IL-6 In Macrophages Research Articles

Inhibition of Mitochondrial Succinate Dehydrogenase with Dimethyl Malonate Promotes M2 Macrophage Polarization by Enhancing STAT6 Activation.

Macrophages exhibit diverse phenotypes depending on environment status, which contribute to physiological and pathological processes of immunological diseases, including sepsis, asthma, multiple sclerosis and colitis. The alternative activation of macrophages is tightly regulated to avoid excessive activation and damage of tissues and organs. Certain works characterized that succinate dehydrogenase (SDH) altered function of macrophages and promoted inflammatory response in M1 macrophages via mitochondrial reactive oxygen species (ROS). However, the effect of succinate dehydrogenase on M2 macrophage polarization remains incompletely understood. We employed dimethyl malonate (DMM) to inhibit succinate dehydrogenase activity and took use of RNA-seq to analyze the changes of inflammatory response of LPS-activated M1 macrophages or IL 4-activated M2 macrophages. Our data revealed that inhibition of SDH with DMM increased expression of M2 macrophages-associated signature genes, including Arg1, Ym1 and Mrc1. Consistent with previous work, we also observed that inhibition of SDH decreased the expression of IL-1β and enhanced the levels of IL-10 in M1 macrophages. Additionally, inhibition of SDH with DMM inhibited the production of chemokines, such as Cxcl3, Cxcl12, Ccl20 and Ccl9. DMM also amplified the M2 macrophages-related signature genes in IL-13-activated M2 macrophages. Mechanistic studies revealed that DMM promoted M2 macrophages polarization through mitochondrial ROS dependent STAT6 activation. Blocking ROS with mitoTEMPO or inhibiting STAT6 activation with ruxolitinib abrogated the promotion effect of DMM on M2 macrophages. Finally, dimethyl malonate treatment promoted peritoneal M2 macrophages differentiation and exacerbated OVA-induced allergy asthma in vivo. Collectively, we identified SDH as a braker to suppress M2 macrophage polarization via mitochondrial ROS, suggesting a novel strategy to treatment of M2 macrophages-mediated inflammatory diseases.

Isolation of New Strains of Lactic Acid Bacteria from the Vaginal Microbiome of Postmenopausal Women and their Probiotic Characteristics

Lactic acid bacteria (LAB), traditionally consumed as fermented foods, are now being applied to the medical field beyond health-functional food as probiotics. Therefore, it is necessary to continuously discover and evaluate new strains with suitable probiotic characteristics, mainly focusing on safety. In this study, we isolated eight new strains from postmenopausal vaginal fluid using culturomics approaches, an emerging area of interest. Data showed that most strains possessed significant cell surface hydrophobicity (≥ 76%), auto-aggregation capacity (17 to 61%), strong adhesion activity (8 to 34%), and excellent resistance to gastric acid, bile salt, and digestive enzyme, enhancing their survival in the gastrointestinal tract. Moreover, the strains exhibited functional characteristics, including substantial antibacterial activity with a minimal inhibitory concentration (MIC) ranging from 12.5 to 50%. They also harbored bacteriocins genes, produced short-chain fatty acids (acetate and propionate), exhibited significant phagocytic activity, possessed high antioxidative properties, rapidly depleted sodium nitrite, and exhibited proteolysis and β-glucosidase activity. In addition, heat-killed LAB strains significantly reduced the gene expressions of proinflammatory cytokines such as IL-β, IL-6, and iNOS in macrophages. Safety assessment revealed no cytotoxicity in macrophage cell lines. All strains tested negative for biogenic amine or H2O2 production, displayed no gelatinase or hemolytic activity, lacked virulence genes or detrimental enzymes, and displayed antibiotic susceptibility. In summary, these newly isolated strains demonstrate excellent probiotic functionality with a strong focus on safety, making them promising candidates for future drug development in the relevant fields.

5-methoxytryptophan protects against toll-like receptor 2 mediated renal tissue inflammation and fibrosis in a murine unilateral ureteral obstruction model.

