Activated THP-1 Macrophages Research Articles

Fasciola hepatica Excretory-Secretory Products (Fh-ES) Either Do Not Affect miRNA Expression Profile in THP-1 Macrophages or the Changes Are Undetectable by a Microarray Technique.

Fasciola hepatica is a liver fluke that resides in the bile ducts of various mammals. The parasitosis leads to economic losses in animal production estimated at USD 3.2 billion annually. It is also considered a zoonosis of great significance and a problem for public health affecting 2.4 million people worldwide. Nevertheless, besides the negative aspects of infestation, the antigens released by the fluke, F. hepatica Excretory-Secretory Products (Fh-ES) contain several immunomodulatory molecules that may be beneficial during the course of type I diabetes, multiple sclerosis, ulcerative colitis, or septic shock. This phenomenon is based on the natural abilities of adult F. hepatica to suppress proinflammatory responses. To underline the molecular basis of these mechanisms and determine the role of microRNA (miRNA) in the process, lipopolysaccharide (LPS)-activated THP-1 macrophages were stimulated with Fh-ES, followed by miRNA microarray analyses. Surprisingly, no results indicating changes in the miRNA expression profile were noted (p < 0.05). We discuss potential reasons for these results, which may be due to insufficient sensitivity to detect slight changes in miRNA expression or the possibility that these changes are not regulated by miRNA. Despite the negative data, this work may contribute to the future planning of experiments by other researchers.

Activated THP-1 Macrophage-Derived Factors Increase the Cytokine, Fractalkine, and EGF Secretions, the Invasion-Related MMP Production, and Antioxidant Activity of HEC-1A Endometrium Cells.

Endometrium receptivity is a multifactor-regulated process involving progesterone receptor-regulated signaling, cytokines and chemokines, and additional growth regulatory factors. In the female reproductive system, macrophages have distinct roles in the regulation of receptivity, embryo implantation, immune tolerance, and angiogenesis or oxidative stress. In the present study, we investigated the effects of PMA-activated THP-1 macrophages on the receptivity-related genes, cytokines and chemokines, growth regulators, and oxidative stress-related molecules of HEC-1A endometrium cells. We established a non-contact co-culture in which the culture medium of the PMA-activated macrophages exhibiting the pro-inflammatory phenotype was used for the treatment of the endometrial cells. In the endometrium cells, the expression of the growth-related factors activin and bone morphogenetic protein 2, the growth hormone EGF, and the activation of the downstream signaling molecules pERK1/2 and pAkt were analyzed by ELISA and Western blot. The secretions of cytokines and chemokines, which are involved in the establishment of endometrial receptivity, and the expression of matrix metalloproteinases implicated in invasion were also determined. Based on the results, the PMA-activated THP-1 macrophages exhibiting a pro-inflammatory phenotype may play a role in the regulation of HEC-1A endometrium cells. They alter the secretion of cytokines and chemokines, as well as the protein level of MMPs of HEC-1A cells. Moreover, activated THP-1 macrophages may elevate oxidative stress protection of HEC-1A endometrium cells. All these suggest that pro-inflammatory macrophages have a special role in the regulation of receptivity-related and implantation-related factors of HEC-1A cells.

Exosomes derived from periodontitis induce hepatic steatosis through the SCD-1/AMPK signaling pathway

AimTo investigate the impact of exosomes released by Porphyromonas gingivalis-Lipopolysaccharide activated THP-1 macrophages and human periodontal ligament fibroblasts on hepatocyte fat metabolism. ResultsThe liver of rats with experimental periodontitis showed obvious steatosis and inflammation compared with control rats. The culture supernatant of macrophages and human periodontal ligament fibroblasts (hPDLFs), when stimulated with Pg-LPS, induced lipogenesis in HepG2 cells. Furthermore, the lipid-promoting effect was effectively inhibited by the addition of the exosome inhibitor GW4869. Subsequently, we isolated exosomes from cells associated with periodontitis. Exosomes released by Pg-LPS-stimulated macrophages and hPDLFs are taken up by hepatocytes, causing mRNA expression related to fat synthesis, promoting triglyceride synthesis, and aggravating NAFLD progression. Finally, two sets of exosomes were injected into mice through the tail vein. In vivo experiments have also demonstrated that periodontitis-associated exosomes promote the development of hepatic injury and steatosis, upregulate SCD-1 expression and inhibit the AMPK signaling pathway. ConclusionsIn conclusion, we found that exosomes associated with periodontitis promote hepatocyte adipogenesis by increasing the expression of SCD-1 and suppressing the AMPK pathway, which indicates that close monitoring of the progression of stomatopathy associated extra-oral disorders is important and establishes a theoretical foundation for the prevention and management of fatty liver disease linked to periodontitis.

