Evaluation of the immunotoxicity potential of nanomaterials using THP-1 cells.

With the expansion of nanomaterials (NMs) usage, concerns about their toxicity are increasing, and the wide variety of NMs makes it difficult to assess their toxicity. Therefore, the development of a high-throughput, accurate, and certified method to evaluate the immunotoxicity of NMs is required. In this study, we assessed the immunotoxicity potential of various NMs, such as nanoparticles of silver, silica, and titanium dioxide, using the human Cell Line Activation Test (h-CLAT) at the cellular level. After exposure to silver nanoparticle dispersions, the expression levels of CD86 and CD54 increased, suggesting the activation of antigen-presenting cells (APCs) by silver nanoparticles. Quantification of silver ions eluted from silver nanoparticles and the activation of APCs by silver ions suggested that it was due to the release of silver ions. Silica nanoparticles also increased the expression of CD86 and/or CD54, and their activation ability correlated with the synthesis methods and hydrodynamic diameters. The ability of titanium dioxide to activate APCs differed depending on the crystal type and hydrodynamic diameter. These results suggest a potential method to evaluate the immunotoxicity potential of various NMs based on their ability to activate APCs using human monocytic THP-1 cells. This method will be valuable in assessing the immunotoxicity potential and elucidating the immunotoxic mechanisms of NMs.

Biflavonoid Methylchamaejasmin and Khaya grandifoliola Extract Inhibit NLRP3 Inflammasome in THP-1 Cell Model of Neuroinflammation.

Neuroinflammation is a common hallmark of Alzheimer's disease (AD), with NLRP3 inflammasome proven to be activated in microglia of AD patients' brains. In this study, a newly isolated biflavonoid (7,7'-di-O-methylchamaejasmin/M8) and a crude extract of the plant Khaya grandifoliola (KG) were investigated for their inhibitory effect on inflammasome activation. In preliminary experiments, M8 and KG showed no cytotoxicity on human macrophage-like differentiated THP-1 cells and exhibited anti-inflammatory inhibition of nitric oxide produced following lipopolysaccharide stimulation. Furthermore, M8 and KG blocked IL-1ß and IL-18 production by reducing NLRP3 inflammasome components including NFκB, NLRP3, Caspase-1, pro-IL-1ß, and pro-IL-18 at the mRNA and protein levels. Regarding the formation of ASC (apoptosis-associated speck-like protein containing a CARD) specks during inflammasome activation, the size and fluorescent intensity of the existing specks were unchanged across all treatment conditions. However, M8 and KG treatments were shown to prevent further speck formation. In addition, experiments on amyloid ß phagocytosis showed that M8 and KG pretreatments can restore the phagocytic activity of THP-1 cells, which was impaired following inflammasome activation. Altogether, our findings describe for the first time a promising role of biflavonoids and KG extract in preventing inflammasome activation and protecting against neuroinflammation, a key factor in AD development.

Sacha Inchi (Plukenetia volubilis L.) Protein Hydrolysate as a New Ingredient of Functional Foods.

Sacha inchi (Plukenetia volubilis L.) is an under-exploited crop with great potential due to its nutritional and medicinal characteristics. A Sacha inchi protein isolate (SII), obtained from defatted Sacha inchi flour (SIF), was hydrolyzed by Bioprotease LA 660 under specific conditions. The hydrolysates were characterized chemically, and their digestibility and antioxidant capacity were evaluated by in vitro cell-free experiments to select the hydrolysate with major antioxidant activity. Sacha inchi protein hydrolysate at 20 min (SIH20B) was selected, and the anti-inflammatory capacity was evaluated by RT-qPCR and ELISA techniques, using two different doses in monocytes THP-1 stimulated with lipopolysaccharide (LPS). The results obtained showed that the in vitro administration of SIH20B down-regulated the TNF-α gene and reduced the release of this cytokine, whereas the anti-inflammatory cytokines IL-10 and IL-4 were up-regulated in LPS-stimulated monocytes and co-administrated with SIH20B. The peptides contained in SIH20B were identified, and the 20 more relatively abundant peptides with a mass by 1 kDa were subjected to in silico analysis to hypothesize those that could be responsible for the bioactivity reported in the hydrolysate. From the identified peptides, the peptides AAGALKKFL and LGVKFKGGL, among others, are proposed as the most biologically actives. In conclusion, SIH20B is a novel, natural source of high-value-added biopeptides that could be used as an ingredient in formulations of food or nutraceutical compounds.

