantly affecting women. Although the pathogenesis of HT is incompletely understood, some studies have found that macrophage polarization plays a role. Puerarin is a soy isoflavone compound that has anti-inflammatory and immunomodulatory effects and regulates macrophage immune activity. This study aimed to verify the therapeutic effect of puerarin on HT and explored its regulatory effect on macrophage polarization imbalance in HT. Through bioinformatics analysis and molecular biology methods, it was found that macrophages increased significantly in HT patients and model mice. Immunological staining showed that puerarin intervention could reduce tissue inflammatory cell infiltration. Molecular biological examination displayed that puerarin could inhibit local and systemic inflammation levels, and the expression of marker thyroglobulin and thyroid peroxidase Abs. In vivo experimental results indicated that puerarin regulated macrophage polarity and reduced inflammatory damage, possibly by inhibiting the pyroptosis signaling pathway. In vivo macrophage clearance experiments demonstrated that puerarin relied on macrophages to exert its mechanism of action in treating HT. The results of this study indicate that macrophages are important mediators in the development of HT, and puerarin can regulate macrophage polarity and inflammatory status to provide thyroid tissue protection, which provides a new idea for the treatment of HT.

Rabeprazole mitigates obesity-induced chronic inflammation and insulin resistance associated with increased M2-type macrophage polarization.

Macrophage polarization is closely associated with obesity-induced chronic inflammation and insulin resistance. Proton pump inhibitor Rabeprazole has long been used to treat gastritis and gastric ulcers. However, whether Rabeprazole plays a role in macrophage polarization during obesity is unknown. Here, we show that Rabeprazole suppresses M1-type macrophage-mediated inflammation, leads to increased M2-type macrophages and alters the polarization status from M1 to M2 in vitro. Mechanistically, Rabe-regulated macrophage polarization is associated with inhibition of NF-κB and activation of STAT6 signaling pathways. Furthermore, Rabeprazole induces M2-type adipose tissue macrophages and alleviates chronic inflammation, improving glucose tolerance and insulin sensitivity in high-fat diet-fed mice. In addition, Rabeprazole increases CD206+ M2-type liver macrophages and relieves liver inflammation, alleviating liver injury and lipid accumulation. Thus, our findings show that Rabeprazole effectively regulates macrophage polarization and controls obesity-associated chronic inflammation and insulin resistance, thus providing a potential therapeutic strategy against obesity-associated metabolic diseases.

Widespread S-persulfidation in activated macrophages as a protective mechanism against oxidative-inflammatory stress.

Acute inflammatory responses often involve the production of reactive oxygen and nitrogen species by innate immune cells, particularly macrophages. How activated macrophages protect themselves in the face of oxidative-inflammatory stress remains a long-standing question. Recent evidence implicates reactive sulfur species (RSS) in inflammatory responses; however, how endogenous RSS affect macrophage function and response to oxidative and inflammatory insults remains poorly understood. In this study, we investigated the endogenous pathways of RSS biogenesis and clearance in macrophages, with a particular focus on exploring how hydrogen sulfide (H2S)-mediated S-persulfidation influences macrophage responses to oxidative-inflammatory stress. We show that classical activation of mouse or human macrophages using lipopolysaccharide and interferon-γ (LPS/IFN-γ) triggers substantial production of H2S/RSS, leading to widespread protein persulfidation. Biochemical and proteomic analyses revealed that this surge in cellular S-persulfidation engaged ∼2% of total thiols and modified over 800 functionally diverse proteins. S-persulfidation was found to be largely dependent on the cystine importer xCT and the H2S-generating enzyme cystathionine γ-lyase and was independent of changes in the global proteome. We further investigated the role of the sulfide-oxidizing enzyme sulfide quinone oxidoreductase (SQOR), and found that it acts as a negative regulator of S-persulfidation. Elevated S-persulfidation following LPS/IFN-γ stimulation or SQOR inhibition was associated with increased resistance to oxidative stress. Upregulation of persulfides also inhibited the activation of the macrophage NLRP3 inflammasome and provided protection against inflammatory cell death. Collectively, our findings shed light on the metabolism and effects of RSS in macrophages and highlight the crucial role of persulfides in enabling macrophages to withstand and alleviate oxidative-inflammatory stress.

