Expression Of Macrophage Markers Research Articles

Glycolytic enzyme PGK1 promotes M1 macrophage polarization and induces pyroptosis of acute lung injury via regulation of NLRP3

Acute lung injury (ALI) is characterized by an unregulated inflammatory reaction, often leading to severe morbidity and ultimately death. Excessive inflammation caused by M1 macrophage polarization and pyroptosis has been revealed to have a critical role in ALI. Recent study suggests that glycolytic reprogramming is important in the regulation of macrophage polarization and pyroptosis. However, the particular processes underlying ALI have yet to be identified. In this study, we established a Lipopolysaccharide(LPS)-induced ALI model and demonstrated that blocking glycolysis by using 2-Deoxy-D-glucose(2-DG) significantly downregulated the expression of M1 macrophage markers and pyroptosis-related genes, which was consistent with the in vitro results. Furthermore, our research has revealed that Phosphoglycerate Kinase 1(PGK1), an essential enzyme in the glycolysis pathway, interacts with NOD-, LRR- and pyrin domain-containing protein 3(NLRP3). We discovered that LPS stimulation improves the combination of PGK1 and NLRP3 both in vivo and in vitro. Interestingly, the absence of PGK1 reduces the phosphorylation level of NLRP3. Based on in vitro studies with mice bone marrow-derived macrophages (BMDMs), we further confirmed that siPGK1 plays a protective role by inhibiting macrophage pyroptosis and M1 macrophage polarization. The PGK1 inhibitor NG52 suppresses the occurrence of excessive inflammation in ALI. In general, it is plausible to consider a therapeutic strategy that focuses on modulating the relationship between PGK1 and NLRP3 as a means to mitigate the activation of inflammatory macrophages in ALI.

Irisin alleviates CFA-induced inflammatory pain by modulating macrophage polarization and spinal glial cell activation

Although the potent anti-inflammatory effects of irisin have been documented in various inflammatory disorders, its efficacy against inflammatory pain remains unexplored. Herein, we examined the therapeutic effects of irisin in a mouse model of inflammatory pain induced by complete Freund’s adjuvant (CFA). Mice were divided into three groups: normal control, CFA-injected (CFA), and CFA plus irisin-treated (CFA+Irisin). The irisin-treated group exhibited a gradual reduction in mechanical allodynia and thermal hyperalgesia when compared with the CFA group. Moreover, treatment with irisin significantly upregulated the expression of M2 macrophage markers (interleukin [IL]-4 and IL-10) and downregulated M1 macrophage markers (IL-1β, IL-6, and tumor necrosis factor-α) in the local paw tissue, dorsal root ganglion, and spinal cord tissue. However, there was no significant difference in the total number of F4/80+ macrophages in the paw tissue and dorsal root ganglion, indicating phenotypic exchange. Treatment with irisin also downregulated the expression of the glial cell activation-related markers Iba-1 and GFAP in the spinal cord tissue. To elucidate the underlying mechanisms, we detected the expression of Toll-like receptor 4 (TLR4), MyD88, and interferon regulatory factor 5 (IRF5) in paw tissues, dorsal root ganglion, and spinal tissues, revealing that irisin could downregulate the expression of these proteins. Irisin alleviated inflammatory pain by modulating local tissue inflammation and peripheral and central neuroinflammation and reducing glial cell activation and M2 macrophage polarization by modulating the TLR4–MyD88–IRF5 signaling pathway. Accordingly, irisin is a promising candidate for treating inflammatory pain in various diseases.

Effect of high fat diet (HFD)-induced obesity on gene expression of adipose tissue macrophage markers in male rats with different ages: Role of AMPK/SIRT1 pathway.

Effect of high fat diet (HFD)-induced obesity on gene expression of adipose tissue macrophage markers in male rats with different ages: Role of AMPK/SIRT1 pathway.

Characterisation of LPS+ bacterial extracellular vesicles along the gut-hepatic portal vein-liver axis.

