Macrophage Secretion Research Articles

Gasdermin D mediates host cell death but not interleukin-1\u03b2 secretion in Mycobacterium tuberculosis-infected macrophages

Necrotic cell death represents a major pathogenic mechanism of Mycobacterium tuberculosis (Mtb) infection. It is increasingly evident that Mtb induces several types of regulated necrosis but how these are interconnected and linked to the release of pro-inflammatory cytokines remains unknown. Exploiting a clinical cohort of tuberculosis patients, we show here that the number and size of necrotic lesions correlates with IL-1β plasma levels as a strong indicator of inflammasome activation. Our mechanistic studies reveal that Mtb triggers mitochondrial permeability transition (mPT) and subsequently extensive macrophage necrosis, which requires activation of the NLRP3 inflammasome. NLRP3-driven mitochondrial damage is dependent on proteolytic activation of the pore-forming effector protein gasdermin D (GSDMD), which links two distinct cell death machineries. Intriguingly, GSDMD, but not the membranolytic mycobacterial ESX-1 secretion system, is dispensable for IL-1β secretion from Mtb-infected macrophages. Thus, our study dissects a novel mechanism of pathogen-induced regulated necrosis by identifying mitochondria as central regulatory hubs capable of delineating cytokine secretion and lytic cell death.

The protein 4.1R downregulates VEGFA in M2 macrophages to inhibit colon cancer metastasis

M2 macrophages are crucial components of the tumour microenvironment and have been shown to be closely related to tumour progression. Co-culture with 4.1R−/− M2 macrophages enhances the malignancy of colon cancer (CC), but the mechanism remains unclear. Here, we report that protein 4.1R knockout reduced the phagocytosis of M2 macrophages (M-CSF/IL-4-treated bone marrow cells) and promoted MC38 colon cancer cell proliferation, migration, invasion, tumour formation and epithelial-mesenchymal transition (EMT), which are regulated by M2 macrophages. Further mechanistic dissection revealed that the 4.1R knockout upregulated vascular endothelial growth factor A (VEGFA) secreted by M2 macrophages and promoted colon cancer progression by activating the PI3K/AKT signalling pathway. In summary, our present study identified that 4.1R downregulates VEGFA secretion in M2 macrophages and delays the malignant potential of colon cancer by inhibiting the PI3K/AKT signalling pathway.

Aspartate Metabolism Facilitates IL-1β Production in Inflammatory Macrophages.

Increasing evidence support that cellular amino acid metabolism shapes the fate of immune cells; however, whether aspartate metabolism dictates macrophage function is still enigmatic. Here, we found that the metabolites in aspartate metabolism are depleted in lipopolysaccharide (LPS) plus interferon gamma (IFN-γ)-stimulated macrophages. Aspartate promotes interleukin-1β (IL-1β) secretion in M1 macrophages. Mechanistically, aspartate boosts the activation of hypoxia-inducible factor-1α (HIF-1α) and inflammasome and increases the levels of metabolites in aspartate metabolism, such as asparagine. Interestingly, asparagine also accelerates the activation of cellular signaling pathways and promotes the production of inflammatory cytokines from macrophages. Moreover, aspartate supplementation augments the macrophage-mediated inflammatory responses in mice and piglets. These results uncover a previously uncharacterized role for aspartate metabolism in directing M1 macrophage polarization.

Quebecol Shows Potential to Alleviate Periodontal Tissue Damage and Promote Bone Formation in In Vitro Models.

Quebecol is a polyphenolic compound initially isolated from Canadian maple syrup in 2011. Recently, our group demonstrated in a macrophage model that quebecol inhibits the secretion of pro-inflammatory cytokines and reduces the activation of the NF-κB transcription factor. In this study, we further explored the therapeutic potential of quebecol against periodontal disease, an inflammatory disorder of bacterial origin affecting tooth-supporting tissues. More specifically, the effects of this natural compound on matrix metalloproteinase (MMP) activity and macrophage secretion, as well as on the mineralization activity of osteoblasts (bone-forming cells), were investigated. Results showed that exposing lipopolysaccharide (LPS)-treated macrophages to quebecol led to a significant decrease in the secretion of MMP-8 and MMP-9. In addition, quebecol dose dependently inhibited the catalytic activity of MMP-9. Quebecol also enhanced the mineralization activity of osteoblasts. This study brought forward additional evidence to support the potential of quebecol as a nutraceutical agent against periodontitis.

