Macrophage-like THP-1 Cells Research Articles

Priming Mesenchymal Stem Cells with Lipopolysaccharide Boosts the Immunomodulatory and Regenerative Activity of Secreted Extracellular Vesicles

Background: Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) have been proposed as an alternative to live-cell administration for Acute Respiratory Distress Syndrome (ARDS). MSC-EVs can be chiefly influenced by the environment to which the MSCs are exposed. Here, lipopolysaccharide (LPS) priming of MSCs was used as a strategy to boost the natural therapeutic potential of the EVs in acute lung injury (ALI). Methods: The regenerative and immunemodulatory effect of LPS-primed MSC-EVs (LPS-EVs) and non-primed MSC-EVs (C-EVs) were evaluated in vitro on alveolar epithelial cells and macrophage-like THP-1 cells. In vivo, ALI was induced in adult male rats by the intrapulmonary instillation of HCl and LPS. Rats (n = 8 to 22/group) were randomized to receive a single bolus (1 × 108 particles) of LPS-EVs, C-EVs, or saline. Lung injury severity was assessed at 72 h in lung tissue and bronchoalveolar lavage. Results: In vitro, LPS-EVs improved wound regeneration and attenuated the inflammatory response triggered by the P. aeruginosa infection, enhancing the M2 macrophage phenotype. In in vivo studies, LPS-EVs, but not C-EVs, significantly decreased the neutrophilic infiltration and myeloperoxidase (MPO) activity in lung tissue. Alveolar macrophages from LPS-EVs-treated animals exhibited a reduced expression of CXCL-1, a key neutrophil chemoattractant. However, both C-EVs and LPS-EVs reduced alveolar epithelial and endothelial permeability, mitigating lung damage. Conclusions: EVs from LPS-primed MSCs resulted in a better resolution of ALI, achieving a greater balance in neutrophil infiltration and activation, while avoiding the complete disruption of the alveolar barrier. This opens new avenues, paving the way for the clinical implementation of cell-based therapies.

Non-cyclic nucleotide EPAC1 activators suppress lipopolysaccharide-regulated gene expression, signalling and intracellular communication in differentiated macrophage-like THP-1 cells

This study explores the anti-inflammatory effects of non-cyclic nucleotide EPAC1 activators, PW0577 and SY007, on lipopolysaccharide (LPS)-induced responses in differentiated THP-1 macrophage-like cells. Both activators were found to selectively activate EPAC1 in THP-1 macrophages, leading to the activation of the key down-stream effector, Rap1. RNA sequencing analysis of LPS-stimulated THP-1 macrophages, revealed that treatment with PW0577 or SY007 significantly modulates gene expression related to fibrosis and inflammation, including the suppression of NLRP3, IL-1β, and caspase 1 protein expression in LPS-stimulated cells. Notably, these effects were independent of p65 NFκB phosphorylation at Serine 536, indicating a distinct mechanism of action. The study further identified a shared influence of both activators on LPS signalling pathways, particularly impacting extracellular matrix (ECM) components and NFκB-regulated genes. Additionally, in a co-culture model involving THP-1 macrophages, vascular smooth muscle cells, and human coronary artery endothelial cells, EPAC1 activators modulated immune-vascular interactions, suggesting a broader role in regulating cellular communication between macrophages and endothelial cells. These findings enhance our understanding of EPAC1's role in inflammation and propose EPAC1 activators as potential therapeutic agents for treating inflammatory and fibrotic conditions through targeted modulation of Rap1 and associated signalling pathways.

Differentially Induced Autophagy by Engineered Nanomaterial Treatment Has an Impact on Cellular Homeostasis and Cytotoxicity.