The dual role of FSP1 in programmed cell death: resisting ferroptosis in the cell membrane and promoting necroptosis in the nucleus of THP-1 cells.

BACKGROUND: Acute monocytic leukemia-M5 (AML-M5) remains a challenging disease due to its high morbidity and poor prognosis. In addition to the evidence mentioned earlier, several studies have shown that programmed cell death (PCD) serves a critical function in treatment of AML-M5. However, the role and relationship between ferroptosis and necroptosis in AML-M5 remains unclear. METHODS: THP-1 cells were mainly treated with Erastin and IMP-366. The changes of ferroptosis and necroptosis levels were detected by CCK-8, western blot, quantitative real-time PCR, and electron microscopy. Flow cytometry was applied to detect the ROS and lipid ROS levels. MDA, 4-HNE, GSH and GSSG were assessed by ELISA kits. Intracellular distribution of FSP1 was studied by immunofluorescent staining and western blot. RESULTS: The addition of the myristoylation inhibitor IMP-366 to erastin-treated acute monocytic leukemia cell line THP-1 cell not only resulted in greater susceptibility to ferroptosis characterized by lipid peroxidation, glutathione (GSH) depletion and mitochondrial shrinkage, as the FSP1 position on membrane was inhibited, but also increased p-RIPK1 and p-MLKL protein expression, as well as a decrease in caspase-8 expression, and triggered the characteristic necroptosis phenomena, including cytoplasmic translucency, mitochondrial swelling, membranous fractures by FSP1 migration into the nucleus via binding importin α2. It is interesting to note that ferroptosis inhibitor fer-1 reversed necroptosis. CONCLUSION: We demonstrated that inhibition of myristoylation by IMP-366 is capable of switching ferroptosis and ferroptosis-dependent necroptosis in THP-1 cells. In these findings, FSP1-mediated ferroptosis and necroptosis are described as alternative mechanisms of PCD of THP-1 cells, providing potential therapeutic strategies and targets for AML-M5.

1,25-dihydroxyvitamin D3 augments low-dose PMA-based monocyte-to-macrophage differentiation in THP-1 cells.

The human monocytic THP-1 cell line is the most routinely employed in vitro model for studying monocyte-to-macrophage differentiation. Despite the wide use of this model, differentiation protocols using phorbol 12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 (1,25D3) vary drastically between studies. Given that differences in differentiation protocols have the potential to impact the characteristics of the macrophages produced, we aimed to assess the efficacy of three different THP-1 differentiation protocols by assessing changes in morphology and gene- and cell surface macrophage marker expression. THP-1 cells were differentiated with either 5 nM PMA, 10 nM 1,25D3, or a combination thereof, followed by a rest period. The results indicated that all three protocols significantly increased the expression of the macrophage markers, CD11b (p < 0.001) and CD14 (p < 0.010). Despite this, THP-1 cells exposed to 1,25D3 alone did not adopt the morphological and expression characteristics associated with macrophages. PMA was required to produce these characteristics, which were found to be more pronounced in the presence of 1,25D3. Both PMA- and PMA with 1,25D3-differentiated THP-1 cells were capable of M1 and M2 macrophage polarization, though the gene expression of polarization-associated markers was most pronounced in PMA with 1,25D3-differentiated THP-1 cells. Moreover, the combination of PMA with 1,25D3 appeared to support the process of commitment to a particular polarization state.

Exposing kinetic disparities between inflammasome readouts using time-resolved analysis.

The NLRP3 inflammasome is an intracellular multiprotein complex described to be involved in both an effective host response to infectious agents and various diseases. Investigation into the NLRP3 inflammasome has been extensive in the past two decades, and often revolves around the analysis of a few specific readouts, including ASC-speck formation, caspase-1 cleavage or activation, and cleavage and release of IL-1ß and/or IL-18. Quantification of these readouts is commonly undertaken as an endpoint analysis, where the presence of each positive outcome is assessed independently of the others. In this study, we apply time-resolved analysis of a human macrophage model (differentiated THP-1-ASC-GFP cells) to commonly accessible methods. This approach yields the additional quantifiable metrics time-resolved absolute change and acceleration, allowing comparisons between readouts. Using this methodological approach, we reveal (potential) discrepancies between inflammasome-related readouts that otherwise might go undiscovered. The study highlights the importance of time-resolved data in general and may be further extended as well as incorporated into other areas of research.