Cardamonin inhibits LPS-induced inflammatory responses and prevents acute lung injury by targeting myeloid differentiation factor 2

BackgroundAcute lung injury (ALI) is a systemic inflammatory process, which has no pharmacological therapy in clinic. Accumulating evidence has demonstrated that natural compounds from herbs have potent anti-inflammatory efficacy in several disease models, which could be the potential candidates for the treatment of ALI. Hypothesis/PurposeAnti-inflammatory screening from natural product bank may provide new anti-inflammatory compounds for therapeutic target discovery and ALI treatment. Methods165 natural compounds were screened for their anti-inflammatory activity in LPS-stimulated macrophages. PCR array, SPR and ELISA were used to determine the potential target of the most active compound, Cardamonin (CAR). The pharmacological effect of CAR was further evaluated in both LPS-stimulated macrophages and ALI mice model. ResultsOut of the screened 165 compounds, CAR significantly inhibited LPS-induced inflammatory cytokine secretion in macrophages. We further showed that CAR significantly inhibited NF-κB and JNK signaling activation, and thereby inflammatory cytokine production via directly interacting with MD2 in vitro. In vivo, our data show that CAR treatment inhibited LPS-induced lung damage, systemic inflammatory cytokine production, and reduced macrophage infiltration in the lungs, accompanied with reduced TLR4/MD2 complex in lung tissues, Treatment with CAR also dose-dependently increased survival in the septic mice induced by DH5α bacterial infection. ConclusionWe demonstrate that a natural product, CAR, attenuates LPS-induced lung injury and sepsis by inhibiting inflammation via interacting with MD2, leading to the inactivation of the TLR4/MD2-MyD88-MAPK/NF-κB pathway.

MiR-195-3p alleviates homocysteine-mediated atherosclerosis by targeting IL-31 through its epigenetics modifications.

Atherosclerosis is a serious age‐related disease, which has a tremendous impact on health care globally. Macrophage inflammation is crucial for the initiation and progression of atherosclerosis, and microRNAs (miRNAs) recently have emerged as potent modulators of inflammation, while the underlying mechanisms of its involvement in homocysteine (Hcy)‐mediated macrophage inflammation of atherosclerosis remain largely unknown. Here, we demonstrated that elevated Hcy inhibits the expression of miR‐195‐3p, which in turn enhances IL‐31 expression and thereby causes the secretion of macrophages pro‐inflammatory factors IL‐1β, IL‐6 and TNF‐α and accelerate atherosclerosis. Furthermore, we identified that Hcy can induce DNA hypermethylation and H3K9 deacetylation of miR‐195‐3p promoter due to the increased the binding of DNMT3a and HDAC11 at its promoter. More importantly, Sp1 interacts with DNMT3a suppressed the binding of HDAC11 at miR‐195‐3p promoter and promoted its transcription. In summary, our results revealed a novel mechanism that transcriptional and epigenetic regulation of miR‐195‐3p inhibits macrophage inflammation through targeting IL‐31, which provides a candidate diagnostic marker and novel therapeutic target in cardiovascular diseases induced by Hcy.

Pathogenic Roles of CXCL10 in Experimental Autoimmune Prostatitis by Modulating Macrophage Chemotaxis and Cytokine Secretion.

Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) is an inflammatory immune disease characterized by intraprostatic leukocyte infiltration and pelvic or perineal pain. Macrophages play vital roles in the pathogenesis of CP/CPPS. However, the mechanisms controlling the activation and chemotaxis of macrophages in CP/CPPS remain unclear. This study aimed to investigate the roles of the CXCL10/CXCR3 pathway in the activation and chemotaxis of macrophages in CP/CPPS patients. The serums of CP/CPPS patients and healthy volunteers were collected and measured. Results showed that CXCL10 expression was significantly elevated and correlated with the severity of CP/CPPS patients. The experimental autoimmune prostatitis (EAP) model was generated, and adeno-associated virus and CXCR3 inhibitors were used to treat EAP mice. Immunofluorescence, flow cytometry, and Western blotting were used to analyze the functional phenotype and regulation mechanism of macrophages. Results showed that CXCL10 deficiency ameliorates EAP severity by inhibiting infiltration of macrophages to prostate. Moreover, CXCL10 could induce macrophage migrations and secretions of proinflammatory mediators via CXCR3, which consequently activated the downstream Erk1/2 and p38 MAPK signaling pathways. We also showed that prostatic stromal cell is a potential source of CXCL10. Our results indicated CXCL10 as an important mediator involved in inflammatory infiltration and pain symptoms of prostatitis by promoting the migration of macrophages and secretion of inflammatory mediators via CXCR3-mediated ERK and p38 MAPK activation.

