Human Monocytic Cell Line THP-1 Research Articles

Chinese Herbal Medicine 'Diwu' Suppresses Myeloma Bone Disease Via ERK/MAPK Signaling Pathway By Targeting CCL3

Background： Multiple myeloma (MM) associated bone disease (MBD) is largely dependent on the bone marrow microenvironment (BME). The cross-talk between MM cells, bone marrow mesenchymal cells and myeloid progenitors affects the osteoclastogenesis. Traditional Chinese medicine 'Diwu' is effective for patients with arthritis and inflammation, also occasionally uses as one ingredient treating MM. But the effects of 'Diwu' in MBD are largely unknown. The aim of this study is to investigate the role and underlying mechanism of 'Diwu' in regulating BME and osteoclast differentiation. Methods： Myeloma cell lines RPMI-8226, NCI-H929, U266 and MM1 were treated with 'Diwu' extract. Killing effects was evaluated by the half inhibitory dose (IC50) value and proportion of apoptotic MM cells. RNA-seq was applied in 'Diwu' treated myeloma cells to identify MBD associated signaling pathways. RPMI-8226 was co-cultured with mesenchymal stromal cell (MSC) line HS5. Human monocyte cell line THP-1 was treated with CCL3 and M-CSF to form osteoclasts determined by tartrate-resistant acid phosphatase (TRACP) staining. Primary human MSCs were cultured into osteogenic or adipogenic differentiation, determined by alizarin red staining and oil red staining, respectively. RNAi technology was applied to knock down CCL3. The expression of osteoclast-specific genes TRACP and MAPK pathway-related genes was tested using quantitative real-time PCR. Phosphorylation and expression of MEK1, ERK, FOS were measured by Western Blot. The myeloma-bearing NOD/SCID mice were treated with intragastric 'Diwu'. Serum C-terminal telopeptide of type 1 collagen (CTXI) and propeptide of type I procollagen (PINP) was tested by ELISA. The tibia of mice was fixed and stained to analyze the differentiation and maturation of osteoblasts and osteoclasts. Micro-CT was used for bone destruction measurement. Results： 'Diwu' inhibited MM cells proliferation (IC50, 29.22-364.7μg/ml) in a dose-dependent manner and promoted apoptosis by upregulating protein expression of CASP3, BCL2. 'Diwu' decreased the secretion of chemokine (C-C motif) ligand 3 (CCL3) and receptor activator of nuclear factor-kappa B ligand (RANKL) in myeloma-MSC co-cultured system by diminishing the direct attachment and interaction of these two cells. The mRNA and protein levels of osteoclast-specific genes such as TRACP, and the phosphorylation and expression of MEK1, ERK, FOS in osteoclast were all reduced after 'Diwu' treatment. We observed improved osteogenic activation rather than lipid formation in hMSC differentiation. The level of TRACP isoform 5b (TRACP-5b) was significantly downregulated in mouse serum after 'Diwu' treatment for 14 days. Conclusions: Altogether, our results demonstrated that effective extract of Chinese herbal medicine 'Diwu' could simultaneously inhibit myeloma cells and suppress osteoclast formation, indicating an important role of 'Diwu' in regulating BM microenvironment. It is possible to translate 'Diwu' into clinical application as an oral agent for myeloma bone disease. Key words： 'Diwu'; Myeloma Bone Disease; Bone Marrow Microenvironment; CCL3; ERK/MAPK

Lipoxin A4, Maresin 1, and Resolvin D1 As Key Factors for the Actions of Plasminogen/Plasmin on Inflammation Resolution