5-methoxytryptophan (5-MTP) is an anti-inflammatory metabolite. Several recent reports indicate that 5-MTP protects against post-injury tissue fibrosis. It was unclear how 5-MTP controls tissue fibrosis. We postulated that 5-MTP attenuates renal interstitial fibrosis by blocking toll-like receptor 2 (TLR2) and transforming growth factor β (TGFβ) signaling pathways. The effect of 5-MTP on renal fibrosis was evaluated by pretreatment of wild-type UUO mice with intraperitoneal administration of 5-MTP. To determine whether 5-MTP attenuates fibrosis by inhibiting TLR2 and TGFβ signaling pathways, we evaluated the effect of 5-MTP on TLR2-induced fibroblast phenotypic switch in NRK-49F fibroblasts and TLR2 and TGFβ signaling pathways in human proximal tubular epithelial cells (HPTEC) and RAW264.7 macrophages stimulated with Pam3CSK4 (Pam3) or TGFβ1. UUO-induced renal fibrosis was abrogated in tlr2-/- mice consistent with a crucial role of TLR2 in UUO-induced renal fibrosis. UUO-induced macrophage infiltration and pro-fibrotic cytokine production in renal tissues were suppressed by tlr2 knockout. 5-MTP attenuated renal tissue fibrosis accompanied by reduction of macrophage infiltration and IL-6 and TGFβ levels. 5-MTP inhibits TLR2 upregulation and blocks TLR2-MyD88-TRAF6 signaling pathway in macrophages. Furthermore, 5-MTP blocked Pam3- and TGFβ1-induced phenotypic switch of NRK-49F to myofibroblasts and inhibited Pam3- and TGFβ1-induced signaling pathways in HPTECs and RAW264.7 cells. 5-MTP effectively protects against UUO-induced renal interstitial fibrosis by blocking TLR2 and TGFβ signaling pathways.

Structurally diverse meroterpenoids from Arnebia euchroma and their anti-inflammatory effects through NF-κB pathway.

Meroterpenoids in the traditional Chinese medicine Arnebia euchroma are thought to be key components in its anti-inflammatory activity. In the present study, 17 meroterpenoids including four types of structural skeletons (1-17), together with a monoterpenoid (18), were isolated from the roots of A. euchroma. HRESIMS, 1D and 2D NMR, electronic circular dichroism, and quantum computing-assisted methods were employed to determine the structures of four previously undescribed compounds (1-3, and 14). Zicaomeroterin B (2) feature with a novel benzo[b]oxepin moiety was confirmed by X-ray analysis based on the crystalline mate method. Meroterpenoids 2, 5, 6, 13, and 17 exhibited significant inhibition against the production of NO, IL-6, and TNF-α in LPS-stimulated macrophages without obvious cytotoxic effects. Furthermore, compounds 2, 5, and 6 significantly inhibited the phosphorylation activation of NF-κB p65 and its nuclear translocation in luciferase reporter test, immunoblotting, and immunofluorescence imaging, which in turn inhibited the NF-κB pathway and exerted anti-inflammatory effects. These findings suggested that meroterpenoids 2, 5, and 6 in A. euchroma are the potential lead compounds for anti-inflammatory agents based on NF-κB signaling pathway.

Tetrahydrocurcumin alleviates di-(2-ethylhexyl) phthalate-induced adipose tissue dysfunction and testicular toxicity in adult mice: possible involvement of adiponectin-adipoR signaling in the testis.

Widespread exposure to endocrine disruptors is associated with metabolic dysfunction and reproductive toxicity. Tetrahydrocurcumin (THC) has attracted attention as it offers protection against obesity and metabolic disorders due to its potent antioxidative and diverse biological properties but its influence and underlying mechanism of action on adipose tissue function and DEHP-induced testicular injury remain unknown. Our results showed that THC (100 mg kg-1 day-1) administration for 27 weeks enlarged adipocytes while attenuating macrophage infiltration and IL-6 expression in the adipose tissue of male C57BL/6J mice exposed to 5 mg kg-1 day-1 of DEHP. Moreover, THC ameliorated DEHP-induced deregulation of adiponectin but not leptin. DEHP caused testicular histological damage, spermatogenesis impairment, apoptosis, inflammation, and AGE, which were improved by THC. THC treatment elevated Nrf2/HO-1 and decreased Glut1 in interstitial Leydig cells, which may contribute to its beneficial effects on the testis. Our results further demonstrated that THC also ameliorated circulating adiponectin and testicular adipoR1-AMPK signaling, partially accounting for the improvement of DEHP-caused testicular dysfunction. The finding of this study revealed that THC is a promising candidate for improving adipose and testicular dysfunction caused by DEHP.

