Inflammatory Activation Of Macrophages Research Articles

Methionine-choline deficient diet deteriorates DSS-induced murine colitis through disturbance of gut microbes and infiltration of macrophages.

Ulcerative colitis (UC) is associated with changed dietary habits and mainly linked with the gut microbiota dysbiosis, necroptosis of epithelial cells, and mucosal ulcerations. Liver dysfunction and abnormal level of liver metabolism indices were identified in UC patients, suggesting a close interaction between gut and liver disorders. Methionine-choline deficient diet (MCD) has been shown to induce persistent alterations of gut microbiota and metabolome during hepatitis. In this study we further explored the disease phenotypes in UC patients and investigated whether MCD functioned as a trigger for UC susceptibility. After assessing 88 serum specimens from UC patients, we found significant liver dysfunction and dyslipidemia including abnormal ALT, AST, TG, TC, LDL-c and HDL-c. Liver dysfunction and dyslipidemia were confirmed in DSS-induced colitis mice. We fed mice with MCD for 14 days to cause mild liver damage, and then treated with DSS for 7 days. We found that MCD intake significantly exacerbated the pathogenesis of mucosal inflammation in DSS-induced acute, progressive, and chronic colitis, referring to promotion of mucosal ulcers, colon shortening, diarrhea, inflammatory immune cell infiltration, cytokines release, and abnormal activation of inflammatory macrophages in colon and liver specimens. Intraperitoneal injection of clodronate liposomes to globally delete macrophages dramatically compromised the pathogenesis of MCD-triggering colitis. In addition, MCD intake markedly changed the production pattern of short-chain fatty acids (SCFAs) in murine stools, colons, and livers. We demonstrated that MCD-induced colitis pathogenesis largely depended on the gut microbes and the disease phenotypes could be transmissible through fecal microbiota transplantation (FMT). In conclusion, this study supports the concept that intake of MCD predisposes to experimental colitis and enhances its pathogenesis via modulating gut microbes and macrophages in mice.

Isoliquiritigenin limits inflammasome activation of macrophage via docking into Syk to alleviate murine non-alcoholic fatty liver disease.

Isoliquiritigenin (ISL) is a chalcone-type flavonoid derived from the root of licorice with antioxidant, anti-inflammatory, anti-tumour and neuroprotective properties. ISL has been proven to downregulate the productions of IL-1β, TNF-α and IL-6 by macrophages. However, detailed molecular mechanisms of this modulation remain elusive. Here, ISL suppressed Syk phosphorylation and CD80, CD86, IL-1β, TNF-α and IL-6 expressions in lipopolysaccharide-stimulated macrophages exvivo. ApoC3-transgenic (ApoC3TG) mice had more activated macrophages. ISL was also able to downregulate the inflammatory activities of macrophages from ApoC3TG mice. Administration of ISL inhibited Syk activation and inflammatory activities of macrophages in ApoC3TG mice invivo. The treatment of ISL further alleviated MCD-induced non-alcoholic fatty liver disease (NAFLD) in wild-type and ApoC3TG mice, accompanied by less recruitment and activation of liver macrophages. Due to the inhibition of Syk phosphorylation, ISL-treated macrophages displayed less production of cytoplasmic ROS, NLRP3, cleaved-GSDMD and cleaved-IL-1β, suggesting less inflammasome activation. Finally, the molecular docking study demonstrated that ISL bound to Syk directly with the Kd of 1.273 × 10-8 M. When the Syk expression was knocked down by its shRNA, the inhibitory effects of ISL on activated macrophages disappeared, indicating that Syk was at least one of key docking-molecules of ISL. Collectively, ISL could alleviate MCD-induced NAFLD in mice involved with the inhibition of macrophage inflammatory activity by the blockade of Syk-induced inflammasome activation.

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.

The association of TNF-alpha secretion and mtDNA copy number in CD14+ monocytes of patients with obesity and CHD.

