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Cyanidin‐3‐O‐glucoside (C3G) is a kind of water‐soluble pigment widely existing in many plants. It has strong antioxidant and anti‐inflammatory activities. However, C3G cannot exist stably for a long time because of the phenolic hydroxyl groups in its structure. Liposome technology could improve the stability and bioavailability of compounds. Based on our previous studies, C3G liposomes prepared by ethanol injection method have a certain stability in two weeks of storage. In this study, THP‐1 macrophages treated with C3G and C3G liposomes can reduce the levels of inflammatory‐related factors, such as tumor necrosis factor‐a (TNF‐a), interleukin (IL)‐1β, IL‐6, and IL‐8, stimulated by lipopolysaccharide (LPS). Further studies showed that the LPS induction could increase the level of phosphorylated nuclear transcription factor NF‐κB and phosphorylated IkBa, while C3G and C3G liposomes could inhibit the expression of phosphorylated proteins. Moreover, C3G and C3G liposomes could protect macrophages from apoptosis. In conclusion, C3G prepared by liposome technology exhibits anti‐inflammatory activity, which provides a theoretical basis for the food industry to study functional food., In this work, the effects of C3G and C3G liposomes on the levels of cytokines, such as TNF‐a, IL‐1β, IL‐6, and IL‐8 in the inflammatory cell model were studied and the expression of inflammatory‐related proteins were analyzed to further explore their anti‐inflammatory mechanisms. In addition, we also studied the effects of C3G and C3G liposomes on cell apoptosis in LPS‐stimulated THP‐1 macrophages.

BACKGROUND/AIM Effective ex vivo maturation of dendritic cells (DCs) can increase the efficiency of cancer immunotherapy. We aimed to identify novel chemicals with the potential to differentiate and activate immature DCs (iDCs) to mature DCs (mDCs). MATERIALS AND METHODS The expression of surface markers on THP-1 monocytes treated with the screened compounds was analyzed using FACS. Subsequent DC subset analysis and secreted cytokine profiling were also performed. RESULTS FACS analysis showed that THP-1 cells treated with amsacrine hydrochloride, a DNA topoisomerase II inhibitor, exhibited the typical phenotype of conventional DCs (cDCs). The expression of DC activation markers was also increased after amsacrine treatment. The profile of cytokines produced by THP-1 cells treated with amsacrine was similar to that of mDCs. CONCLUSION Amsacrine has an ex vivo capability of differentiating THP-1 monocytes into cDCs. As amsacrine has been used as a stable chemotherapeutic agent in humans, it can be useful for producing mDCs for cancer immunotherapy.

PURPOSE Doxorubicin (DOX) is a common chemotherapeutic agent, with toxic side effects, and chemoresistance. Combination chemotherapy is a successful approach to overcome these limitations. Here, we investigated the effects of pioglitazone (PGZ), a PPARγ agonist, and/or DOX on the viability, cell cycle, apoptosis on THP-1 cells and normal human monocytes (NHMs). METHODS MTT assay was used to evaluate the cytotoxicity of DOX and/or PGZ. Cell cycle progression and apoptosis induction were examined by PI or Annexin V-PI double staining, and analyzed by flow cytometry. Quantitative RT-PCR was used to evaluate the changes in the mRNA expression of cell cycle progression or apoptosis-associated genes including P27, P21, CDK2, P53, BCL2 and FasR. RESULTS DOX, PGZ and DOX + PGZ exerted their cytotoxic effects in a dose- and time-dependent manner with low toxicity on NHMs. The cell growth inhibitory effects of DOX were in association with G2/M arrest, while PGZ executed S phase arrest. PGZ treatment enhanced G2/M among DOX-treated combinations with moderate elevation in the S phase. DOX, PGZ and combined treatments induced apoptosis (mostly late phase) in a dose-dependent manner. All treatments resulted in the significant overexpression of p21, p27, p53 and FasR genes and downregulation of CDK2. DOX + PGZ combined treatments exhibited the most significant changes in mRNA expression. CONCLUSION We demonstrated that the antiproliferative, cell cycle regulation and apoptosis-inducing capacity of DOX was enhanced by PGZ in THP-1 leukemia cells in a dose-dependent manner. Therefore, the combination of DOX + PGZ could be used as a novel combination to target AML.

Cytokine storm induced by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a major pathological feature of Coronavirus Disease 2019 (COVID-19) and a crucial determinant in COVID-19 prognosis. Understanding the mechanism underlying the SARS-CoV-2-induced cytokine storm is critical for COVID-19 control. Here, we identify that SARS-CoV-2 ORF3a and host hypoxia-inducible factor-1α (HIF-1α) play key roles in the virus infection and pro-inflammatory responses. RNA sequencing shows that HIF-1α signaling, immune response, and metabolism pathways are dysregulated in COVID-19 patients. Clinical analyses indicate that HIF-1α production, inflammatory responses, and high mortalities occurr in elderly patients. HIF-1α and pro-inflammatory cytokines are elicited in patients and infected cells. Interestingly, SARS-CoV-2 ORF3a induces mitochondrial damage and Mito-ROS production to promote HIF-1α expression, which subsequently facilitates SARS-CoV-2 infection and cytokines production. Notably, HIF-1α also broadly promotes the infection of other viruses. Collectively, during SARS-CoV-2 infection, ORF3a induces HIF-1α, which in turn aggravates viral infection and inflammatory responses. Therefore, HIF-1α plays an important role in promoting SARS-CoV-2 infection and inducing pro-inflammatory responses to COVID-19.

Acute pancreatitis is a disease associated with suffering and high lethality. Although the disease mechanism is unclear, phospholipase A2 (PLA2) produced by pancreatic acinar cells is a known pathogenic trigger. Here, we show macrophage membrane-coated nanoparticles with a built-in ‘lure and kill’ mechanism (denoted ‘MΦ-NP(L&K)’) for the treatment of acute pancreatitis. MΦ-NP(L&K) are made with polymeric cores wrapped with natural macrophage membrane doped with melittin and MJ-33. The membrane incorporated melittin and MJ-33 function as a PLA2 attractant and a PLA2 inhibitor, respectively. These molecules, together with membrane lipids, work synergistically to lure and kill PLA2 enzymes. These nanoparticles can neutralize PLA2 activity in the sera of mice and human patients with acute pancreatitis in a dose-dependent manner and suppress PLA2-induced inflammatory response accordingly. In mouse models of both mild and severe acute pancreatitis, MΦ-NP(L&K) confer effective protection against disease-associated inflammation, tissue damage and lethality. Overall, this biomimetic nanotherapeutic strategy offers an anti-PLA2 treatment option that might be applicable to a wide range of PLA2-mediated inflammatory disorders., Phospholipase A2 (PLA2) inhibitors have been shown to be able to treat acute pancreatitis, but are toxic with systemic application. Here the authors design a nanoparticle with macrophage membrane components to carry PLA2 inhibitor to macrophages and treat pancreatitis in mice, with no evidence of toxicity.