The plasminogen/plasmin (Plg/Pla) system plays a fundamental role in modulating fibrinolysis and many other biological processes, including extracellular matrix degradation, cell migration, and tissue repair. Recent studies highlight the role of the Plg/Pla system in key steps of inflammation resolution, which is mediated, at least in part, by the glucocorticoid-induced protein annexin A1. However, it is unclear whether Plg/Pla might also modulate specialized proresolving mediators (SPMs) derived from omega-3 and omega-6 fatty acids, including lipoxin A4 (LXA4), maresin 1 (MaR1), and resolvin D1 (RvD1). Therefore, we investigated the ability of Plg/Pla to modulate the synthesis of LXA4, MaR1, and RvD1, as well as the involvement of these SPMs in the proresolving effects elicited by Plg or Pla using both in vitro and in vivo strategies. Treatment of bone marrow-derived macrophages (BMDMs) with Plg/Pla (4 μg/mL) induced the release of LXA4, MaR1, and RvD1 as well as increased mRNA levels of their lipoxygenase (LO) synthesizing enzymes 5-LO and 15-LO. Plg treatment also increased the levels of the anti-inflammatory cytokine IL-10 and reduced the levels of the pro-inflammatory cytokine TNF-α in BMDMs. Moreover, Plg decreased the levels of TNF-α and IL-1β induced by lipopolysaccharide (LPS) stimulus of the human monocytic cell line THP-1. Interestingly, LXA4 was able to induce Plg release in THP-1 cells. Notably, Pla (4 μg, i.p.) was able to increase the efferocytosis of apoptotic neutrophils in wild-type (SV129) mice, but not in 5-LO deficient mice (5-LO-/-). Taken together, these findings suggest that Plg and Pla increase the levels of SPMs and IL-10, and modulate the levels of LPS-elicited pro-inflammatory cytokines. Mechanistically, the action of Plg/Pla on efferocytosis was 5-LO dependent, suggesting that SPMs mediate this proresolving effect of Plg/Pla. Moreover, the reciprocal modulation between the Plg/Pla system and SPMs pathways elucidated should be explored in future studies on candidate targets for therapies for inflammatory diseases. Keywords: Inflammation resolution; plasminogen/plasmin system; pro-resolving lipid mediators. Financial support: FAPEMIG, CNPq, CAPES.

Efficient Knock-out of Serine Protease Cathepsin G Enhances Chemosensitivity to Cytarabine By Modulating the Cytarabine Metabolic Pathway Genes in Acute Myeloid Leukemia

Relapse and refractoriness to chemotherapy may exist prior to the exposure to chemotherapeutic agents or can develop and increase during the treatment leading to acquired resistance. Despite considerable progress in developing new therapeutic agents to be administered singly or in combination with the old regimen, drug resistance poses a serious concern preventing adequate response to Acute Myeloid Leukemia (AML) treatment with cytarabine (AraC) and daunorubicin (DNR). Differentially expression analysis of the RNA sequencing data between the naive and the AraC-resistant human monocytic MLL-AF9 cell line THP1 revealed cathepsin G (CTSG) as one of the top hits among the upregulated genes with Log2Fc 4.2. This was further validated in the AML cell line by q-RT-PCR with a fold change ~5 and immunoblot, confirming increased CTSG expression in THP1 Ara-C resistant cell line. Although the role of (CTSG) in host defence, granulopoiesis has been intensively investigated, its role in acquired cytarabine resistance has not been explored yet. To investigate the functional role of CTSG in the acquired resistant model, we performed CRISPR/Cas9-based editing of CTSG in THP1 Ara-C resistant cell line using dual gRNAs, which resulted in a robust deletion of the targeted region and complete loss of protein expression as measured by immunoblotting. CTSG KO cells showed a reduced cell proliferation rate with a significant reduction at 72hrs (compared to the scrambled gRNA (ScrG) transduced cells doubling time CTSG KO=30.3 vs ScrG=22.39, p= 0.0049). We also checked the sensitivity of the CTSG KO cells to AraC and other chemo drugs (DNR, ATO and Venetoclax). In vitro cytotoxicity assay (72hrs) for the CTSG KO cells treated with AraC showed sensitivity to Ara-C with a reduction in IC50 as low as the parental IC50 (IC50 ScrG: 1457 µM; IC50 CTSG KO: 38.13 µM; IC50 Parental: 56.6 µM). CTSG KO cells also sensitized the cells to DNR (IC50 ScrG: 288nM vs and CTSG KO: 58nM but not to ATO and venetoclax. Further, we measured the intracellular DNR by flow cytometry in the CTSG KO cells. There was a considerable increase in the intracellular DNR at the end of the treatment compared to the ScrG cells (~1.5-fold, p <0.0001). Our results show that CTSG KO re-sensitized the resistant cells to cytarabine and showed significant sensitivity to DNR. To further understand the molecular mechanism contributing to the increased sensitivity of the CTSG KO cells to AraC, we checked our basal RNA sequencing data for the expression of AraC metabolizing genes. The THP1 Ara-C R cell line showed decreased expression of DCK and increased expression of NT5C2 and CDA compared to the parental cell line. We checked the RNA expression of Ara-C metabolizing genes post CTSG KO by RT-q PCR. There was a 6-fold increase in DCK and a 5-fold decrease in NT5C2 with a further increase in SAMHD1 and CDA genes with no changes in the RRM1 and ENT1 genes, suggesting that CTSG KO increased the sensitivity to by rewiring the AraC metabolic pathway. Further studies are ongoing to assess the in-vivo efficacy of molecular and pharmacological inhibition of CTSG in reversing AraC resistance in the AML mouse model.