Gene expression profiles and protein-protein interaction networks in THP-1 cells exposed to metal-based nanomaterials.

We analyzed gene expression in THP-1 cells exposed to metal-based nanomaterials (NMs) [TiO2 (NM-100), ZnO (NM-110), SiO2 (NM-200), Ag (NM-300 K)]. A functional enrichment analysis of the significant differentially expressed genes (DEGs) identified the key modulated biological processes and pathways. DEGs were used to construct protein-protein interaction networks. NM-110 and NM-300 K induced changes in the expression of genes involved in oxidative and genotoxic stress, immune response, alterations of cell cycle, detoxification of metal ions and regulation of redox-sensitive pathways. Both NMs shared a number of highly connected protein nodes (hubs) including CXCL8, ATF3, HMOX1, and IL1B. NM-200 induced limited transcriptional changes, mostly related to the immune response; however, several hubs (CXCL8, ATF3) were identical with NM-110 and NM-300 K. No effects of NM-100 were observed. Overall, soluble nanomaterials NM-110 and NM-300 K exerted a wide variety of toxic effects, while insoluble NM-200 induced immunotoxicity; NM-100 caused no detectable changes on the gene expression level.

Deciphering Staphylococcus aureus-host dynamics using dual activity-based protein profiling of ATP-interacting proteins.

The utilization of ATP within cells plays a fundamental role in cellular processes that are essential for the regulation of host-pathogen dynamics and the subsequent immune response. This study focuses on ATP-binding proteins to dissect the complex interplay between Staphylococcus aureus and human cells, particularly macrophages (THP-1) and keratinocytes (HaCaT), during an intracellular infection. A snapshot of the various protein activity and function is provided using a desthiobiotin-ATP probe, which targets ATP-interacting proteins. In S. aureus, we observe enrichment in pathways required for nutrient acquisition, biosynthesis and metabolism of amino acids, and energy metabolism when located inside human cells. Additionally, the direct profiling of the protein activity revealed specific adaptations of S. aureus to the keratinocytes and macrophages. Mapping the differentially activated proteins to biochemical pathways in the human cells with intracellular bacteria revealed cell-type-specific adaptations to bacterial challenges where THP-1 cells prioritized immune defenses, autophagic cell death, and inflammation. In contrast, HaCaT cells emphasized barrier integrity and immune activation. We also observe bacterial modulation of host processes and metabolic shifts. These findings offer valuable insights into the dynamics of S. aureus-host cell interactions, shedding light on modulating host immune responses to S. aureus, which could involve developing immunomodulatory therapies. IMPORTANCE: This study uses a chemoproteomic approach to target active ATP-interacting proteins and examines the dynamic proteomic interactions between Staphylococcus aureus and human cell lines THP-1 and HaCaT. It uncovers the distinct responses of macrophages and keratinocytes during bacterial infection. S. aureus demonstrated a tailored response to the intracellular environment of each cell type and adaptation during exposure to professional and non-professional phagocytes. It also highlights strategies employed by S. aureus to persist within host cells. This study offers significant insights into the human cell response to S. aureus infection, illuminating the complex proteomic shifts that underlie the defense mechanisms of macrophages and keratinocytes. Notably, the study underscores the nuanced interplay between the host's metabolic reprogramming and immune strategy, suggesting potential therapeutic targets for enhancing host defense and inhibiting bacterial survival. The findings enhance our understanding of host-pathogen interactions and can inform the development of targeted therapies against S. aureus infections.

Imeglimin attenuates NLRP3 inflammasome activation by restoring mitochondrial functions in macrophages.

Imeglimin is a novel oral antidiabetic drug for treating type 2 diabetes. However, the effect of imeglimin on NLRP3 inflammasome activation has not been investigated yet. Here, we aimed to investigate whether imeglimin reduces LPS-induced NLRP3 inflammasome activation in THP-1 macrophages and examine the associated underlying mechanisms. We analyzed the mRNA and protein expression levels of NLRP3 inflammasome components and IL-1ß secretion. Additionally, reactive oxygen species (ROS) generation, mitochondrial membrane potential, and mitochondrial permeability transition pore (mPTP) opening were measured by flow cytometry. Imeglimin inhibited NLRP3 inflammasome-mediated IL-1ß production in LPS-stimulated THP-1-derived macrophages. In addition, imeglimin reduced LPS-induced mitochondrial ROS production and mitogen-activated protein kinase phosphorylation. Furthermore, imeglimin restored the mitochondrial function by modulating mitochondrial membrane depolarization and mPTP opening. We demonstrated for the first time that imeglimin reduces LPS-induced NLRP3 inflammasome activation by inhibiting mPTP opening in THP-1 macrophages. These results suggest that imeglimin could be a promising new anti-inflammatory agent for treating diabetic complications.