Disulfiram treatment suppresses antibody-producing reactions by inhibiting macrophage activation and B cell pyrimidine metabolism.

Antibody responses, involving B cells, CD4 + T cells, and macrophages, are implicated in autoimmune diseases and organ transplant rejection. We have previously shown that inhibiting FROUNT with disulfiram (DSF) suppresses macrophage activation and migration, effectively treating inflammatory diseases. In this study, we investigated the effectiveness of DSF in antibody-producing reactions. Using a heart transplantation mouse model with antibody-mediated rejection, we administered anti-CD8 antibody to exclude cellular rejection. DSF directly inhibited B cell responses in vitro and significantly reduced plasma donor-specific antibodies and graft antibody deposition in vivo, resulting in prolonged survival of the heart graft. DSF also mediated various effects, including decreased macrophage infiltration and increased Foxp3+ regulatory T-cells in the grafts. Additionally, DSF inhibited pyrimidine metabolism-related gene expression induced by B-cell stimulation. These findings demonstrate that DSF modulates antibody production in the immune response complexity by regulating B-cell and macrophage responses.

Targeting BRD4 with PROTAC degrader ameliorates LPS-induced acute lung injury by inhibiting M1 alveolar macrophage polarization.

OBJECTIVES: Acute lung injury (ALI) is a highly inflammatory condition with the involvement of M1 alveolar macrophages (AMs) polarization, eventually leading to the development of non-cardiogenic edema in alveolar and interstitial regions, accompanied by persistent hypoxemia. Given the significant mortality rate associated with ALI, it is imperative to investigate the underlying mechanisms of this condition so as to identify potential therapeutic targets. The therapeutic effects of the inhibition of bromodomain containing protein 4 (BRD4), an epigenetic reader, has been proven with high efficacy in ameliorating various inflammatory diseases through mediating immune cell activation. However, little is known about the therapeutic potential of BRD4 degradation in acute lung injury. METHODS: This study aimed to assess the protective efficacy of ARV-825, a novel BRD4-targeted proteolysis targeting chimera (PROTAC), against ALI through histopathological examination in lung tissues and biochemical analysis in bronchoalveolar lavage fluid (BALF). Additionally, the underlying mechanism by which BRD4 regulated M1 AMs was elucidated by using CUT & Tag assay. RESULTS: In this study, we found the upregulation of BRD4 in a lipopolysaccharide (LPS)-induced ALI model. Furthermore, we observed that intraperitoneal administration of ARV-825, significantly alleviated LPS-induced pulmonary pathological changes and inflammatory responses. These effects were accompanied by the suppression of M1 AMs. In addition, our findings revealed that the administration of ARV-825 effectively suppressed M1 AMs by inhibiting the expression of IRF7, a crucial transcriptional factor involved in M1 macrophages. CONCLUSION: Our study suggested that targeting BRD4 using ARV-825 is a potential therapeutic approach for ALI.

Hepatocyte mitochondrial DNA mediates macrophage immune response in liver injury induced by trichloroethylene.

We have previously shown that excessive activation of macrophage proinflammatory activity plays a key role in TCE-induced immune liver injury, but the mechanism of polarization is unclear. Recent studies have shown that TLR9 activation plays an important regulatory role in macrophage polarization. In the present study, we demonstrated that elevated levels of oxidative stress in hepatocytes mediate the release of mtDNA into the bloodstream, leading to the activation of TLR9 in macrophages to regulate macrophage polarization. In vivo experiments revealed that pretreatment with SS-31, a mitochondria-targeting antioxidant peptide, reduced the level of oxidative stress in hepatocytes, leading to a decrease in mtDNA release. Importantly, SS-31 pretreatment inhibited TLR9 activation in macrophages, suggesting that hepatocyte mtDNA may activate TLR9 in macrophages. Further studies revealed that pharmacological inhibition of TLR9 by ODN2088 partially blocked macrophage activation, suggesting that the level of macrophage activation is dependent on TLR9 activation. In vitro experiments involving the extraction of mtDNA from TCE-sensitized mice treated with RAW264.7 cells further confirmed that hepatocyte mtDNA can activate TLR9 in mouse peritoneal macrophages, leading to macrophage polarization. In summary, our study comprehensively confirmed that TLR9 activation in macrophages is dependent on mtDNA released by elevated levels of oxidative stress in hepatocytes and that TLR9 activation in macrophages plays a key role in regulating macrophage polarization. These findings reveal the mechanism of macrophage activation in TCE-induced immune liver injury and provide new perspectives and therapeutic targets for the treatment of OMDT-induced immune liver injury.