Citrus pectins impact the function of chicken macrophages

The restrictions on excessive use of antimicrobials in the poultry industry have led to the search for alternative strategies including nutritional interventions to enhance gut health with the ultimate aim to prevent gut infections. Pectins as prebiotics have shown beneficial effects on gut health in humans and mice by improving the gut barrier function, altering the gut microbiota, and by modulating the gut immune response. However, little is known about immunomodulatory properties of pectins in chickens. The present in vitro study assessed the effect of three pectins (SPE6, SPE7, SPE8) differing in methyl esterification, on responsiveness of the chicken macrophage cell line HD11 cells and primary monocyte derived macrophage from the blood, through interaction with chicken TLRs. All three pectins increased gene expression of iNOS and IL10 in chicken macrophages, Differences in immunomodulatory activity between the three pectins were observed in other assays. The low methoxyl pectin (SPE8) interacted with TLR4 leading to the production of NO, but also to increased phagocytosis of E. coli, while high methoxyl pectins SPE6 and SPE7 did not activate TLR4. All three pectins were able to attenuate PAM3CSK4 induced activation of chicken macrophages as measured by a decreased NO production and phagocytosis. Additional studies using ITC and flow cytometry suggest that the inhibiting properties of pectins (SPE6, SPE7) on macrophages is due to pectins occupying TLR2 and blocking PAM3CSK4 to activate chicken macrophages, whereas SPE8 actually binds to the TLR2 ligand and that way attenuates the PAM3CSK4 induced activation. Based on these immunomodulatory properties observed in this study, these pectins may in the future be suitable as feed additive for the treatment and prevention of inflammatory disorders in poultry.

Class B Scavenger Receptor CD36 as a Potential Therapeutic Target in Inflammation Induced by Danger-Associated Molecular Patterns.

The class B scavenger receptor CD36 is known to bind and mediate the transport of lipid-related ligands and it functions as a pattern recognition receptor (PRR) for a variety of pathogens, including bacteria and viruses. In this study, we assessed CD36's role as a PRR mediating pro-inflammatory effects of several known Danger-Associated Molecular Patterns (DAMPs) used either as a single preparation or as a combination of DAMPs in the form of total cell/skeletal muscle tissue lysates. Our data demonstrated that multiple DAMPs, including HMGB1, HSPs, histone H3, SAA, and oxPAPC, as well as cell/tissue lysate preparations, induced substantially higher (~7-10-fold) IL-8 cytokine responses in HEK293 cells overexpressing CD36 compared to control WT cells. At the same time, DAMP-induced secretion of IL-6 in bone marrow-derived macrophages (BMDM) from CD36-/- mice was markedly (~2-3 times) reduced, as compared to macrophages from normal mice. Synthetic amphipathic helical peptides (SAHPs), known CD36 ligands, efficiently blocked CD36-dependent inflammatory responses induced by both cell and tissue lysates, HMGB1 and histone H3 in CD36+ cells. IP injection of total cellular lysate preparation induced inflammatory responses that were assessed by the expression of liver and lung pro-inflammatory markers, including IL-6, TNF-α, CD68, and CXCL1, and was reduced by ~50% in CD36-deficient mice compared to normal mice. Our findings demonstrate that CD36 is a PRR contributing to the innate immune response via mediating DAMP-induced inflammatory signaling and highlight the importance of this receptor as a potential therapeutic target in DAMP-associated inflammatory conditions.

Sesamol protects against LPS-induced inflammation in rat peritoneal macrophages by promoting SIRT1-induced repression of NF-κB.

Sesamol, a polyphenolic compound isolated from roasted sesame seeds exhibits significant anti-inflammatory effect, but the molecular mechanism is poorly understood. Peritoneal macrophages play a pivotal role in the control of infections and inflammatory pathologies and are also found in injured tissuesalong with resident macrophages. The present study aimed to examine the anti-inflammatory effectof sesamol and the molecular mechanisms involved, particularly the role of sesamol in modulating SIRT1- and SIRT1-mediated deacetylation of NF-κB p65 using in vivo activated peritoneal macrophages. Sprague Dawley rats were injected with LPS to induce inflammation and sesamol was intraperitoneally administered to study its anti-inflammatory effect. ELISA and real time PCR were used to study the expression of proinflammatory cytokines. Effects of sesamol on iNOS and COX-2 were studied with activity assays and ELISA. ICAM-1, MMP-9 and TIMP-1 expressions were analysed by ELISA, RT PCR and zymography. Western blot analysis was performed to determine p65 acetylation. Nuclear translocation of p65 was evaluated by ELISA. The gene and protein expression of SIRT1 was analysed with ELISA and real time PCR. Sesamol downregulated the expression of proinflammatory markers TNF-α, IL-6, iNOS, COX-2, TLR-4, ICAM-1 and MMP-9 in rat peritoneal macrophages. Additionally, sesamol upregulated SIRT1expression and attenuated the nuclear translocation of NF-κB p65 by promoting its deacetylation. Inhibition of SIRT1 by its specific inhibitor EX527 diminished the inhibitory effect of sesamol on TNF-α and IL-6. Moreover, EX527 reduced the suppressive impact of sesamol on p65 acetylation and subsequent nuclear translocation. Our findings suggest that the anti-inflammatory effect of sesamol involves upregulation of SIRT-1, leading to the downregulation of the nuclear translocation of NF-κB p65 through its deacetylation. Therefore, the dietary bioactive compound sesamol shows potential as a promising strategy for preventing inflammatory diseases by modulating SIRT1 expression.