Gut microbiome dysbiosis is a major contributing factor to several pathological conditions. However, the mechanistic understanding of the communication between gut microbiota and extra-intestinal organs remains largely elusive. Extracellular vesicles (EVs), secreted by almost every form of life, including bacteria, could play a critical role in this inter-kingdom crosstalk and are the focus of present study. Here, we present a novel approach for isolating lipopolysaccharide (LPS)+ bacterial extracellular vesicles (bEVLPS) from complex biological samples, including faeces, plasma and the liver from lean and diet-induced obese (DIO) mice. bEVLPS were extensively characterised using nanoparticle tracking analyses, immunogold labelling coupled with transmission electron microscopy, flow cytometry, super-resolution microscopy and 16S sequencing. In liver tissues, the protein expressions of TLR4 and a few macrophage-specific biomarkers were assessed by immunohistochemistry, and the gene expressions of inflammation-related cytokines and their receptors (n=89 genes) were measured using a PCR array. Faecal samples from DIO mice revealed a remarkably lower concentration of total EVs but a significantly higher percentage of LPS+ EVs. Interestingly, DIO faecal bEVLPS showed a higher abundance of Proteobacteria by 16S sequencing. Importantly, in DIO mice, a higher number of total EVs and bEVLPS consistently entered the hepatic portal vein and subsequently reached the liver, associated with increased expression of TLR4, macrophage markers (F4/80, CD86 and CD206), cytokines and receptors (Il1rn, Ccr1, Cxcl10, Il2rg and Ccr2). Furthermore, a portion of bEVLPS escaped liver and entered the peripheral circulation. In conclusion, bEV could be the key mediator orchestrating various well-established biological effects induced by gut bacteria on distant organs.

Broncho-Vaxom Attenuates Lipopolysaccharide-Induced Inflammation in a Mouse Model of Acute Lung Injury.

Acute lung injury (ALI) is a condition associated with acute respiratory failure, resulting in significant morbidity and mortality. It involves cellular changes such as disruption of the alveolar-capillary membrane, excessive neutrophil migration, and release of inflammatory mediators. Broncho-Vaxom® (BV), a lyophilized product containing cell membrane components derived from eight bacteria commonly found in the respiratory tract, is known for its potential to reduce viral and bacterial lung infections. However, the specific effect of BV on ALI has not been clearly defined. This study explored the preventive effects of BV and its underlying mechanisms in a lipopolysaccharide (LPS)-induced ALI mouse model. Oral BV (1 mg/kg) gavage was administered one hour before the intratracheal injection of LPS to evaluate its preventive effect on the ALI model. The pre-administration of BV significantly mitigates inflammatory parameters, including the production of inflammatory mediators, macrophage infiltration, and NF-κB activation in lung tissue, and the increase in inflammatory cells in bronchoalveolar lavage fluid (BALF). Moreover, BV (3 μg/mL) pretreatment reduced the expression of M1 macrophage markers, interleukins (IL-1β, IL-6), tumor necrosis factor α, and cyclooxygenase-2, which are activated by LPS, in both mouse alveolar macrophage MH-S cells and human macrophage THP-1 cells. These findings showed that BV exhibits anti-inflammatory effects by suppressing inflammatory mediators through the NF-κB pathway, suggesting its potential to attenuate bronchial and pulmonary inflammation.

Dietary sugar kelp (Saccharina latissima) consumption did not attenuate atherosclerosis in low-density lipoprotein receptor knockout mice.

We previously demonstrated the beneficial effects of U.S.-grown sugar kelp (Saccharina latissima), a brown seaweed, on reducing serum triglycerides (TG) and total cholesterol (TC) and protecting against inflammation and fibrosis in the adipose tissue of diet-induced obesity mice. In this current study, we aimed to explore whether the dietary consumption of sugar kelp can prevent atherosclerosis using low-density lipoprotein receptor knockout (Ldlr KO) mice fed an atherogenic diet. Eight-week-old male Ldlr KO mice were fed either an atherogenic high-fat/high-cholesterol control (HF/HC) diet or a HF/HC diet supplemented with 6% (w/w) sugar kelp (HF/HC-SK) for 16 weeks. Consumption of sugar kelp significantly increased the body weight gain without altering fat mass and lean mass. Also, there were no significant differences in energy expenditure and physical activities between the groups. The two groups did not show significant differences in serum and hepatic TG and TC levels or the hepatic expression of genes involved in cholesterol and lipid metabolism. Although serum alanine aminotransferase (ALT) activity did not differ significantly between the two groups, there were significant increases in the expression of macrophage markers, including adhesion G protein-coupled receptor E1 and cluster of differentiation 68, as well as tumor necrosis factor alpha in the HF/HC-SK group compared to the HF/HC mice. The consumption of sugar kelp did not elicit a significant effect on the development of aortic lesions. Moreover, lipopolysaccharide-stimulated splenocytes isolated from HF/HC-SK-fed mice showed no significant changes in the mRNA levels of pro-inflammatory genes compared with those from the HF/HC mice. In summary, the consumption of dietary sugar kelp did not elicit anti-atherogenic and hepatoprotective effects in Ldlr KO mice.

