Human Monocytic Cell Line THP-1 Research Articles

Systemic redox imbalance in severe COVID-19 patients.

The aim of this study was to evaluate the systemic redox state and inflammatory markers in intensive care unit (ICU) or non‐ICU severe COVID‐19 patients during the hospitalization period. Blood samples were collected at hospital admission (T1) (Controls and COVID‐19 patients), 5–7 days after admission (T2: 5–7 days after hospital admission), and at the discharge time from the hospital (T3: 0–72 h before leaving hospital or death) to analyze systemic oxidative stress markers and inflammatory variables. The reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) were analyzed in peripheral granulocytes and monocytes. THP‐1 human monocytic cell line was incubated with plasma from non‐ICU and ICU COVID‐19 patients and cell viability and apoptosis rate were analyzed. Higher total antioxidant capacity, protein oxidation, lipid peroxidation, and IL‐6 at hospital admission were identified in both non‐ICU and ICU COVID‐19 patients. ICU COVID‐19 patients presented increased C‐reactive protein, ROS levels, and protein oxidation over hospitalization period compared to non‐ICU patients, despite increased antioxidant status. Granulocytes and monocytes of non‐ICU and ICU COVID‐19 patients presented lower MMP and higher ROS production compared to the healthy controls, with the highest values found in ICU COVID‐19 group. Finally, the incubation of THP‐1 cells with plasma acquired from ICU COVID‐19 patients at T3 hospitalization period decreased cell viability and apoptosis rate. In conclusion, disturbance in redox state is a hallmark of severe COVID‐19 and is associated with cell damage and death.

Anti - Inflammatory Activity of Quercetin and Kaempferol Is Limited to Tlr4 Stimulation in Monocytes

Flavonoids are known to exert anti-inflammatory activity but their mechanism at receptor level and their comparative potencies are less studied. Objective of the study is to evaluate, rank order few flavonoids for their cytokine inhibitory activity in monocytes (THP-1) and explore the inhibitory signature among toll-like receptors (TLRs). We tested flavonoids for their potential to inhibit LPS induced cytokines IL-6, TNF alpha in human monocyte cell line THP-1 and mouse macrophage cell line using Enzyme linked immunosorbent assay (ELISA) assays. Quercetin and Kaempferol reduced LPS induced cytokine secretion in monocyte and macrophage cell lines. Quercetin and Kaempferol demonstrated dose dependent inhibition of cytokines IL-6 and TNF alpha. Quercetin demonstrated IC50 of 6.9µM whereas Kaempferol demonstrated IC50 of 14.3µM in cytokine reduction. In THP-1, NF-kB activity is measured using secreted embryonic alkaline phosphatase (SEAP) reporter with different toll-like receptor activation. TLR4 stimulated NF-kB activity was inhibited by Quercetin and Kaempferol in THP-1 cell line, while these flavonoids did not impact signaling of other TLR ligands Pam3CSK4 (TLR1/2) FLS (TLR2/6), Flagellin (TLR5) and R848 (TLR7/8). I. Quercetin and Kaempferol modulate NF-kB activity when stimulated with TLR4, but not with TLR1/2, TLR2/6, TLR5 and TLR7/8 in Monocytes. Understanding the inhibition profile of flavonoids against Toll receptors will help in design better inhibitor which can alleviate inflammatory diseases

Heat Shock Protein 90's Mechanistic Role in Contact Hypersensitivity.