Corticosteroid effects on IL-10 and IL-1β in U937-derived macrophages: A model for Kawasaki disease-associated inflammation

BackgroundKawasaki disease (KD) is a pediatric vasculitis that has a predilection for coronary artery involvement. Activated macrophages play an important role in the destruction of the coronary arteries in KD. Although intravenous immunoglobulin (IVIG) is standard therapy, corticosteroids are sometimes given to patients at a higher risk of IVIG non-responsiveness. In this study, we examined the effect of IVIG and corticosteroids in U937 derived M1 and M2 a macrophages. MethodsA total of 40 children with KD and 30 healthy controls were enrolled. U937-derived macrophages were stimulated with patient plasma to examine its effect on macrophage polarization. U937 derived M1 and M2 macrophages were then stimulated with IVIG and methylprednisolone. RNA was extracted from cell cultures and the expression levels of STAT1, interleukin (IL)-1β, PPARγ and IL-10 were determined by RT-PCR. ResultsIVIG was effective at suppressing IL-10 expression in M2 macrophages (relative mRNA expression mean ± SE, high dose IVIG Vs. untreated 0.304 ± 0.095 Vs. 2.541 ± 0.157, p = 0.002), but did not suppress the production of IL-1β in M1 macrophages. In contrast, methylprednisolone both suppressed the IL-1β in M1 macrophages and also enhanced IL-10 in M2 macrophages even at low doses (relative mRNA expression mean ± SE, low dose methylprednisolone Vs. untreated IL-1β 6.353 ± 0.414 Vs. 93.838 ± 1.321, p < 0.001, IL-10 61.117 ± 2.319 Vs. 46.867 ± 2.893, p = 0.005). DiscussionIn this study we found that methylprednisolone was effective at suppressing the inflammatory cytokine IL-1β in M1 macrophages, and enhanced the production of anti-inflammatory IL-10 in M2 macrophages, an effect that could not be produced by IVIG. Our findings provide further mechanistic evidence that corticosteroid therapy, even at low doses may be a cost-effective adjuvant to IVIG therapy in patients with high-risk KD.

Design and synthesis of C-17 benzylidene derivatives of 14-deoxyandrographolide (14-DAG) and their TNF-α and IL-6 expression inhibitory activities

Benzylidene derivatives of 14-deoxyandrographolide (14-DAG) have been synthesised in good to excellent yield. All the synthesised compounds were screened for anti-inflammatory activity. The results of the cell viability assay suggested that most of the compounds were non-toxic and were further selected for investigation of NO inhibition potential. The selected compounds were investigated for their NO inhibition potential in comparison to andrographolide using LPS induced RAW 264.7 cells. The results demonstrated significant NO inhibitory activity of compound 4e in comparison to andrographolide. Based on these results, compound 4e was further investigated for inhibition of pro-inflammatory cytokines, including cytokines TNF-α and IL-6 in LPS-induced murine macrophages. Inhibitory effects of compound 4e, on pro-inflammatory cytokines TNF-α and IL-6 were compared to the parent compound, andrographolide, wherein the results demonstrated that compound 4e exhibited an IC50 (TNF- α) of 8.07 µM in comparison to andrographolide which exhibited an IC50 of 15.86 µM. Further molecular docking studies of the most potent compound 4e was also performed, which showed that the chemical forms substantial hydrogen and alkyl bonds with TNF-α, demonstrating a greater binding affinity than IL-6. The overall findings of compound 4e suggested that it can possibly be a promising structural lead for the development of new anti-inflammatory agents.

Harnessing β-glucan conjugated quercetin nanocomplex to function as a promising anti-inflammatory agent via macrophage-targeted delivery