F1 fraction isolated from Mesobuthus eupeus scorpion venom induces macrophage polarization toward M1 phenotype and exerts anti-tumoral effects on the CT26 tumor cell line.

Scorpion venoms identified as agents with anti-tumor and anti-angiogenic features. Tumor microenvironment (TME) plays a pivotal role in the process of tumorigenesis, tumor development, and polarization of M2 phenotype tumor associated macrophages (TAMs). M2 polarized cells are associated with tumor growth, invasion, and metastasis. The fractionation process was performed by gel filtration chromatography on a Sephadex G50 column. To elucidate whether scorpion venom can alter macrophage polarization, we treated interleukin (IL)-4-polarized M2 cells with isolated fractions from Mesobuthus eupeus. Next, we evaluated the cytokine production and specific markers expression for M2 and M1 phenotype using enzyme linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR), respectively. The phagocytic capacity of macrophages was also assessed. In addition, the migration assay and MTT analysis were performed to investigate the effects of reprogrammed macrophages on the CT-26 colon cancer cells. The results indicated that F1 fraction of venom significantly upregulated the levels and expression of M1-associated cytokines and markers, including tumor necrosis factor-alpha (TNF-α) (p < 0.001), IL-1 (p < 0.01), interferon regulatory factor 5 (IRF5) (p < 0.0001), induced nitric oxide synthase (iNOS) (p < 0.0001), and CD86 (p < 0.0001), and downregulated M2-related markers, including transforming growth factor-beta (TGF-ß) (p < 0.05), IL-10 (p < 0.05), Fizz1 (p < 0.0001), arginase-1 (Arg-1) (p < 0.0001), and CD206 (p < 0.001). The macrophage phagocytic capacity was enhanced after treatment with F1 fraction (p < 0.01). Moreover, incubation of CT-26 cell line with conditioned media of F1-treated macrophages suppressed migration (p < 0.0001) and proliferation (p < 0.01) of tumor cells. In conclusion, our findings demonstrated the potential of Mesobuthus eupeus venom in M2-to-M1 macrophage polarization as a promising therapeutic approach against proliferation and metastasis of colon cancer cells.

CCR2 antagonist attenuates calcium oxalate-induced kidney oxidative stress and inflammation by regulating macrophage activation.

C-C chemokine receptor type 2 (CCR2) is a monocyte chemokine associated with oxidative stress and inflammation. Kidney stones (KS) are composed of calcium oxalate (CaOx), which trigger renal oxidative stress and inflammatory. This study aims to evaluate the effects of CCR2 on KS in vivo and in vitro. Eight-week-old male C57BL/6J mice were intraperitoneally injected with glyoxylate (GOX) daily to establish a KS model, and along with CCR2 antagonist (INCB3344) treatment on days 2, 4, and 6. The results showed that CCR2 antagonist reduced renal injury markers (blood urea nitrogen and serum creatinine), alleviated renal tubular injury and CaOx crystal deposition. CCR2 antagonist also decreased CCR2 expression induced by GOX treatment and increased Nrf2 expression. GOX treatment promoted malondialdehyde (MDA) production, decreased glutathione (GSH) content, and inhibited catalase (CAT) and superoxide dismutase (SOD) activity, however, CCR2 antagonist attenuated the above effects of GOX. CCR2 antagonist had inhibitory effects on GOX-induced inflammatory cytokine expression (IL1B, IL6 and MCP1), and inhibited apoptosis by increasing Bcl-2 expression and decreasing Bax and cleaved-caspase 3 expression. In vitro experiments were performed by co-culture model of CaOx-induced damaged HK-2 cells and macrophage-like THP-1 cells. CCR2 antagonist inhibited CaOx-induced THP-1 cell M1 polarization by decreasing the TNF-α, IL6 and iNOS levels, and further alleviated CaOx-induced oxidative stress damage, inflammatory response and apoptosis of HK-2 cells. The study suggests that CCR2 antagonist may be resistant to CaOx crystals-induced oxidative stress and inflammation by inhibiting macrophage M1 polarization.