SHARPIN contributes to sevoflurane-induced neonatal neurotoxicity through up-regulating HMGB1 to repress M2 like-macrophage polarization.

Sevoflurane exposure can result in neurotoxicity especially among children, which remains an important complication after surgery. However, its related mechanisms remain unclear. Here, we investigated the biological roles of SHARPIN in sevoflurane-induced neurotoxicity. As detected by qPCR, Western blotting and immunohistochemical staining, SHARPIN and HMGB1 expression was elevated in sevoflurane-stimulated mice as compared with the control mice. SHARPIN depletion attenuated hippocampus injury, repressed the expression of HMGB1 and M1-like macrophage markers (iNOS, TNF-α, IL-1β, IL-6), but enhanced the expression of M2-like macrophage markers (ARG-1, IL-10). GST pull-down and Co-IP assays demonstrated that SHARPIN directly interacted with HMGB1 to enhance HMGB1 expression in SH-SY5Y cells. The inhibitory effects of SHARPIN silencing on inflammatory reaction and M1-like macrophages were counteracted by HMGB1 overexpression. Finally, SHARPIN-HMGB1 pathway affected neuroinflammation triggered by sevoflurane via modulating macrophage polarization. Collectively, our data suggested that SHARPIN stimulated sevoflurane-induced neurotoxicity via converting M2-like macrophages to M1-like macrophages by enhancing HMGB1 expression. SHARPIN intervention may be a promising therapeutic method to relieve sevoflurane-induced neurotoxicity.

Study of late toxicity biomarkers of locally advanced head and neck cancer patients treated with radiotherapy plus cisplatin or cetuximab points to the relevance of skin macrophages (TOX-TTCC-2015-01).

Radiotherapy (RT) with concomitant cisplatin (CRT) or cetuximab (ERT) are accepted treatment options for locally advanced squamous cell carcinoma of the head and neck (LA-SCCHN). Long-term adverse events (AEs) have a vast impact on patients' quality of life. This study explored tissue biomarkers which could help predict late toxicity. Single-institution prospective study including patients aged ≥ 18 with histologically confirmed newly diagnosed LA-SCCHN treated with RT and either concomitant cisplatin q3w or weekly cetuximab, according to institutional protocols. All patients underwent pre- and post-treatment skin biopsies of neck regions included in the clinical target volume. Angiogenesis, macrophages, and extracellular matrix (ECM) markers were evaluated by immunohistochemistry (IHC). From April 15, 2016, to December 11, 2017; 31 patients were evaluated [CRT = 12 (38.7%) and ERT = 19 (61.3%)]. 27 patients (87%) had received induction chemotherapy. All patients finished RT as planned. IHC expression of vasculature (CD34) and collagen (Masson's Trichrome) did not differ significantly between and within CRT and ERT arms. Conversely, an increased CD68 and CD163 macrophage infiltration expression was observed after treatment, without significant impact of treatment modality. Patients with higher late toxicity showed lower expression of macrophage markers in pre-treatment samples compared with those with lower late toxicity, with statistically significant differences for CD68. Angiogenesis and ECM biomarkers did not differ significantly between CRT and ERT. Macrophage markers increased after both treatments and deserve further investigation as predictors of late toxicity in LA-SCCHN patients. [Protocol code: TOX-TTCC-2015-01/Spanish registry of clinical studies (REec): 2015-003012-21/Date of registration: 27/01/2016].

BACH1 transcriptionally upregulates FOSL2 to induce M2 macrophages phenotype by activating TGFβ/SMAD signaling to promote the transformation of lung fibroblasts into myofibroblasts

Pulmonary fibrosis is a chronic and progressive lung disorder. The pro-fibrosis factors induced by M2 macrophage phenotype promote the differentiation of fibroblasts into myofibroblasts, which is essential for pulmonary fibrosis. We aimed to explore the role and mechanism of BTB domain and CNC homology 1 (BACH1) in pulmonary fibrosis. BACH1 was knocked down in THP-1 polarized M2 macrophages with or without FOS-like antigen 2 (FOSL2) overexpression, the expression of M2 macrophage markers was detected. Cell viability, migration, invasion and extracellular matrix (ECM) accumulation were estimated by CCK-8, wound healing, transwell, western bot and immunofluorescence staining. Luciferase reporter and chromatin immunoprecipitation assays were used to verify the binding of BACH1 to FOSL2 promotor region. In vivo, a bleomycin (BLM)-induced pulmonary fibrosis mice model was established to evaluate the effect of BACH1 silencing on the histopathological changes, M2 macrophage phenotype and extracellular matrix (ECM) deposition. Expression of proteins was assessed with western blot. Results indicated that BACH1 expression was upregulated in M2 macrophages polarized from THP-1 cells. BACH1 deficiency inhibited the polarization of THP-1 to the M2 macrophage phenotype to promote the transformation of lung fibroblasts into myofibroblasts. Additionally, BACH1 could transcriptionally activate FOSL2 expression in THP-1-derived macrophages to upregulate TGFβ/SMAD signaling in HFL-1 cells. The animal experiments indicated that BACH1 knockdown alleviated BLM-induced pulmonary fibrosis, M2 macrophage polarization and inactivated FOSL2/TGFβ/SMAD signaling in mice lung tissues. Together, this finding suggests BACH1/FOSL2 may be useful therapeutic targets for the treatment of pulmonary fibrosis.