Effect of galectin-3 inhibition on hepatocyte nuclear factors 4α and 1α expression in liver after acetaminophen long-term induced toxicity.

Acetaminophen (APAP) induced liver injury can occur through acute or repeated administration of high doses. Hepatocyte nuclear factors HNF4α and HNF1α are key transcription factors involved in regulating the expression of drug metabolism enzymes and are affected by proinflammatory cytokines. Meanwhile galectin-3 (Gal-3) is a β-galactoside-binding lectin implicated in the regulation of macrophage activation and secretion of inflammatory mediators. This study aimed to evaluate the impact of galectin-3 inhibition on HNF4α and HNF1α gene expression after long-term administration of APAP, and to investigate the resulted effect on APAP induced hepatotoxicity. The study was performed on thirty two Wistar rats divided equally into four groups; control group, modified citrus pectin (MCP) (Gal-3 inhibitor) receiving group, APAP receiving group, and APAP plus MCP receiving group. APAP induced inhibition of liver HNF4α and HNF1α gene expression, and elevation of liver Gal-3 and tumor necrosis factor- α levels (TNF-α), depletion of reduced glutathione and its maintaining enzymes, besides elevation in levels of serum liver function parameters and appearance of necrotic areas in the liver. Gal-3 inhibition preserved HNF4α and HNF1α gene expression, TNF-α, and each of reduced glutathione and its maintaining enzymes near normal levels. It also significantly reduced APAP induced hepatotoxicity.

Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1β Secretion in Human Macrophages.

Interleukin 1β (IL-1β) plays a major role in inflammation and is secreted by immune cells, such as macrophages, upon recognition of danger signals. Its secretion is regulated by the inflammasome, the assembly of which results in caspase 1 activation leading to gasdermin D (GSDMD) pore formation and IL-1β release. During inflammation, danger signals also activate the complement cascade, resulting in the formation of the membrane attack complex (MAC). Here, we report that stimulation of LPS-primed human macrophages with sub-lytic levels of MAC results in activation of the NOD-like receptor 3 (NLRP3) inflammasome and GSDMD-mediated IL-1β release. The MAC is first internalized into endosomes and then colocalizes with inflammasome components; adapter protein apoptosis associated speck-like protein containing a CARD (ASC) and NLRP3. Pharmacological inhibitors established that MAC-triggered activation of the NLRP3 inflammasome was dependent on MAC endocytosis. Internalization of the MAC also caused dispersion of the trans-Golgi network. Thus, these data uncover a role for the MAC in activating the inflammasome and triggering IL-1β release in human macrophages.

Phytochemicals and Immunomodulatory Effect of Nelumbo nucifera Flower Extracts on Human Macrophages.

This research characterizes phytochemicals inherent in lotus flower and investigates the antioxidant and immunomodulatory activity of ethyl acetate (EA) and ethyl alcohol (ET) lotus petal extracts. In the experiment, human monocytes-derived macrophages were stimulated by lipopoly-saccharide to mimic bacteria-induced inflammation. The results showed that ferulic acid, couma-rin, and chlorogenic acid were three dominant polyphenols. The EA and ET lotus petal extracts also possessed high antioxidant capability. Furthermore, the extracts exhibited immunomodulatory properties by suppressing TNF-α secretion in inflammatory-induced human macrophages by in-hibiting NF-κB-dependent inflammatory response. In essence, the lotus petal extracts possess reme-dial attributes beneficial to individuals afflicted with declined immune functions.

Itaconate regulates macrophage function through stressful iron-sulfur cluster disrupting and iron metabolism rebalancing.