Mitochondrial dysfunction may be one of the causes of inflammatory activation of monocytes and macrophages, which leads to excessive secretion of inflammatory mediators and the development of chronic inflammation. The study was aimed to evaluate the secretion of inflammatory cytokine tumor necrosis factor-α (TNF-α) in the primary culture of monocytes, and to analyze its relationship with the number of mitochondrial DNA (mtDNA) copies in the blood of patients with coronary heart disease (CHD) and obesity. 108 patients with obesity and concomitant CHD and a control group of 25 participants were included in the study. CD14+ monocytes were isolated by a standard method in a ficoll-urographin gradient, followed by separation using magnetic particles. The number of mtDNA copies was estimated using qPCR. It was demonstrated that the number of mtDNA copies was significantly increased in groups of patients with CHD and obesity + CHD in comparison with control group. mtDNA copy number positively correlated with basal and LPS-stimulated TNF-α secretion, the most significant correlation was found in the group of patients with CHD and obesity. Thus, the change in mtDNA copy number in CD14+ monocytes which indicates the presence of mitochondrial dysfunction, confirm the direct involvement of mitochondria in the violation of the inflammatory response of monocytes revealed in this study as an increased secretion of inflammatory cytokine TNF-α.

Modulatory actions of Echinococcus granulosus antigen B on macrophage inflammatory activation.

Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1β, IL-6, IL-12p40, IFN-β) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid's effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages.

Inhibition of IL-17 signaling in macrophages underlies the anti-arthritic effects of halofuginone hydrobromide: Network pharmacology, molecular docking, and experimental validation

BackgroundRheumatoid arthritis (RA) is a prevalent autoimmune disease marked by chronic synovitis as well as cartilage and bone destruction. Halofuginone hydrobromide (HF), a bioactive compound derived from the Chinese herbal plant Dichroa febrifuga Lour., has demonstrated substantial anti-arthritic effects in RA. Nevertheless, the molecular mechanisms responsible for the anti-RA effects of HF remain unclear.MethodsThis study employed a combination of network pharmacology, molecular docking, and experimental validation to investigate potential targets of HF in RA.ResultsNetwork pharmacology analyses identified 109 differentially expressed genes (DEGs) resulting from HF treatment in RA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses unveiled a robust association between these DEGs and the IL-17 signaling pathway. Subsequently, a protein-protein interaction (PPI) network analysis revealed 10 core DEGs, that is, EGFR, MMP9, TLR4, ESR1, MMP2, PPARG, MAPK1, JAK2, STAT1, and MAPK8. Among them, MMP9 displayed the greatest binding energy for HF. In an in vitro assay, HF significantly inhibited the activity of inflammatory macrophages, and regulated the IL-17 signaling pathway by decreasing the levels of IL-17 C, p-NF-κB, and MMP9.ConclusionIn summary, these findings suggest that HF has the potential to inhibit the activation of inflammatory macrophages through its regulation of the IL-17 signaling pathway, underscoring its potential in the suppression of immune-mediated inflammation in RA.

Platelets induce endoplasmic reticulum stress in macrophages in vitro

BackgroundEndoplasmic reticulum (ER) stress is a key feature of lipid-laden macrophages and contributes to the development of atherosclerotic plaques. Blood platelets are known to interact with macrophages and fine-tune effector functions such as inflammasome activation and phagocytosis. However, the effect of platelets on ER stress induction is unknown. ObjectivesThe objective of this study is to elucidate the potential of platelets in regulating ER stress in macrophages in vitro. MethodsBone marrow–derived macrophages and RAW 264.7 cells were incubated with isolated murine platelets, and ER stress and inflammation markers were determined by reverse transcription-quantitative polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. ER morphology was investigated by electron microscopy. Cell viability, lipid accumulation, and activation were measured by flow cytometry. To gain mechanistic insights, coincubation experiments were performed with platelet decoys/releasates as well as lipopolysaccharide, blocking antibodies, and TLR4 inhibitors. ResultsCoincubation of platelets and macrophages led to elevated levels of ER stress markers (BIP, IRE1α, CHOP, and XBP1 splicing) in murine and human macrophages, which led to a pronounced enlargement of the ER. Macrophage ER stress was accompanied by increased release of proinflammatory cytokines and intracellular lipid accumulation, but not cell death. Platelet decoys, but not platelet releasates or lysate from other cells, phenocopied the effect of platelets. Blocking TLR4 inhibited inflammatory activation of macrophages but did not affect ER stress induction by platelet coincubation. ConclusionTo our knowledge, this study is the first to demonstrate that platelets induce ER stress and unfolded protein response in macrophages by heat-sensitive membrane proteins, independent of inflammatory activation of macrophages.

Unraveling the complex roles of macrophages in obese adipose tissue: an overview.