N,N-Diarylsulfonamide Reduces Proinflammatory Cytokine Interleukin-6 Levels in Cells through Nuclear Factor-κB Regulation.

The arylsulfonamides were synthesized from aryl sulfonyl chloride and aromatic amines in dichloromethane in the presence of pyridine. The aryne chemistry was used to prepare diarylsulfonamide from arylsulfonamides and O-silylaryl triflate with CsF in acetonitrile at room temperature for 30 min. The synthesized compounds were evaluated for cytotoxicity followed by the cytokine/inflammatory marker's inhibition capability and its mechanism of action in RAW-264.7 cells. Elevated interleukin-6 (IL-6) levels have been reported in inflammatory conditions and inflammation-associated disorders. Hence, reducing the IL-6 levels in inflammatory conditions can serve as an attractive therapeutic target in dealing the inflammation. Among 42 compounds, seven compounds showed significant inhibition of IL-6 levels in lipopolysaccharide (LPS) challenged RAW-264.7 cells at 12.5 μM concentration. Further, investigation revealed that the IC50 value of these compounds for reducing IL-6 levels was found to be in the range of 2.6 to 9.7 μM. The promising compounds 5y (IC50 of 2.6 μM) and 5n (IC50 of 4.1 μM) along with other derivatives fulfil drug-likeness parameters laid down by Lipinski's rule of five. Further, RT-qPCR and Western-blot analysis revealed that treatment with 5n significantly reduced the expression of pro-inflammatory, inflammatory and macrophage marker's expression (IL-1β, CCL2, COX2 and CD68) compared to LPS control. The mechanistic evaluation showed that 5n exhibited anti-inflammatory properties by modulating the nuclear factor-κB (NF-κB) activation. The identified compound can be a promising candidate for further discovery efforts to generate a preclinical candidate effective in inflammation.

Exposure to Extracellular Mitochondrial DNA During Pregnancy Induces a Pro-Inflammatory Response in Rat Placenta