Lipopolysaccharide (LPS)-stimulated macrophages express an aconitate decarboxylase (IRG1, also called ACOD1), leading toaccumulationof the endogenous metabolite itaconate. However, the precise mechanisms by which elevated itaconate levels alter macrophage function are not clear. Our hypothesis is itaconate affects macrophage function through some uncertain mechanism. Based on this, we established a transcriptional and proteomic signature of macrophages stimulated by itaconate and identified the pathways of IL-1β secretion and altered iron metabolism. Consistently, the effect of IRG1 deficiency on IL-1β secretion and iron metabolism was confirmed in IRG1 knockout THP-1 cell lines. Several common inhibitors and other compounds were used to examine the molecular mechanisms involved.Only cysteine and antioxidants (catechin hydrate) could inhibit caspase-1 activation and IL-1β secretion in itaconate-stimulated macrophages. We further found that aconitase activity was decreased by itaconate stimulation. Our results demonstrate the counteracting effects of overexpression of mitochondrial aconitase (ACO2,a tricarboxylic acidcycle enzyme) or cytosolic aconitase (ACO1, an iron regulatory protein) on IL-1β secretion and altered iron metabolism. Both enzyme activities were inhibited by itaconate because of iron-sulfur (Fe-S)cluster destruction. Our findings indicate that the immunoregulatory functions of IRG1 and itaconate in macrophages are stressful Fe-S cluster of aconitases disrupting and iron metabolism rebalancing.

7S,15R-Dihydroxy-16S,17S-Epoxy-Docosapentaenoic Acid, a Novel DHA Epoxy Derivative, Inhibits Colorectal Cancer Stemness through Repolarization of Tumor-Associated Macrophage Functions and the ROS/STAT3 Signaling Pathway.

Colorectal cancer is a highly malignant cancer that is inherently resistant to many chemotherapeutic drugs owing to the complicated tumor-supportive microenvironment (TME). Tumor-associated macrophages (TAM) are known to mediate colorectal cancer metastasis and relapse and are therefore a promising therapeutic target. In the current study, we first confirmed the anti-inflammatory effect of 7S,15R-dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA), a novel DHA dihydroxy derivative synthesized in our previous work. We found that diHEP-DPA significantly reduced lipopolysaccharide (LPS)-induced inflammatory cytokines secretion of THP1 macrophages, IL-6, and TNF-α. As expected, diHEP-DPA also modulated TAM polarization, as evidenced by decreased gene and protein expression of the TAM markers, CD206, CD163, VEGF, and TGF-β1. During the polarization process, diHEP-DPA treatment decreased the concentration of TGF-β1, IL-1β, IL-6, and TNF-α in culture supernatants via inhibiting the NF-κB pathway. Moreover, diHEP-DPA blocked immunosuppression by reducing the expression of SIRPα in TAMs and CD47 in colorectal cancer cells. Knowing that an inflammatory TME largely serves to support epithelial-mesenchymal transition (EMT) and cancer stemness, we tested whether diHEP-DPA acted through polarization of TAMs to regulate these processes. The intraperitoneally injected diHEP-DPA inhibited tumor growth when administered alone or in combination with 5-fluorouracil (5-FU) chemotherapy in vivo. We further found that diHEP-DPA effectively reversed TAM-conditioned medium (TCCM)-induced EMT and enhanced colorectal cancer stemness, as evidenced by its inhibition of colorectal cancer cell migration, invasion and expression of EMT markers, as well as cancer cell tumorspheres formation, without damaging colorectal cancer cells. DiHEP-DPA reduced the population of aldehyde dehydrogenase (ALDH)-positive cells and expression of colorectal stemness marker proteins (CD133, CD44, and Sox2) by modulating TAM polarization. Additionally, diHEP-DPA directly inhibited cancer stemness by inducing the production of reactive oxygen species (ROS), which, in turn, reduced the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). These data collectively suggest that diHEP-DPA has the potential for development as an anticancer agent against colorectal cancer.