Macrophages, a heterogeneous population of innate immune cells, exhibit remarkable plasticity and play pivotal roles in coordinating immune responses and maintaining tissue homeostasis within the context of metabolic diseases. The activation of inflammatory macrophages in obese adipose tissue leads to detrimental effects, inducing insulin resistance through increased inflammation, impaired thermogenesis, and adipose tissue fibrosis. Meanwhile, adipose tissue macrophages also play a beneficial role in maintaining adipose tissue homeostasis by regulating angiogenesis, facilitating the clearance of dead adipocytes, and promoting mitochondrial transfer. Exploring the heterogeneity of macrophages in obese adipose tissue is crucial for unraveling the pathogenesis of obesity and holds significant potential for targeted therapeutic interventions. Recently, the dual effects and some potential regulatory mechanisms of macrophages in adipose tissue have been elucidated using single-cell technology. In this review, we present a comprehensive overview of the intricate activation mechanisms and diverse functions of macrophages in adipose tissue during obesity, as well as explore the potential of drug delivery systems targeting macrophages, aiming to enhance the understanding of current regulatory mechanisms that may be potentially targeted for treating obesity or metabolic diseases.

Diosmetin alleviates neuropathic pain by regulating the Keap1/Nrf2/NF-κB signaling pathway

BackgroundNeuropathic pain, a chronic condition with a high incidence, imposes psychological burdens on both patients and society. It is urgent to improve pain management and develop new analgesic drugs. Traditional Chinese medicine has gained popularity as a method for pain relief. Diosmetin (Dio) is mainly found in Chinese herbal medicines with effective antioxidant, anti-cancer, and anti-inflammatory properties. There are few known mechanisms underlying the effectiveness of Dio in treating neuropathic pain. However, the complete understanding of its therapeutic effect is missing. PurposeThis study aimed to evaluate Dio's therapeutic effects on neuropathic pain models and determine its possible mechanism of action. We hypothesized that Dio may activate antioxidants and reduce inflammation, inhibit the activation of Kelch-like epichlorohydrin-associated protein 1 (Keap1) and nuclear factor-k-gene binding (NF-κB), promote the metastasis of nuclear factor erythroid 2-related factor 2 (Nrf2) and the expression of heme oxygenase 1 (HO-1), thus alleviating the neuropathic pain caused by spinal nerve ligation. MethodsChronic nociceptive pain mouse models were established in vivo by L4 spinal nerve ligation (SNL). Different dosages of Dio (10, 50, 100 mg/kg) were intragastrically administered daily from the third day after the establishment of the SNL model. Allodynia, caused by mechanical stimuli, and hyperalgesia, caused by heat, were assessed using the paw withdrawal response frequency (PWF) and paw withdrawal latency (PWL), respectively. Cold allodynia were assessd by acetone test. RT-PCR was used to detect the content of interleukin-(IL)− 1β, IL-6 and tumor necrosis factor (TNF)-a. Immunofluorescence and western blotting were employed to assess the expression levels of Glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule (Iba1), Keap1, Nrf2, HO-1, and NF-κB p-p65 protein. ResultsDio administration relieved SNL-induced transient mechanical and thermal allodynia in mice. The protective effect of Dio in the SNL model was associated with its anti-inflammatory and anti-glial responses in the spinal cord. Dio inhibited both inflammatory factors and macrophage activation in the DRG. Furthermore, Dio regulated the Keap1/Nrf2/NF-κB signaling pathway. HO-1 and Nrf2 were upregulated following Dio administration, which also decreased the levels of Keap1 and NF-κB p65 protein. ConclusionMice with SNL-induced neuropathic pain were therapeutically treated with Dio. Dio may protect against pain by inhibiting inflammatory responses and improved Keap1/Nrf2/NF-κB pathway. These results highlight the potential therapeutic effect of Dio for the development of new analgesic drugs.