Regulation of iron metabolism in HEC-1A endometrium cells by macrophage-derived factors and fractalkine.

Macrophages in the endometrium promote receptivity and implantation by secreting proinflammatory cytokines and other factors like fractalkine (FKN). Macrophages are closely linked to regulating iron homeostasis and can modulate iron availability in the tissue microenvironment. It has been revealed that the iron metabolism of the mother is crucial in fertility. Iron metabolism is strictly controlled by hepcidin, the principal iron regulatory protein. The inflammatory cytokines can modulate hepcidin synthesis and, therefore, the iron metabolism of the endometrium. It was proven recently that FKN, a unique chemokine, is implicated in maternal-fetal communication and may contribute to endometrial receptivity and implantation. In the present study, we investigated the effect of activated THP-1 macrophages and FKN on the iron metabolism of the HEC-1A endometrial cells. We established a noncontact coculture with or without recombinant human FKN supplementation to study the impact of the macrophage-derived factors and FKN on the regulation of hepcidin synthesis and iron release and storage of endometrial cells. Based on our findings, the conditioned medium of the activated macrophages could modify hepcidin synthesis via the nuclear factor kappa-light-chain-enhancer of activated B cells, the signal transducer and activator of transcription 3, and the transferrin receptor 2/bone morphogenetic protein 6/suppressor of mothers against decapentaplegic 1/5/8 signaling pathways, and FKN could alter this effect on the endometrial cells. It was also revealed that the conditioned macrophage medium and FKN modulated the iron release and storage of HEC-1A cells. FKN signaling may be involved in the management of iron trafficking of the endometrium by the regulation of hepcidin. It can contribute to the iron supply for fetal development at the early stage of the pregnancy.

Inflammasome Activation in the Hip Synovium of Rapidly Destructive Coxopathy Patients and Its Relationship with the Development of Synovitis and Bone Loss

Rapidly destructive coxopathy (RDC), a rare disease of unknown etiology, is characterized by the rapid destruction of the hip joint. In the current study, the potential involvement of inflammasome signaling in the progression of RDC was investigated. Histopathologic changes and the gene expression of inflammasome activation markers in hip synovial tissues collected from patients with RDC were evaluated and compared with those of osteoarthritis and osteonecrosis of the femoral head patients. The synovial tissues of patients with RDC exhibited remarkable increases in the number of infiltrated macrophages and osteoclasts, and the expression of inflammasome activation markers was also increased compared with those of osteoarthritis and osteonecrosis of the femoral head patients. To further understand the histopathologic changes in the joint, a co-culture model of macrophages and synoviocytes that mimicked the joint environment was developed. Remarkably, the gene expression levels of NLRP3, GSDMD, IL1B, TNFA, ADMTS4, ADMTS5, MMP3, MMP9, and RANKL were significantly elevated in the synoviocytes that were co-cultured with activated THP-1 macrophages, suggesting the association between synovitis and inflammasome activation. Consistent with these findings, osteoclast precursor cells that were co-cultured with stimulated synoviocytes exhibited an increased number of tartrate-resistant acid phosphatase-positive cells, compared with cells that were co-cultured with non-stimulated synoviocytes. These findings suggest that the activation of inflammasome signaling in the synovium results in an increase in local inflammation and osteoclastogenesis, thus leading to the rapid bone destruction in RDC.

Moderate Beer Intake Downregulates Inflammasome Pathway Gene Expression in Human Macrophages.