Considering the increasing production of engineered nanomaterials (ENMs), new approach methodologies (NAMs) are essential for safe-by-design approaches and risk assessment. Our aim was to enhance screening strategies with a focus on reactivity-triggered toxicities. We applied in vitro tests to 10 selected benchmark ENMs in two cell models, lung epithelial A549 and differentiated THP-1 macrophage-like cells. Previously, we categorized ENMs based on surface reactivity. Here we elucidated their reactivity-triggered cytotoxicity and mode of action using the WST-1 assay (metabolic activity), LDH assay (cell membrane integrity), autophagosome detection, and proteomics. Nonreactive SiO2 NM-200 showed no significant impact on cell viability. Conversely, highly reactive CuO and ZnO (NM-110 and NM-111) disrupted cell homeostasis. Interestingly, moderately reactive TiO2 (NM-101 and NM-105) and CeO2 (NM-211 and NM-212), apparently without an adverse effect, induced autophagosome formation, evidencing autophagy as a defensive mechanism. Our improved in vitro testing strategy, combined with state-of-the-art reactivity information, screens ENMs for potential reactivity-triggered toxicity.

Towards characterization of cell culture conditions for reliable proteomic analysis: in vitro studies on A549, differentiated THP-1, and NR8383 cell lines

Proteomic investigations result in high dimensional datasets, but integration or comparison of different studies is hampered by high variances due to different experimental setups. In addition, cell culture conditions can have a huge impact on the outcome. This study systematically investigates the impact of experimental parameters on the proteomic profiles of commonly used cell lines—A549, differentiated THP-1 macrophage-like cells, and NR8383—for toxicity studies. The work focuses on analyzing the influence at the proteome level of cell culture setup involving different vessels, cell passage numbers, and post-differentiation harvesting time, aiming to improve the reliability of proteomic analyses for hazard assessment. Mass-spectrometry-based proteomics was utilized for accurate protein quantification by means of a label-free approach. Our results showed that significant proteome variations occur when cells are cultivated under different setups. Further analysis of these variations revealed their association to specific cellular pathways related to protein misfolding, oxidative stress, and proteasome activity. Conversely, the influence of cell passage numbers on the proteome is minor, suggesting a reliable range for conducting reproducible biological replicates. Notable, substantial proteome alterations occur over-time post-differentiation of dTHP-1 cells, particularly impacting pathways crucial for macrophage function. This finding is key for the interpretation of experimental results. These results highlight the need for standardized culture conditions in proteomic-based evaluations of treatment effects to ensure reliable results, a prerequisite for achieving regulatory acceptance of proteomics data.

Immunomodulatory potential of cytokine-licensed human bone marrow-derived mesenchymal stromal cells correlates with potency marker expression profile.

Cytokine(s) pre-activation/licensing is an effective way to enhance the immunomodulatory potency of mesenchymal stromal cells (MSCs). Currently, IFN-γ licensing received the most attention in comparison with other cytokines. After licensing human bone marrow-derived MSCs with pro-/anti-inflammatory cytokines IFN-γ, IL-1β, TNF-α, TGF-β1 alone or in combination, the in-vitro immunomodulatory potency of these MSCs was studied by incubating with allogeneic T cells and macrophage-like THP-1 cells. In addition, immunomodulation-related molecules filtered by bioinformatics, complement 1 subcomponent (C1s) and interferon-induced GTP-binding protein Mx2 (MX2), were studied to verify whether to reflect the immunomodulatory potency. Herein, we reported that different cytokines cause different effects on the function of MSC. While TGF-β1 licensing enhances the capacity of MSCs to induce T cells with an immunosuppressive phenotype, IFN-γ-licensing strengthens the inhibitory effect of MSC on T cell proliferation. Both TGF-β1 and IFN-γ licensing can enhance the effect of MSC on reducing the expression of pro-inflammatory cytokines by M1 macrophage-like THP-1 cells. Interestingly, IFN-γ upregulates potential potency markers extracellular C1s and kynurenine (KYN) and intracellular MX2. These three molecules have the potential to reflect mesenchymal stromal cell immunomodulatory potency. In addition, we reported that there is a synergistic effect of TGF-β1 and IFN-γ in immunomodulation.