Targeted modulation and enhancement of macrophages via sonodynamic therapy-driven cupferroptosis-like stress for implant-associated biofilm infections

As the vanguard against implant-associated infections (IAIs), macrophages facilitate the phagocytosis and pro-inflammatory polarization of planktonic and biofilm infections, but indirectly serve as a “Trojan horse” for intracellular bacterial infections. Effective drug delivery and macrophage immunomodulation to eliminate intracellular infections will contribute to the treatment of refractory IAIs and prevention of relapse. Herein, we developed an ultrasound-responsive copper-based nanoparticle targeting macrophages by encapsulating copper in a porphyrin metal-organic framework and surface-grafting D-Mannosamine to synthesize CuTCPP@MOF nanodots@ Mannosamine (CTMM) with macrophage-targeting functionality. Concurrent generation of hydroxyl radicals and singlet oxygen in biofilm microenvironment mediated by ultrasound disrupts the biofilm structure, achieving CTMM overload and interfering with bacterial biofilm growth and metabolism. By regulating macrophage oxidative stress metabolism, CTMM induced a cupferroptosis-like stress, characterized by concurrent cuproptosis and ferroptosis, to eliminate intracellular infections. Transcriptomic analysis verified that the cupferroptosis-like stress based on GPX4 axis modulation further promotes macrophage inflammatory activation. This CTMM+US-mediated bacterial clearance in biofilms and macrophage immunomodulation informs the "Trojan War" strategy to combat IAIs clinically.

Analogs of FTY720 inhibit TRPM7 but not S1PRs and exert multimodal anti-inflammatory effects.

TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine-1-phosphate (S1P) receptors. In this study, we test non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo, for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, though this likely occurrs through multiple molecular targets. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct anti-inflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo. Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting which demonstrate potent, polymodal anti-inflammatory activities ex vivo and in vivo.

Protein Tyrosine Phosphatase SHP2 in Macrophages Acts as an Antiatherosclerotic Regulator in Mice.

Macrophages have versatile roles in atherosclerosis. SHP2 (Src homology 2 containing protein tyrosine phosphatase 2) has been demonstrated to play a critical role in regulating macrophage activation. However, the mechanism of SHP2 regulation of macrophage function in an atherosclerotic microenvironment remains unknown. APOE (apolipoprotein E) or LDLR (low-density lipoprotein receptor) null mice treated with SHP099 were fed a Western diet for 8 weeks, while Shp2MKO:ApoE-/- or Shp2MKO:Ldlr-/- mice and exo-AAV8-SHP2E76K/ApoE-/- mice were fed a Western diet for 12 weeks. In vitro, levels of proinflammatory factors and phagocytic function were then studied in mouse peritoneal macrophages. RNA sequencing was used to identify PPARγ (peroxisome proliferative activated receptor γ) as the key downstream molecule. A PPARγ agonist was used to rescue the phenotypes observed in SHP2-deleted mice. Pharmacological inhibition and selective deletion in macrophages of SHP2 aggravated atherosclerosis in APOE and LDLR null mice with increased plaque macrophages and apoptotic cells. In vitro, SHP2 deficiency in APOE and LDLR null macrophages enhanced proinflammatory polarization and its efferocytosis was dramatically impaired. Conversely, the expression of gain-of-function mutation of SHP2 in mouse macrophages reduced atherosclerosis. The SHP2 agonist lovastatin repressesed macrophage inflammatory activation and enhanced efferocytosis. Mechanistically, RNA sequencing analysis identified PPARγ as a key downstream transcription factor. PPARγ was decreased in macrophages upon SHP2 deletion and inhibition. Importantly, PPARγ agonist decreased atherosclerosis in SHP2 knockout mice, restored efferocytotic defects, and reduced inflammatory activation in SHP2 deleted macrophages. PPARγ was decreased by the ubiquitin-mediated degradation upon SHP2 inhibition or deletion. Finally, we found that SHP2 was downregulated in atherosclerotic vessels. Overall, SHP2 in macrophages was found to act as an antiatherosclerotic regulator by stabilizing PPARγ in APOE/LDLR null mice.

The transcription factor ELF4 alleviates inflammatory bowel disease by activating IL1RN transcription, suppressing inflammatory TH17 cell activity, and inducing macrophage M2 polarization.