Objectives Salvia verbenaca is a medicinal plant that has been traditionally used in Algeria for the treatment of wounds and emptied abscesses. The present study aimed to evaluate the cytotoxicity of methanolic extract of S. verbenaca roots and explore its ability to bestow protection against oxidative damage induced by H2O2 (200 μM). Materials and Methods The cytotoxic effects and protective properties of S. verbenaca on human monocytic leukemia cells (THP-1) was studied using thiazolyl blue tetrazolium bromide assay. The protective effects of the extract against H2O2-induced oxidative damage was evaluated using single cell gel electrophoresis (comet) assay and 2,7-dichlorodihydrofluorescien diacetate (H2DCFDA) assay. Results S. verbenaca extract was found to be non-cytotoxic at concentrations

Accumulation of cholesterol‐laden macrophage foam cells characteristic of early stage atherosclerotic lesions. Phenethyl isothiocyanate (PEITC) is a naturally occurring isothiocyanate found in cruciferous vegetables that has reported a variety of activities including antioxidant and anti‐inflammatory properties. However, the protective effect of PEITC on foam cell formation and its precise mechanism is not yet clear. Therefore, we investigated whether PEITC suppresses foam cell formation and regulates the expression of genes related to lipid accumulation, cholesterol efflux, and inflammation in THP‐1 derived‐macrophages. We exposed THP‐1 derived‐macrophages to oxidized low‐density lipoprotein (ox‐LDL) (20 μg/mL) and lipopolysaccharide (LPS) (500 ng/ml) to mimic foam cell formation. Here, PEITC downregulated the expression of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), cluster of differentiation 36 (CD36), scavenger receptor A1 (SR‐A1), and nuclear factor‐κB (NF‐κB), while upregulated ATP binding cassette subfamily A member 1 (ABCA1)/liver‐X‐receptor α (LXR‐α)/peroxisome proliferator‐activated receptor gamma (PPARγ) and sirtuin 1 (SIRT1) expression compared to co‐treated with ox‐LDL and LPS. Taken together, PEITC, at least in part, inhibits foam cell formation and reduces lipid accumulation in foam cells. Therefore, we suggest that PEITC may be a potential candidate for the treatment and prevention of vascular inflammation and atherosclerosis., PEITC control foam cell formation by promoting cholesterol efflux, reducing lipid accumulation, and modulating the SIRT1‐NF‐κB signaling pathway.

Leptospira interrogansare pathogenic bacteria responsible for leptospirosis, a zoonosis impacting 1 million peopleperyear worldwide. Leptospires can infect all vertebrates, but not all hosts develop similar symptoms. Human and cattle may suffer from mild to acute illnesses and are therefore considered as sensitive to leptospirosis. In contrast, mice and rats remain asymptomatic upon infection, although they get chronically colonized in their kidneys. Upon infection, leptospires are stealth pathogens that partially escape the recognition by the host innate immune system. Although leptospires are mainly extracellular bacteria, it was suggested that they could also replicate within macrophages. However, contradictory data in the current literature led us to reevaluate these findings. Using a gentamicin–protection assay coupled to high-content (HC) microscopy, we observed that leptospires were internalizedin vivoupon peritoneal infection of C57BL/6J mice. Additionally, three different serotypes of pathogenicL. interrogansand the saprophyticL. biflexaactively infected both human (PMA differentiated) THP1 and mouse RAW264.7 macrophage cell lines. Next, we assessed the intracellular fate of leptospires using bioluminescent strains, and we observed a drastic reduction in the leptospiral intracellular load between 3 h and 6 h post-infection, suggesting that leptospires do not replicate within these cells. Surprisingly, the classical macrophage microbicidal mechanisms (phagocytosis, autophagy, TLR–mediated ROS, and RNS production) were not responsible for the observed decrease. Finally, we demonstrated that the reduction in the intracellular load was associated with an increase of the bacteria in the supernatant, suggesting that leptospires exit both human and murine macrophages. Overall, our study reevaluated the intracellular fate of leptospires and favors an active entrance followed by a rapid exit, suggesting that leptospires do not have an intracellular lifestyle in macrophages.

Introduction THP-1 cells, a human leukemia monocytic cell line, differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages. Differentiated THP-1 cells acquire macrophage-like characteristics including more adherence and altered cell function. Nitric oxide (NO), an intracellular messenger, is critical in regulating cell differentiation. Here we elucidated whether NO relates to PMA-induced monocyte-to-macrophage differentiation of THP-1 cells. The mutual regulation of calcium and NO was also investigated. Material & methods THP-1 cells were incubated with PMA for 24 h, followed by assay of adherence, morphological change, migration or IL-1β release. L-NG-Nitroarginine methyl ester ( l -NAME, a nitric oxide synthase inhibitor) or BAPTA-AM (a calcium chelator) was added before PMA stimulation, and levels of calcium and NO were measured. Furthermore, a selective inhibitor of inducible nitric oxide synthase (iNOS) activity was employed to study the role of iNOS. Results and Discussion Effects of PMA on upregulation of adherence, lipopolysaccharide-triggered IL-1β, and migration ability of THP-1 cells were consistent with NO concentrations. Both l -NAME and BAPTA-AM mitigated effects of PMA on THP-1 cells differentiation. BAPTA-AM decreased levels of NO, while l -NAME had no effect on calcium levels. Of note, inhibition of iNOS activity decreased PMA-triggered upregulation of NO. Conclusion PMA induced differentiation of THP-1 cells partially in a NO-dependent manner. The calcium signaling may mediate PMA-triggered upregulation of NO.

COVID-19 has been diffusely pandemic around the world, characterized by massive morbidity and mortality. One of the remarkable threats associated with mortality may be the uncontrolled inflammatory processes, which were induced by SARS-CoV-2 in infected patients. As there are no specific drugs, exploiting safe and effective treatment strategies is an instant requirement to dwindle viral damage and relieve extreme inflammation simultaneously. Here, highly biocompatible glycyrrhizic acid (GA) nanoparticles (GANPs) were synthesized based on GA. In vitro investigations revealed that GANPs inhibit the proliferation of the murine coronavirus MHV-A59 and reduce proinflammatory cytokine production caused by MHV-A59 or the N protein of SARS-CoV-2. In an MHV-A59-induced surrogate mouse model of COVID-19, GANPs specifically target areas with severe inflammation, such as the lungs, which appeared to improve the accumulation of GANPs and enhance the effectiveness of the treatment. Further, GANPs also exert antiviral and anti-inflammatory effects, relieving organ damage and conferring a significant survival advantage to infected mice. Such a novel therapeutic agent can be readily manufactured into feasible treatment for COVID-19.