Small Extracellular Vesicles Loaded with Immunosuppressive miRNAs Leads to an Inhibition of Dendritic Cell Maturation

In particular conditions, inhibition of an immune response is required to prevent tissue damage. Among these conditions are diseases caused by an over-reactive immune response, such as autoimmune or allergic disorders, or imminent organ rejection after transplantation. To avoid tissue damage, drug-mediated systemic immune suppression is an option, but it comes with high costs in the form of susceptibility to viral and bacterial infections. Thus, the induction of antigen-specific tolerance is preferable. Extracellular vesicles (EVs) are capable of delivering antigen together with immunosuppressive signals and may be used to specifically induce antigen-specific tolerance. However, naturally occurring EVs are heterogeneous and not all of them show immunosuppressive character. In our trials to engineer cell culture derived EVs to increase their tolerogenic potential, we equipped them with immunosuppressive miRNA mimics. Small EVs (sEVs) were isolated and purified from the human monocytic THP-1 cell line or from healthy donor peripheral blood mononuclear cells, and electroporated with miR-494 and miR-146a mimics. The acquired immunosuppressive potential of the modified sEVs was demonstrated by their ability to alter the major histocompatibility complex molecules and co-stimulatory receptors present on dendritic cells (DCs). To avoid allogeneic responses, the same cells that produced the sEVs served also as recipient cells. In contrast to the treatment with unmodified sEVs, the tolerogenic sEVs impeded lipopolysaccharide-induced maturation and kept DCs in a more immature developmental stage. Our experiments show that simple manipulations of sEVs using immunosuppressive cargo can lead to the inhibition of DC maturation.

Antileishmanial Activities of (Z)-2-(Nitroimidazolylmethylene)-3(2H)-Benzofuranones: Synthesis, In Vitro Assessment, and Bioactivation by NTR 1 and 2.

The antileishmanial activity of a series of (Z)-2-(heteroarylmethylene)-3(2H)-benzofuranone derivatives, possessing 5-nitroimidazole or 4-nitroimidazole moieties, was investigated against Leishmania major promastigotes and some analogues exhibited prominent activities. Compounds with IC50 values lower than 20 μM were further examined against L. donovani axenic amastigotes. Evaluated analogues in 5-nitroimidazole subgroup demonstrated significantly superior activity (~17-88-folds) against L. donovani in comparison to L. major. (Z)-7-Methoxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5n) showed the highest L. donovani anti-axenic amastigote activity with IC50 of 0.016 μM. The cytotoxicity of these analogues was determined using PMM peritoneal mouse macrophage and THP-1 human leukemia monocytic cell lines and high selectivity indices of 26 to 431 were obtained for their anti-axenic amastigote effect over the cytotoxicity on PMM cells. Further studies on their mode of action showed that 5-nitroimidazole compounds were bioactivated predominantly by nitroreductase 1 (NTR1) and 4-nitroimidazole analogues by both NTR1 and 2. It is likely that this bioactivation results in the production of nitroso and hydroxylamine metabolites that are cytotoxic for the Leishmania parasite.