Abstract MP41: Myeloid Ccn3 Protects Against Aortic Valve Calcification

Objective: CCN3, an inhibitor in osteoblast differentiation, can modulate calcium signaling, yet, whether CCN3 can regulate valvular calcification is unknown. While it has been suggested that macrophages are important in the regulation of valvular calcification, the molecular and cellular mechanisms of this process remain poorly understood. In the present study, we investigated the specific role that macrophage-derived CCN3 plays during the progression of calcific aortic valve disease (CAVD). Methods and Results: Myeloid-specific knockout of CCN3 (Mye-CCN3-KO) and control mice were subjected to a single injection of AAV encoding mutant mPCSK9 (rAAV8/D377Y-mPCSK9) to induce hyperlipidemia. AAV-injected mice were then fed a high fat diet (HFD) for 40 weeks. At the conclusion of HFD feeding, tissues were harvested for subsequent histological and pathological analysis. Echocardiography revealed that both male and female mye-CCN3-KO animals displayed compromised aortic valvular function accompanied by exacerbated valve thickness and cardiac dysfunction. Histologically, alizarin red and OsteoSense staining revealed a marked increase of aortic valve calcification in mye-CCN3-KO animals when compared to controls. In vitro, CCN3 deficiency augmented bmp2 production and secretion from bone marrow derived macrophages (BMDMs). Further, human valvular interstitial cells (VICs) cocultured with conditional media from CCN3-deficient BMDMs resulted in exaggerated pro-calcifying gene expression and the attendant calcification as revealed by alizarin red staining. Conclusions: Our data uncovered a novel role of myeloid CCN3 in the regulation of aortic valve calcification. Modulation of bmp2 production and secretion in macrophages might serve as a key mechanism for CCN3’s anti-calcification function in the development of CAVD.

Effect of curcumin on the non-alcoholic steatohepatitis via inhibiting the M1 polarization of macrophages.

Non-alcoholic steatohepatitis (NASH) is a global medical problem and macrophages' activation is closely related to the pathogenesis of NASH. Curcumin is a polyphenol from turmeric with significant anti-inflammatory activity. The objective of present study was to observe the effect of curcumin on macrophages' activation and secretion of pro-inflammatory cytokines in NASH. Hematoxylin and eosin and TUNEL staining were used to observe the hepatic function. RT-PCR was conducted to evaluate the hepatic mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Flow cytometry was adopted to detect the M1 polarization of macrophages. The RAW264.7 macrophage was pretreated with different doses of curcumin, and then lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were given to activate the M1 macrophage. The activation ratio of M1 macrophage was observed by flow cytometry, and IL-1β and TNF-α expression was detected by RT-PCR and ELISA. After treatment with curcumin, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the mRNA expression of TNF-α and IL-1β, and M1 polarization of macrophages were significantly decreased. Hematoxylin and eosin and TUNEL staining showed that inflammation and apoptosis in the liver were improved. What is more, curcumin can effectively inhibit M1 macrophage activation induced by lipopolysaccharide and IFN-γ and reduce the secretion of IL-1β and TNF-α. Curcumin can effectively improve NASH and reduce hepatic cell necrosis by inhibiting the M1 polarization of macrophages and the secretion of inflammatory factors.

Abstract P183: FFAR4: A Novel Target In Preventing Atherosclerosis?

Free fatty acid receptor 4 (FFAR4), also known as G-protein coupled receptor 120 (GPR120), is a long-chain unsaturated fatty acid receptor expressed in adipocytes, endothelial cells, and macrophages. Activation of FFAR4 helps maintain metabolic homeostasis by regulating adipogenesis, insulin sensitivity, and inflammation. While FFAR4 is best known for its role its role in preventing obesity and diabetes, recent studies have demonstrated that FFAR4 may also play an important role in the development of atherosclerosis and cardiovascular disease (CVD). Given FFAR4’s importance in anti-inflammatory signaling and high expression levels in macrophages, we designed experiments to test the hypothesis that FFAR4 prevents the development of atherosclerosis by reversing macrophage foam cell formation, a hallmark of early atherogenesis. In these studies, we isolated peritoneal macrophages from wild-type C57/Bl6 mice and incubated them with 20 μg/ml oxidized low-density lipoprotein (oxLDL) to generate foam cells. We then investigated the effects of FFAR4 activation on lipid accumulation, cytokine secretion, and cholesterol efflux. We found that activation of FFAR4 with synthetic agonist GW9508 reduced lipid accumulation as observed by decreased Oil Red O staining and reduced cellular cholesterol content. Activation of FFAR4 by GW9508 also decreased macrophage secretion of pro-inflammatory cytokines interleukin 1 beta (IL-1β) by 5.3-fold, interleukin 6 (IL-6) by 2.4-fold, monocyte chemoattractant protein-1 (MCP-1) by 44.1-fold, and tumor necrosis factor alpha (TNFα) by 2.4-fold. Additionally, activation of FFAR4 by GW9508 significantly increased [ 3 H] cholesterol efflux to high-density lipoprotein (HDL) from peritoneal macrophages. Taken together, our results support an exciting and novel protective role for FFAR4 in the reversal of foam cell formation and could emerge this receptor as a new target for treating CVD by preventing accumulation of atherosclerotic plaque.