Leukocytes play an important role in vascular inflammation prior to atherosclerosis. In particular, monocyte adhesion and migration to the endothelium contribute to the development of vascular inflammation. Previously, we showed the importance of neutrophils and complement C5a in the early phase of vascular inflammation in mice fed a high‐fat diet. However, the relationship between monocytes and neutrophils is not well understood. In this study, we elucidated the involvement of neutrophils in the migration of monocytes. We observed that C5a induces CCL2 expression in neutrophil‐like dHL‐60 cells. To investigate the physiological significance of CCL2 secretion, we performed a chemotaxis assay. Interestingly, dHL‐60 culture supernatant in the presence of C5a enhanced the migration of THP‐1 in comparison with the absence of C5a. Furthermore, CCL2 expression and secretion significantly increased in C5a‐stimulated dHL‐60 through the phosphorylation of NF‐κB p65. Actin polymerization on THP‐1 was enhanced by the presence of C5a compared with the absence of C5a when stimulated by a dHL‐60‐cultured medium. These results suggest that crosstalk between neutrophils and monocytes via CCL2 may play an important role in vascular inflammation., In this study, we found that neutrophil‐like dHL‐60 stimulated with C5a induces the migration of monocytic THP‐1 through phosphorylation of p65 NF‐κB and that dHL‐60‐derived CCL2 plays an important role in the crosstalk between these cells. These results suggest that there may be a transition process from acute to chronic inflammation in atherosclerotic vascular inflammation.

Background: Ionizing radiation plays a significant role in cancer treatment. Despite recent advances in radiotherapy approaches, the existence of irradiation-resistant cancer cells is still a noteworthy challenge. Therefore, developing novel therapeutic approaches are still warranted in order to increase the sensitivity of tumor cells to radiation. Many types of research rely on the role of mitochondria in radiation protection. Objective: Here, we aimed to target the mitochondria of monocyticleukemia (THP-1) radio-resistant cell line cells by a mitochondrial disrupting peptide, D (KLAKLAK)2, and investigate the synergistic effect of Gamma-irradiation and KLA for tumor cells inhibition in vitro. Material and Methods: In this experimental study, KLA was delivered into THP-1 cells using a Cell-Penetrating Peptide (CPP).The cells were then exposed to gamma-ray radiation both in the presence and absence of KLA conjugated with CPP. The impacts of KLA, ionizing radiation or combination of both were then evaluated on the cell proliferation and apoptosis of THP-1 cells using MTT assay and flow cytometry, respectively. Results: The MTT assay indicated the anti-proliferative effects of combined D (KLAKLAK)2 peptide with ionizing radiation on THP-1cells. Moreover, synergetic effects of KLA and ionizing radiation reduced cell viability and consequently enhanced cell apoptosis. Conclusion: Using KLA peptide in combination with ionizing irradiation increases the anticancer effects of radio-resistant THP-1 cells. Therefore, the combinational therapy of (KLAKLAK)2 and radiation is a promising strategy for cancer treatment the in future.

Non-pathogenic Rickettsia species LON strains closely related to an agent of Japanese spotted fever (JSF), R. japonica, were isolated in Japan from Haemaphysalis longicornis ticks in 2001. However, the biological properties of LONs in mammalian host cells are poorly understood. In this study, microscopic analysis showed that LONs in a mouse-derived L929 host cell line were rod shaped with sizes of 0.3-0.5 × 0.5-2.0 μm. Molecular analysis revealed the existence of a LON-specific disrupted open reading frame in R. japonica-related group-specific DNA regions. Growth kinetics of LON-2 and LON-13 strains analyzed by a quantitative real-time PCR showed 100-fold or more increment of LONs cultured in L929 host cells at 30°C and slightly less increment at 33°C, and 25-fold increment in human-derived THP-1 host cells at 35°C on day 7 (168 h) post infection. The generation times of the two LON strains cultured in L929 and THP-1 were estimated to be 9.4-12.9 h and 9.6-10.9 h, respectively. To our knowledge, this is the first report on the biological characteristics of Rickettsia sp. LON strains in mammalian cells, which may provide significant information for the experimental approaches for other rickettsiae.

Background The activation of macrophages and the release of inflammatory cytokines are the main reasons for the progress of systemic lupus erythematosus (SLE). MicroRNA (miRNA)-124 is involved in the regulation of macrophages and is a key regulator of inflammation and immunity. Objective To explore whether paeoniflorin (PF) regulates the biological functions of macrophages depends on miR-124. Methods RT-PCR, WB, ELISA, CCK-8 and flow cytometry were used to evaluate that PF regulated the biological functions of THP-1 cells through miR-124. Results PF significantly inhibited the proliferation while promotes the apoptosis of THP-1 cells, and inhibited the release of IL-6, TNF-α and IL-1βin THP-1 cells. RT-PCR results shown that PF up-regulated the expression of miR-124 in THP-1 cells. Functional recovery experiments showed that compared with the LPS + mimic-NC group, LPS + miR-124 mimic significantly inhibited the proliferation and the release of IL-6, TNF-α and IL-1β, but promoted the apoptosis of THP-1 cells. In addition, compared with the LPS + PF + inhibitor-NC group, LPS + PF + miR-124 inhibitor significantly promoted the proliferation and the release of IL-6, TNF-α and IL-1β, but inhibited the apoptosis of THP-1 cells. Conclusions By down-regulating miR-124, PF inhibits the proliferation and inflammation of THP-1 cells, and promotes the apoptosis of THP-1 cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental and food contaminant generated as a byproduct of various industrial activities. It is found in a lot of foods, especially in dairy products, eggs, fish, and meat. Autophagy is a highly conserved cellular degradation and cycling process, which plays an important role in lipid metabolism. This study aimed to explore the effects of TCDD on autophagic flux and lipid metabolism in THP-1 macrophages. The data showed that TCDD promoted the accumulation of autophagosomes in THP-1 macrophages, and subsequent findings revealed that this autophagosome accumulation was caused by the inhibition of autophagic flux by testing the expression of LC3II, p62 levels, and mRFP-GFP-LC3. Further, we found that TCDD treatment significantly increased the amount of triglyceride (TG) and total cholesterol (TC) in THP-1 macrophages. Meanwhile, pretreatment with autophagy activator (rapamycin, Rapa) efficiently relieved TCDD-induced lipid accumulation. On the contrary, pretreatment with autophagy inhibitor (Chloroquine, CQ) promoted TCDD-induced lipid accumulation. In the experiment of co-localization of LC3 and lipid droplets, the co-localization of LC3 and lipid droplets increased after TCDD induction. These results indicated that TCDD promoted lipid accumulation in THP-1 macrophages by inhibiting autophagic flux. Our findings revealed new insights into the toxicity mechanisms of TCDD.

The NLRP3 inflammasome is a critical component of the innate immune response to sterile inflammation. Its regulation involves a priming step, required for up-regulation of inflammasome protagonists and an activation step leading to NLRP3 inflammasome complex assembly, which triggers caspase-1 activity. The IκKβ kinase regulates canonical NF-κB, a key pathway involved in transcriptional priming. We found that IκKβ also regulates the activation and function of the NLRP3 inflammasome beyond the priming step. Two unrelated IκKβ inhibitors, AFN700 and TPCA-1, when applied after priming, fully blocked IL-1β secretion triggered by nigericin in THP-1 cells. Both inhibitors prevented neither inflammasome assembly, as monitored by measuring the formation of ASC specks, nor the generation of caspase-1 p20, a hallmark of caspase-1 activity, but they impaired the initial cleavage and activation of procaspase-1. These data thus indicate that IκKβ activity is required for efficient activation of NLRP3, suggesting that IκKβ may fulfill a dual role in coupling priming and activation of the NLRP3 inflammasome.