Simple SummaryModerate consumption of fermented beverages is associated with prevention against diseases involving inflammation and immunity. Conversely, for high drinking levels, an increase of inflammatory mediators and the susceptibility to infections occur, which tend to offset the benefits in terms of health. Unfortunately, this area remains poorly understood. Inflammation is now recognized as an overwhelming burden for public health. Thus, subcellular molecular complexes such as the “inflammasomes” have been identified as key players in cellular stress and tissue damage, and as regulators of immune and inflammatory responses. Here, we investigated the impact of moderate intake of alcohol-free and traditional beer in humans on the inflammasome pathway of activated pro-inflammatory human macrophages. The results of this study showed that macrophages submitted to a pro-inflammatory stimulus to activate the inflammosome had a mitigated inflammatory response in the presence of blood serum obtained from healthy volunteers after consuming alcohol-free or traditional beer for four weeks (women one can and men two cans per day) compared to the response found in the presence of blood serum obtained before beer intake. This was shown by a decrease in end components of the inflammasome cascade (IL-1β and TNF) at gene expression and protein level as found with alcohol-free and traditional beer, respectively.Inflammasomes are key components of the innate immunity system that trigger the inflammatory response. Inappropriate activity of the inflammasome system has been linked to onset and perpetuation of inflammation in atherosclerotic plaques and cardiovascular disease. Low-to-moderate beer consumption is inversely associated with cardiovascular event presentation, while high levels of alcohol intake are associated with increased cardiovascular risk. Although fermented beverages have been suggested to exert their beneficial effects through their anti-oxidant and anti-inflammatory properties, little is known regarding the capacity of beer to modulate innate immunity cell responses. To this aim, primed or activated THP-1 macrophages were conditioned with human serum obtained from a prospective two-arms longitudinal crossover study to investigate the effect of a moderate and regular daily intake of beer, either alcohol-free or traditional, in the regulation of TLR-mediated inflammatory responses in healthy but overweight individuals. Conditioned macrophages with serum obtained after four-week intervention with alcohol-free beer significantly reduced the transcription of pro-inflammatory interleukins such as IL-1β and TNF. The serum of traditional beer consumers did not exhibit the same capacity as the serum of alcohol-free beer consumers to reduce gene expression of pro-inflammatory interleukins; however, serum from traditional beer consumers showed a regulatory effect at the protein level by significantly decreasing the intracellular protein levels of pro-IL-1β in primed macrophages and preventing cleaved-IL-1β protein release.

Pro-inflammatory response and genotoxicity caused by clay and graphene nanomaterials in A549 and THP-1 cells

Nanoclays and graphene oxide nanomaterials represent a class of materials sharing similar shapes constituted of high aspect ratio platelets. The increased production of these materials for various industrial applications increases the risk of occupational exposure, consequently with elevated risk of adverse reactions and development of pulmonary diseases, including lung cancer. In this study, pro-inflammatory responses and genotoxicity were assessed in alveolar epithelial cells (A549) and activated THP-1 macrophages (THP-1a) after exposure to three nanoclays; a pristine (Bentonite) and two surface modified (benzalkonium chloride-coated Nanofil9, and dialkyldimethyl-ammonium-coated NanofilSE3000); graphene oxide (GO) and reduced graphene oxide (r-GO) nanomaterials. The pro-inflammatory response in terms of IL-8 expression was strongest in cells exposed to Bentonite, whereas surface modification resulted in decreased toxicity in both cell lines when exposed to Nanofil9 and NanofilSE3000. GO and r-GO induced a pro-inflammatory response in A549 cells, while no effect was detected with the two nanomaterials on THP-1a cells. The pro-inflammatory response was strongly correlated with in vivo inflammation in mice after intra-tracheal instillation when doses were normalized against surface area. Genotoxicity was assessed as DNA strand breaks, using the alkaline comet assay. In A549 cells, an increase in DNA strand breaks was detected only in cells exposed to Bentonite, whereas Bentonite, NanofilSE3000 and GO caused an increased level of genotoxicity in THP-1a cells. Genotoxicity in THP-1a cells was concordant with the DNA damage in bronchoalveolar lavage fluid cells following 1 and 3 days after intra-tracheal instillation in mice. In conclusion, this study shows that surface modification of pristine nanoclays reduces the inflammatory and genotoxic response in A549 and THP-1a cells, and these in vitro models show comparable toxicity to what seen in previous mouse studies with the same materials.

P2X7 receptor-targeted regulation by tetrahydroxystilbene glucoside in alcoholic hepatosteatosis: A new strategy towards macrophage-hepatocyte crosstalk.