The role of Pim-1 kinases in inflammatory signaling pathways.

This observational study investigated the regulatory mechanism of Pim-1 in inflammatory signaling pathways. THP-1, RAW 264.7, BV2, and Jurkat human T cell lines were used. None. Lipopolysaccharide (LPS) was used to induce inflammation, followed by PIM1 knockdown. Western blot, immunoprecipitation, immunofluorescence, and RT-PCR assays were used to assess the effect of PIM1 knockdown on LPS-induced inflammation. PIM1 knockdown in macrophage-like THP-1 cells suppressed LPS-induced upregulation of pro-inflammatory cytokines, inducible nitric oxide synthase, cyclooxygenase-2, phosphorylated Janus kinase, signal transducer and activator of transcription 3, extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, and nuclear factor kappa B p65 (NF-κB p65). It also suppressed upregulation of inhibitor of NF-κB kinase α/β and enhanced the nuclear translocation of NF-κB p65. Moreover, it inhibited the upregulation of Nod-like receptor family pyrin domain-containing 3 (NLRP3) and cleavage of caspase-1 induced by co-treatment of LPS with adenosine triphosphate. Additionally, p-transforming growth factor-β-activated kinase 1 (TAK1) interacted with Pim-1. All three members of Pim kinases (Pim-1, Pim-2, and Pim-3) were required for LPS-mediated inflammation in macrophages; however, unlike Pim-1 and Pim-3, Pim-2 functioned as a negative regulator of T cell activity. Pim-1 interacts with TAK1 in LPS-induced inflammatory responses and is involved in MAPK/NF-κB/NLRP3 signaling pathways. Additionally, considering the negative regulatory role of Pim-2 in T cells, further in-depth studies on their respective functions are needed.

Cellular Effects of Cationic Copper(II) Schiff Base Complexes: Anti-Inflammatory and Antiproliferative Properties.

A series of potassium isothiocyanato-(N-salicylidene-aminoacidato) cuprates (1-5) with the general formula of the monomeric unit K[Cu(sal-aa)(NCS)] ⋅ xH2O (x=0 or 2), containing a Schiff-base ligand (H2sal-aa) derived from natural amino acids such as glycine, DL-α-alanine, DL-valine, DL-phenylalanine and β-alanine, and salicylaldehyde, was screened for in vitro antiradical and major cellular effects against selected cancerous and normal cells. The complexes exhibited strong antioxidant properties against superoxide in vitro and a protective effect on DNA under Fenton-like reaction conditions. Screening of their cellular effects revealed moderate in vitro cytotoxicity against human cancer cell lines (A2780, A2780R and MCF-7), with IC50 values of 25-35 μM, and relatively low toxicity to normal fibroblast MRC-5 cells (with IC50 values>50 μM). Additional experiments performed on A2780 cells revealed that the most potent complex 5 significantly increased the number of A2780 cells arrested in the G2/M phase of the cell cycle and triggered intracellular oxidative stress. The selected flow cytometry experiments (detection of apoptosis/autophagy and activation of caspases 3/7 and depletion of mitochondrial membrane potential) did not reveal the dominant mechanism underlying the cytotoxicity of the complexes but clearly differentiated their molecular effects from those of the reference drug cisplatin. All the complexes exerted anti-inflammatory effects by modulating the levels of the proinflammatory cytokines TNF-α and IL-1β in LPS-activated THP-1 macrophage-like cells. Complex 5 also slightly influenced the activity of the upstream NF-κB transcription factor, while no effect on PPARγ activation was detected.

Caspase-8 activity mediates TNFα production and restricts Coxiella burnetii replication during murine macrophage infection.