Despite the known dangers of contact allergens and their long-lasting use as models in immunology, their molecular mode of action largely remains unknown. In this study, we report that a contact allergen, 1-chloro-2,4-dinitrobenzene (DNCB), elicits contact hypersensitivity through binding the protein we identify. Starting from an unbiased sampling of proteomics, we found nine candidate proteins with unique DNCB-modified peptide fragments. More than half of these fragments belonged to heat shock protein 90 (HSP90), a common stress-response protein and a damage-associated molecular pattern, and showed the highest probability of incidence. Inhibition and short hairpin RNA knockdown of HSP90 in human monocyte cell line THP-1 suppressed the potency of DNCB by >80%. Next, we successfully reduced DNCB-induced contact hypersensitivity in HSP90-knockout mice, which confirmed our findings. Finally, we hypothesized that DNCB-modified HSP90 activates the immune cells through HSP90's receptor, CD91. Pretreatment of CD91 in THP-1 cell lines and BALB/c mice attenuated the potency of DNCB, consistent with the result of HSP90-knockout mice. Altogether, our data show that DNCB-HSP90 binding plays a role in mediating DNCB-induced contact hypersensitivity, and the activation of CD91 by DNCB-modified HSP90 proteins could mediate this process.

Insight Into the Properties and Immunoregulatory Effect of Extracellular Vesicles Produced by Candida glabrata, Candida parapsilosis, and Candida tropicalis Biofilms.

Currently, non-albicans Candida species, including C. tropicalis, C. glabrata, and C. parapsilosis, are becoming an increasing epidemiological threat, predominantly due to the distinct collection of virulence mechanisms, as well as emerging resistance to antifungal drugs typically used in the treatment of candidiasis. They can produce biofilms that release extracellular vesicles (EVs), which are nanometric spherical structures surrounded by a lipid bilayer, transporting diversified biologically active cargo, that may be involved in intercellular communication, biofilm matrix production, and interaction with the host. In this work, we characterize the size and protein composition of these structures for three species of non-albicans Candida fungi forming biofilm, indicating considerable heterogeneity of the investigated population of fungal EVs. Examination of the influence of EVs on cytokine production by the human monocytic cell line THP-1 differentiated into macrophage-like cells revealed that the tested vesicles have a stimulating effect on the secretion of tumor necrosis factor α and interleukin 8, while they reduce the production of interleukin 10. This may indicate the proinflammatory nature of the effect of EVs produced by these species on the host immune cells. Moreover, it has been indicated that vesicles may be involved in C. tropicalis biofilm resistance to fluconazole and caspofungin. This reveals the important role of EVs not only in the physiology of C. tropicalis, C. glabrata, and C. parapsilosis fungi but also in the pathogenesis of infections associated with the production of fungal biofilm.

Quantitative Analysis of the Transcriptome of Two Commonly Used Human Monocytic Cell Lines—THP-1 and Mono Mac 6—Reveals Their Arrest during Early Monocyte/Neutrophil Differentiation

Cell lines of monocyte/macrophage origin are often used as model systems to study monocyte/macrophage biology. A relevant question is how similar these cell lines are to their in vivo counterparts? To address this issue, we performed a detailed analysis of the transcriptome of two commonly used human monocyte/macrophage cell lines, Mono Mac 6 and THP-1. Both of these cell lines originate from leukemic cells with myelo-monocytic characteristics. We found that both Mono Mac 6 and THP-1 represent cells of very immature origin. Their transcriptomes show more similarities to immature neutrophils than cells of the monocyte/macrophage lineage. They express significant levels of N-elastase, proteinase 3, cathepsin G, and azurocidin but very low levels of CD14, ficolin, and complement factor P. All major MHC class II genes are also expressed at low levels. They show high levels of lysozyme and low levels of one of the immunoglobulin Fc receptors, FCGRIIA, which is characteristic of both neutrophils and monocytes. THP-1, but not Mono Mac 6, also expresses the high-affinity receptor for IgG, FCGRIA. Both cell lines lack the expression of the connective tissue components fibronectin, proteoglycan 4, and syndecan 3, which are characteristics of tissue macrophages but are absent in blood monocytes, indicating that they originate from bone marrow precursors and not yolk sac-derived hematopoietic cells. Both of these cell lines seem, therefore, to represent cells arrested during early myelo-monocytic development, at a branch point between neutrophil and monocyte differentiation. Their very immature phenotype indicates that great care should be taken when using these cell lines as models for normal monocyte/macrophage biology.