Introduction: Mitochondrial DNA (mtDNA) is released in the cytosol or the extracellular space due to mitochondrial dysfunction or cell death. Extracellular mtDNA is recognized by the innate immune receptor Toll-like receptor 9 (TLR9) and induces pro-inflammatory and immunogenic responses. Circulating cell-free mtDNA (ccf-mtDNA) increases with advancing gestational age and abnormal concentrations have been reported in obstetric complications such as preeclampsia (PE). Placentas from women with PE exhibit signs of extensive inflammation; however, the contribution of ccf-mtDNA to placental inflammation during pregnancy has not been investigated. We hypothesized that in vivo exposure to extracellular mtDNA during pregnancy would induce an inflammatory response in rat placenta via activation of TLR9 signaling. Methods: We isolated mtDNA from liver of Sprague Dawley pregnant rats (gestational day: 14-15, term=22-23 days) and treated gestational age-matched dams with mtDNA (350ng/kgBW) or saline (control) via intravenous injection. Following 4 hours after treatment, maternal organs and fetoplacental units were collected and gene expression of cytokines and TLR9 were measured. Results: Plasma levels of mtDNA 4 hours after treatment were not different between saline and mtDNA-treated rats (2−ΔCT, median (IQR); saline: 2.75 (1.61) vs. mtDNA: 2.64 (1.29), p>0.05). Treatment with mtDNA increased mRNA expression of pro-inflammatory tumor necrosis factor-alpha (TNF-α) (saline: 0.65 (1.1) vs. mtDNA: 2.84 (1.9), p=0.015), interleukin (IL)-6 (saline: 0.32 (0.2) vs. mtDNA: 3.37 (4.3), p=0.008), and IL-1β (saline: 0.2 (0.93) vs. mtDNA: 4.7 (6.0), p=0.01) and decreased expression of anti-inflammatory IL-10 (saline: 1.6 (0.8) vs. mtDNA: 0.6 (0.8), p=0.001) in rat placentas. Expression of macrophage marker F4/80 (saline: 0.4 (1.0) vs. mtDNA: 2.41 (4.7), p=0.04) and monocyte chemoattractant protein-1 (MCP-1) (saline: 0.68 (0.54) vs. mtDNA: 2.0(1.96), p=0.041) also increased in rat placentas in response to mtDNA treatment. To assess whether this inflammatory response was tissue-specific, we evaluated mRNA expression of inflammatory genes in maternal kidneys. Treatment with mtDNA increased the levels of IL-1β (2−ΔCT, median (IQR); saline: 1.05 (1.1) vs. mtDNA: 10.17 (8.0), p=0.008) and IL-6 (saline: 0.98 (1.76) vs. mtDNA: 6.8 (3.5), p=0.002). mtDNA treatment did not affect kidney TNF-α and IL-10 expression (p>0.05). In contrast, expression of F4/80 (saline: 0.87 (2.2) vs. mtDNA: 12.5 (12.4), p=0.003) and MCP-1 (saline: 0.9 (1.7) vs. mtDNA: 7.4 (8.3), p=0.011) was greater in maternal kidneys from mtDNA-treated rats vs. controls. TLR9 expression increased in placentas (saline: 1.29 (0.5) vs. mtDNA: 4.0 (7.8), p<0.0001) and maternal kidneys (saline: 1.2 (1.8) vs. mtDNA: 4.7 (4.6), p=0.045) in response to mtDNA treatment. Conclusion: Exposure to an in vivo challenge with extracellular mtDNA during pregnancy induces a pro-inflammatory response in placentas and kidneys from pregnant rats. These responses may be mediated by TLR9 signaling activation. NIH R01-HL0146562. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1053: The Role Of Bile Acid-initiated Signaling In Vascular Smooth Muscle Cell Phenotypic Switching And Atherosclerosis

Atherosclerosis is a chronic inflammatory disease in which foam cells (lipid-laden macrophage-like cells) accumulate in the arterial wall. Vascular smooth muscle cells (VSMCs) are not terminally differentiated; they switch to a foam cell-like phenotype in response to various stimuli, such as cholesterol (CHOL), increasing susceptibility to vascular diseases. Modulated SMCs are the main contributors to atherosclerotic plaque formation. The intracellular metabolism of CHOL via peroxisomes in hepatocytes produces bile acids like cholic or deoxycholic acid, which agonize liver X receptor (LXR). LXR facilitates the first step of reverse CHOL transport. It is unknown if VSMCs synthesize bile acids and whether peroxisomes and the bile acids they produce play a role in VSMC-derived foam cell formation. Using human aortic SMCs (HASMCs), we found that CHOL treatment decreased expression of several peroxisomal proteins. Peroxisomal loss-of-function experiments demonstrated increased intracellular lipid accumulation, as visualized with Oil Red O (ORO) staining. We have shown that HASMCs synthesize bile acids after exposure to CHOL, which led us to hypothesize that bile acid-initiated signaling participates in the phenotypic modulation initiated by CHOL in VSMCs. Treatment of HASMCs with LXR agonists or bile acids increased expression of the efflux CHOL transporter ABCA1, bile acid-related peroxisomal enzymes, and differentiation markers; they decreased expression of macrophage and foam cell markers and ORO staining. These data show that VSMCs have the metabolic capacity to synthesize bile acids, which is impaired if peroxisomes are non-functional or absent. Our data indicates that activation of bile acid receptors upregulates ABCA1, favoring a differentiated VSMC phenotype. Together, these data suggest that activation of LXR plays a role in CHOL-induced VSMC to foam cell transition and that LXR is a potential therapeutic target which promotes VSMC differentiation and decreases atherosclerotic plaque formation.