Inflammatory bowel disease (IBD) is a chronic immune-mediated disorder affecting millions worldwide. Due to the complexity of its pathogenesis, the treatment options for IBD are limited. This study focuses on ELF4, a member of the ETS transcription factor family, as a target to elucidate its role in IBD and investigate its mechanism of action in alleviating IBD symptoms by activating IL1RN transcription to suppress the activity of inflammatory TH17 cells. Using the GEO database, this study examined LPS-induced intestinal inflammatory genes and their regulation mechanisms. We examined the colon length of LPS-treated mice and derived the Disease Activity Index (DAI). H&E staining, ELISA, and flow cytometry were used to detect mice colon tissue damage, inflammatory factor levels in mouse serum, mouse macrophage types and inflammatory TH17 cell activity. RT-qPCR and Western blot detected ELF4, IL1RN, M1, and M2 polarization markers. In Vitro, using dual-luciferase and ChIP assays, we tested mouse bone marrow-derived macrophages (BMDMs) and mouse intestinal epithelial cells for IL1RN promoter activity and ELF4 enrichment. Bioinformatics showed that LPS-induced colitis animals have reduced ELF4 expression in their colon tissue. In vivo tests confirmed reduced ELF4 expression in mice with LPS-induced colitis. ELF4 overexpression reduced mouse intestinal inflammation. ELF4 activated IL1RN transcription in bioinformatics and in vitro tests. ELF4 promoted IL1RN transcription and macrophage M2 polarization to limit intestinal epithelial cell death and inflammation and reduce mouse intestinal inflammation in vitro. ELF4 also reduced the Th17/Treg ratio by increasing IL1RN transcription. ELF4 activates IL1RN transcription, suppresses inflammatory TH17 cells, and induces macrophage M2 polarization to treat IBD.

Metformin reverses impaired osteogenesis due to hyperglycemia-induced neutrophil extracellular traps formation.

Diabetic patients suffer from delayed fracture healing and impaired osteogenic function, but the underlying pathophysiological mechanisms are not fully understood. Neutrophil extracellular traps (NETs) formed by neutrophils in high glucose microenvironments affect the healing of wounds and other tissues. Some evidence supports that NETs may inhibit osteogenic processes in the microenvironment through sustained inflammatory activation. In this study, we observed that high glucose-induced NETs led to sustained inflammatory activation of macrophages. Pro-inflammatory NETs inhibited the osteogenic function of osteoblasts in vitro. A bone defect healing model based on diabetic rat animal models confirmed that bone healing was impaired in a high glucose environment, but this process could be reversed by DNase I, a NETs clearance agent. More importantly, the classic hypoglycemic drug metformin had a similar antagonistic effect as DNase I and could reverse the inhibitory effect of NETs on osteogenesis in a high-glucose environment. In summary, we found that NETs formation induced by high glucose microenvironment is a potential cause of osteogenic dysfunction in diabetic patients, and metformin can reverse this osteogenic disadvantage.

Macrophages derived from LPS-stimulated monocytes from individuals with subclinical atherosclerosis were characterized by increased pro-inflammatory activity

ObjectiveAtherosclerosis is characterized by chronic inflammation in the vascular wall. Currently the violation of immune tolerance of innate immune cells is considered as a possible mechanism of chronification of inflammation. The aim of this study is to assess the inflammatory activity and tolerance of monocytes and macrophages in subclinical atherosclerosis. MethodsA total of 55 individuals free from clinical manifestations of atherosclerosis-associated cardiovascular disease with a presence or absence of atherosclerotic plaques in the carotid arteries were included in this study. CD14+ monocytes were isolated from individuals' blood and stimulated with a single dose of lipopolysaccharide (LPS) on day 1 or with double doses of LPS on day 1 and day 6. The secretion of cytokines TNF, IL-1β, IL-6, IL-8, IL-10 and CCL2 were evaluated using ELISA. ResultsOur findings demonstrate that macrophages derived from LPS-stimulated monocytes in individuals with subclinical atherosclerosis exhibited increased secretion of IL-6, IL-10 and CCL2, which was associated with intima-media thickness, body mass index, but not with individuals’ age. Moreover, macrophages from individuals with atherosclerotic plaques exhibited impaired tolerance towards the second LPS stimulation manifested by elevated secretion of the chemoattractant CCL2. ConclusionIncreased secretion of these cytokines by macrophages may contribute to chronic local inflammation in the vascular wall by recruiting other immune cells.