Potential application for antimicrobial and antileukemic therapy of a flavonoid-rich fraction of Camellia sinensis

The antimicrobial and antileukemic effect of a purified fraction of flavonoids from the leaves of Camellia sinensis was evaluated. An extraction yield of 9.77 mg.g-1 total flavonoids was recovered through a pressurized liquid extraction associated with solid-phase extraction. This fraction was tested against pathogenic microorganisms (Staphylococcus, Salmonella, and Enterococcus), considering the minimum inhibitory concentration. In addition, the human monocyte cell line THP-1, derived from a patient with acute monocytic leukemia, was used for the antitumor assay. The results show that the flavonoid-rich fraction obtained by coupling a Pressurized Liquids Extraction in-line with a Solid Phase Extraction (PLE-SPE) has a high antimicrobial effect and resulted in cell cycle blockage G0 / G1, increased DNA fragmentation, and altered leukemic cell morphology. These results suggest that a flavonoid-rich fraction obtained from Camellia sinensis can be applied as potential adjuvants in chemotherapy treatment to mitigate the side effects caused by chemotherapy or even as a supplement to cancer therapy.

Interferon inducible guanylate binding protein 1 restricts the growth of Leishmania donovani by modulating the level of cytokines/chemokines and MAP kinases

Visceral Leishmaniasis (VL) is a zoonotic chronic endemic infectious disease caused by Leishmania donovani infection and a well-studied model for intracellular parasitism. Guanylate binding proteins (GBPs) are induced by interferons (IFNs), and play a crucial role in cell autonomous immunity and the regulation of inflammation. Guanylate-binding protein 1 (GBP1) has been shown vital for the host immune response against various pathogens. However, the role of GBP1 during VL is undefined. In the present study, we have investigated the role of GBP1 in Leishmania donovani infection using in vitro model. For that, knock down of the Gbp1 gene was carried out in both PMA differentiated human monocyte cell line THP-1 and mouse macrophages RAW264.7 cell line using siRNA based RNA interference. Infection of these cell lines revealed a high parasite load in knock down cells at 24 and 48h post infection as compared to control cells. A significant increase was observed in the level of different cytokines (IL-4, IL-10, IL-12b, IFN-γ, TNF-α) and chemokines (CXCL9, CXCL 10, and CXCL 11) in GBP1 knock down cell lines after post-infection. In GBP1 knock down cells the expression level of IFN effector molecules (iNOS and PKR) was found to be elevated in THP1 cells and remained almost unchanged in RAW264.7 cells after Leishmania donovani infection as compared to the control cells. Moreover, interestingly, the level of MAPK activated ERK1/2, and p38 MAPK were considerably induced by the parasite in knock down cells as compared to control after 24 h post-infection. This study, first time reported the involvement of GBP1 in Leishmania donovani infection by modulating the level of important cytokines, chemokines, IFN effector molecules, and MAP kinases.

α1B‐Adrenoceptor is Required for Chemokine (C‐X‐C Motif) Receptor 4 Mediated Chemotaxis in THP1 Cells