P311 Influences Macrophage Polarization

Obesity is a pandemic disease that has become a serious concern worldwide. In United States, recently self-reported obesity rates of adults were escalated to 40%. In the early phase of obesity, excessive macrophage infiltration and disequilibrium of macrophage phenotypes (M1 vs. M2 or vice versa) play a critical role in the microenvironment of adipose tissue. It often leads to chronic inflammation and is a major cause for metabolic disorders including insulin resistance, type-2 diabetes, and cardiovascular diseases. P311 is an intrinsically disordered protein, contributes to adipogenesis as transcription regulator through activation of PPARγ2, master regulator of adipogenesis. However, the P311 role on the macrophage polarization was not known. Here we studied the macrophage polarization in presence of P311 using Raw 264.7 cells. Raw cells were treated with either interleukin-4 (IL4) or lipopolysaccharide (LPS) for macrophage polarization towards M2 and M1 phenotype respectively in presence and absence of P311 overexpression. Macrophage phenotypic switch was assessed using CD206 (M2 marker) and CD86 (M1 marker) expression. The dose and time dependent effects of IL4 and LPS on the expression of P311 and macrophage markers was also studied. A significant increase in endogenous P311 expression observed with IL4 treatment whereas LPS treatment showed the opposite effect. Our findings showed that the P311 overexpression promotes anti-inflammatory macrophage phenotype (M2) as indicated by the significant increase in mRNA and protein expression of CD206 when compared to empty vector (EV, pCMV) transfected control group. Further, P311 overexpression in LPS treated RAW cells significantly increased the expression of CD206, on contrary to the classical M1 polarization of LPS, compared to EV transfected cells. In addition, P311 suppressed the expression of CD86 there by facilitating macrophage switch towards anti-inflammatory phenotype in presence of LPS treatment. IL4 treatment alone increased macrophage activation towards M2 phenotype however IL4 treated P311 overexpressed cells showed further increase in CD206 expression compared to EV transfected cells. Our studies indicate that the P311 expression plays an important role in promoting macrophage polarization toward M2 phenotype. This study was supported by research funding to KB, from NIH/NIGMS #SC1GM141937. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PGRN is involved in macrophage M2 polarization regulation through TNFR2 in periodontitis

Background and objectiveProgranulin (PGRN), a multifunctional growth factor, plays indispensable roles in the regulation of cancer, inflammation, metabolic diseases, and neurodegenerative diseases. Nevertheless, its immune regulatory role in periodontitis is insufficiently understood. This study attempts to explore the regulatory effects of PGRN on macrophage polarization in periodontitis microenvironment.MethodsImmunohistochemical (IHC) and multiplex immunohistochemical (mIHC) stainings were performed to evaluate the expression of macrophage-related markers and PGRN in gingival samples from periodontally healthy subjects and periodontitis subjects. RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were polarized towards M1 or M2 macrophages by the addition of LPS or IL-4, respectively, and were treated with or without PGRN. Real-time fluorescence quantitative PCR (qRT-PCR), immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), and flow cytometry were used to determine the expressions of M1 and M2 macrophage-related markers. Co-immunoprecipitation was performed to detect the interaction between PGRN and tumor necrosis factor receptor 2 (TNFR2). Neutralizing antibody was used to block TNFR2 to confirm the role of TNFR2 in PGRN-mediated macrophage polarization.ResultsThe IHC and mIHC staining of human gingival slices showed a significant accumulation of macrophages in the microenvironment of periodontitis, with increased expressions of both M1 and M2 macrophage markers. Meanwhile, PGRN was widely expressed in the gingival tissue of periodontitis and co-expressed mainly with M2 macrophages. In vitro experiments showed that in RAW264.7 cells and BMDMs, M1 markers (CD86, TNF-α, iNOS, and IL-6) substantially decreased and M2 markers (CD206, IL-10, and Arg-1) significantly increased when PGRN was applied to LPS-stimulated macrophages relatively to LPS stimulation alone. Besides, PGRN synergistically promoted IL-4-induced M2 markers expression, such as CD206, IL-10, and Arg1. In addition, the co-immunoprecipitation result showed the direct interaction of PGRN with TNFR2. mIHC staining further revealed the co-localization of PGRN and TNFR2 on M2 macrophages (CD206+). Blocking TNFR2 inhibited the regulation role of PGRN on macrophage M2 polarization.ConclusionsIn summary, PGRN promotes macrophage M2 polarization through binding to TNFR2 in both pro- and anti-inflammatory periodontal microenvironments.

Effect of Low-Intensity Pulsed Ultrasound on Macrophage Properties and Fibrosis in the Infrapatellar Fat Pad in a Carrageenan-Induced Knee Osteoarthritis Rat Model.