Coxiella burnetii is an obligate intracellular bacteria that causes the global zoonotic disease Q Fever. Treatment options for chronic infection are limited, and the development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii, but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii-infected tumor necrosis factor alpha (TNFα)/cycloheximide-treated THP-1 macrophage-like cells and TNFα/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNFα induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8-/- bone marrow-derived macrophages (BMDMs) to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii, coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNFα production and reduced cell death. As TNFα is associated with the control of C. burnetii, this lack of a TNFα response may allow for the unchecked bacterial growth we saw in caspase-8-/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors.

Differential effects of acute and chronic hydrocortisone treatment on pyroptosis

Pyroptosis is a programmed and inflammation-inducing cell death that occurs predominantly in macrophages. It is characterized by the inflammasome-mediated activation of caspase-1, leading to cell lysis. During pyroptosis, pro-inflammatory mediators such as IL-1β are released extracellularly to further recruit and activate other immune cells. Thus, pyroptosis plays a crucial role in the prevention of the spread of pathogens. The clinically applied synthetic glucocorticoid, hydrocortisone (HC), has strong immunoregulatory properties. It may act as an immunosuppressive agent by negatively regulating pro-inflammatory gene transcription but has also shown immune-sensitizing properties. The conditions that determine the immunosuppressive or immune-sensitizing actions of HC during an infection are not fully clear. We hypothesized that the outcome may differ depending on the onset and duration of its administration. Therefore, we investigated the impact of acute (treatment upon infection) and chronic (24 h pre-treatment before infection) HC treatment on pyroptosis induction and execution in THP-1 macrophage-like cells. The focus was on pyroptosis-associated signaling pathways, inflammasome assembly and activation, IL-1β, and cell death. Physiological HC concentration and HC deprivation were used as controls. Compared to the physiological concentration, cells displayed augmented inflammasome activation and IL-1β release following acute HC treatment. Conversely, the whole pyroptosis machinery was suppressed by chronic HC administration.These in vitro investigations demonstrate pro-inflammatory actions of acute HC exposure and the immunosuppressive effects of chronic treatment. These differential effects on pyroptosis emphasize the importance of individualized HC medication in patients upon infection, and suggest the inclusion of IL-1β as a marker for current immune capacities.

NOD2 Responds to Dengue Virus Type 2 Infection in Macrophage-like Cells Interacting with MAVS Adaptor and Affecting IFN-α Production and Virus Titers.

In pathogen recognition, the nucleotide-binding domain (NBD) and leucine rich repeat receptors (NLRs) have noteworthy functions in the activation of the innate immune response. These receptors respond to several viral infections, among them NOD2, a very dynamic NLR, whose role in dengue virus (DENV) infection remains unclear. This research aimed to determine the role of human NOD2 in THP-1 macrophage-like cells during DENV-2 infection. NOD2 levels in DENV-2 infected THP-1 macrophage-like cells was evaluated by RT-PCR and Western blot, and an increase was observed at both mRNA and protein levels. We observed using confocal microscopy and co-immunoprecipitation assays that NOD2 interacts with the effector protein MAVS (mitochondrial antiviral signaling protein), an adaptor protein promoting antiviral activity, this occurring mainly at 12 h into the infection. After silencing NOD2, we detected increased viral loads of DENV-2 and lower levels of IFN-α in supernatants from THP-1 macrophage-like cells with NOD2 knock-down and further infected with DENV-2, compared with mock-control or cells transfected with Scramble-siRNA. Thus, NOD2 is activated in response to DENV-2 in THP-1 macrophage-like cells and participates in IFN-α production, in addition to limiting virus replication at the examined time points.

Peptide selection via phage display to inhibit Leishmania-macrophage interactions.