Regulating macrophage-hepatocyte crosstalk through P2X7 receptors has led to new pharmacological strategies to reverse alcoholic hepatosteatosis. We investigated how tetrahydroxystilbene glucoside (2354glu), isolated from Polygonum multiflorum, modulates macrophage-hepatocyte crosstalk during alcoholic hepatosteatosis. A model of alcoholic hepatosteatosis was established by giving ethanol intragastrically to C57BL/6 mice. HepG2 cells were incubated in conditioned medium from LPS+ATP-activated THP-1 human macrophages with silenced or overexpressed P2X7 receptors. THP-1 macrophages or mouse peritoneal macrophages were pretreated with 2354glu for 1 hr prior to LPS+ATP stimulation. Western blots, RT-PCR and immunohistochemical analysis were used, along with over-expression and silencing of P2X7 receptors. Knockdown or overexpression of P2X7 receptors in THP-1 macrophages affected release of mature IL-1β and, subsequently, modulated lipid metabolism in HepG2 cells via the LKB-AMPK pathway. 2354glu ameliorated alcoholic hepatosteatosis in mice by regulating LKB1-AMPK-SREBP1 pathway and its target genes. Suppression of P2X7 receptor activation by 2354glu inhibited IL-1β release and reduced macrophage and neutrophil infiltration. In macrophages stimulated with LPS+ATP, expression of P2X7 receptors, caspase-1 and NF-κB, release of IL-1β, calcium influx and PI uptake were reduced by 2354glu. SIRT1-LKB1-AMPK-SREBP1 axis-mediated lipid accumulation in HepG2 cells was reduced when they were cultured with conditioned media from LPS+ATP-activated THP-1 macrophages pretreated with 2354glu. Modulation of P2X7 receptors in macrophages regulated lipid accumulation in hepatocytes during alcoholic hepatosteatosis. 2354glu might be a promising candidate that targets P2X7 receptors in macrophages interacting with hepatocytes during alcoholic hepatosteatosis.

Forskolin inhibited the secretion of interleukin-1β in activated human THP-1 macrophages

Objective To investigate the effects of Forskolin on the activation of nucleotide-binding oligomerization domain like receptor family, pyrin domain-containing 3(NLRP-3) inflammasome and the secretion of interleukin-1β(IL-1β) in activated human THP-1 macrophages, which can provide evidence for clinical treatment of inflammatory diseases in children. Methods Human monocyte cell line THP-1 cells were induced to differentiate into macrophages by Phorbol-12-myristate-13-acetate(PMA), and Forskolin(10, 50, 100 μmol/L) stimulated activated macrophages by nigericin.The mRNA of the inflammasome markers NLRP-3, IL-1β and Caspase-1 were detected by real-time quantitative polymerase chain reaction(qPCR) method.The protein of NLRP-3, pro-IL-1β, pro-Caspase-1 and Caspase-1 were detected by Western blot.The secretion of inflammatory cytokines IL-1β was detected by enzyme-linked immuno-sorbent assay(ELISA). Results Nigericin activated the cells and the mRNA expression of NLRP-3, IL-1β and Caspase-1 increased by 7.59 times(P 0.05). Western blot showed that Forskolin also had no effect on the protein expression of pro-Caspase-1 and pro-IL-1β in activated THP-1 macrophages(P>0.05), but the expression of NLRP-3 and Caspase-1 decreased significantly (P<0.001). The results of ELISA showed that the IL-1β secretion increased from basal 584.0 nmol/L to activated 2 695.6 nmol/L(t=16.031 1, P<0.001) on THP-1 macrophages by nigericin, but Forskolin(10, 50, 100 μmol/L) reduced it to 1 858.4 nmol/L(t=5.365 5, P<0.001), 1 467.9 nmol/L(t=8.047 5, P<0.001) and 1 246.7 nmol/L(t=10.199 0, P<0.001) on the activated THP-1 macrophages. Conclusions Forskolin can not affect the expression of pro-cytokines at the gene transcription and protein translation levels in activated THP-1 cells, but inhibit the secretion of inflammatory cytokines IL-1β, so as to inhibit inflammatory response, which can be used to treat pediatric inflammatory diseases caused by IL-1β. Key words: Inflammation; Forskolin; Macrophage; Inflammasome; Interleukin-1β

Abstract 158: Indoleamine 2,3-dioxygenase 1 is Expressed in Macrophages in Human Coronary Atherosclerotic Plaque and is Associated With Tissue Factor Expression in Activated THP-1 Macrophages