Conjugation of Listeriolysin O to Anti-CD47 mAb: Harnessing Listeria Toxin to Weaponize Macrophages against Tumor Cells

<h3>Purpose/Objective(s)</h3> CD47 is expressed on various tumor types including breast, prostate, glioblastoma, non-Hodgkin lymphoma, and hepatocellular carcinoma. Interrupting the CD47-SIRP alpha axis by monoclonal antibodies fosters macrophage-mediated phagocytosis of solid and hematopoietic tumor cells. The pore-forming toxin listeriolysin O (LLO) is activated in the acidic vacuoles of bone marrow-derived macrophages. We hypothesize that LLO conjugation to the anti-CD47 mAb enhances the release of autophagic cargo within macrophages thereby augmenting cGAS-STING activation, antigen presentation, and tumor phagocytosis. <h3>Materials/Methods</h3> The anti-CD47 mAb was conjugated to the LLO toxin using a water-soluble SPDP crosslinker and purified by affinity chromatography. The cytotoxicity of anti-CD47-LLO mAb relative to anti-CD47 mAb was tested in the mouse breast cancer cell line E0771, the human breast cancer cell line MDA-MB-468, the mouse lung cancer cell line LLC, and the human leukemia monocytic cell line THP-1. C57B6 mouse bone marrow-derived macrophages (BMDM) co-cultured with tumor cell lines were analyzed in the presence of anti-CD47-LLO mAb and anti-CD47 mAb to determine the efficiency of BMDM tumor cell phagocytosis, STING activation, and antigen presentation. Tumor growth of E0771-bearing wildtype mice was measured following intratumoral injection of anti-CD47 mAb and anti-CD47-LLO mAB. <h3>Results</h3> The anti-CD47-LLO mAb conjugate enhanced BMDM phagocytosis of LLC and E0771 tumor cells in a dose dependent manner that was significantly higher than cells treated with the anti-CD47 mAb alone. The anti-CD47-LLO mAb significantly increased BMDM levels of phosphorylated STING, interferon-stimulated genes, and antigen presentation relative to anti-CD47 mAb treatment. At doses compatible with preserved animal total body weight, red blood cell counts, and lymphocyte counts, intratumoral injection of anti-CD47-LLO mAb significantly suppressed tumor growth of E0771-bearing wildtype mice relative to the anti-CD47 mAb. <h3>Conclusion</h3> Conjugation of the LLO toxin to anti-CD47 mAb enhances macrophage cGAS-STING activation, antigen presentation, and tumor cell phagocytosis. This novel CD47 antibody builds on clinical interest in targeting macrophages for the treatment of malignancy and may be studied as a supplemental therapy for patients with tumors resistant or refractory to checkpoint therapy.

17β-neriifolin from unripe fruits of Cerbera manghas suppressed cell proliferation via the inhibition of HOXA9-dependent transcription and the induction of apoptosis in the human AML cell line THP-1.