A flow cytometry-based assay to determine the ability of anti-Streptococcus pyogenes antibodies to mediate monocytic phagocytosis in human sera.

Streptococcus pyogenes, commonly referred to as Group A Streptococcus (Strep A), causes a spectrum of diseases, with the potential to progress into life-threatening illnesses and autoimmune complications. The escalating threat of antimicrobial resistance, stemming from the prevalent reliance on antibiotic therapies to manage Strep A infections, underscores the critical need for the development of disease control strategies centred around vaccination. Phagocytes play a critical role in controlling Strep A infections, and phagocytosis-replicating assays are essential for vaccine development. Traditionally, such assays have employed whole-blood killing or opsonophagocytic methods using HL-60 cells as neutrophil surrogates. However, assays mimicking Fcγ receptors- phagocytosis in clinical contexts are lacking. Therefore, here we introduce a flow cytometry-based method employing undifferentiated THP-1 cells as monocytic/macrophage model to swiftly evaluate the ability of human sera to induce phagocytosis of Strep A. We extensively characterize the assay's precision, linearity, and quantification limit, ensuring robustness. By testing human pooled serum, the assay proved to be suitable for the comparison of human sera's phagocytic capability against Strep A. This method offers a valuable complementary assay for clinical studies, addressing the gap in assessing FcγR-mediated phagocytosis. By facilitating efficient evaluation of Strep A -phagocyte interactions, it may contribute to elucidating the mechanisms required for the prevention of infections and inform the development of future vaccines and therapeutic advancements against Strep A infections.

Resolvin D2 attenuates LPS-induced macrophage exhaustion.

Early in sepsis, a hyperinflammatory response is dominant, but later, an immunosuppressive phase dominates, and the host is susceptible to opportunistic infections. Anti-inflammatory agents may accelerate the host into immunosuppression, and few agents can reverse immunosuppression without causing inflammation. Specialized pro-resolving mediators (SPMs) such as resolvin D2 (RvD2) have been reported to resolve inflammation without being immunosuppressive, but little work has been conducted to examine their effects on immunosuppression. To assess the effects of RvD2 on immunosuppression, we established a model of macrophage exhaustion using two lipopolysaccharide (LPS) treatments or hits. THP-1 monocyte-derived macrophages were first treated with RvD2 or vehicle for 1 h. One LPS hit increased NF-κB activity 11-fold and TNF-α release 60-fold compared to unstimulated macrophages. RvD2 decreased LPS-induced NF-κB activity and TNF-α production but increased bacterial clearance. Two LPS hits reduced macrophage bacterial clearance and decreased macrophage NF-κB activity (45%) and TNF-α release (75%) compared to one LPS hit, demonstrating exhaustion. RvD2 increased NF-κB activity, TNF-α release, and bacterial clearance following two LPS hits compared to controls. TLR2 inhibition abolished RvD2-mediated changes. In a mouse sepsis model, splenic macrophage response to exogenous LPS was reduced compared to controls and was restored by in vivo administration of RvD2, supporting the in vitro results. If RvD2 was added to monocytes before differentiation into macrophages, however, RvD2 reduced LPS responses and increased bacterial clearance following both one and two LPS hits. The results show that RvD2 attenuated macrophage suppression in vitro and in vivo and that this effect was macrophage-specific.

Antibodies from chlamydia-infected individuals facilitate phagocytosis via Fc receptors.

Non-neutralizing functions of antibodies, including phagocytosis, may play a role in Chlamydia trachomatis (CT) infection, but these functions have not been studied and assays are lacking. We utilized a flow-cytometry-based assay to determine whether serum samples from a well-characterized cohort of CT-infected and naïve control individuals enhanced phagocytosis via Fc-receptor-expressing THP-1 cells, and whether this activity correlated with antibody titers. Fc-receptor-mediated phagocytosis was detected only in CT+ donors. Phagocytosis generally did not correlate well with antibody titer. In addition, we found that complement from both CT+ and negative individuals enhanced phagocytosis of CT into primary neutrophils. These results suggest that anti-CT antibodies can have functions that are not reflected by titer. This method could be used to quantitively measure Fc-receptor-mediated function of anti-CT antibodies or complement activity and could reveal new immune correlates of protection.