Divergent regulation of inflammatory cytokines by mTORC1 in THP-1–derived macrophages and intestinal epithelial Caco-2 cells

AimsThe sustained activation of intestinal mechanistic target of rapamycin complex 1 (mTORC1) brought about by repeated mucosal insult or injury has been linked to escalation of gut inflammatory response, which may progress to damage the epithelium if not controlled. This study investigated the role of mTORC1 in the response of macrophage and enterocyte to inflammatory stimuli. Materials and methodsWe genetically manipulated human THP-1 monocytes and epithelial intestinal Caco-2 cells to generate stable cell lines with baseline, low or high mTORC1 kinase activity. The effects of THP-1 macrophage secretions onto Caco-2 cells were investigated by means of conditioned media transfer experiments. Key findingsThe priming of mTORC1 for activation promoted lipopolysaccharide (LPS)-mediated THP-1 macrophage immune response as evidenced by the stimulation of inflammatory mediators (TNFα, IL-6, IL-8, IL-1β and IL-10). The treatment of THP-1 macrophages with LPS more than the manipulated level of mTORC1 activity of macrophages determined whether cytokine gene expression was induced in Caco-2 cells. LPS carry over was not responsible for the stimulation of Caco-2 cells' cytokine response. Knocking down Raptor in Caco-2 cells or treating Caco-2 cells with rapamycin enhanced Caco-2 TNFα gene expression revealing the anti-inflammatory role of a functional mTORC1 in intestinal epithelial cells exposed to macrophage-derived pro-inflammatory stimuli. SignificanceTaken together, mTORC1 differentially impacts the immune responses of THP-1–derived macrophages and Caco-2 epithelial cells when placed in a pro-inflammatory microenvironment.

HCV Core Protein Induces Chemokine CCL2 and CXCL10 Expression Through NF-κB Signaling Pathway in Macrophages.

HCV core protein is the first structural protein synthesized during hepatitis C virus (HCV) infection and replication. It is released from virus infected liver cells and mediates multiple functions to affect host cell response. The innate immune response is the first line of defense against viral infection. After HCV infection, Kupffer cells (KCs) which are liver macrophages play an important role in host innate immune response. Kupffer cells act as phagocytes and release different cytokines and chemokines to counter viral infection and regulate inflammation and fibrosis in liver. Earlier, we have demonstrated that HCV core protein interacts with gC1qR and activates MAPK, NF-κB and PI3K/AKT pathways in macrophages. In this study, we explored the effect of HCV core protein on CCL2 and CXCL10 expression in macrophages and the signaling pathways involved. Upon silencing of gC1qR, we observed a significant decrease expression of CCL2 and CXCL10 in macrophages in the presence of HCV core protein. Inhibiting NF-κB pathway, but not P38, JNK, ERK and AKT pathways greatly reduced the expression of CCL2 and CXCL10. Therefore, our results indicate that interaction of HCV core protein with gC1qR could induce CCL2 and CXCL10 secretion in macrophages via NF-κB signaling pathway. These findings may shed light on the understanding of how leukocytes migrate into the liver and exaggerate host-derived immune responses and may provide novel therapeutic targets in HCV chronic inflammation.

Abiotrophia defectiva DnaK Promotes Fibronectin-Mediated Adherence to HUVECs and Induces a Proinflammatory Response