Silicosis is a serious occupational disease with the highest incidence in China. However, its pathogenesis has not been fully elucidated. Studies have shown that the sphingomyelin signaling pathway may play an important role in different fibrotic diseases but its role in silicosis-mediated fibrosis is still unclear. In this study, the supernatant of human peripheral blood mononuclear cell line (THP-1)-derived macrophages exposed to silica (SiO2 ) was used to stimulate the transformation of human embryonic lung fibroblast cell line (HFL-1) into myofibroblasts, and the intervention effect of recombinant human acid ceramidase (rAC) was observed. The results showed that SiO2 stimulated the production of reactive oxygen species and malondialdehyde in the supernatant of THP-1-derived macrophages and increased the secretion of TGF-β1, TNF-α, and IL-8. In addition, we found that the expression levels of α-SMA, FN, Col I, and Col III in HFL-1 cells increased. Meanwhile, the activities of ASMase and ACase and the expression levels of Cer, Sph, and S1P were increased. Intervention by rAC can suppress these changes to different degrees. In conclusion, the present study shows that SiO2 dust poisoning may stimulate HFL-1 cell differentiation into myofibroblasts by inducing oxidative stress in THP-1-derived macrophages, thereby promoting the secretion of a variety of inflammatory factors and activating the sphingolipid signaling pathway in HFL-1 cells. Exogenous rAC can effectively interfere with the stimulation of HFL-1 cells by silica in vitro.

Background The Human T-cell Lymphotropic Virus Type-1 (HTLV-1) is a blood-borne pathogen and etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 has currently infected up to 10 million globally with highly endemic areas in Japan, Africa, the Caribbean and South America. We have previously shown that Extracellular Vesicles (EVs) enhance HTLV-1 transmission by promoting cell–cell contact. Results Here, we separated EVs into subpopulations using differential ultracentrifugation (DUC) at speeds of 2 k (2000×g), 10 k (10,000×g), and 100 k (100,000×g) from infected cell supernatants. Proteomic analysis revealed that EVs contain the highest viral/host protein abundance in the 2 k subpopulation (2 k > 10 k > 100 k). The 2 k and 10 k populations contained viral proteins (i.e., p19 and Tax), and autophagy proteins (i.e., LC3 and p62) suggesting presence of autophagosomes as well as core histones. Interestingly, the use of 2 k EVs in an angiogenesis assay (mesenchymal stem cells + endothelial cells) caused deterioration of vascular-like-tubules. Cells commonly associated with the neurovascular unit (i.e., astrocytes, neurons, and macrophages) in the blood–brain barrier (BBB) showed that HTLV-1 EVs may induce expression of cytokines involved in migration (i.e., IL-8; 100 k > 2 k > 10 k) from astrocytes and monocyte-derived macrophages (i.e., IL-8; 2 k > 10 k). Finally, we found that EVs were able to promote cell–cell contact and viral transmission in monocytic cell-derived dendritic cell. The EVs from both 2 k and 10 k increased HTLV-1 spread in a humanized mouse model, as evidenced by an increase in proviral DNA and RNA in the Blood, Lymph Node, and Spleen. Conclusions Altogether, these data suggest that various EV subpopulations induce cytokine expression, tissue damage, and viral spread.

Di Yang,1,* Lin-xia Shen,1,2,* Ri-feng Chen,1,* Yu Fu,1 Hong-yan Xu,1 Li-na Zhang,1 Dong-hua Liu1,3 1Department of Dermatology, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, People’s Republic of China; 2Department of Dermatology and Venereology, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of China; 3Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Nanning, 530021, People’s Republic of China*These authors contributed equally to this workCorrespondence: Dong-hua LiuDepartment of Dermatology, The First Affiliated Hospital of Guangxi Medical University, No.6, Shuangyong Road, Qingxiu District, Nanning, Guangxi Province, 530021, People’s Republic of ChinaEmail ldhgxmu@163.comBackground: Talaromyces marneffei (T. marneffei) is a destructive opportunistic dimorphic fungal which can cause lethiferous Talaromycosis, but the clearance of T. marneffei mainly depends on the innate immune response.Objective: To investigate whether T. marneffei can inhibit the expression of CD86 in THP-1 cells after infection and discuss the potential mechanisms.Methods: Western blot and immunoelectron microscopy were used to detect the CD86 expression on T. marneffei cultured on BHI medium at 37°C. Western blot, enzyme-linked immunoassay and immunofluorescence were used to detect the change of CD86 expression on macrophages incubating with T. marneffei. Enzyme-linked immunoassay was used to detect the content of CD86 in supernatant in the co-culture system. Immunohistochemistry and immunoelectron microscopy were used to detect the expression of CD86 on T. marneffei incubating with macrophages.Results: T. marneffei did not express CD86 when cultured separately at 37°C detected by Western blot and immunoelectron microscopy, but it did express CD86 when incubated with macrophages detected by immunohistochemistry and immunoelectron microscopy. The CD86 expression of macrophages significantly decreased at 72 hours when infected with T. marneffei while the content of CD86 in supernatant significantly increased at 72 hours compared with the control group which were detected by Western blot, enzyme-linked immunoassay and immunofluorescence.Conclusion: 1) After T. marneffei infection, CD86 expression on THP-1 decreased, and with the progression of infection, insufficient polarization of M1 macrophages gradually appeared; 2) T. marneffei may adsorb or uptake CD86 in supernatant produced by macrophages during the contact with THP-1 cells, thus leading to the consumption of CD86 in macrophages.Keywords: Talaromyces marneffei, T. marneffei, macrophages, CD86, THP-1, immunoelectron microscopy

Monocytes and muscles demonstrate functionally contrasting behavior under conditions of zinc deficiency with relation to zinc storage system (muscle retain zinc in contrast to monocytes). We aimed to understand the effects of zinc status and HIV-1 Tat mediated inflammation on expression of zinc transporters in these types of cells. Expression of zinc transporters [ZnTs, ZIPs, and metallothionein (MT)] was quantified by qRT-PCR in RD, THP-1 cells separately and in co-cultured THP-1-RD cells. ZnT1 protein expression levels were confirmed by Western blot. Significant increase of MT and ZnT1 mRNA in response to zinc supplementation and decrease during zinc deficiency indicates significance of the genes encoding transporters in maintaining zinc homeostasis in these tissues. In the RD cells ZIP10 exhibited inverse relation to zinc status whereas no correlation was found in the THP-1 cells. Tat-induced inflammation resulted in the significant elevation of MT, IL6, ZIP7, ZIP8, ZIP9 transcripts in the co-cultured RD cells, whereas THP-1 cells demonstrated increased IL-1β levels and reduced levels of ZIP7 and ZIP14. Zinc status and HIV-1Tat induced inflammation appear to influence differential expression of MT, ZnTs, and ZIPs in the muscle and monocyte cells.