Background: Recently, several clinical studies have found that changes in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism are associated with cardiovascular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a rate-limiting enzyme of the Kyn pathway and is induced by cytokines, particularly IFNγ. However, the roles of the Kyn pathway on vascular wall thrombogenicity remain unknown. Objective: The present study aimed to localize IDO1 in human coronary atherosclerotic plaques from patients with stable angina pectoris (SAP) and unstable angina pectoris (UAP) and define its role in plaque thrombogenicity. Methods: Immunohistochemical methods were applied to localize IDO1 in coronary atherosclerotic plaques from patients with SAP and UAP. We examined the role of IDO1 in tissue factor (TF) expression in THP-1 macrophages activated by interferon (IFN)γ and tissue necrosis factor (TNF)α. Results: We localized IDO1 mainly in CD68-positive macrophages in atherosclerotic plaques, and in close association with TF. Areas that were immunopositive for IDO1, TF, and CD3-positive T lymphocytes were significantly larger in plaques from patients with UAP than SAP. THP-1 macrophages activated by IFNγ and TNFα upregulated IDO1 expression, increased the Kyn/Trp ratio and enhanced TF expression and activity, but not TF pathway inhibitor expression. The IDO1 inhibitor epacadostat significantly reduced the Kyn/Trp ratio, TF expression and activity, as well as NF-κB (p65) binding activity in activated THP-1 macrophages. Inhibition of the aryl hydrocarbon receptor (AHR) that binds to Kyn, also reduced Kyn-induced TF expression in activated THP-1 macrophages. Kynurenine dose-dependently increased TF expression under IDO1 inhibition whereas hydroxyanthranilic and quinolinic acids did not. Conclusion: The IDO1 is expressed in coronary atherosclerotic plaques and might contribute to thrombus formation through TF upregulation via NF-κB (p65) and AHR in activated macrophages.

Abstract 4089: Thyroid tumor microenvironment: mutual interaction between cancer and inflammatory cells

Abstract Thyroid cancer is the most prevalent endocrine malignancy in the United States with an unacceptably high recurrence rate of 20-30%. Tumor associated macrophages (TAMs), one of the most critical component of solid tumor microenvironments, promote cancer initiation, growth, progression, metastasis and angiogenesis. These TAMs release cytokines as well as other secretory components like exosomes in the tumor microenvironment. Previously, we found that M1 polarized TAMs modulate thyroid cancer phenotype by inducing epithelial-mesenchymal transition (EMT), facilitating tissue metastasis and dissemination. In our present study, we used an in vitro model system to assess the crosstalk between the secretory components of macrophages and the epithelial cells in the thyroid tumor microenvironment. We used THP-1 monocyte/macrophage cell line along with thyroid cancer cell lines: consisting of two papillary cancer cell lines (BCPAP and TPC-1), one anaplastic cancer cell line (8505C) and one follicular cancer cell line (CGTHW-1). We observed that activated THP-1 macrophages are polarized towards M1 phenotype, secreting pro-inflammatory cytokines such as TGF-β, IL6, TNF-α, IL-1β, amongst others. These cytokines are responsible for halt in proliferation and change in morphology to mesenchymal phenotype promoting EMT in thyroid cancer cells. Similar pattern in phenotypical changes were noted in thyroid cancer cells treated with activated THP-1 macrophage exosomes. We also observed that EMT markers, such as vimentin and NFκ-B, are modulated in response to activated macrophage secreted exosomes. Moreover, secretory components from anaplastic thyroid cancer cells led to enhanced activation of THP-1 cells. These findings support a mutual cooperation between thyroid cancer cells and inflammatory cells in tumor microenvironment in defining thyroid cancer phenotype. Such correlation can identify early markers and prevent thyroid cancer differentiation, and are putative targets for therapy. Citation Format: Neha Yashpal Tuli, Robert B. Bednarczyk, Ghada M. Ben Rahoma, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Thyroid tumor microenvironment: mutual interaction between cancer and inflammatory cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4089.

Graphene quantum dots induce apoptosis, autophagy, and inflammatory response via p38 mitogen-activated protein kinase and nuclear factor-κB mediated signaling pathways in activated THP-1 macrophages.