Transcriptome analysis of T cells from Ldlr−/− mice and effects of in vitro vitamin D treatment

Vitamin D is known for its immunosuppressive effects on T cells, suppressing Th1 and Th17 and promoting Treg differentiation. Th1 cells contribute to inflammatory responses such as inflammatory cytokine production and macrophage activation, which accelerate the progression of atherosclerosis. However, the mechanisms underlying the modulation of T cell functions by vitamin D in atherosclerosis have not been investigated. This study analyzed the gene expression profiles of T cells, using RNA-seq transcriptome analysis, to investigate the effects of in vitro vitamin D treatment on T cell differentiation and signal transduction pathways in Ldlr knock-out (Ldlr−/−) mice. C57BL/6 mice were randomly assigned to two groups and fed a control diet (CON) or a Western diet (WD) for 16 weeks, while Ldlr−/- mice (LDLR−/−) were fed a Western diet. Splenic T cells were isolated and stimulated with anti-CD3e and anti-CD28 mAb for 48 h with or without 10 nM 1,25(OH)2D3. RNA sequencing was performed, followed by KEGG and GO enrichment analyses. Populations of T cell subsets and cytokine production were measured to assess T cell lineage differentiation. The JAK-STAT, HIF-1, and calcium signaling pathways of Ldlr−/− mice significantly differed from those of control mice, and 1,25(OH)2D3 treatment affected MAPKKK binding molecular function of Ldlr−/− mice. Percentages of Th1 cells and IL-17 production were significantly reduced by 1,25(OH)2D3 treatment in all three mouse groups. These results suggest that 1,25(OH)2D3 has anti-inflammatory effects in atherosclerosis and is involved in cell signaling pathways that could prevent disease progression by regulating T cell differentiation and effector functions.

Keratinocyte derived HMGB1 aggravates psoriasis dermatitis via facilitating inflammatory polarization of macrophages and hyperproliferation of keratinocyte

Psoriasis is one of the most common immune-mediated chronic inflammatory skin diseases, involving excessive proliferation of keratinocyte and infiltration of immune cells. There are many factors that cause the onset of psoriasis, so the exact pathogenesis of psoriasis still needs to be determined. High mobility group box-1 (HMGB1), a pro-inflammatory cytokine, is closely related to the pathogenesis of various inflammatory diseases. However, there are few studies investigating the effects of HMGB1 on inflammatory dermatoses. Here, we found that keratinocyte in the the IMQ-treated skin lesions of psoriasis model mice expressed more HMGB1. Notably, HMGB1 produced by keratinocyte could promote the activation of inflammatory type macrophages without affecting the polarization of anti-inflammatory type macrophages. Meanwhile, the proportion of M1 type macrophages in the skin lesions is significantly increased. Moreover, local clearance of macrophages in the skin could alleviate psoriasis like inflammation. Finally, keratinocyte-derived HMGB1 could also act on itself in turn, promoting the excessive proliferation and the mRNA expression of inflammatory cytokines of keratinocyte. Therefore, this study not only found the effect of HMGB1 on the hyperproliferation of keratinocyte, but also revealed that keratinocyte could communicate with macrophages through HMGB1, thereby facilitating macrophage inflammatory polarization. Collectively, these findings have clinical significance for the research and treatment of psoriasis, HMGB1 may become a potential target for the treatment of psoriasis.

Effect of Pp2cm Gene Silencing on Mouse Macrophage Resistance Against Staphylococcus aureus Infection via TLR Pathway

To investigate the effect of silencing protein phosphatase 2cm ( Pp2cm) gene on the expression of inflammatory factors in macrophages infected with Staphylococcus aureus ( S. aureus) and the mechanisms involved. The effects of Pp2cm knockdown on inflammatory factors, proliferation, apoptosis, and Toll-like receptor (TLR) signaling were analyzed in RAW 264.7 cells, a murine macrophage cell line, transfected with adenovirus (Ad). The cells were divided into four groups, including Ad-Ctrl group, Ad- Pp2cm group, Ad-Ctrl+ S. aureus group and Ad- Pp2cm+ S. aureus group. Cell transfection was achieved by separately introducing control adenovirus (Ad-Ctrl) or adenovirus targeting the Pp2cm gene (Ad- Pp2cm) and inflammation or the absence of inflammation was induced by applying or not applying S. aureus. The expression of tumor necrosis factor-alpha ( TNF-α), interleukin-1β ( IL-1 β), TLR2, TLR4, Toll-like receptor adaptor protein ( Tirap) and myeloid differentiation factor 88 ( Myd88) was determined by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). PP2Cm protein expression was determined by Western blot. Cell proliferation was determined by cell counting kit-8 (CCK-8) assay and cell apoptosis was measured by flow cytometry. The expression of Pp2cmgene and PP2Cm protein was downregulated in the Ad- Pp2cm group when compared to the Ad-Ctrl group, with the diference showing statistical significance ( P<0.05). When compared to those of the Ad-Ctrl+ S. aureus group, macrophages in the Ad- Pp2cm+ S. aureus group showed significantly increase in the TNF- α and IL-1 β gene levels ( P<0.01). Furthermore, the Ad- Pp2cm group demonstrated elevated gene expression levels of TLR2, TLR4, Tirap and Myd88 in macrophages when compared to the Ad-Ctrl group, with the difference showing statistical significance ( P<0.05). There were no statistically significant differences in cell apoptosis and proliferation between the Ad-Ctrl and Ad- Pp2cm groups. Silencing Pp2cm gene promotes the inflammatory response of macrophages to S. aureus infection. Moreover, the TLR pathway plays an important role in the inflammatory activation of macrophages.