Trichinella spiralis is a foodborne zoonotic nematode, which causes trichinellosis. During the infection, parasite evades the host immune responses by direct and indirect (through excretory-secretory products) contact with host immune cells. One of the main targets for immunomodulation induced by helminths are macrophages. In this study, we examined whether direct contact of different stages of T. spiralis can affect the polarization of human THP-1 macrophages. Co-culture of adult parasite stage and cells in direct contact without LPS addition had a significant impact on TNFα levels. Interestingly, in settings with the addition of LPS, the levels of IL-1β and TNFα significantly increased in adult parasite and newborn larvae (NBL) but not for muscle larvae (ML). While we tested muscle larvae ESP products to compare its effect with whole ML parasite, we detect an increase of pro-inflammatory cytokines like IL-1β and TNFα in no LPS conditions. Whereas, muscle larvae ESP significantly suppressed the inflammatory response measured by IL-1β, TNFα, and IL-6 levels and anti-inflammatory IL-10 compared to LPS control. Our findings indicate the anti-inflammatory potential of T. spiralis muscle larvae excretory-secretory products and propose signaling pathways which might be engaged in the mechanism of how muscle larvae ESP affect human macrophages.

Previously, we found a significant increase in TRAIL (TNFα-related apoptosis-inducing ligand) resistance of acute myeloid leukemia (AML) cells in multicellular aggregates in vitro that simulate the location of leukemia cells in the bone marrow. In this study, we tried to assess whether the increased resistance of the AML cells to the cytotoxic effect of TRAIL in multicellular aggregates in vitro can be associated with the launch of mechanisms mediated by cell differentiation or the acquisition of a pro-inflammatory phenotype. The phenotype of THP-1 cells in aggregated and disaggregated cultures was studied in comparison with the phenotype of a disaggregated THP-1 cell culture exposed to factors that induce monocyte-like differentiation or pro-inflammatory phenotype. It was found that an increase in the TRAIL resistance of the THP-1 cells in the aggregated culture was accompanied by the appearance of signs characteristic of monocyte-like differentiation and a pro-inflammatory phenotype. The results are of interest for developing approaches for suppressing TRAIL resistance of AML cells in human bone marrow.

The overactivation of macrophages causes chronic inflammatory diseases. Short-chain fatty acids (SCFAs), potential drugs for clinical treatment, are modulators of macrophage inflammatory reaction. Therefore, the modulation of macrophage-mediated cell activity is expected to become a new therapeutic strategy for inflammatory diseases caused by Mycoplasma pneumoniae. In this study, 2 kinds of SCFAs (propionate and butyrate) were found to have anti-inflammatory effects in M. pneumoniae-stimulated THP-1 cells inflammatory. They inhibited the expressions of IL-4, IL-6, ROS, and NLRP3 inflammasome, while enhancing the expressions of IL-10 and IFN-γ. Our study revealed these 2 agents to repress transcriptional activities of NF-κB, which are important modulators of inflammation. Meanwhile, SCFAs can significantly enhance the autophagy induced by M. pneumoniae. Considering that SCFAs have few side effects, they might be the promising adjuvant therapy for the prevention and/or treatment of various inflammatory diseases.

Mitochondrial dysfunction plays a crucial role in the central pathogenesis of insulin resistance and type 2 diabetes mellitus. Macrophages play important roles in the pathogenesis of insulin resistance. Lauric acid is a 12-carbon medium chain fatty acid (MCFA) found abundantly in coconut oil or palm kernel oil and it comes with multiple beneficial effects. This research objective was to uncover the effects of the lauric acid on glucose uptake, mitochondrial function and mitochondrial biogenesis in insulin-resistant macrophages. THP-1 monocytes were differentiated into macrophages and induce insulin resistance, before they were treated with increasing doses of lauric acid (5 μM, 10 μM, 20 μM, and 50 μM). Glucose uptake assay, cellular ROS and ATP production assays, mitochondrial content and membrane potential assay were carried out to analyse the effects of lauric acid on insulin resistance and mitochondrial biogenesis in the macrophages. Quantitative RT-PCR (qRT-PCR) and western blot analysis were also performed to determine the expression of the key regulators. Insulin-resistant macrophages showed lower glucose uptake, GLUT-1 and GLUT-3 expression, and increased hallmarks of mitochondrial dysfunction. Interestingly, lauric acid treatment upregulated glucose uptake, GLUT-1 and GLUT-3 expressions. The treatment also restored the mitochondrial biogenesis in the insulin-resistant macrophages by improving ATP production, oxygen consumption, mitochondrial content and potential, while it promoted the expression of mitochondrial biogenesis regulator genes such as TFAM, PGC-1α and PPAR-γ. We show here that lauric acid has the potential to improve insulin sensitivity and mitochondrial dysregulation in insulin-resistant macrophages.

Background Allicin (ACN), a sulfoxide in freshly crushed garlic, is known for its diverse bioactive properties. Among the most notable effects of ACN is its antitumor activity against a wide array of cancer types. Thus, ACN may be a promising anticancer therapeutic. Nevertheless, chemotherapy-induced anemia is a major obstacle in cancer management with a prevalence of up to 70%. Although the pathophysiology behind it remains elusive, a number of medications known to cause anemia in patients have been shown to induce premature programmed cell death in red blood cells (RBCs) known as eryptosis. This study, thus, investigates the anticancer potential of ACN against THP-1 monocytic leukemia cells, its toxic effects on human RBCs, and delineate the underlying biochemical mechanisms. Methods Cytotoxicity was detected using the MTT assay, while hemoglobin leakage was used as a surrogate for hemolysis which was photometrically measured. Major eryptotic events were examined using flow cytometry with fluorescent probes. Phosphatidylserine (PS) exposure was detected by Annexin-V-FITC, cytosolic calcium with Fluo4/AM, and reactive oxygen species with H2DCFDA. Results Our results show that ACN induces hemolysis in a dose-dependent fashion, which is significantly abrogated in absence of extracellular calcium. Moreover, ACN stimulates PS exposure, intracellular calcium overload, and oxidative stress. Using small-molecule inhibitors, we demonstrate that the pro-eryptotic activity of ACN is ameliorated in presence of zVAD(OMe)-FMK, SB203580, and D4476. Conclusion ACN possesses both hemolytic and eryptotic properties mediated through elevated intracellular calcium levels, oxidative stress, caspase, p38 MAPK, and CK1α.