IntroductionChemokine (C‐X‐C motif) receptor 4 (CXCR4) is a key regulator of leukocyte trafficking under physiological conditions and in numerous inflammatory disease processes. Previously, we demonstrated that CXCR4 hetero‐oligomerizes with α1B‐adrenoceptors (ARs), through which the receptors cross‐talk. The existence and function of these hetero‐oligomers in leukocytes, however, is unknown. Thus, we tested whether such heteromeric complexes are detectable in the human monocytic cell line THP1 and in freshly isolated human monocytes, and evaluated how α1B‐AR knockdown by CRISPR/Cas9 gene editing in THP1 cells affects CXCR4‐mediated chemotaxis.MethodsPeripheral blood mononuclear cells were isolated by density gradient centrifugation from human whole blood; monocytes were then isolated via negative selection using MACS LS, an indirect magnetic labeling system. Proximity ligation assays (PLA) were used to visualize individual receptors and receptor‐receptor interactions in THP1 cells and freshly isolated monocytes. CRISPR/Cas9 gene editing was utilized to create a α1B‐AR knockout strain (ADRA1BKO); wild type THP1 cells were used as a negative control (ADRA1Bwt). Cell migration toward the cognate CXCR4 agonist chemokine (C‐X‐C motif) ligand 12 (CXCL12) was tested employing a 96 well Boyden chamber. Transmigrated cells were counted automatically utilizing high contrast bright field and post‐imaging particle analyses. The chemotactic index (ChI) was calculated as the ratio of cells that transmigrated in the presence versus the absence of CXCL12. Data were analyzed with ANOVA/Tukey’s post‐hoc. Data are mean ± SE from n=4 experiments performed on different days. A 2‐tailed p<0.05 was considered significant.ResultsPLA confirmed the presence of endogenously and constitutively expressed CXCR4:α1B‐AR heteromers in freshly isolated monocytes and THP1 cells. PLA signals for α1B‐AR and CXCR4:α1B‐AR heteromers were not detectable in ADRA1BKO cells. CXCR4 signals were not affected by CRISPR/Cas9 gene knockout. ADRA1Bwtcells showed a bell‐shaped chemotactic dose response to CXCL12 with a maximal ChI of 9.1±1.1 at 100 nM. CRISPR/Cas9 knockout of α1B‐AR reduced the chemotactic response to CXCL12 by 89±52% (p<0.05). ADRA1BKO results were replicated in an additional α1B‐AR knockout strain to rule out clonal effects.ConclusionOur observations suggest that CXCR4 heteromerizes with α1B‐AR in human monocytes and that CXCL12‐mediated chemotaxis depends on the formation of CXCR4:α1B‐AR heteromers. These data provide evidence for a novel molecular mechanism by which CXCR4‐mediated chemotaxis in monocytes/leukocytes is controlled.

High-resolution crystal structure of LpqH, an immunomodulatory surface lipoprotein of Mycobacterium tuberculosis reveals a distinct fold and a conserved cleft on its surface

Tuberculosis, caused by Mycobacterium tuberculosis, is predominantly a disease of the lungs acquired by inhaling mycobacteria from infected individuals via airborne droplets. In order to facilitate their entry into the alveolar macrophages, mycobacteria have a collection of pathogen-associated molecular patterns (PAMPs) on their surface that are known to detect certain pattern recognition receptors present on the surface of host cells. A major group of these PAMPs includes mycobacterial lipoproteins, of which, the 19 kDa surface antigen LpqH, has been reported to play a critical role in both host-pathogen interactions as well as pleiotropic immune regulation. Despite its crucial involvement in tuberculosis, the detailed structure-function relationship of this protein remains to be explored. Here, we report the high-resolution crystal structure of the non-acylated LpqH (LpqH48–159) at a resolution of 1.26 Å, which adopts a unique fold. Flow cytometry-based experiments show that the protein can bind and induce apoptosis in PMA-activated human monocytic cell line THP-1, indicative of the preservation of functionality of the protein. Furthermore, analysis of conservation of LpqH sequences from Mycobacterium species reveals a patch of conserved residues on the surface which may play a role in its binding partner recognition and hence in host-pathogen interaction.

Andrographolide, isolated from Andrographis paniculata, induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production.

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC). Results: Andro reduced the viability of THP-1 and H929 in a concentration-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors.The high toxicity forH929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

YTHDF1 Negatively Regulates Treponema pallidum-Induced Inflammation in THP-1 Macrophages by Promoting SOCS3 Translation in an m6A-Dependent Manner.