Liposome-encapsulated zoledronate increases inflammatory macrophage population in TNBC tumours

BackgroundTumour associated macrophages (TAMs) are important players in breast tumour progression and metastasis. Clinical and preclinical evidence suggests a role for zoledronate (ZOL) in breast cancer metastasis prevention. Further, zoledronate is able to induce inflammatory activation of monocytes and macrophages, which can be favourable in cancer treatments. The inherent bone tropism of zoledronate limits its availability in soft tissues and tumours. In this study we utilised an orthotopic murine breast cancer model to evaluate the possibility to use liposomes (EMP-LIP) to target zoledronate to tumours to modify TAM activation. MethodsTriple-negative breast cancer 4T1 cells were inoculated in the 4th mammary fat pad of female Balb/c mice. Animals were divided according to the treatment: vehicle, ZOL, EMP-LIP and liposome encapsulated zoledronate (ZOL-LIP). Treatment was done intravenously (with tumour resection) and intraperitoneally (without tumour resection). Tumour growth was followed by bioluminescence in vivo imaging (IVIS) and calliper measurements. Tumour-infiltrating macrophages were assessed by immunohistochemical and immunofluorescence staining. Protein and RNA expression levels of inflammatory transcription factors and cytokines were measured by Western Blotting and Taqman RT-qPCR. ResultsLiposome encapsulated zoledronate (ZOL-LIP) treatment suppressed migration of 4T1 cell in vitro. Tumour growth and expression of the angiogenic marker CD34 were reduced upon both ZOL and ZOL-LIP treatment in vivo. Long-term ZOL-LIP treatment resulted in shift towards M1-type macrophage polarization, increased CD4 T cell infiltration and activation of NF-κB indicating changes in intratumoural inflammation, whereas ZOL treatment showed similar but non-significant trends. Moreover, ZOL-LIP had a lower bisphosphonate accumulation in bone compared to free ZOL. ConclusionResults show that the decreased bisphosphonate accumulation in bone promotes the systemic anti-tumour effect of ZOL-LIP by increasing inflammatory response in TNBC tumours via M1-type macrophage activation.

A Model of iPSC-Derived Macrophages with TNFAIP3 Overexpression Reveals the Peculiarities of TNFAIP3 Protein Expression and Function in Human Macrophages.

Macrophages play a crucial role in the development and control of inflammation. Understanding the mechanisms balancing macrophage inflammatory activity is important to develop new strategies for treating inflammation-related diseases. TNF-α-induced protein 3 (TNFAIP3, A20) is a negative regulator of intracellular inflammatory cascades; its deficiency induces hyper-inflammatory reactions. Whether A20 overexpression can dampen macrophage inflammatory response remains unclear. Here, we generated human-induced pluripotent stem cells with tetracycline-inducible A20 expression and differentiated them into macrophages (A20-iMacs). A20-iMacs displayed morphology, phenotype, and phagocytic activity typical of macrophages, and they displayed upregulated A20 expression in response to doxycycline. A20 overexpression dampened the A20-iMac response to TNF-α, as shown by a decreased expression of IL1B and IL6 mRNA. A dynamic analysis of A20 expression following the generation of A20-iMacs and control iMacs showed that the expression declined in iMacs and that iMacs expressed a lower molecular weight form of the A20 protein (~70 kDa) compared with less differentiated cells (~90 kDa). A low-level expression of A20 and the predominance of a low-molecular-weight A20 form were also characteristic of monocyte-derived macrophages. The study for the first time developed a model for generating macrophages with an inducible expression of a target gene and identified the peculiarities of A20 expression in macrophages that likely underlie macrophage preparedness for inflammatory reactivity. It also suggested the possibility of mitigating inflammatory macrophage responses via A20 overexpression.