AIM To assess the expression of Retinoic acid-related orphan receptor beta (Ror β) in human inflamed dental pulp stem cells (hI-DPSCs) and during macrophage phenotypic conversion. METHODOLOGY Commercially procured THP-1 monocytes conversion to macrophages was judged by their morphology, the percentage of adherent cells and the expression of CD-14 surface marker. THP-1 macrophage cell viability following LPS, IFN-γ/IL-4, IL-13 stimulus was evaluated at 24 and 48h. The phenotypic conversion of macrophages to M1 and M2 was confirmed by flow cytometry and Western blot analysis. Cytokine release following polarization was estimated by the BD cytokine flex kit. The expression of Ror β in THP-1 macrophages and hI-DPSCs following LPS, IFN-γ/IL-4, IL-13 stimulus was assessed by Western blot analysis. Statistical significance was analysed using one-way Anova followed by Tukey's Post hoc test. RESULTS THP-1 monocytes pretreated with PMA (100 ng mL-1 ) for 48 h followed by culturing in PMA-free media for another 48 h yielded cells with morphological characteristics similar to macrophages with a high percentage of adherence capability and CD-14 expression. Macrophages treated with LPS 100 ng mL-1 and IFN-γ 20 ng mL-1 or IL-4 20 ng mL-1 had high expression of the respective M1 and M2 CD markers in flow cytometry and Western blot analysis. Cytokine release studies demonstrated the expression of IL-1β, TNF-α and IL-10 in the M1-polarized macrophages (P

Amyloid-β (Aβ), the influence of which is considered the pathomechanism of Alzheimer’s disease, is also present in healthy people. The microbiome’s impact is also taken into account, where bacterial lipopolysaccharide (LPS) activates inflammatory processes and stimulates microglia via TLRs. Molecules of bacterial origin can co-create senile plaques with Aβ. This study evaluated the activity of physiological Aβ concentrations on neuronal and microglial cells after preincubation with LPS. Two cell lines were used in the study: PC12 cells differentiated with NGF and THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA). Cells were incubated with LPS at concentrations of 1–100 μM for 24 h and then with Aβ25–35 at a concentration of 0.001 μM or 1.0 μM for another 24 h. The viability of the culture and free oxygen radicals and the number of DNA strand breaks in both cell lines were evaluated. Additionally, for PC12 cells, neural features were assessed. Stimulation of repair processes in the presence of Aβ was observed for both studied cell lines. There was a decrease in free radical level and DNA damage number compared to control cultures (cells treated with LPS and without Aβ). The neurotrophic activity of Aβ was observed—the effect on neurites’ growth even after the preincubation of PC12 cells with LPS. At the lowest concentration of LPS used, the increase in neurite length was about 50% greater than in the negative control. At low concentrations, Aβ has a protective effect on neuron-like PC12 cells pretreated with LPS.

Fermented dairy products have become attractive functional foods for the delivery of probiotics and their biologically active metabolites. The aim of this study was to examine the immunomodulatory activity of milk fermented with the probiotic lactic acid bacterium Lactobacillus rhamnosus R0011 (LrF) on macrophages challenged with lipopolysaccharide (LPS), a potent pro-inflammatory stimulus. To this end, human THP-1 or U937 monocytes were differentiated into resting macrophages then stimulated with LPS and co-incubated with the LrF or with milk controls. Levels of pro-inflammatory and immunoregulatory cytokines were determined by enzyme-linked immunosorbent assays. Culturing of LPS-stimulated U937 macrophages with either the whole or filtered LrF resulted in an increase in Interleukin (IL)-1Ra production relative to the negative control. THP-1 macrophages cultured with the LrF demonstrated an increase in LPS-induced IL-10 and IL-1β production, while production of LPS-induced IL-6, sCD54, IL-8, IL-1β, TNF-α, IL-12p70 and Transforming Growth Factor-β (TGF-β) was unaffected. Further, the LrF induced the expression of DC-SIGN and CD206, markers of immunoregulatory M2 macrophage polarization, in LPS-challenged THP-1 macrophages. Taken together, milk fermented with L. rhamnosus R0011 increased regulatory cytokine production from LPS-challenged U937 and THP-1 macrophages, while simultaneously up-regulating the production of IL-1β and expression of DC-SIGN and CD206, a profile characteristic of polarization into the immunoregulatory M2 macrophage phenotype., Graphical abstract Image created with Biorender.Image 1, Highlights • Milk fermented with Lactobacillus rhamnosus R0011 (Lrf) induces a regulatory macrophage phenotype. • Modulation of cytokine profiles induced by lipopolysaccharide challenge consistent with an alternatively activated (M2) macrophage phenotype. • Fermented milk conditioning induced macrophage expression of genes characteristic of M2 macrophage polarization (DC-SIGN and CD206).

Because of a large number of macrophages and its secreted pro-inflammatory factors in the synovial fluid of patients with rheumatoid arthritis, the present study aimed to investigate the effect and mechanism of 630-nm LED exposure on monocytes/macrophages and its anti-inflammatory effect. The THP-1 monocytes and PMA-induced THP-1 macrophages (THP-1 macrophages) were employed and irradiated by 630-nm LED for different time and times, and then measure the pro-inflammatory cytokines production by RT-qPCR and Milliplex MAP Multiplex assay, the proteins involved in inflammation pathway and reactive oxygen species (ROS) levels in the cells were detected by Western blot and DCFH-DA method. The exposure dose of red LED (15.3 J/cm2, 30.6 J/cm2) were determined as no-influence on the cell proliferation, the pro-inflammatory factors TNF-α and IL-1β mRNAs, and secretions in supernatant of THP-1 macrophages were significantly decreased after LED exposure. The ROS production was blocked in THP-1 monocytes and THP-1 macrophages after treatment of LED. Finally, the phosphorylated NF-κB proteins which involved in inflammation pathway significantly decreased, and its inhibitors Nrf2 were slightly upregulated. The effects of LED anti-inflammation response are dependent on the mechanism of inhibiting ROS level and regulating NF-κB signaling pathways by increasing Nrf2 expression in the cells. It is suggested that 630-nm LED could decrease pro-inflammation in immune cells, and it may be a beneficial adjunct therapy in relieving inflammation of patients with rheumatoid arthritis.

Fungal keratitis (FK) is a keratopathy caused by pathogenic fungal infection. The aim of this work is to explore the role of thymic stromal lymphopoietin (TSLP) in FK. Human corneal epithelial cells (HCECs) were treated with Aspergillus fumigatus hyphae, and we found that TSLP was highly expressed and secreted in the hyphae-treated HCECs. Hyphae-treated HCECs or TSLP treatment enhanced the expression of caspase-1 P20, GSDMD-N (p30), IL-1β, and IL-18 in the human THP-1 macrophages. The influence conferred by hyphae-treated HCECs or TSLP treatment was rescued by TSLP neutralizing antibody or VX-765 (caspase-1 inhibitor) treatment. Moreover, TSLP treatment promoted the expression of NLRP3, ASC, caspase-1 P20, GSDMD-N (p30), IL-1β, and IL-18 in the THP-1 macrophages, which was abolished by NLRP3 knockdown. Furthermore, TSLPR silencing suppressed the expression of NLRP3, ASC, caspase-1 P20, GSDMD-N (p30), IL-1β, and IL-18 in the TSLP-treated THP-1 macrophages. In conclusion, our article confirms that Aspergillus fumigatus-stimulated HCECs induce pyroptosis of THP-1 macrophages by secreting TSLP. TSLP/TSLPR induces caspase-1-dependent pyroptosis through activation of NLRP3 inflammasome. Thus, our work suggests that TSLP may be a potential target for FK treatment.