In the progression of knee osteoarthritis (KOA), fibrosis of the infrapatellar fat pad (IFP) is a key pathological change. Low-intensity pulsed ultrasound (LIPUS) inhibits IFP fibrosis by decreasing the gene expression and activity of hypoxia-inducible factor (HIF-1α), which is a protein involved in IFP fibrosis in KOA rat models. On the other hand, macrophages play an important role in the progression of fibrosis in various tissues, and LIPUS irradiation suppresses macrophage infiltration and inflammatory cytokine secretion. However, whether LIPUS suppresses macrophage polarity and IFP fibrosis in KOA remains unclear. Therefore, we investigated the effect of LIPUS on macrophage polarity and IFP fibrosis. A KOA model was created by injecting carrageenin into the bilateral knee joints of Wistar rats (eight weeks old). Tissues were harvested over time for histological and molecular biological analysis. The KOA model was also subjected to LIPUS irradiation for two weeks following the injection of carrageenin. RM-4-positive cells were widely distributed in IFP two weeks after carrageenin administration, but M2 macrophages were significantly increased, and the Sirius red area was decreased in the LIPUS-irradiated group compared with those in the non-irradiated group. The gene expression of M1 macrophage markers was significantly decreased and that of M2 macrophage markers was significantly increased in the LIPUS-irradiated group. The expression of transforming growth factor-β (TGF-β) and type 1 collagen was also significantly decreased. These results suggest that LIPUS may serve as a novel approach for the treatment of KOA through its effect on M1 macrophages and suppression of TGF-β expression.

Vitamin A enhanced periosteal osteoclastogenesis is associated with increased number of tissue-derived macrophages/osteoclast progenitors

A deleterious effect of elevated levels of vitamin A on bone health has been reported in numerous clinical studies. Mechanistic studies in rodents have shown that numbers of periosteal osteoclasts are increased, while endocortical osteoclasts are simultaneously decreased by vitamin A treatment. These observations indicate that osteoclastogenesis on the endocortical and periosteal surfaces of bone is differentially controlled by vitamin A. The present study investigated the in vitro and in vivo effect of all-trans retinoic acid (ATRA), the active metabolite of vitamin A, on periosteal osteoclast progenitors. Mouse calvarial bone cells were cultured in media containing ATRA, with or without the osteoclastogenic cytokine RANKL, on plastic dishes or bone discs. Whereas ATRA did not stimulate osteoclast formation alone, the compound robustly potentiated the formation of RANKL-induced bone resorbing osteoclasts. This effect was due to stimulation by ATRA (EC50 ∼3nM) on the numbers of macrophages/osteoclast progenitors in the bone cell cultures, as assessed by mRNA and protein expression of several macrophage and osteoclast progenitor cell markers, such as M-CSF receptor, RANK, F4/80 and CD11b, as well as by FACS-analysis of CD11b+/F480+/Gr1- cells. The stimulation of macrophage numbers in the periosteal cell cultures was not mediated by increased M-CSF or IL-34. In contrast, ATRA did not enhance macrophages in bone marrow cell cultures. Importantly, ATRA treatment upregulated the mRNA expression of several macrophage-related genes also in the periosteum of tibia in adult mice. These observations demonstrate a novel mechanism by which vitamin A enhances osteoclast formation specifically on periosteal surfaces.

Genistein alleviates dextran sulfate sodium-induced colitis in mice through modulation of intestinal microbiota and macrophage polarization.

Ulcerative colitis (UC) is a colonic immune system disorder, manifested with long duration and easy relapse. Genistein has been reported to possess various biological activities. However, it remains unclear whether genistein can ameliorate UC by modulating the homeostasis of the intestinal bacterial community. The dextran sodium sulfate (DSS)-induced UC mice were administrated with genistein (20mg/kg/day) or genistein (40mg/kg/day) for ten days. The general physical condition of the mice was monitored. After sacrifice, the changes in colon length and colonic pathological morphology were observed. The expression of intestinal barrier proteins, inflammatory cytokines, and macrophage markers in the colon was detected. The composition and metabolic products of the intestinal microbiota were analyzed. Genistein treatment visibly improved body weight change and disease activity index in DSS-induced mice. Genistein treatment ameliorated colonic pathological alterations and promoted the expression of mucin-2 and tight junction proteins. Genistein administration inhibited myeloperoxidase activity and colonic inflammatory cytokines. Furthermore, genistein administration improved the structure of the intestinal microbial community, promoted the production of short-chain fatty acids, and modulated macrophage polarization. These results revealed that genistein mediated macrophage polarization balance by improving intestinal microbiota and its metabolites, thereby alleviating DSS-induced colitis.

STING-Pathway Inhibiting Nanoparticles (SPINs) as a Platform for Treatment of Inflammatory Diseases.