Elucidating miR-146a-3p as a key player in autophagy and lipid metabolism in Leishmania major infection.

Autophagy is a key step involved in many unicellular eukaryotic diseases, including leishmaniasis, for cellular remodelling and differentiation during parasite's lifecycle. Lipids play a significant role in the infection process that begins with Leishmania major invading host cells. MicroRNAs (miRNAs), a family of small, 22-24 nucleotide noncoding regulatory RNAs, target mRNAs to modify gene expression and, subsequently, proteome output may have a regulatory role in altering the host cell processes. We observed miR-146a-3p expression increases in a time-dependent manner post Leishmania major infection. Transfecting miR-146a-3p mimic increases the expression of ATG7, an autophagy gene that encodes an E1-like enzyme in two ubiquitin-like conjugation systems required for autophagosome progression. HPGD (15-hydroxyprostaglandin dehydrogenase) operates as an enzyme, converting prostaglandin to its non-active form. Microarray data and western studies reveal that miR-146a-3p targets and inhibits HPGD, thereby increasing prostaglandin activity in lipid droplets. Herein, our research focuses on miR-146a-3p, which boosts ATG7 expression while reducing HPGD post Leishmania major infections helping us comprehend the intricate network of microRNA, autophagy, and lipid metabolism in leishmaniasis.

rBet v 1a-BanLecwt induce upregulation of IL-10 and IFN-γ gene expression in Caco-2/THP-1 co-culture and secretion of IL-10 and IFN-γ/IL-4 levels in PBMCs of birch pollen allergic donors.

Novel allergen immunotherapy (AIT) approaches necessitate the use of more effective and safe therapeutics, which can be accomplished by employing novel adjuvants for improved innate immune cell activation, as well as hypoallergenic allergen forms. In this study, we investigate the immunomodulatory effects of a chimera rBet v 1a-BanLecwt (rBv1a-BLwt; Cwt) composed of the major birch pollen allergen Bet v 1a and banana lectin (BanLecwt; BLwt) and two novel chimeras, rBv1l-BLH84T (rBet v 1l-BanLecH84T; C1) and rBLH84T-Bv1l (rBanLecH84T-Bet v 1l; C2), both composed of BLH84T and hypoallergenic birch pollen allergen Bv1l in the co-culture model Caco-2/THP-1, and PBMCs from donors with birch pollen allergy. The chimeric molecules rBv1l-BLH84T (C1) and rBLH84T-Bv1l (C2) were created in silico and then produced in E. coli using recombinant DNA technology. Real-time PCR analysis of gene expression following compound treatment in the co-culture model revealed that all three chimeras have the potential to induce the anti-inflammatory cytokine IL-10 gene expression in Caco-2 cells and IFN-γ gene expression in THP-1 cells. Sandwich ELISA revealed that Cwt increased IL-10 secretion and IFN-/IL-4 levels in PBMCs from birch pollen allergic donors, whereas C1 and C2 were less effective. The findings suggest that Cwt should be analyzed further due to its potential benefit in AIT.

Anti-inflammatory effects of α-humulene on the release of pro-inflammatory cytokines in lipopolysaccharide-induced THP-1 cells.

While acute inflammation is an essential physical response to harmful external influences, the transition to chronic inflammation is problematic and associated with the development and worsening of many deadly diseases. Until now, established pharmaceutical agents have had many side effects when used for long periods. In this study, a possible anti-inflammatory effect of the sesquiterpene α-humulene on lipopolysaccharide (LPS) induction was tested. Herein, human THP-1-derived macrophages were used and their pro-inflammatory interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interleukin-1ß (IL-1ß) cytokine release was measured by means of enzyme-linked immunosorbent assay. A dose-dependent effect of α-humulene on IL-6 release was observed at 0.5 and 100 µM α-humulene, with a maximum IL-6 inhibition of 60% compared to the LPS reference value after the addition of 100 µM α-humulene. TNF-α as well as IL-1ß cytokine concentrations were not reduced by the addition of 0.5 and 100 µM α-humulene. This study suggests that α-humulene has potential as a promising natural alternative to established pharmaceuticals for the treatment of elevated IL-6 levels and chronic inflammation in humans.