Leishmaniasis comprises a complex group of diseases caused by protozoan parasites from the Leishmania genus, presenting a significant threat to human health. Infection starts by the release into the skin of metacyclic promastigote (MP) form of the parasite by an infected sand fly. Soon after their release, the MPs enter a phagocytic host cell. This study focuses on finding peptides that can inhibit MP-phagocytic host cell interaction. We used a phage display library to screen for peptides that bind to the surface of L. amazonensis (causative agent for cutaneous leishmaniasis) and L. infantum (causative agent for cutaneous and visceral leishmaniasis) MPs. Candidate peptide binding to the MP surface and inhibition of parasite-host cell interaction were tested in vitro. Peptide Inhibition of visceral leishmaniasis development was assessed in BALB/c mice. The selected L. amazonensis binding peptide (La1) and the L. infantum binding peptide (Li1) inhibited 44% of parasite internalization into THP-1 macrophage-like cells in vitro. While inhibition of internalization by La1 was specific to L. amazonensis, Li1 was effective in inhibiting internalization of both parasite species. Importantly, Li1 inhibited L. infantum spleen and liver infection of BALB/c mice by 84%. We identified one peptide that specifically inhibits L. amazonensis MP infection of host cells and another that inhibits both, L. amazonensis and L. infantum, MP infection. Our findings suggest a promising path for the development of new treatments and prevention of leishmaniasis.

Luteolin Is a Potential Immunomodulating Natural Compound against Pulpal Inflammation.

The present study evaluated the therapeutic effects of luteolin in alleviating pulpitis of dental pulp- (DP-) derived microvesicles (MVs) via the inhibition of protein kinase R- (PKR-) mediated inflammation. Methodology. Proteomic analysis of immortalized human dental pulp (DP-1) cell-derived MVs was performed to identify PKR-associated molecules. The effect of luteolin on PKR phosphorylation in DP-1 cells and the expression of tumor necrosis factor-α (TNF-α) in THP-1 macrophage-like cells were validated. The effect of luteolin on cell proliferation was compared with that of chemical PKR inhibitors (C16 and 2-AP) and the unique commercially available sedative guaiacol-parachlorophenol. In the dog experimental pulpitis model, the pulps were treated with (1) saline, (2) guaiacol-parachlorophenol, and (3) luteolin. Sixteen teeth from four dogs were extracted, and the pulp tissues were analyzed using hematoxylin and eosin staining. Immunohistochemical staining was performed to analyze the expression of phosphorylated PKR (pPKR), myeloperoxidase (MPO), and CD68. Experimental endodontic-periodontal complex lesions were established in mouse molar through a silk ligature and simultaneous MV injection. MVs were prepared from DP-1 cells with or without pretreatment with 2-AP or luteolin. A three-dimensional microcomputed tomography analysis was performed on day 7 (n = 6). Periodontal bone resorption volumes were calculated for each group (nonligated-ligated), and the ratio of bone volume to tissue volume was measured. Proteomic analysis identified an endogenous PKR activator, and a protein activator of interferon-induced PKR, also known as PACT, was included in MVs. Luteolin inhibited the expressions of pPKR in DP-1 cells and TNF-α in THP-1 cells with the lowest suppression of cell proliferation. In the dog model of experimental pulpitis, luteolin treatment suppressed the expression of pPKR-, MPO-, and CD68-positive cells in pulp tissues, whereas guaiacol-parachlorophenol treatment caused coagulative necrosis and disruption. In a mouse model of endodontic-periodontal complex lesions, luteolin treatment significantly decreased MV-induced alveolar bone resorption. Luteolin is an effective and safe compound that inhibits PKR activation in DP-derived MVs, enabling pulp preservation.

CCR2 antagonist attenuates calcium oxalate-induced kidney oxidative stress and inflammation by regulating macrophage activation.