Abiotrophia defectiva is a nutritionally variant streptococci that is found in the oral cavity, and it is an etiologic agent of infective endocarditis. We have previously reported the binding activity of A. defectiva to fibronectin and to human umbilical vein endothelial cells (HUVECs). However, the contribution of some adhesion factors on the binding properties has not been well delineated. In this study, we identified DnaK, a chaperon protein, as being one of the binding molecules of A. defectiva to fibronectin. Recombinant DnaK (rDnaK) bound immobilized fibronectin in a concentration-dependent manner, and anti-DnaK antiserum reduced the binding activity of A. defectiva with both fibronectin and HUVECs. Furthermore, DnaK were observed on the cell surfaces via immune-electroscopic analysis with anti-DnaK antiserum. Expression of IL-8, CCL2, ICAM-1, and VCAM-1 was upregulated with the A. defectiva rDnaK treatment in HUVECs. Furthermore, TNF-α secretion of THP-1 macrophages was also upregulated with the rDnaK. We observed these upregulations in rDnaK treated with polymyxin B, but not in the heat-treated rDnaK. The findings show that A. defectiva DnaK functions not only as an adhesin to HUVECs via the binding to fibronectin but also as a proinflammatory agent in the pathogenicity to cause infective endocarditis.

The Role of Adenosine Receptor A2A in the Regulation of Macrophage Exosomes and Vascular Endothelial Cells During Bone Healing.

BackgroundMacrophage exosomes and vascular endothelial cells (VECs) are critical to bone healing. However, few studies explore the molecular regulation of them in the bone fracture microenvironment.MethodsIn this study, we explored the effects of adenosine receptor A2A (ADA2AR) in macrophage exosomes and VECs during bone healing. CGS21680 (an ADA2AR agonist) and ZM241385 (an ADA2AR antagonist) were used. First, the effects of the ADA2AR on VECs during bone healing were studied in vivo in a rat tibial fracture model. Second, the effects of ADA2AR on VECs and in the regulation of VECs by macrophages were examined in the bone fracture microenvironment. Third, the effects of ADA2AR on the regulation of macrophage exosomes on VECs were analyzed. Finally, the genes and long non-coding RNAs (lncRNAs) associated with the regulation of VECs by the ADA2AR were examined by high-throughput sequencing and bioinformatics analysis.ResultsCGS21680 accelerated VEC proliferation in the early stage of bone healing and that ZM241385 suppressed VEC proliferation in vivo. ZM241385 inhibited cell viability and tube formation in vitro. However, CGS21680 did not promote tube formation, cell proliferation, or cell migration in vitro. The inhibition of macrophage exosomes could suppress tube formation and VEC migration. CGS21680 had no effects on tube formation in a macrophage-VEC co-culture. The macrophage exosomes were purified and CGS21680 promoted the macrophage secretion of exosomes. In contrast, ZM241385 inhibited the macrophage secretion exosomes. Finally, the lncRNA and mRNA involved in the activation of the ADA2AR in VECs were analyzed. CGS21680 upregulated 3274 mRNAs and downregulated 2236 mRNAs, and upregulated 1696 lncRNAs and downregulated 1882 lncRNAs. The hub genes involved in angiogenesis were Flt1, Fgf2, Mapk14, Fn1, and Jun.ConclusionThe activation of ADA2AR was essential for angiogenesis and the secretion of exosomes by macrophages during bone healing; moreover, the inactivation of the ADA2AR led to poor angiogenesis and bone nonunion.

Ethanolic Extract of Black Cumin Seed Reduced Radical Reactive from Dimethylbenzanthracene Compounds

Dimethylbenzantracene is a compound that is radical reactive, genotoxic, and immunotoxic. Black cumin seeds have antioxidative properties. The study aimed to determine the effect of the black cumin seed ethanolic extract as an antioxidant by observing the secretion activity of ROI and NO peritoneal macrophages in Sprague Dawley (SD) rats induced by DMBA. This controlled experiment used 30 male SD rats. The test animals were randomly divided into six groups. Group I was given standard food and drink. Group II was given DMBA. Groups III, IV, and V were given EEBCS doses of 5, 25, and 125 mg orally for seven weeks, respectively. Group VI was given Imboost. From week 3 to week seven, all test animals were induced 10x20 DMBA, 2x per week.At week 16, all test animals were sacrificed and isolated and cultured peritoneal macrophage cells. The activity of ROI secretion and NO macrophage secretion was tested. Data on ROI secretion and NO levels were tested for ANOVA, followed by LSD test with a confidence level of 95%. The results showed that DMBA induction increased ROI secretion by macrophages and decreased NO.EEBCS secretion decreased ROI secretion and grew NO. The DMBA group had the lowest NO levels. This study concluded that EEBCS at doses 5 and 125 reduced the flow of ROI peritoneal macrophages in SD rats induced by DMBA but reduced NO levels.