Extracellular Vesicles from Leishmania (Leishmania) infantum Contribute in Stimulating Immune Response and Immunosuppression in Hosts with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a chronic systemic disease. In Brazil this infection is caused by Leishmania (Leishmania) infantum. Extracellular vesicles (EVs) released by Leishmania species have different functions like the modulation of host immune systems and inflammatory responses, among others. This study evaluated the participation of EVs from L. (L.) infantum (Leish-EVs) in recognition of the humoral and cellular immune response of hosts with VL. Promastigotes were cultivated in 199 medium and, in the log phase of growth, they were centrifuged, washed, resus-pended in RPMI medium, and incubated for 2 to 24 h, at 25 °C or 37 °C to release Leish-EVs. This dynamic was evaluated using transmission (TEM) and scanning (SEM) electron microscopies, as well as nanoparticle tracking analysis (NTA). The results suggested that parasite penetration in mammal macrophages requires more Leish-EVs than those living in insect vectors, since promastigotes incubated at 37 °C released more Leish-EVs than those incubated at 25 °C. Infected THP-1 cells produced high EV concentration (THP-1 cells-EVs) when compared with those from the control group. The same results were obtained when THP-1 cells were treated with Leish-EVs or a crude Leishmania antigen. These data indicated that host-EV concentrations could be used to distinguish infected from uninfected hosts. THP-1 cells treated with Leish-EVs expressed more IL-12 than control THP-1 cells, but were unable to express IFN-γ. These same cells highly expressed IL-10, which inhibited TNF-α and IL-6. Equally, THP-1 cells treated with Leish-EVs up-expressed miR-21-5p and miR-146a-5p. In conclusion, THP-1 cells treated with Leish-EVs highly expressed miR-21-5p and miR-146a-5p and caused the dysregulation of IL-10. Indirectly, these results suggest that high expression of these miRNAs species is caused by Leish-EVs. Consequently, this molecular via can contribute to immunosuppression causing enhanced immunopathology in infected hosts.

sTREM2 Differentially Affects Cytokine Expression in Myeloid-Derived Cell Models via MAPK-JNK Signaling Pathway.

TREM2 is a critical innate immune receptor primarily expressed on myeloid-derived cells, such as microglia and macrophages. Mutations in TREM2 are linked to several neurodegenerative diseases including Alzheimer's disease (AD). TREM2 can be cleaved from the cell membrane and released as soluble TREM2 (sTREM2). sTREM2 levels are shown to peak prior to AD, with its levels fluctuating throughout disease progression. However, the mechanism by which sTREM2 may affect innate immune responses is largely uncharacterized. In this study, we investigated whether sTREM2 can induce inflammatory response in myeloid-derived THP-1 monocytes and macrophages and characterized the signaling mechanisms involved. Our results show that sTREM2 was capable of stimulating the expression of several inflammatory cytokines in THP-1 cells throughout the time course of 2 h to 8 h but inducing anti-inflammatory cytokine expression at later time points. A TREM2 antibody was capable of inhibiting the expression of some cytokines induced by sTREM2 but enhancing others. The complex of sTREM2/TREM2 antibody was shown to enhance IL-1ß expression, which was partially blocked by an NLRP3 specific inhibitor, indicating that the complex activated the NRLP3 inflammasome pathway. sTREM2 was also shown to have differential effects on cytokine expression in M0, M1, and M2 macrophages differentiated from THP-1 cells. sTREM2 has a more stimulating effect on cytokine expression in M0 macrophages, less of an effect on M2 macrophages, and some inhibitory effects on cytokine expression in M1 macrophages at early time points. Analyses of several signaling pathways revealed that sTREM2-induced expression of cytokines occurs mainly through MAPK-JNK signaling. Our work reveals differential effects of sTREM2 on cytokine expression profiles of THP-1 cells and macrophages and demonstrates that the MAPK-JNK signaling pathway is mainly responsible for sTREM2-induced cytokine expression.

Soluble urate-induced effects on cytokine production in vitro - Assessment of methodologies and cell types.