Aberrant activation of the cyclic GMP-AMP synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway has been implicated in the development and progression of a myriad of inflammatory diseases including colitis, nonalcoholic steatohepatitis, amyotrophic lateral sclerosis (ALS), and age-related macular degeneration. Thus, STING pathway inhibitors could have therapeutic application in many of these inflammatory conditions. The cGAS inhibitor RU.521 and the STING inhibitor H-151 have shown promise as therapeutics in mouse models of colitis, ALS, and more. However, these agents require frequent high-dose intraperitoneal injections, which may limit translatability. Furthermore, long-term use of systemically administered cGAS/STING inhibitors may leave patients vulnerable to viral infections and cancer. Thus, localized or targeted inhibition of the cGAS/STING pathway may be an attractive, broadly applicable treatment for a variety of STING pathway-driven ailments. Here we describe STING-Pathway Inhibiting Nanoparticles (SPINS)-poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with RU.521 and H-151-as a platform for enhanced and sustained inhibition of cGAS/STING signaling. We demonstrate that SPINs are equally or more effective at inhibiting type-I interferon responses induced by cytosolic DNA than free H-151 or RU.521. Additionally, we describe a SPIN formulation in which PLGA is coemulsified with poly(benzoyloxypropyl methacrylamide) (P(HPMA-Bz)), which significantly improves drug loading and allows for tunable release of H-151 over a period of days to over a week by varying P(HPMA-Bz) content. Finally, we find that all SPIN formulations were as potent or more potent in inhibiting cGAS/STING signaling in primary murine macrophages, resulting in decreased expression of inflammatory M1-like macrophage markers. Therefore, our study provides an in vitro proof-of-concept for nanoparticle delivery of STING pathway inhibitors and positions SPINs as a potential platform for slowing or reversing the onset or progression of cGAS/STING-driven inflammatory conditions.

SFRP1 decreases WNT-Mediated M2 macrophage marker expression in breast tissue

The Wnt family of secreted proteins are involved in mammary gland development and tumorigenesis. It has recently been shown that Wnt ligands promote M2 macrophage polarization and so we sought to determine the effects of a Wnt signaling antagonist, Secreted Frizzled Related Protein 1 (SFRP1), on M2 marker expression. We measured a murine M2 marker (Arg1) in mice with a targeted deletion of Sfrp1 during different stages of mammary gland development including puberty, pregnancy, and lactation, as well as in response to obesity. Next, to determine whether Wnt signaling/antagonism affects human M2 markers (CD209 and CCL18), we treated a human patient derived explant (PDE) breast tissue sample with exogenous Wnt3a in the presence and absence of rSFRP1. Finally, we expanded our PDE study to 13 patients and performed bulk RNAseq analysis following the treatment described above. We found that in loss of Sfrp1 in the murine mammary gland increased Arg1 expression. Moreover, we showed that Wnt3a increases CD209 and CCL18 mRNA and protein expression in breast PDEs and that their expression is decreased in response to rSFRP1. Our RNAseq analysis unveiled novel genes that were affected by Wnt3a treatment and subsequently reversed when rSFRP1 was added. Validation of these data exhibited that chemokines involved in promoting macrophage polarization and cancer metastasis, including CCL11 and CCL26, were stimulated by Wnt3a signaling and their expression was abrogated by treatment with rSFRP1. Our data suggest that SFRP1 may be an important mediator that tempers Wnt signaling in the tumor microenvironment.

Single-cell RNA sequencing analysis of vestibular schwannoma reveals functionally distinct macrophage subsets

BackgroundVestibular schwannomas (VSs) remain a challenge due to their anatomical location and propensity to growth. Macrophages are present in VS but their roles in VS pathogenesis remains unknown.ObjectivesThe objective was to assess phenotypic and functional profile of macrophages in VS with single-cell RNA sequencing (scRNAseq).MethodsscRNAseq was carried out in three VS samples to examine characteristics of macrophages in the tumour. RT-qPCR was carried out on 10 VS samples for CD14, CD68 and CD163 and a panel of macrophage-associated molecules.ResultsscRNAseq revealed macrophages to be a major constituent of VS microenvironment with three distinct subclusters based on gene expression. The subclusters were also defined by expression of CD163, CD68 and IL-1β. AREG and PLAUR were expressed in the CD68+CD163+IL-1β+ subcluster, PLCG2 and NCKAP5 were expressed in CD68+CD163+IL-1β− subcluster and AUTS2 and SPP1 were expressed in the CD68+CD163−IL-1β+ subcluster. RT-qPCR showed expression of several macrophage markers in VS of which CD14, ALOX15, Interleukin-1β, INHBA and Colony Stimulating Factor-1R were found to have a high correlation with tumour volume.ConclusionsMacrophages form an important component of VS stroma. scRNAseq reveals three distinct subsets of macrophages in the VS tissue which may have differing roles in the pathogenesis of VS.