Quercetin, a promising anti-inflammatory agent, faces challenges related to poor bioavailability and limited practical applications. β-glucan, a natural polysaccharide, can be specifically recognized by macrophages, making it an ideal targeting carrier to enhance therapeutic efficacy for macrophage-related dysfunctions. In this study, β-glucan conjugated quercetin nano-complexes (CM-Cur@QT) were developed to target macrophage and alleviate pro-inflammatory response in M1-like macrophages. The results demonstrated that CM-Cur@QT exhibited a spheric shape with an average diameter around 200 nm. FT-IR, 1H NMR, XRD and XPS analyses confirmed the complexation of CM-Cur@QT. This complex showed excellent stability during stimulated digestion, protecting QT from degradation while maintaining favorable antioxidant activity. After complexation, CM-Cur@QT displayed sustained uptake kinetics and enhanced accumulation in macrophages, with a 61.88 % increase compared to individual quercetin after 5 h of incubation. Meanwhile, CM-Cur@QT administration induced evidently cell cycle phases transitions and altered phagocytotic activity in M1-like macrophages. Furthermore, CM-Cur@QT reduced intracellular ROS accumulation, achieving a ROS scavenging rate of up to 49.92 %, compared to 25.59 % in quercetin group. This complex also effectively modulated TNF-a, IL-6 and TGF-β secretion profiles in pro-inflammatory macrophages, outperforming individual QT treatment. Notably, CM-Cur@QT facilitated anti-inflammatory effects while minimizing impacts on inactivated M0 macrophages. These findings underscore the potential of CM-Cur@QT as a promising agent for mitigating inflammatory disorders.

Abstract 4135790: Carotid Plaque Inflammation Is Associated with Atrial Fibrillation

Introduction: Atrial fibrillation (AF) is prevalent in patients with atherosclerotic coronary and carotid artery disease and is associated with elevated plasma inflammatory markers, such as Interleukin (IL)-6. In carotid artery atherosclerosis, macrophages are considered a local marker of inflammation. Hypothesis: We hypothesize that AF is independently associated with carotid plaque inflammation. Aims: In patients undergoing carotid endarterectomy (CEA), we aimed to determine whether carotid plaques inflammation is associated with AF. Approach: In this prospective cohort study, consecutive patients with carotid artery disease undergoing CEA were enrolled. From the plaques removed during surgery, fixed in formalin and embedded in paraffin blocks, sections were stained with antibodies against CD68 to detect macrophages (Fig.1A) . Digital copies of the slides were created by Motic Easy Scan and histologically annotated with QuPath. The percentage of CD68+ cells was defined as (CD68+ positive cells/total nuclei) x 100. Flash-frozen plaque segments were analyzed by ELISA for IL-6 concentration. Baseline demographics and clinical data, including AF diagnosis, were extracted from the electronic health records. Results: A total of 106 patients undergoing CEA, with a median age of 73 [67-78] years and comprising 83 (78.3%) males, were included in the study. AF was diagnosed prior to CEA in 13 (12.3%) patients. Univariate logistic regression showed that plaque CD68+ cell content was associated with AF (OR 1.048 [95% CI 1.010 – 1.088]; p = 0.013). Additionally, in Mann-Whitney test IL-6 levels were significantly over twofold higher in plaques of patients with AF compared to those without AF (82.4 [64.9-111.7] pg/mg vs 37.0 [18.4-93.3] pg/mg; p=0.020) (Fig. 1B) . Conclusions: The current study demonstrates an association between carotid atherosclerotic plaque macrophage content, IL6 levels and AF, supporting the role of inflammation in the pathogenesis of AF in these patients. Further studies are needed to determine the potential of plaque inflammation as a therapeutic target in AF.

Licochalcone A loaded multifunctional chitosan hyaluronic acid hydrogel with antibacterial and inflammatory regulating effects to promote wound healing

The wound healing process is characterized by persistent infection and long-term inflammation. The licochalcone A (LicA) has the potential for skin wound healing and needs a good drug-loading platform to apply its antibacterial and anti-inflammatory effects. In this study, the LicA@chitosan (CS) -hyaluronic acid (HA) hydrogel with antibacterial and anti-inflammatory was developed for wound healing in mice. The SEM displayed that the hydrogel had an obvious porous structure and was very suitable to be used as a delivery carrier for LicA. The FTIR results suggested that the LicA can be effectively loaded in the CS-HA hydrogel. Variable strain scanning, frequency scanning and temperature scanning indicated that the LicA@CS-HA hydrogel can maintain the gel state. The LicA@CS-HA hydrogel had good biological safety, can inhibit the activity of Escherichia coli and Staphylococcus aureus, and can release LicA stably. The LicA@CS-HA hydrogel also has good adhesion and hemostatic properties. Finally, the LicA@CS-HA hydrogel significantly accelerated wound healing in mice skin injury model, and reduced inflammation and orderly collagen deposition were observed by HE and Masson staining. The immunohistochemistry indicated that the LicA@CS-HA hydrogel induced the positive expression of CD31, VEGF, and HIF-1α promoted neovascularization. The LicA@CS-HA hydrogel also down-regulated the expression of M1 macrophage markers CD86, IL-6, and TNF-α, and increased the expression of M2 macrophage markers CD206, IL-4, and IL-10 proteins. The molecular docking demonstrated that the target proteins had better binding activity to LicA. Collectively, the LicA@CS-HA hydrogel has broad application prospects in promoting wound healing.