C-C chemokine receptor type 2 (CCR2) is a monocyte chemokine associated with oxidative stress and inflammation. Kidney stones (KS) are composed of calcium oxalate (CaOx), which trigger renal oxidative stress and inflammatory. This study aims to evaluate the effects of CCR2 on KS in vivo and in vitro. Eight-week-old male C57BL/6J mice were intraperitoneally injected with glyoxylate (GOX) daily to establish a KS model, and along with CCR2 antagonist (INCB3344) treatment on days 2, 4, and 6. The results showed that CCR2 antagonist reduced renal injury markers (blood urea nitrogen and serum creatinine), alleviated renal tubular injury and CaOx crystal deposition. CCR2 antagonist also decreased CCR2 expression induced by GOX treatment and increased Nrf2 expression. GOX treatment promoted malondialdehyde (MDA) production, decreased glutathione (GSH) content, and inhibited catalase (CAT) and superoxide dismutase (SOD) activity, however, CCR2 antagonist attenuated the above effects of GOX. CCR2 antagonist had inhibitory effects on GOX-induced inflammatory cytokine expression (IL1B, IL6 and MCP1), and inhibited apoptosis by increasing Bcl-2 expression and decreasing Bax and cleaved-caspase 3 expression. In vitro experiments were performed by co-culture model of CaOx-induced damaged HK-2 cells and macrophage-like THP-1 cells. CCR2 antagonist inhibited CaOx-induced THP-1 cell M1 polarization by decreasing the TNF-α, IL6 and iNOS levels, and further alleviated CaOx-induced oxidative stress damage, inflammatory response and apoptosis of HK-2 cells. The study suggests that CCR2 antagonist may be resistant to CaOx crystals-induced oxidative stress and inflammation by inhibiting macrophage M1 polarization.

Sebacic acid, a royal jelly-containing fatty acid, decreases LPS-induced IL-6 mRNA expression in differentiated human THP-1 macrophage-like cells.

Macrophages produce many inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in innate immune responses. However, excess production of these mediators by activated macrophages triggers deleterious effects, leading to disorders associated with inflammation. Royal jelly (RJ), a milky-white substance secreted by worker bees, contains unique fatty acids, including 10-hydroxy-2-decenoic acid (10H2DA) and sebacic acid (SA). 10H2DA has been reported to have various biological functions, such as anti-inflammation. However, the anti-inflammatory effect of SA is not fully understood. In this study, we investigated the effects of SA on lipopolysaccharide (LPS)-induced cytokine expression using differentiated human THP-1 macrophage-like cells. SA dose-dependently decreased LPS-induced mRNA expression of IL-6, but not TNF-α and IL-1β. SA suppressed the phosphorylation of signal transducers and activators of transcription 1 (STAT1) and STAT3, but hardly affected the activation of JNK, p38, or NF-κB. In addition, SA decreased LPS-induced interferon-β (IFN-β) expression, and the addition of IFN-β restored the inhibition by SA of LPS-induced STAT activation and IL-6 expression. Furthermore, SA suppressed LPS-induced nuclear translocation of interferon regulatory factor 3 (IRF3), a transcription factor responsible for IFN-β expression. Taken together, we conclude that SA selectively decreases LPS-induced expression of IL-6 mRNA through inhibition of the IRF3/IFN-β/STAT axis.

Exploring hydrophilic 2,2-di(indol-3-yl)ethanamine derivatives against Leishmania infantum.

Herein we report the design and the synthesis of a library of new and more hydrophilic bisindole analogues based on our previously identified antileishmanial compound URB1483 that failed the preliminary in vivo test. The novel bisindoles were phenotypically screened for efficacy against Leishmania infantum promastigotes and simultaneously for toxicity on human macrophage-like THP-1 cells. Among the less toxic compounds, eight bisindoles showed IC50 below 10 μM. The most selective compound 1h (selectivity index = 10.1, comparable to miltefosine) and the most potent compound 2c (IC50 = 2.7 μM) were tested for their efficacy on L. infantum intracellular amastigotes. The compounds also demonstrated their efficacy in the in vitro infection model, showing IC50 of 11.1 and 6.8 μM for 1h and 2c, respectively. Moreover, 1h showed a better toxicity profile than the commercial drug miltefosine. For all these reasons, 1h could be a possible new starting point for hydrophilic antileishmanial agents with low cytotoxicity on human macrophage-like cells.

Differential survival of Staphylococcal species in macrophages.