The biomedical application of graphene quantum dots (GQDs) is a new emerging area. However, their safety data are still in scarcity to date. Particularly, the effect of GQDs on the immune system remains unknown. This study aimed to elucidate the interaction of GQDs with macrophages and the underlying mechanisms. Our results showed that GQDs slightly affected the cell viability and membrane integrity of macrophages, whereas GQDs significantly increased reactive oxygen species (ROS) generation and apoptotic and autophagic cell death with an increase in the expression level of Bax, Bad, caspase 3, caspase 9, beclin 1, and LC3-I/II and a decrease in that of Bcl-2. Furthermore, low concentrations of GQDs significantly increased the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-8, whereas high concentrations of GQDs elicited opposite effects on the cytokines production. SB202190, a selective inhibitor of p38 mitogen-activated protein kinase (MAPK), abolished the cytokine-inducing effect of GQDs in macrophages. Moreover, GQDs significantly increased the phosphorylation of p38 MAPK and p65, and promoted the nuclear translocation of nuclear factor-κB (NF-κB). Taken together, these results show that GQDs induce ROS generation, apoptosis, autophagy, and inflammatory response via p38MAPK and NF-κB mediated signaling pathways in THP-1 activated macrophages.

A novel p65 inhibitor suppresses glutamate release and proliferation of macrophages: therapeutic implications for chronic neurological diseases. (THER3P.887)

Abstract Activated nuclear factor-κB (NFκB) plays critical role in the pathogenesis of chronic neurological diseases constituting an attractive therapeutic target. Based on the cross talk between NFκB and glucocorticoid signaling pathways, we developed a novel strategy to selectively target p65, the functionally critical subunit of NFκB. Glucocorticoid induced leucine zipper (GILZ) is a “divergence” hub linked to multiple proteins in the NFκB signaling pathway. Structurally, proline rich carboxy terminus of GILZ binds the transactivation domain of p65. An immunomodulatory peptide derived from the p65 binding region of GILZ, the GILZ-P ameliorated autoimmune disease in mice. Here we determine the secondary structural characteristics and investigate the efficacy of GILZ-P in lipopolysaccharide (LPS) activated ThP-1 macrophages used as model systems for human microglial cells. GILZ-P treatment did not adversely affect cell viability as determined by vital staining and flow cytometry. LPS stimulated mature macrophages treated with GILZ-P exhibited reduced proliferation and inflammatory cytokine secretion as assessed by the MTT assay and ELISA respectively. Mechanistically the inhibitory potential of GILZ-P was substantiated by the reduced nuclear translocation of p65 and decreased glutamate release. Collectively, our data support anti-inflammatory and neuroprotective effects for the PPII helical GILZ-P suggesting therapeutic applications in neuroinflammatory-neurodegenerative conditions.

MiR-613 regulates cholesterol efflux by targeting LXRα and ABCA1 in PPARγ activated THP-1 macrophages

Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Although PPARγ is known to be a potent sterol sensor that play a fundamental role in cholesterol metabolism, the potential effects of PPARγ responsive miRNA still need to be revealed. In this study, we found that miR-613 is inversely correlated with LXRα and ABCA1 in PPARγ activated THP-1 cells. PPARγ negatively regulates the expression of miR-613 at transcriptional level, and miR-613 suppressed LXRα and ABCA1 by targeting the 3′-UTR of their mRNAs. Furthermore, downregulation of LXRα and ABCA1 by miR-613 inhibited cholesterol efflux from PPARγ activated THP-1 macrophages. These results revealed an alternative mechanism for PPARγ regulation and provided a potential target for the treatment of cholesterol metabolic diseases.

Plant-Derived Human Collagen Scaffolds for Skin Tissue Engineering

Tissue engineering scaffolds are commonly formed using proteins extracted from animal tissues, such as bovine hide. Risks associated with the use of these materials include hypersensitivity and pathogenic contamination. Human-derived proteins lower the risk of hypersensitivity, but possess the risk of disease transmission. Methods engineering recombinant human proteins using plant material provide an alternate source of these materials without the risk of disease transmission or concerns regarding variability. To investigate the utility of plant-derived human collagen (PDHC) in the development of engineered skin (ES), PDHC and bovine hide collagen were formed into tissue engineering scaffolds using electrospinning or freeze-drying. Both raw materials were easily formed into two common scaffold types, electrospun nonwoven scaffolds and lyophilized sponges, with similar architectures. The processing time, however, was significantly lower with PDHC. PDHC scaffolds supported primary human cell attachment and proliferation at an equivalent or higher level than the bovine material. Interleukin-1 beta production was significantly lower when activated THP-1 macrophages where exposed to PDHC electrospun scaffolds compared to bovine collagen. Both materials promoted proper maturation and differentiation of ES. These data suggest that PDHC may provide a novel source of raw material for tissue engineering with low risk of allergic response or disease transmission.