Macrophages overexpressing interleukin-10 target and prevent atherosclerosis: regression of plaque formation and reduction in necrotic core

Abstract Background Atherosclerosis, a slowly progressing inflammatory disease, is characterized by the presence of monocyte-derived macrophages. Interventions targeting the inflammatory characteristics of atherosclerosis hold promising potential. Although interleukin (IL)-10 is widely acknowledged for its anti-inflammatory effects, systemic administration of IL-10 has limitations due to its short half-life and significant systemic side effects. Purpose We aimed to investigate the effectiveness of an approach designed to overexpress IL-10 in macrophages and subsequently introduce these genetically modified cells into ApoE-/- mice to promote atherosclerosis regression. Methods We engineered RAW264.7 cells to overexpress IL-10 (referred to as IL-10M) using lentivirusvectors. The ApoE-/- mice were divided into two groups based on the timing of the intervention. The early intervention group (n = 45) received intravenous tail injections from the beginning of Western diet (WD) at six weeks old. The late intervention group (n = 45) started receiving injections at 16 weeks old, 2 months after being fed the WD. Both the early and late intervention group stopped receiving injections after 5 months of being fed the WD. Results The IL-10M exhibited robust IL-10 secretion, maintained phagocytic function, improved mitochondrial membrane potentials, reduced superoxide production and showed a tendency toward the M2 phenotype when exposed to inflammatory stimuli. IL-10M can selectively target plaques in ApoE-/- mice, and oil red staining of the entire aorta unequivocally demonstrated that intervention with IL-10M elicited a substantial reduction in plaque area. H&amp;E staining revealed that mice treated with IL-10M in the early intervention group showed a significant reduction in the ratio of plaque area to lumen area. However, the late intervention group did not exhibit statistically significant differences. Notably, mice treated with IL-10M exhibited significantly smaller necrotic cores and reduction in matrix metalloproteinase 9 within the aortic plaques in both the early and late intervention groups. Additionally, the administration of IL-10M showed no obvious side effects. Conclusion We developed an anti-inflammatory protein delivery system based on engineered macrophages that can target atherosclerotic plaques and reduce the plaque area and necrotic core. This approach has a promising safety profile and has the potential to be a therapeutic strategy for the intervention of atherosclerosis.

Toll-like receptor 2-mediated ERK activation significantly upregulates interleukin-6 expression in M2-polarized macrophages

Abstract Objectives M2-polarized macrophages and interleukin (IL)-6 significantly alter the tumor microenvironment and promote the malignant behaviors of tumor cells. This study aimed to establish M2-type macrophages from THP-1 cells, which are human leukemia monocytes, and investigate the significance of toll-like receptor 2 (TLR2) signaling in IL-6 production. Methods THP-1 cells were treated with phorbol 12-myristate 13-acetate, IL-4, and IL-13 to stimulate their differentiation into M2 macrophages. Cell differentiation was confirmed by cytokine production, marker expression, and morphological alterations. Treatment with TLR agonists induced TLR stimulation in M2 macrophages. Subsequently, secretion and expression levels of IL-6 in M2 macrophages were measured using enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, and western blotting. Results Myeloid differentiation factor 88, tumor necrosis factor-associated factor 6, and IL-1 receptor-associated kinase-1/4 signaling pathways contributed to IL-6 production upon TLR2 activation in M2 macrophages. While both TLR2 and TLR4 activated NF-κB in M2 macrophages, IL-6 production was mainly dependent on TLR2, not TLR4, suggesting the involvement of major mechanisms other than NF-κB in IL-6 production. Notably, TLR2-stimulated extracellular signal-regulated kinase (ERK) was necessary for abundant IL-6 production, indicating that TLR2-mediated ERK signaling plays an essential role in M2 macrophages. Conclusions These results highlight the significance of TLR2 signaling in IL-6 production by M2 macrophages and provide insights into the underlying regulatory mechanisms.