Homeobox A9 (HOXA9) is a transcription factor that is overexpressed in acute myeloid leukemia (AML). It is associated with the pathogenesis and progression of AML, and is a factor responsible for a poor prognosis. Therefore, the development of HOXA9-targeting molecules may contribute to not only better understanding of the mechanism of HOXA9 regulation, but also the development of therapeutic applications. We constructed a reporter assay system using the promoter region of the KBTBD10 gene, to which HOXA9 directly binds and regulates transcription, in the human acute monocytic leukemia cell line THP-1. Using this luciferase gene assay, we screened 1120 plant extracts and a methanol extract of the unripe fruits of Cerbera manghas was found to suppress the reporter gene expression mediated by the KBTBD10 promoter. From the extract, five steroid-type compounds were identified as the active constituents: 7α-neriifolin (1), 17β-neriifolin (2), 17α-digitoxigenin β-D-glucosyl-(1 → 4)-α-L-thevetoside (3), 17β-digitoxigenin β-D-glucosyl-(1 → 4)-α-L-thevetoside (4), and acetylthevetin B (5). Among the five compounds, 17β-neriifolin most potently inhibited HOXA9-dependent gene expression without affecting the HOXA9 mRNA levels, and suppressed cell proliferation by inducing apoptosis. The findings on the structure-activity relationships of the compounds from C. manghas may contribute to the development of small molecule inhibitors of HOXA9.

Therapeutic effect of chinese herbal medicine gu-ben-hua-shi (AESS) formula on atopic dermatitis through regulation of yes-associated protein

Background: Atopic dermatitis (AD) is a chronic and recurrent skin disease. At present, there is a lack of sufficiently effective and safe medicines that can be used for a prolonged time and reduce the recurrence of AD. The Gu-Ben-Hua-Shi (AESS) formula has been used for many years with a good clinical effect on AD but its specific treatment mechanism is unknown. Methods: The main components of AESS were analyzed using ultra-high performance liquid chromatography (UPLC). The composition of AESS compounds in the serum from rats was analyzed using ultra-high performance liquid chromatography-mass spectrometry. An AD mouse model was constructed using 2,4-dinitrofluorobenzene stimulation in Balb/C mice and the effect on the reduction of skin lesions and Th1/Th2/Th17/Treg balance after AESS administration were measured. The effects of AESS serum on the proliferation and apoptosis of keratinocyte cell line HaCaT and adhesion of HaCaT to human monocyte cell line THP-1 were detected in an IFN-γ/TNF-α stimulated AD-like inflammatory cell model. The effects of Yes-associated protein (YAP) expression on the therapeutic effect and a related signaling pathway were also investigated. Results: In total, 10 components were confirmed using UPLC, namely five organic acids, three flavonoids, and two chromogenic ketones. Additionally, the similarity of the three batches of samples (S1–3) was above 0.98, indicating that the formula samples have good uniformity. These 10 compounds were also detected in rat serum, suggesting that they are absorbed into rat blood as prototype components. Furthermore, AESS effectively reduced the skin lesions in the AD mouse model, regulated the Th1/Th2/Th17/Treg imbalance, improved the proliferation ability of the AD-like cell model, and inhibited HaCaT apoptosis and adhesion to THP-1 cells. It also reduced the expression of YAP in Th17 and Treg cells of the mouse spleen and increased YAP expression in the skin. The change in YAP expression in keratinocytes weakened the curative effect of AESS, and AESS exerted its effects through the NF-κB signaling pathway. Conclusion: AESS may play a role in the treatment of AD by affecting the expression of YAP. These findings can be used to promote its use as an alternative medication for prolonged use with fewer side effects.

Suppressive Effects of 4-(Phenylsulfanyl) Butan-2-One on CCL-1 Production via Histone Acetylation in Monocytes.