Molecular cloning and characterisation of in vitro immune response against astacin-like metalloprotease Ace-MTP-2 from Ancylostoma ceylanicum

Ancylostoma ceylanicum belongs to the group of parasites commonly known as hookworms, blood-sucking nematodes which infect around 576 million people and hundreds of millions of animals. The interactions between these parasites and host immune systems are complicated and yet to be determined. Hookworm infections are usually long lasting and recurrent, due in part to their ability to synthesize macromolecules capable of modulating the host immune response. The interaction of parasite proteins with host immune systems has been proven, but so far there is no data describing the influence of astacin-like metalloproteases (expressed among different parasitic nematodes) on the human immune system. The cDNA encoding A. ceylanicum metalloprotease 2 (Ace-mtp-2) was cloned using RACE-PCR. Computational analysis was used to examine the immunogenicity and recombinant Ace-MTP-2 was used to investigate its influence on human THP-1 monocytes and macrophages. The Ace-mtp-2 gene encodes an astascin-like metalloprotease, with a theoretical molecular mass of 26.7 kDa. The protease has a putative signal peptide, 11 potential phosphorylation sites, and two disulfide bridges revealed by computational analysis. Maximal expression of Ace-mtp-2 by A. ceylanicum occurs in the adult stage of the parasite, and Western blot indicates the secretory nature of the protease. This suggests the protease is working at the host-parasite interface and would likely be exposed to the hosts immune response. Recombinant protein were expressed in Escherichia coli and Pichia pastoris. Recombinant Ace-MTP-2 amplified the in vitro release of TNFα and induced release of IFNγ by lipopolysaccharide activated THP-1 macrophages. The presence of Ace-MTP-2 in secretory products of the adult parasite and the induction of IFNγ release may suggest an important role for Ace-MTP-2 in host-parasite interactions since IFNγ is suggested to be responsible for the protective immune response against adult hookworms.

Effect of Lipopolysaccharide Mediating Early- and Late- Activated THP-1 Macrophages on ECV304 Endothelial Cell Dysfunction: Dysregulation of Secretion of VEGF and Proliferation and Migration of ECV304

Objective: To investigate the different effects of lipopolysaccharide (LPS) mediating early and late activated THP-1 macrophages (Mφ) on ECV304 endothelial cell dysfunction: dysregulation of secretion of VEGF and proliferation, and migration of ECV304. Methods: The inflammatory Mφ was divided into early phase (2 h) group and late phase (24 h) group according the different exposure time to LPS. Then the inflammatory Mφ and ECV304 were co-cultured via transwell chambers in both non-contacting and contacting systems. The levels of VEGF were determined by ELISA, and the proliferation index and apoptosis of ECV304 were analyzed by FACSCalibur. The migration of ECV304 was tested by modified Boyden chamber assay. Results: The level of VEGF and the proliferation of ECV304 cell were increased more apparently in early-phase Mφ-treated group. But the proportion of early apoptotic and late apoptotic/necrotic cells in late-phase Mφ-treated group were higher than that of the former. Migration rate of ECV304 was enhanced in early-phase Mφ-treated group. All those effects were more significant in contacting system comparing with no-contacting system. Conclusion: Early-activated macrophages (mediated by LPS) could increase the secretion of VEGF and promote the proliferation and migration of ECV304; while the late-activated macrophages could promote/enhance the apoptosis of ECV304 more significant in contacting system when (it was) compared with no-contacting system.

CD200 Positive Human Mesenchymal Stem Cells Suppress TNF-Alpha Secretion from CD200 Receptor Positive Macrophage-Like Cells

Human mesenchymal stem cells (hMSCs) display immunosuppressive properties in vitro and the potential has also been transferred successfully to clinical trials for treatment of autoimmune diseases. OX-2 (CD200), a member of the immunoglobulin superfamily, is widely expressed in several tissues and has recently been found from hMSCs. The CD200 receptor (CD200R) occurs only in myeloid-lineage cells. The CD200-CD200R is involved in down-regulation of several immune cells, especially macrophages. The present study on 20 hMSC lines shows that the CD200 expression pattern varied from high (CD200Hi) to medium (CD200Me) and low (CD200Lo) in bone marrow-derived mesenchymal stem cell (BMMSC) lines, whereas umbilical cord blood derived mesenchymal stem cells (UCBMSCs) were constantly negative for CD200. The role of the CD200-CD200R axis in BMMSCs mediated immunosuppression was studied using THP-1 human macrophages. Interestingly, hMSCs showed greater inhibition of TNF-α secretion in co-cultures with IFN-γ primed THP-1 macrophages when compared to LPS activated cells. The ability of CD200Hi BMMSCs to suppress TNF-α secretion from IFN-γ stimulated THP-1 macrophages was significantly greater when compared to CD200Lo whereas UCBMSCs did not significantly reduce TNF-α secretion. The interference of CD200 binding to the CD200R by anti-CD200 antibody weakened the capability of BMMSCs to inhibit TNF-α secretion from IFN-γ activated THP-1 macrophages. This study clearly demonstrated that the efficiency of BMMSCs to suppress TNF-α secretion of THP-1 macrophages was dependent on the type of stimulus. Moreover, the CD200-CD200r axis could have a previously unidentified role in the BMMSC mediated immunosuppression.