Impact of proNGF/NGF-NGFR-axis on the regulation of arterial remodelling

Abstract Background Peripheral blood mononuclear cells (MNCs) contribute to the pathogenesis of arteriosclerosis. Nerve growth factor receptor (NGFR) is present in peripheral blood and the ischemic coronary artery. NGFR transduces neurotrophin signals towards cell survival or apoptosis in different cell types dependent on coupling co-receptors. However, the role of NGFR-positive (NGFR+) MNCs in arterial remodelling is unknown. Purpose To investigate the functional mechanisms under which NGFR+ MNCs are involved in arterial remodelling. Methods Adult C57BL/6J male NGFR-wild-type (WT) or -bone marrow (BM)-specific (KO) mice were subjected to unilateral carotid artery ligation. The ligated and the opposite-sided, sham-operated arteries were assessed by immunohistology and gene expression analysis using a PCR on day 28. Also, human NGFR+ MNCs from a healthy volunteer were sorted using fluorescence-activated cell sorting and characterised using RNA sequencing. The cell apoptosis (AnnexinV+7AAD-) and chemotaxis in response to the ligands, NGF and its precursor proNGF, were assessed using flow cytometry and a Transwell migration. Results In WT mice, BM-derived NGFR+ cells accumulated in the neointima after ligation. The neointimal area was significantly greater in the BM-specific depletion of NGFR than in WT mice. NGFR+ cells in the neointima exhibited apoptosis (TUNEL+ and cleaved caspase-3+) accompanied by promoted F4/80+ macrophage and anti-inflammatory IL-10. By contrast, in the BM-specific NGFR-KO model, non-BM-derived SMCs increased in the neointima with augmented proliferation, and the accumulation of BM-derived cells and the expression of IL-10 were decreased. The expressions of proNGF/NGF and inflammatory cytokines, including IL-6, were not changed in a ligated artery, irrespective of NGFR+ depletion. Human NGFR+ MNCs expressed TrkB, TrkC, and sortilin co-receptors but not macrophage markers. Human NGFR+ MNCs, but not NGFR- MNCs, co-cultured with artery SMCs were susceptible to apoptosis in response to proNGF. Additionally, PDGF stimulated proNGF/NGF expression in SMCs, and proNGF/NGF did not affect the proliferation of SMCs in vitro. Furthermore, NGFR+ MNCs, but not NGFR- MNCs, increased migration toward NGF and did not show chemotaxis toward proNGF. Conclusions These data imply that local NGF might draw NGFR+ MNCs to the injured artery, in which proNGF induces NGFR+ MNCs apoptosis. Apoptotic NGFR+ cells promoted macrophage chemotaxis to resolve inflammation and decrease neointimal formation. Thus, we propose the proNGF/NGF-NGFR axis as potentially contributing to the suppression of arterial remodelling.

Unique transcriptomic profile of peripheral blood monocytes in rheumatoid arthritis-associated interstitial lung disease.

Though interstitial lung disease (ILD) contributes to excess morbidity and mortality in rheumatoid arthritis (RA), RA-ILD pathogenesis remains incompletely defined. As intermediate, non-classical and suppressed CD14+ monocytes are expanded in RA-ILD, this study sought to characterize gene expression profiles of circulating monocytes in RA-ILD. Peripheral blood mononuclear cells were collected from patients with RA without lung disease (N = 5), RA-ILD (N = 5), idiopathic pulmonary fibrosis (IPF; N = 5), and controls without lung and autoimmune disease (N = 4). RNA was extracted from CD14+ isolated monocytes and subjected to transcriptional analysis of 1365 genes. Gene enrichment and pathway analyses were performed. Unsupervised clustering grouped patients with RA-ILD together with IPF for myeloid innate genes. For fibrosis genes, patients with RA-ILD clustered independent of comparator groups. There were 103, 66, and 64 upregulated and 66, 14, and 25 downregulated genes for RA-ILD, RA, and IPF, vs controls, respectively. For RA-ILD, there was increased expression of genes involved in regulating inflammation and fibrosis (SOCS3, CECAM1, LTB4R2, CLEC7A, IRF7, PHYKPL, GBP5, RAPGEF), epigenetic modification (KDM5D, KMT2D, OGT), and macrophage activation. Top canonical pathways included macrophage differentiation-activation, IL-12, neuroinflammatory, glucocorticoid receptor, and IL-27 signalling. Circulating monocytes in RA-ILD patients demonstrate unique gene expression profiles with innate immune gene features more aligned with IPF as opposed to RA in the absence of clinical lung disease with fibrosis gene expression that was distinct from RA and IPF. These studies are important for understanding disease pathogenesis and may provide information for future therapeutic targets in RA-ILD.

Macrophages overexpressing interleukin-10 target and prevent atherosclerosis: Regression of plaque formation and reduction in necrotic core.