The 4-(phenylsulfanyl) butan-2-one (4-PSB-2), a marine-derived compound from soft coral, was proven to have multiple biological activities including neuroprotection and potent anti-inflammatory effects. CC chemokine ligand (CCL)-1 belongs to T helper (Th)2-related chemokines that are involved in the recruitment of Th2 inflammatory cells. Histone acetylation has been recognized as a critical mechanism underlying the regulated cytokine and chemokine production. Our study tried to investigate the anti-inflammatory effect of 4-PSB-2 on CCL-1 production in human monocytes and explore possible underlying intracellular processes, including epigenetic regulation. To confirm our hypothesis, human monocyte THP-1 cell line and primary CD14+ cells were pretreated with various concentrations of 4-PSB-2 and then were stimulated with lipopolysaccharide (LPS). The CCL-1 concentration was measured by enzyme-linked immunosorbent assays, and the intracellular signaling pathways and epigenetic regulation of 4-PSB-2 were investigated by using Western blotting and chromatin immunoprecipitation analysis. In this study, we found that 4-PSB-2 had a suppressive effect on LPS-induced CCL-1 production. Moreover, this suppressive effect of 4-PSB-2 was mediated via intracellular signaling such as the mitogen-activated protein kinase and nuclear factor-κB pathways. In addition, 4-PSB-2 could suppress CCL-1 production by epigenetic regulation through downregulating histone H3 and H4 acetylation. In short, our study demonstrated that 4-PSB-2 may have a potential role in the treatment of allergic inflammation.

Monocytes as suitable carriers for dissemination of dengue viral infection

Dengue viruses (DENVs) exploit monocytes and macrophages for tropism and replication, therefore, establishing a long-term reservoir. However, their roles in dengue pathogenesis remains unclear. Here, using the human monocytic cell line THP-1, human primary monocytes, and non-human primate models, we show that DENV-infected monocytes represent suitable carriers for circulatory viral dissemination. Monocyte-derived macrophages expressing M2 surface markers at the gene level efficiently replicated, while the productivity of monocyte replication was low. However, attachment of DENVs to the cellular surface of monocytes was similar to that of macrophages. Furthermore, after differentiation with type-2 cytokines, DENV-attached monocytes could replicate DENVs. Productive DENV infection was confirmed by intravenous injection of DENVs into nonhuman primate model, in which, DENV attachment to monocytes was positively correlated with viremia. These results provide insight into the role of circulating monocytes in DENV infection, suggesting that monocytes directly assist in DENV dissemination and replication during viremia and could be applied to design antiviral intervention.

Study of Biological Activities and ADMET-Related Properties of Salicylanilide-Based Peptidomimetics.

A series of eleven benzylated intermediates and eleven target compounds derived from salicylanilide were tested against Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 as reference strains and against three clinical isolates of methicillin-resistant S. aureus (MRSA) and three isolates of vancomycin-resistant E. faecalis. In addition, the compounds were evaluated against Mycobacterium tuberculosis H37Ra and M. smegmatis ATCC 700084. The in vitro cytotoxicity of the compounds was assessed using the human monocytic leukemia cell line THP-1. The lipophilicity of the prepared compounds was experimentally determined and correlated with biological activity. The benzylated intermediates were found to be completely biologically inactive. Of the final eleven compounds, according to the number of amide groups in the molecule, eight are diamides, and three are triamides that were inactive. 5-Chloro-2-hydroxy-N-[(2S)- 4-(methylsulfanyl)-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino}butan-2-yl]benzamide (3e) and 5-chloro-2-hydroxy-N-[(2S)-(4-methyl-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino)pentan-2-yl)benzamide (3f) showed the broadest spectrum of activity against all tested species/isolates comparable to the used standards (ampicillin and isoniazid). Six diamides showed high antistaphylococcal activity with MICs ranging from 0.070 to 8.95 μM. Three diamides showed anti-enterococcal activity with MICs ranging from 4.66 to 35.8 μM, and the activities of 3f and 3e against M. tuberculosis and M. smegmatis were MICs of 18.7 and 35.8 μM, respectively. All the active compounds were microbicidal. It was observed that the connecting linker between the chlorsalicylic and 4-CF3-anilide cores must be substituted with a bulky and/or lipophilic chain such as isopropyl, isobutyl, or thiabutyl chain. Anticancer activity on THP-1 cells IC50 ranged from 1.4 to >10 µM and increased with increasing lipophilicity.