Preclinical Evaluation of New Indomethacin-Eluting Biodegradable Urethral Stent

To evaluate the effect of an indomethacin-eluting biodegradable urethral stent on the production of inflammatory cytokines in vitro and the degradation and biocompatibility of the new stent in vivo. The effects of an indomethacin and indomethacin-eluting biodegradable stent on monocyte chemoattractant protein (MCP)-1, RANTES (regulated on activation, normal T-cell expressed and secreted), and transforming growth factor-ß were measured in THP-1 cells by enzyme-linked immunosorbent assay. Stents (copolymer of L-lactide and glycolide acid) that were coated with 50L/50D polylactic acid and two different concentrations of indomethacin were inserted into the rabbit urethra. Stents without the drug were used as controls. Scanning electron microscopy (SEM) was used to assess the degradation of the stents. Biocompatibility was evaluated using histologic analyses of the urethral specimen. The measurements were performed at 3 weeks and 3 months. Indomethacin and indomethacin-releasing stent material inhibited MCP-1 and RANTES production in activated THP-1 macrophages. SEM analysis revealed that indomethacin coating had no effect on the degradation process of the stents and less epithelial polyposis had developed in the indomethacin stent group. In histologic analyses at 3 weeks, indomethacin-eluting stents caused more calcification but no significant differences in other tissue reactions. At 3 months, the indomethacin-eluting stents caused less inflammatory reaction and calcification compared with the control stents. Indomethacin-eluting property can be safely added to biodegradable stents without major influence on the degradation time. The development of epithelial polyposis in the urethra can be potentially reduced by the new indomethacin-eluting urethral stents.

Dual use of amphiphilic macromolecules as cholesterol efflux triggers and inhibitors of macrophage athero-inflammation

Activated vascular wall macrophages can rapidly internalize modified lipoproteins and escalate the growth of atherosclerotic plaques. This article proposes a biomaterials-based therapeutic intervention for depletion of non-regulated cholesterol accumulation and inhibition of inflammation of macrophages. Macromolecules with high scavenger receptor (SR)-binding activity were investigated for SR-mediated delivery of agonists to cholesterol-trafficking nuclear liver-X receptors. From a diverse feature space of a family of amphiphilic macromolecules of linear and aromatic mucic acid backbones modified with varied aliphatic chains and conjugated with differentially branched poly(ethylene glycol), a key molecule (carboxyl-terminated, C12-derivatized, linear mucic acid backbone) was selected for its ability to preferentially bind scavenger receptor A (SR-A) as the key target. At a basal level, this macromolecule suppressed the pro-inflammatory signaling of activated THP-1 macrophages while competitively lowering oxLDL uptake in vitro through scavenger receptor SRA-1 targeting. To further deplete intracellular cholesterol, the core macromolecule structure was exploited to solubilize a hydrophobic small molecule agonist for nuclear Liver-X Receptors, which regulate the efflux of intracellular cholesterol. The macromolecule-encapsulated agonist system was found to reduce oxLDL accumulation by 88% in vitro in comparison to controls. in vivo studies were designed to release the macromolecules (with or without encapsulated agonist) to injured carotid arteries within Sprague Dawley rats fed a high fat diet, conditions that yield enhanced cholesterol accumulation and macrophage recruitment. The macromolecules lowered intimal levels of accumulated cholesterol (50% for macromolecule alone; 70% for macromolecule-encapsulated agonist) and inhibited macrophage retention (92% for macromolecule; 96% for macromolecule-encapsulated agonist; 4 days) relative to non-treated controls. Thus, this study highlights the promise of designing bioactive macromolecule therapeutics based on scavenger receptor targeting, for potential management of vascular arterial disease.