BackgroundPrevious studies have confirmed that the bacterium Treponema pallidum (TP) or its proteins provide signals to macrophages that induce an inflammatory response; however, little is known about the negative regulation of this macrophage-mediated inflammatory response during syphilis infection or the underlying mechanism. Recent evidence suggests the role of the RNA modification, N6-adenosine methylation (m6A), in regulating the inflammatory response and pathogen-host cell interactions. Therefore, we hypothesized that m6A plays a role in the regulation of the inflammatory response in macrophages exposed to TP.MethodsWe first assessed m6A levels in TP-infected macrophages differentiated from the human monocyte cell line THP-1. The binding and interaction between the m6A “writer” methyltransferase-like 3 (METTL3) or the m6A “reader” YT521-B homology (YTH) domain-containing protein YTHDF1 and the suppressor of cytokine signaling 3 (SOCS3), as a major regulator of the inflammatory response, were explored in differentiated TP-infected THP-1 cells as well as in secondary syphilitic lesions from patients. The mechanisms by which YTHDF1 and SOCS3 regulate the inflammatory response in macrophages were assessed.Results and ConclusionAfter macrophages were stimulated by TP, YTHDF1 was upregulated in the cells. YTHDF1 was also upregulated in the syphilitic lesions compared to adjacent tissue in patients. YTHDF1 recognizes and binds to the m6A methylation site of SOCS3 mRNA, consequently promoting its translation, thereby inhibiting the JAK2/STAT3 pathway, and reducing the secretion of inflammatory factors, which results in anti-inflammatory regulation. This study provides the first demonstration of the role of m6A methylation in the pathological process of syphilis and further offers new insight into the pathogenesis of TP infection.

Gene Silencing of Toll-like Receptor 2 Gene Expression as a Tactic to Control Mycobacterium Tuberculosis and Granuloma Formation.

Mycobacterium tuberculosis (MT) is the causative agent of tuberculosis (TB) in humans. Tuberculosis is one of the top 10 causes of mortality worldwide, resulting in 1.8 million deaths and 10.4 million new cases in 2016. Understanding the fundamental features of MT biology is critical to the eradication of MT in the future. Due to the increasing frequency of antimicrobial treatment resistance and problems in vaccine development, the pathogenesis of TB for its survival and growth is highly dependent on host lipids and stimulated-lipid droplets formation. Toll-like receptor 2 (TLR2) forms heterophilic dimers with TLR1 and TLR6, therefore, recognizing many MT components. Both of these receptors identify the invading antigen and activate downstream protein kinases. Some studies demonstrated that the cyclooxygenase-2 (COX-2) promoter-driven gene expression includes connecting sites for transcription factors, such as nuclear factor-kappa B, CREB, NFAT, and c/EBPβ. The current study aimed to investigate the role of the TLR2 receptor in positively regulating prostaglandin E2 production in M. bovis (BCG) infected macrophages in vivo using a human monocytic cell line THP-1. Our results revealed that MT infection triggers a time-dependent increase in COX-2 expression via pathways involving TLR2 receptor activation and enhances COX-2 expression, leading to an increase in lipid droplet formation and suppression of macrophage activation.

IL-33 regulates M1/M2 chemokine expression via mitochondrial redox-related mitophagy in human monocytes

Interleukin (IL)-33 is an epithelial-derived cytokine that enhances T helper (Th) 2 responses. Allergens and other agents induce IL-33 in asthma. Excessive production of reactive oxygen species (ROS) leads to airway inflammation. Mitophagy is the selective degradation of mitochondria by autophagy and often occurs in defective mitochondria, followed by ROS production. In the present study, we examined the effects of IL-33 on ROS production and mitophagy in human monocytes, and the detailed mechanisms were investigated. Human monocyte cell line THP-1 was pretreated with different concentrations of IL-33. ROS production was measured by flow cytometry. Mitochondrial involvement and the mitophagy and intercellular pathway activation were evaluated by quantitative real-time PCR, western blotting, and confocal microscopy, and cytokine/chemokine concentrations were detected by ELISA. The data showed that IL-33 alone could induce ROS expression in THP-1 cells. The expression of complex II and V mRNA was increased in the presence of IL-33. The mitophagy-related proteins PINK1, Parkin, and LC3 were regulated by IL-33 through the AMPK pathway. IL-33 significantly decreased M1-related cytokines CXCL-10 and TNF-α production and significantly increased M2-related cytokine CCL-22 production. In conclusion, IL-33 induces ROS production and subsequently influences mitophagy through AMPK activation, altering the macrophage-polarization phenotype of monocytes.