BACKGROUND: Hyperuricemia has been shown to be an inducer of pro-inflammatory mediators by human primary monocytes. To study the deleterious effects of hyperuricemia, a reliable and stable in vitro model using soluble urate is needed. One recent report showed different urate-dissolving methods resulted in either pro-inflammatory or anti-inflammatory properties. The aim of this study was to compare the effect of two methods of dissolving urate on both primary human peripheral blood mononuclear cells (PBMCs) and THP-1 cells. The two methods tested were 'pre-warming' and 'dissolving with NaOH'. METHODS: Primary human PBMCs and THP-1 cells were exposed to urate solutions, prepared using the two methodologies: pre-warming and dissolving with NaOH. Afterwards, cells were stimulated with various stimuli, followed by the measurement of the inflammatory mediators IL-1ß, IL-6, IL-1Ra, TNF, IL-8, and MCP-1. RESULTS: In PBMCs, we observed an overall pro-inflammatory effect of urate, both in the pre-warming and the NaOH dissolving method. A similar pro-inflammatory effect was seen in THP-1 cells for both dissolving methods after restimulation. However, THP-1 cells exhibited pro-inflammatory profile with exposure to urate alone without restimulation. We did not find MSU crystals in our cellular assays. CONCLUSIONS: Overall, the urate dissolving methods do not have critical impact on its inflammatory properties. Soluble urate prepared using either of the two methods showed mostly pro-inflammatory effects on human primary PBMCs and monocytic cell line THP-1. However, human primary PBMCs and the THP-1 differ in their response to soluble urate without restimulation.

Anti-inflammatory mechanisms in cancer research: Characterization of a distinct M2-like macrophage model derived from the THP-1 cell line.

AIMS: Macrophages play an essential role in cancer development. Tumor-associated macrophages (TAMs) have predominantly M2-like attributes that are associated with tumor progression and poor patient survival. Numerous methods have been reported for differentiating and polarizing macrophages in vitro, but there is no standardized and validated model for creating TAMs. Primary cells show varying cytokine responses depending on their origin and functional studies utilizing these cells may lack generalization and validity. A distinct cell line-derived TAM-like M2 subtype is required to investigate the mechanisms mediated by anti-inflammatory TAMs in vitro. Our previous work demonstrated a standardized protocol for creating an M2 subtype derived from a human THP-1 cell line. The cell expression profile, however, has not been validated. The aim of this study was to characterize and validate the TAM-like M2 subtype macrophage created based on our protocol to introduce them as a standardized model for cancer research. METHODS AND RESULTS: Using qRT-PCR and ELISA, we demonstrated that proinflammatory, anti-inflammatory, and tumor-associated marker expression changed during THP-1-derived marcrophage development in vitro, mimicking a TAM-related profile (e.g., TNFα, IL-1ß). The anti-inflammatory marker IL-8/CXCL8, however, is most highly expressed in young M0 macrophages. Flow cytometry showed increased expression of CD206 in the final TAM-like M2 macrophage. Single-cell RNA-sequencing analysis of primary human monocytes and colon cancer tissue macrophages demonstrated that cell line-derived M2 macrophages resembled a TAM-related gene profile. CONCLUSIONS: The THP-1-derived M2 macrophage based on a standardized cell line model represents a distinct anti-inflammatory TAM-like phenotype with an M2a subtype profile. This model may provide a basis for in vitro investigation of functional mechanisms in a variety of anti-inflammatory settings, particularly colon cancer development.

Temporal Profiling of Host Proteome against Different M. tuberculosis Strains Reveals Delayed Epigenetic Orchestration.

Apart from being preventable and treatable, tuberculosis is the deadliest bacterial disease afflicting humankind owing to its ability to evade host defence responses, many of which are controlled by epigenetic mechanisms. Here, we report the temporal dynamics of the proteome of macrophage-like host cells after infecting them for 6, 18, 30, and 42 h with two laboratory strains (H37Ra and H37Rv) and two clinical strains (BND433 and JAL2287) of Mycobacterium tuberculosis (MTB). Using SWATH-MS, the proteins characterized at the onset of infection broadly represented oxidative stress and cell cytoskeleton processes. Intermediary and later stages of infection are accompanied by a reshaping of the combination of proteins implicated in histone stability, gene expression, and protein trafficking. This study provides strain-specific and time-specific variations in the proteome of the host, which might further the development of host-directed therapeutics and diagnostic tools against the pathogen. Also, our findings accentuate the importance of proteomic tools in delineating the complex recalibration of the host defence enabled as an effect of MTB infection. To the best of our knowledge, this is the first comprehensive proteomic account of the host response to avirulent and virulent strains of MTB at different time periods of the life span of macrophage-like cells. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD022352.