Staphylococcus aureus is considered an extracellular pathogen, yet the bacterium is able to survive within and escape from host cells. An agr/sae mutant of strain USA300 is unable to escape from macrophages but can replicate and survive within. We questioned whether such "non-toxic" S. aureus resembles the less pathogenic coagulase-negative Staphylococcal (CoNS) species like S. epidermidis, S. carnosus, S. lugdunensis, S. capitis, S. warneri, or S. pettenkoferi. We show that the CoNS are more efficiently killed in macrophage-like THP-1 cells or in human primary macrophages. Mutations in katA, copL, the regulatory system graRS, or sigB did not impact bacterial survival in THP-1 cells. Deletion of the superoxide dismutases impaired S. aureus survival in primary macrophages but not in THP-1 cells. However, expression of the S. aureus-specific sodM in S. epidermidis was not sufficient to protect this species from being killed. Thus, at least in those cells, better bacterial survival of S. aureus could not be linked to higher protection from ROS. However, "non-toxic" S. aureus was found to be insensitive to pH, whereas most CoNS were protected when phagosomal acidification was inhibited. Thus, species differences are at least partially linked to differences in sensitivity to acidification.

Immunomodulatory function of licensed human bone marrow mesenchymal stromal cell-derived apoptotic bodies

BackgroundMesenchymal stromal cells (MSCs) show great potential for immunomodulatory and anti-inflammatory treatments. Clinical trials have been performed for the treatment of Type 1 diabetes, graft-versus-host disease and organ transplantation, which offer a promise of MSCs as an immunomodulatory therapy. Nevertheless, their unstable efficacy and immunogenicity concerns present challenges to clinical translation. It has emerged that the MSC-derived secretome, which includes secreted proteins, exosomes, apoptotic bodies (ABs) and other macromolecules, may have similar therapeutic effects to parent MSCs. Among all of the components of the MSC-derived secretome, most interest thus far has been garnered by exosomes for their therapeutic potential. However, since MSCs were reported to undergo apoptosis after in vivo transplantation and release ABs, we speculated as to whether ABs have immunomodulatory effects. In this study, cytokine licensing was used to enhance the immunomodulatory potency of MSCs and ABs derived from licensed MSCs in vitro were isolated to explore their immunomodulatory effects as an effective non-viable cell therapy. ResultsIFN-γ and IFN-γ/TGF-β1 licensing enhanced the immunomodulatory effect of MSCs on T cell proliferation. Further, TGF-β1 and IFN-γ licensing strengthened the immunomodulatory effect of MSC on reducing the TNF-α and IL-1β expression by M1 macrophage-like THP-1 cells. Additionally, we discovered the immunomodulatory effect mediated by MSC-derived apoptotic bodies. Licensing impacted the uptake of ABs by recipient immune cells and importantly altered their phenotypes. ConclusionABs derived from IFN-γ/TGF-β1-licensed apoptotic MSCs significantly inhibited T cell proliferation, induced more regulatory T cells, and maintained immunomodulatory T cells but reduced pro-inflammatory T cells.

Antioxidant Activity of Sweet Whey Derived from Bovine, Ovine and Caprine Milk Obtained from Various Small-Scale Cheese Plants in Greece before and after In Vitro Simulated Gastrointestinal Digestion.