Picroside II alleviates DSS-induced ulcerative colitis by suppressing the production of NLRP3 inflammasomes through NF-κB signaling pathway

Context Ulcerative colitis (UC) is a common acute or chronic intestinal disease with an imbalance of inflammation. Picroside II (P-II) exerts a protective role in various inflammation-related diseases. However, the effect of P-II on UC is still unclear. Objective To explore the effect of P-II on UC and its potential mechanism. Materials and methods Human monocytic leukemia cell line THP-1 was treated with phorbol ester (PMA) to differentiate into a macrophage. The differentiated THP-1 cells were hatched with LPS combined with ATP or Nigericin to activate the NLRP3 inflammasome in vitro. The UC model was constructed by injection of DSS into mice. Results The maximum nontoxic concentration of P-II on THP-1 cells was 60 μM. LPS combined with ATP or Nigericin stimulated the production of IL-1β, which was antagonized by P-II treatment. Meanwhile, P-II administration interfered with the aggregation of ASC and the assembly of NLRP3 inflammasomes. Also, P-II treatment reduced the LPS and ATP-induced elevation of the relative protein expression of NLRP3, pro-caspase-1, IL-1β and p-p65/p65, and the concentration of TNF-α and IL-6. Besides, the NF-κB specific inhibitor BAY-117082 notably repressed the LPS together with ATP-enhanced the relative protein expression of NLRP3, caspase-1 and IL-1β. Moreover, in vivo results showed that P-II relieved the DDS-induced UC, as evidenced by the improvement of mice weight, DAI and pathological scores. In addition, P-II treatment notably decreased DDS-promoted expression of NLRP3 inflammasomes and inflammatory factors in vivo. Conclusion P-II alleviated DSS-induced UC by repressing the production of NLRP3 inflammasomes via the NF-κB signaling pathway.

MicroRNA-142-3p facilitates inflammatory response by targeting ZEB2 and activating NF-κB signaling in gouty arthritis

ABSTRACT Gouty arthritis (GA) is caused by monosodium urate (MSU) crystal accumulation in the joints. MSU-mediated inflammation is an important inducing factor in gouty arthritis (GA). Recent studies have demonstrated that microRNAs can influence GA progression. Herein, the role and mechanism of miRNA-142-3p in GA were explored. To establish the in vitro and in vivo GA models, MSU was used to induce inflammatory response in human monocyte cell line THP-1 and male C57BL/6 mice. Protein levels, gene expression and proinflammatory cytokine secretion were respectively tested by Western blotting, RT-qPCR, and enzyme-linked immunosorbent assay (ELISA). Pathological changes in sagittal sections of ankle tissues were exhibited by hematoxylin-eosin (HE) staining. Binding relationship between miRNA-142-3p and zinc finger E-box binding homeobox 2 (ZEB2) was predicted and confirmed by bioinformatics analysis and luciferase reporter assay. In this study, MSU induced inflammatory response and upregulated miRNA-142-3p in THP-1 cells. Functionally, miRNA-142-3p knockdown inhibited inflammatory response in MSU-stimulated THP-1 cells and alleviated pathological symptoms of GA mice. Mechanically, miRNA-142-3p targeted ZEB2 in THP-1 cells. ZEB2 expression was elevated in MSU-administrated THP-1 cells and GA mice. ZEB2 downregulation reserved the inhibitory effect of miRNA-142-3p deficiency on inflammatory response in MSU-treated THP-1 cells. In addition, miRNA-142-3p activated NF-κB signaling by binding with ZEB2 in THP-1 cells upon MSU stimulation. Overall, miRNA-142-3p facilitates inflammatory response by targeting ZEB2 and activating NF-κB signaling in GA.