Anti-inflammatory properties of S53P4 bioactive glass implant material

ObjectivesTo assess whether the dissolution products of S53P4 bioactive glass (BG) affect cellular response of macrophages and clinically relevant peri‑implant cell populations to dental implant particles in vitro. Cells chosen were human gingival fibroblasts (HGFs), osteoblasts and bone marrow derived stromal cells (HBMSCs). MethodsMelt-derived S53P4 bioactive glass were prepared. HGFs, Saos-2 human osteoblastic cell line, HBMSCs and macrophages, derived from THP-1 human monocytic cell line, were cultured in the presence of particles from commercially pure titanium (Ti-CP4), grade 5 titanium alloy (Ti-6Al-4V), titanium-zirconium alloy (Ti-15Zr) or zirconia (Zr) (with respective diameters of 34.1 ± 3.8, 33.3 ± 4.4, 97.8 ± 8.2 and 71.3 ± 6.1 µm) with or without S53P4 dissolution products (conditioned media contained 327.30 ± 2.01 ppm Ca, 51.34 ± 0.41 ppm P and 61.48 ± 1.17 ppm Si, pH 8.01 ± 0.21). Inflammatory and macrophage polarisation markers including TNF-ɑ, IL-1, IL-6 and CD206 were quantified using enzyme-linked immunosorbent assay (ELISA). ResultsThe presence of Ti-6Al-4V implant particles significantly induced the expression of pro-inflammatory markers in all tested cell types. S53P4 BG dissolution products regressed the particle induced up-regulation of pro-inflammatory markers and, appeared to suppress M1 macrophage polarisation. ConclusionsImplant particles, Ti-6Al-4V in particular, resulted in significant inflammatory responses from cells. S53P4 BG may possess anti-inflammatory properties and potentially mediate macrophage polarisation behaviour. Clinical SignificanceThe findings highlight that the use and benefits of BG is a promising field of study. Authors believe more collective efforts are required to fully understand the reliability, efficiency and exact mechanisms of action of BG in the search for new generation of treatment modalities in dentistry.

Effects of endocrine disrupting chemicals on the expression of RACK1 and LPS-induced THP-1 cell activation

The existence of a complex hormonal balance among glucocorticoids, androgens and estrogens involved in the regulation of Receptor for Activated C Kinase 1 (RACK1) expression and its related immune cells activation, highlights the possibility to employ this protein as screening tool for the evaluation of the immunotoxic profile of endocrine disrupting chemicals (EDCs), hormone-active substances capable of interfering with the physiologic hormonal signaling. Hence, the aim of this work was to investigate the effect of the exposure of EDCS 17α-ethynylestradiol (EE), diethyl phthalate (DEP) and perfluorooctanesulfonic acid (PFOS) on RACK1 expression and on lipopolysaccharide (LPS)-induced activation of the human monocytic cell line THP-1, a validated model for this investigation. In line with our previous results with estrogen-active compounds, EE treatment significantly induced RACK1 promoter transcriptional activity, mRNA expression, and protein levels, which paralleled an increase in LPS-induced IL-8, TNF-α production and CD86 expression, previously demonstrated to be dependent on RACK1/PKCβ activation. EE mediates its effect on RACK1 expression through G-protein-coupled estrogen receptor (GPER) and androgen receptor (AR) ligand-independent cascade, as also suggested by in silico molecular docking simulation. Conversely, DEP and PFOS induced a dose-dependent downregulation of RACK1 promoter transcriptional activity, mRNA expression, and protein levels, which was mirrored by a reduction of IL-8, TNF-α production and CD86 expression. Mifepristone pre-treatments abolish DEP and PFOS effects, confirming their GR agonist profile, also corroborated by molecular docking. Altogether, our data confirm that RACK1 represents an interesting target of steroid active compounds, which expression offers the opportunity to screen the immunotoxic potential of different hormone-active substances of concerns due to their human exposure and environmental persistence.

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.