Atherosclerosis, a slowly progressing inflammatory disease, is characterized by the presence of monocyte-derived macrophages. Interventions targeting the inflammatory characteristics of atherosclerosis hold promising potential. Although interleukin (IL)-10 is widely acknowledged for its anti-inflammatory effects, systemic administration of IL-10 has limitations due to its short half-life and significant systemic side effects. In this study, we aimed to investigate the effectiveness of an approach designed to overexpress IL-10 in macrophages and subsequently introduce these genetically modified cells into ApoE-/- mice to promote atherosclerosis regression. We engineered RAW264.7 cells to overexpress IL-10 (referred to as IL-10M) using lentivirus vectors. The IL-10M exhibited robust IL-10 secretion, maintained phagocytic function, improved mitochondrial membrane potentials, reduced superoxide production and showed a tendency toward the M2 phenotype when exposed to inflammatory stimuli. IL-10M can selectively target plaques in ApoE-/- mice and has the potential to reduce plaque area and necrotic core at both early and late stages of plaque progression. Moreover, there was a significant reduction in MMP9, a biomarker associated with plaque rupture, in IL-10M-treated plaques from both the early and late intervention groups. Additionally, the administration of IL-10M showed no obvious side effects. This study serves as proof that cell therapy based on anti-inflammatory macrophages might be a promising strategy for the intervention of atherosclerosis.

Daidzein attenuates high glucose-induced inflammatory injury in macrophages by regulating NLRP3 inflammasome signaling pathway

This study aims to explore the inhibitory effect of daidzein on macrophage inflammation induced by high glucose via regulating the NOD-like receptor protein 3(NLRP3) inflammasome signaling pathway. The cell counting kit-8(CCK-8) assay was employed to detect the effects of daidzein at different concentrations on the viability of RAW264.7 cells. Western blot was employed to determine the protein level of tumor necrosis factor(TNF)-α in macrophages exposed to different concentrations of glucose for different time periods as well as the expression levels of proteins involved in the polarization and Toll-like receptor 4(TLR4)-myeloid differentiation factor(MyD88)-NLRP3 inflammasome pathway of the macrophages exposed to high glucose. Enzyme-linked immunosorbent assay was employed to measure the levels of TNF-α, interleukin(IL)-18, and IL-1β secreted by macrophages. The expression level of nuclear factor-kappa B(NF-κB) p65 in macrophages exposed to high glucose was detected by immunofluorescence, and the level of intracellular reactive oxygen species(ROS) was detected by the DCFH-DA fluorescent probe. The mRNA levels of NLRP3, TNF-α, and IL-18 in macrophages were determined by qRT-PCR. The results showed that treatment with 30 mmol·L~(-1) glucose for 48 h was the best condition for the modeling of macrophage injury. Compared with the blank group, the model group showed improved polarization of macrophages, increased secretion of TNF-α, IL-18, and IL-1β, elevated ROS level, and up-regulated expression of NF-κB p65. In addition, the modeling up-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 and the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1β, cleaved IL-1β, and pro-IL-18. Compared with the model group, daidzein(10, 20, and 40 μmol·L~(-1)) lowered the levels of inflammatory cytokines and down-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 as well as the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1β, cleaved IL-1β, and pro-IL-18. In addition, daidzein reduced intracellular ROS. According to the available reports and the experimental results, high glucose can induce the polarization of macrophages and promote the secretion of inflammatory cytokines. Daidzein can inhibit the expression of ROS in macrophages by regulating the NLRP3 inflammasome signaling pathway, thereby reducing the inflammation of macrophages exposed to high glucose.

Levilactobacillus brevis IBARAKI-TS3 Isolated From Pickles Promotes Production of Interleukin-10 via Toll-Like Receptor 2 in Human M2 Macrophages.

M2 macrophages play an important role in food allergy. Several studies have reported that lactic acid bacteria isolated from pickles exert antiallergic effects. We investigated the effects of several strains of lactic acid bacteria on the immune function of M2 macrophages. M2 macrophages differentiated from THP-1 cell line by interleukin-4 (IL-4) and IL-13 strongly expressed CD163, CD206, and HMOX1 mRNA. Levilactobacillus brevis IBARAKI-TS3 (IBARAKI-TS3) isolated from pickles was identified as a lactic acid bacterium that enhances the expressions of IL-10 and EBI3 mRNA in M2 macrophages. IBARAKI-TS3 induced the expression of genes involved in Toll-like receptor (TLR) signaling, such as IRAK, mitogen-activated protein kinases (MAPKs), and NF-κB mRNA. IBARAKI-TS3-induced IL-10 production was suppressed by anti-TLR2-neutralizing antibodies. Furthermore, the IBARAKI-TS3-induced increase in IL-10 levels was significantly reduced in TLR2-knockdown M2 macrophages compared to M2 macrophages. These results suggest that IBARAKI-TS3 promotes of IL-10 production via TLR2 in M2 macrophages.