IL-25 improves diabetic wound healing through stimulating M2 macrophage polarization and fibroblast activation

BackgroundPersistent chronic inflammation is one of the main pathogenic characteristics of diabetic wounds. The resolution of inflammation is important for wound healing and extracellular matrix (ECM) formation. Interleukin (IL)-25 can modulate the function of macrophage and fibroblast, but its role and mechanism of action in the treatment of diabetic wounds remain largely unclear. MethodsThe mice were categorized into diabetic, diabetic + IL-25 and control groups. Human monocytic THP-1 cell line and human dermal fibroblast (HDF) were stimulated under different IL-25 conditions. Then, flow cytometry, real-time quantitative PCR (RT-qPCR), Western blot (WB), and immunofluorescence (IF) assays were carried out. ResultsThe mice in diabetes group (DG) had a slower wound healing rate, more severe inflammation, less blood vessels and more disordered collagen than those in control group (CG). Intradermal injection of IL-25 could improve these conditions. IL-25 promoted M2 macrophage polarization and fibroblast activation in DG and high-glucose environment. The phenomenon, which was dependent on PI3K/AKT/mTOR and TGF-β/SMAD signaling, could be blocked by LY294002 and LY2109761. ConclusionIL-25 may serve as a therapeutic target to improve wound healing in diabetic mice.

Pharmacologic ACE-Inhibition Mitigates Radiation-Induced Pneumonitis by Suppressing ACE-Expressing Lung Myeloid Cells

Radiation-induced lung injury is a major dose-limiting toxicity for thoracic radiation therapy patients. In experimental models, treatment with angiotensin converting enzyme (ACE) inhibitors mitigates radiation pneumonitis; however, the mechanism of action is not well understood. Here, we evaluate the direct role of ACE inhibition on lung immune cells. ACE expression and activity were determined in the lung immune cell compartment of irradiated adult rats after either high dose fractionated radiation therapy to the right lung (5 fractions×9 Gy) or a single dose of 13.5 Gy partial body irradiation. Mitigation of radiation-induced pneumonitis with the ACE-inhibitor lisinopril was evaluated in the 13.5 Gy rat partial body irradiation model. During pneumonitis, we characterized inflammation and immune cell content in the lungs and bronchoalveolar lavage fluid. In vitro mechanistic studies were performed using primary human monocytes and the human monocytic THP-1 cell line. In both the partial body irradiation and fractionated radiation therapy models, radiation increased ACE activity in lung immune cells. Treatment with lisinopril improved survival during radiation pneumonitis (P=.0004). Lisinopril abrogated radiation-induced increases in bronchoalveolar lavage fluid monocyte chemoattractant protein 1 (chemokine ligand 2) and MIP-1a cytokine levels (P < .0001). Treatment with lisinopril reduced both ACE expression (P=.006) and frequency of CD45+ CD11b+ lung myeloid cells (P=.004). In vitro, radiation injury acutely increased ACE activity (P=.045) and reactive oxygen species (ROS) generation (P=.004) in human monocytes, whereas treatment with lisinopril blocked radiation-induced increases in both ACE and ROS. Radiation-induced ROS generation was blocked by pharmacologic inhibition of either NADPH oxidase 2 (P=.012) or the type 1 angiotensin receptor (P=.013). These data demonstrate radiation-induced ACE activation within the immune compartment promotes the pathogenesis of radiation pneumonitis, while ACE inhibition suppresses activation of proinflammatory immune cell subsets. Mechanistically, our in vitro data demonstrate radiation directly activates the ACE/type 1 angiotensin receptor pathway in immune cells and promotes generation of ROS via NADPH oxidase 2.

Mitochondria Fusion upon SERCA Inhibition Prevents Activation of the NLRP3 Inflammasome in Human Monocytes.