Ranitidine as an adjuvant regulates macrophage polarization and activates CTLs through the PI3K-Akt2 signaling pathway.

Adjuvants are an indispensable component of vaccines, but there are few adjuvants for human vaccines. H2 receptor blockers, inhibiting gastric acid secretion, have immune enhancement effects. Ranitidine (RAN) is a water-soluble H2 receptor blocker, and whether it has an immune-enhancing effect is still unknown. In this study, flow cytometry, western blotting, and immunofluorescence methods were used to analyze whether RAN could activate macrophage polarization to the M1 phenotype in vivo and in vitro. Here, we found that the M1 inflammatory cytokine levels and surface markers in RAW264.7 cells were upregulated by NF-κB activation, possibly through the PI3K-Akt2 signaling pathway, after RAN treatment. Endocytic function was also enhanced by feedback regulation of Akt2/GSK3ß/Dynmin1 signaling. Furthermore, to evaluate the adjuvant function of RAN, we used OVA plus RAN as a vaccine to inhibit the growth of B16-OVA tumors in mice. We also found that in the RAN adjuvant group, macrophage polarization to M1, Th1 cell differentiation, and cytotoxic T lymphocyte (CTL) activation were significantly upregulated. The tumor growth of mice was inhibited, and the survival rate of mice was significantly improved. This study provides new evidence for the mechanism by which RAN activates the immune response and is expected to provide a new strategy for the research and development of tumor vaccine adjuvants.

A druggable copper-signalling pathway that drives inflammation.

Inflammation is a complex physiological process triggered in response to harmful stimuli1. It involves cells of the immune system capable of clearing sources of injury and damaged tissues. Excessive inflammation can occur as a result of infection and is a hallmark of several diseases2-4. The molecular bases underlying inflammatory responses are not fully understood. Here we show that the cell surface glycoprotein CD44, which marks the acquisition of distinct cell phenotypes in the context of development, immunity and cancer progression, mediates the uptake of metals including copper. We identify a pool of chemically reactive copper(II) in mitochondria of inflammatory macrophages that catalyses NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with supformin (LCC-12), a rationally designed dimer of metformin, induces a reduction of the NAD(H) pool, leading to metabolic and epigenetic states that oppose macrophage activation. LCC-12 interferes with cell plasticity in other settings and reduces inflammation in mouse models of bacterial and viral infections. Our work highlights the central role of copper as a regulator of cell plasticity and unveils a therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.

Isosteviol Sodium Ameliorates Dextran Sodium Sulfate-Induced Chronic Colitis through the Regulation of Metabolic Profiling, Macrophage Polarization, and NF-κB Pathway.

Inflammatory bowel diseases (IBDs) constitute a group of chronic intestinal conditions prominently featuring deranged metabolism. Effective pharmacological treatments for IBDs are lacking. Isosteviol sodium (STV-Na) exhibits anti-inflammatory activity and may offer therapeutic benefits in chronic colitis. However, the associated mechanism remains unclear. This study is aimed at exploring the therapeutic effects of STV-Na against chronic colitis in terms of metabolic reprogramming and macrophage polarization. Results show that STV-Na attenuated weight loss and colonic pathological damage and restored the hematological and biochemical parameters in chronic colitis mice models. STV-Na also restored intestinal permeability by increasing the goblet cell numbers, which was accompanied by lowered plasma lipopolysaccharide and diamine oxidase levels. Metabolomic analysis highlighted 102 candidate biomarkers and 5 vital pathways that may be crucial in the potential pharmacological mechanism of STV-Na in regulating intestinal inflammation and oxidative stress. These pathways were glycerophospholipid metabolism, phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, the pentose phosphate pathway, and phosphonate and phosphinate metabolism. Furthermore, STV-Na significantly decreased M1 macrophage polarization in the spleen and colon. The mRNA and protein levels of IL-1ß, TNF-α, and NF-κB/p65 in colonic tissue from the colitis mice were decreased after the STV-Na treatment. Overall, STV-Na could alleviate chronic colitis by suppressing oxidative stress and inflammation levels, reprogramming the metabolic profile, inhibiting macrophage polarization, and suppressing the NF-κB/p65 signaling pathway. STV-Na remains a promising candidate drug for treating IBDs.