Cytokine-induced endothelial dysfunction leads to inflammation and vascular adhesion molecule production in retinal pigment epithelium (RPE) cells. Inflammation is a critical mediator in retinal degeneration (RD) diseases, including age-related macular degeneration (AMD), and RD progression may be prevented through anti-inflammatory activity in RPE cells. The flavonoid polyphenol luteolin (LU) has anti-inflammatory and antidiabetes activities, but its effects regarding retinal protection remain unknown. Here, we examined the ability of luteolin to alleviate markers of inflammation related to RD in cytokine-primed APPE-19 cells. We found that luteolin decreased the levels of interleukin- (IL-) 6, IL-8, soluble intercellular adhesion molecule-1 (sICAM-1), and monocyte chemoattractant protein-1 (MCP-1) and attenuated adherence of the human monocytic leukemia cell line THP-1 to IL-1β-stimulated ARPE-19 cells. Luteolin also increased anti-inflammatory protein heme oxygenase-1 (HO-1) levels. Interestingly, luteolin induced protein kinase B (AKT) phosphorylation, thus inhibiting nuclear factor- (NF-) κB transfer from cytoplasm into the nucleus and suppressing mitogen-activated protein kinase (MAPK) inflammatory pathways. Furthermore, cotreatment with MAPK inhibitors and luteolin decreased inflammatory cytokine and chemokine levels, and further suppressed THP-1 adhesion. Overall, these results provide evidence that luteolin protects ARPE-19 cells from IL-1β-stimulated increases of IL-6, IL-8, sICAM-1, and MCP-1 production by blocking the activation of MAPK and NF-κB signaling pathways, thus ameliorating the inflammatory response.

The various functional properties of chitosan have been investigated for use in biomedical applications. Indeed, chitosan or its derivatives have shown anti-inflammatory effect by regulating a range of cytokines, chemokines or growth factors. In the present study, we prepared water-soluble chitosan oligosaccharide (AS-WSC) by the hydrolysis of high molecular weight water-soluble chitosan (HMW-WSC) and examined its anti-inflammatory effect on LPS-stimulated human monocytic THP-1 cells. Our results revealed that LPS-induced NF-κB activity of THP-1 cells was significantly suppressed by treatment with 0.02% AS-WSC. In addition, 0.02% AS-WSC treatment down-regulated the expression of Toll-like receptor 4 and inflammatory cytokines, such as IL-1β and TNF-α, but had no effect on IL-10 and IL-4 expression in LPS-stimulated THP-1 cells. Our results suggest that 0.02% AS-WSC can be considered as a promising anti-inflammatory agent.

Plants and natural compounds are extensively reported for diverse biological activities, including their effects on the inflammation pathway. The annual winter herb Diplotaxis Acris (D.Acris) is found only slopes of sandy and stony valleys in the desert. This research is intended to make a contribution to the literature regarding the employment of the species of plant ethnomedicinally by undertaking FTIR spectroscopic analysis and examining several concentrations of the plant's extract for anti-inflammatory activity in vitro with activated THP-1 human macrophages to examine its mediating effect on inflammation. Cell viability was also evaluated, and there was no severe cytotoxic effect from D.Acris extract with any of the concentrations being assessed on cells. ELISA was used to assess the pro-inflammatory cytokines and chemo kines that were produced. It has been noted that the plant extract led to a significant decrease in levels of the pro-inflammatory cytokines, including interleukin (IL)-1β, tumour necrosis factor (TNF)- α and IL-6. Inhibition of pro-inflammatory cytokines indicates the anti-inflammatory trends of D.Acris. This plant can be investigated further by isolation of natural compounds from the extract and effects of these compounds can be evaluated on the same inflammatory markers to show the main active constituent responsible for anti-inflammatory activities.

BACKGROUND: Berry processing residues called pomaces are rich in polyphenols, sugars, organic acids, and minerals. Polyphenols are reported to reduce the risk of non-communicable diseases, including cardiovascular diseases, cancer, and diabetes mellitus, owing to their anti-inflammatory activity. OBJECTIVE: We aimed to assess the anti-inflammatory properties of five Vaccinium spp. berry pomace extracts using LPS-stimulated THP-1 monocytes and a COX-2 inhibition assay. METHODS: THP-1 monocytes were pre-incubated with chemically characterized bilberry, blueberry, American cranberry, bog cranberry, and lingonberry pomace extracts following LPS stimulation. NF-κB nuclear translocation was assessed by flow cytometry. TNF-α, MMP-9, IL-23, IL-10, IL-1β, CCL22, and IL-8 secretion was analyzed in cell culture supernatant by using Luminex assay and ELISA. The expression of IL-6, TNF-α, IL-10, IL-23, and TGF-β1 was assessed by qPCR, and COX-2 activity was determined by a fluorometric inhibition assay. RESULTS: All berry pomace extracts tested, except for lingonberry, inhibited LPS-induced NF-κB nuclear translocation. All five extracts significantly downregulated the expression of pro-inflammatory cytokines and inhibited COX-2 activity in vitro. CONCLUSIONS: Vaccinium spp. pomace extracts demonstrated anti-inflammatory properties in an LPS-stimulated THP-1 cell inflammation model and inhibited COX-2 activity. Thus, Vaccinium spp. berry pomace extracts could serve as a valuable source of anti-inflammatory compounds.

Fine particulate matter (PM2.5) in the ambient atmosphere is strongly associated with detrimental health effects. However, these particles from various sources and regions are unlikely equally toxic. While animal studies are impractical for high-throughput toxicity testing, appropriate in vitro models are urgently needed. Co-culture of A549 and THP-1 macrophages grown at air–liquid interface (ALI) or under submerged conditions was exposed to same concentrations of ambient PM2.5 to provide accurate comparisons between culture methods. Following 24-h incubation with PM2.5 collected in Harbin in China, biological endpoints being investigated include cytotoxicity, reactive oxygen species (ROS) levels and pro-inflammatory mediators. The co-culture grown under submerged condition demonstrated a significant increase in ROS levels and all tested pro-inflammatory indicators [interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α] in mRNA expression and released protein levels. Similar but a declining response trend was observed using the same PM2.5 incubation after grown at ALI. We further observed a significant increase of PM2.5-induced phosphorylation of p38 MAPK and activation of NF-κB p65 in a dose-dependent trend for co-cultures grown under submerged condition. These results provide important implications that culture conditions (ALI versus submerged) can induce different extents of biological responses to ambient PM2.5; the co-culture grown at ALI is less likely to produce false-positive results than submerged culture. Hence, culture conditions should be discussed when comparing in vitro methods used for high-throughput PM2.5 toxicity assessment in future.

The interaction between Chronic lymphocytic leukemia (CLL) cells and monocyte-derived nurse-like cells (NLCs) is fundamentally important to CLL biology. However, studies of how CLL cells and NLCs interact have been hampered by the need for freshly obtained CLL blood samples, coupled with wide variation in the number of monocytes present in the blood of individual patients. Here, we report the development and validation of a cell-line model of NLCs which overcomes these difficulties. Co-culture of primary CLL cells with THP-1 cells induced to differentiate into macrophages by phorbol 12-myristate 13-acetate (PMA) significantly reduced both spontaneous and fludarabine-induced cell death of leukemic cells. Furthermore, compared with their M1-polarized counterparts, M2-polarized macrophages derived from PMA-differentiated THP-1 cells conferred to CLL cells greater protection from spontaneous and fludarabine-induced apoptosis. Since NLCs resemble M2 tumor-associated macrophages, this cell-line model could be useful for investigating the mechanisms through which NLCs protect CLL cells from spontaneous and drug-induced apoptosis.