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) is a crucial component of the cellular machinery responsible for Ca2+ homeostasis. The selective inhibition of SERCA by thapsigargin (TG) leads to perturbations in Ca2+ signaling, which can trigger endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) pathway is activated in response to ER stress and induces an adaptive response to preserve cell survival or committee cells to programmed death, depending on stress duration and/or level. Early stages of ER stress stimulate mitochondrial metabolism to preserve survival but under chronic ER stress conditions, mitochondrial dysfunction is induced, which, in turn, can enhance inflammation through NLRP3 inflammasome activation. This study was aimed at investigating the role of SERCA inhibition on NLRP3 inflammasome activation in human monocytes, which was evaluated in primary monocytes isolated from healthy individuals and in the THP-1 human monocytic cell line. Findings obtained in both THP-1 and primary monocytes demonstrate that SERCA inhibition triggered by TG does not activate the NLRP3 inflammasome in these innate immune cells since IL-1β secretion was not affected. Results from THP-1 monocytes showing that SERCA inhibition increases mitochondrial Ca2+ content and fusion, in the absence of changes in ROS levels and membrane potential, support the view that human monocytes counteract ER stress that arises from inhibition of SERCA through modulation of mitochondrial morphology towards mitochondria fusion, thus preventing NLRP3 inflammasome activation. Overall, this work contributes to a better understanding of the molecular mechanisms that modulate the activity of the NLRP3 inflammasome leading to sterile inflammation, which are still poorly understood.

Factors secreted by monosodium urate crystal-stimulated macrophages promote a proinflammatory state in osteoblasts: a potential indirect mechanism of bone erosion in gout

BackgroundTophi are lesions commonly present at sites of bone erosion in gout-affected joints. The tophus comprises a core of monosodium urate (MSU) crystals surrounded by soft tissue that contains macrophages and other immune cells. Previous studies found that MSU crystals directly reduce osteoblast viability and function. The aim of the current study was to determine the indirect, macrophage-mediated effects of MSU crystals on osteoblasts.MethodsConditioned medium from the RAW264.7 mouse macrophage cell line cultured with MSU crystals was added to the MC3T3-E1 mouse osteoblastic cell line. Conditioned medium from the THP-1 human monocytic cell line cultured with MSU crystals was added to primary human osteoblasts (HOBs). Matrix mineralization was assessed by von Kossa staining. Gene expression was determined by real-time PCR, and concentrations of secreted factors were determined by enzyme-linked immunosorbent assay.ResultsIn MC3T3-E1 cells cultured for 13 days in an osteogenic medium, the expression of the osteoblast marker genes Col1a1, Runx2, Sp7, Bglap, Ibsp, and Dmp1 was inhibited by a conditioned medium from MSU crystal-stimulated RAW264.7 macrophages. Mineral staining of MC3T3-E1 cultures on day 21 confirmed the inhibition of osteoblast differentiation. In HOB cultures, the effect of 20 h incubation with a conditioned medium from MSU crystal-stimulated THP-1 monocytes on osteoblast gene expression was less consistent. Expression of the genes encoding cyclooxygenase-2 and IL-6 and secretion of the proinflammatory mediators PGE2 and IL-6 were induced in MC3T3-E1 and HOBs incubated with conditioned medium from MSU crystal-stimulated macrophages/monocytes. However, inhibition of cyclooxygenase-2 activity and PGE2 secretion from HOBs indicated that this pathway does not play a major role in mediating the indirect effects of MSU crystals in HOBs.ConclusionsFactors secreted from macrophages stimulated by MSU crystals attenuate osteoblast differentiation and induce the expression and secretion of proinflammatory mediators from osteoblasts. We suggest that bone erosion in joints affected by gout results from a combination of direct and indirect effects of MSU crystals.

Antibody-Dependent THP-1 Cell-Mediated Phagocytosis of Plasmodium falciparum-Infected Erythrocytes.

Antibodies that recognize variant surface antigens (VSAs) expressed on Plasmodium falciparum-infected erythrocytes (IEs) opsonize IEs for phagocytic clearance. The anti-VSA antibodies promote antibody-dependent cellular phagocytosis (ADCP) of IEs by interacting with innate immune cells. ADCP is an important immune effector mechanism of parasite clearance. ADCP can be a tool to assess the efficacy of vaccine-induced antibodies, in addition to measuring the neutralizing ability of antibodies. Here, we developed and validated an efficient and high-throughput plate-based flow cytometric assay to measure ADCP of IEs using the human monocytic THP-1 cell line. This flow cytometric assay can be used to analyze the level of naturally acquired or vaccine-induced opsonic antibodies in large cohorts.