L-Asparaginase (L-ASNase EC 3.5.1.1) is considered as an important biopharmaceutical drug enzyme in the treatment of childhood acute lymphoblastic leukemia (ALL). In the present study, Pyrococcus furiosus L-ASNase gene was cloned into pET26b (+), expressed in E. coli BL21(DE3) pLysS, and purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 5.7 purification fold and 23.9% recovery. The purified enzyme exhibited a molecular weight of ~33,660 Da on SDS-PAGE and showed maximal activity at 50 °C and pH 8.0. It retained 98.3% and 60.7% initial activity after 60 min at 37 °C and 50 °C, respectively. The recombinant enzyme showed highest substrate specificity towards L-ASNase substrate, while no detectable specificity was observed for l -glutamine, urea, and acrylamide at 10 mM concentration. The Km and Vmax of the purified recombinant enzyme as calculated using Lineweaver-Burk plot were determined to be 1.623 mM and 105 μmol min−1 mg−1, respectively. Human leukemia cell line THP-1 treated with recombinant L-ASNase showed significant morphological changes, and the IC50 of the purified enzyme was found to be 0.8 IU. Moreover, the purified recombinant L-ASNase induced cytotoxic effects on lung adenocarcinoma A549 and colorectal adenocarcinoma Caco-2 cell lines with IC50 of 1.78 IU and 30 IU, respectively.

Tumor-associated macrophages (TAMs) have a significant presence in the tumor stroma across multiple human malignancies and are believed to be beneficial to tumor growth. Targeting CSF1R has been proposed as a potential therapy to reduce TAMs, especially the protumor, immune-suppressive M2 TAMs. Additionally, the high expression of CSF1R on tumor cells has been associated with poor survival in certain cancers, suggesting tumor dependency and therefore a potential therapeutic target. The CSF1-CSF1R signaling pathway modulates the production, differentiation, and function of TAMs; however, the discovery of selective CSF1R inhibitors devoid of type III kinase activity has proven to be challenging. We discovered a potent, highly selective, and orally bioavailable CSF1R inhibitor, IACS-9439 (1). Treatment with 1 led to a dose-dependent reduction in macrophages, promoted macrophage polarization toward the M1 phenotype, and led to tumor growth inhibition in MC38 and PANC02 syngeneic tumor models.

Background. Scrophularia dentata is an important medicinal plant and used for the treatment of exanthema and fever in Traditional Tibetan Medicine. Scrodentoids H and I (SHI), a pair of epimerides of C19-norditerpenoids isolated from Scrophularia dentata, could transfer to each other in room temperature and were firstly reported in our previous work. Here, we first reported the anti-inflammatory effects of SHI on LPS-induced inflammation. Purpose. To evaluate the anti-inflammatory property of SHI, we investigated the effects of SHI on LPS-activated THP-1 cells. Methods. THP-1 human macrophages were pretreated with SHI and stimulated with LPS. Proinflammatory cytokines IL-1β and IL-6 were measured by RT-PCR and enzyme-linked immunosorbent assays (ELISA). The mechanism of action involving phosphorylation of ERK, JNK, P38, and STAT3 was measured by western Blot. The NF-κB promoter activity was evaluated by Dual-Luciferase Reporter Assay System in TNF-α stimulated 293T cells. Results. SHI dose-dependently reduced the production of proinflammatory cytokines IL-1β and IL-6. The ability of SHI to reduce production of cytokines is associated with phosphorylation depress of JNK and STAT3 rather than p38, ERK, and NF-κB promoter. Conclusions. Our experimental results indicated that anti-inflammatory effects of SHI exhibit attenuation of LPS-induced inflammation and inhibit activation through JNK/STAT3 pathway in macrophages. These results suggest that SHI might have a potential in treating inflammatory disease.

Scarce attention has been paid to the immunotoxicity of organophosphate flame retardants (PFRs), which poses a challenge to the systematic assessment of their health risks. In this study, a battery of in vitro immunotoxicity screening assays, including adhesion, phagocytosis, and 48 cytokine/chemokine production, was measured after exposing THP-1-derived macrophages to six selected common PFRs (TPHP, TDCPP, TNBP, TOCP, TCEP, and TBOEP) at a noncytotoxic concentration (≤50 μM). Our results showed that TPHP and TBOEP partially attenuated the adhesion and phagocytosis of the THP-1 mφs and that TDCPP caused a functional loss of phagocytosis, implying the potential immunosuppression. In contrast, TNBP and TOCP may cause an immunostimulation by significantly promoting cell adhesion and enhancing phagocytic efficiency. Additionally, the results from a cytokine/chemokine secretion analysis revealed the proinflammatory properties of TDCPP, TPHP, and TBOEP. TOCP was thought to disrupt the inflammatory balance by inhibiting both proinflammatory and antiinflammatory cytokines. TCEP showed no effect on adhesion or phagocytosis and little modulation of cytokine release at this experimental concentration. Overall, this study supports that PFRs can be immunotoxic to macrophages in different ways and provides evidence for developing more sensitive in vitro immunotoxicity bioassay methods.

P2X7 receptor (P2X7R) is an ATP-gated non-selective cation channel which mediates ATP-induced inflammation in macrophages. Transient receptor potential (TRP) receptors are nociceptors in cellular membrane which can perceive the stimuli of environmental irritant. The interaction between TRP channels and P2X7R has been found while the details about inflammation are still unclear. In this study, we suggested that transient receptor potential ankyrin 1 (TRPA1), a member of TRP superfamily, participates in ATP-induced oxidative stress and inflammation in human acute monocytic leukemia cell line (THP-1)-derived macrophage. The co-localization between TRPA1 and P2X7R was detected using immunofluorescence in THP-1-derived macrophage and transfected human embryonic kidney cell line (HEK293T). The mechanism by which ATP or 3'-O-(4-Benzoylbenzoyl)-ATP (BzATP) induces the activation of macrophages was verified by calcium imaging, mitochondrial reactive oxygen species (mtROS) detection, mitochondrial membrane potential (∆Ψm) measurement, flow cytometry, enzyme-linked immunosorbent assay (ELISA), western blotting, CCK-8 assay, and the lactate dehydrogenase (LDH) release cytotoxic assay. The BzATP and ATP induced calcium overload, mitochondria injury, interleukin-1β (IL-1β) secretion, and cytotoxicity can be inhibited by TRPA1 antagonists. These results indicated that TRPA1 can co-localize with P2X7R and mediate ATP-induced oxidative stress and inflammation. Therefore, the inhibition of TRPA1 may provide a potential therapy for ATP-elicited inflammatory diseases, including atherosclerosis.