Whey-derived peptides have been associated with different biological properties, but most peptides are usually further hydrolyzed during the digestive process. In the present study, the antioxidant capacity of 48 samples of sweet whey (SW) derived from cheeses obtained from small-scale cheese plants made with bovine, ovine, caprine or a mixture of ovine/caprine milk was assessed using both cell-free and cell-based assays. SW digestates (SW-Ds) and a fraction (<3 kDa; SW-D-P3) thereof were obtained after in vitro digestion and subsequent ultrafiltration. Antioxidant properties using four different assays were evaluated before and after digestion. Our data showed higher values (p < 0.05) for ORAC, ABTS, FRAP and P-FRAP after in vitro digestion (SW-Ds and SW-D-P3) when compared with the corresponding values before digestion. In the non-digested SW, ORAC values were higher (p < 0.05) for the bovine SW compared with all the other samples. In contrast, the ABTS assay indicated a higher antioxidant activity for the ovine SW both before digestion and for SW-D-P3 compared with the bovine SW. The fraction SW-D-P3 of the ovine SW, using HT29 cells and H2O2 as an oxidizing agent, increased (p < 0.05) the cellular antioxidant activity. Furthermore, the same fraction of the ovine/caprine mixed SW increased, through the NF-κB pathway, the expression of SOD1 and CAT, genes implicated in the oxidative response in macrophage-like THP-1 cells. These findings indicate that SW, and particularly bovine and ovine SW, could be a candidate source for physical antioxidants in human and animal nutrition.

Klebsiella pneumoniae manipulates human macrophages to acquire iron.

Klebsiella pneumoniae (KP) is a major cause of hospital-acquired infections, such as pneumonia. Moreover, it is classified as a pathogen of concern due to sprawling anti-microbial resistance. During infection, the gram-negative pathogen is capable of establishing an intracellular niche in macrophages by altering cellular metabolism. One factor critically affecting the host-pathogen interaction is the availability of essential nutrients, like iron, which is required for KP to proliferate but which also modulates anti-microbial immune effector pathways. We hypothesized, that KP manipulates macrophage iron homeostasis to acquire this crucial nutrient for sustained proliferation. We applied an in-vitro infection model, in which human macrophage-like PMA-differentiated THP1 cells were infected with KP (strain ATCC 43816). During a 24-h course of infection, we quantified the number of intracellular bacteria via serial plating of cell lysates and evaluated the effects of different stimuli on intracellular bacterial numbers and iron acquisition. Furthermore, we analyzed host and pathogen specific gene and protein expression of key iron metabolism molecules. Viable bacteria are recovered from macrophage cell lysates during the course of infection, indicative of persistence of bacteria within host cells and inefficient pathogen clearing by macrophages. Strikingly, following KP infection macrophages strongly induce the expression of the main cellular iron importer transferrin-receptor-1 (TFR1). Accordingly, intracellular KP proliferation is further augmented by the addition of iron loaded transferrin. The induction of TFR1 is mediated via the STAT-6-IL-10 axis, and pharmacological inhibition of this pathway reduces macrophage iron uptake, elicits bacterial iron starvation, and decreases bacterial survival. Our results suggest, that KP manipulates macrophage iron metabolism to acquire iron once confined inside the host cell and enforces intracellular bacterial persistence. This is facilitated by microbial mediated induction of TFR1 via the STAT-6-IL-10 axis. Mechanistic insights into immune metabolism will provide opportunities for the development of novel antimicrobial therapies.

Role of reactive oxygen species in the toxicity of two-dimensional nanomaterials: A study on layered Ti3C2 MXenes

Two-dimensional (2D) materials, such as MXenes and graphene, are tested as catalysts, sensors, and nanomedicine, but their safety and biocompatibility are poorly understood. In this study, Ti3C2 MXenes (single-layer and multilayer) and graphene oxide are prepared to evaluate their inflammatory potential in vivo and in vitro using the read-across approach. Pulmonary exposure of the test materials in mice led to significant dose-dependent increases in several toxicity endpoints of bronchoalveolar lavage fluid. The correlation of the oxidative potential of the test materials and the toxicity endpoints shows that both the intrinsic and intracellular oxidative potentials of 2D materials are key factors for the inflammation and damage of the plasma membrane. Scanning electron microscopy and in vitro studies using the differentiated THP-1 macrophage-like cells support this finding. Furthermore, the toxicity of test items is reduced when treated with N-acetyl cysteine, a scavenger for reactive oxygen species; this finding further supports the oxidative stress paradigm proposed in this study. This oxidative stress paradigm may apply to 2D materials in general and improve the understanding the toxicity mechanism of such materials, which would facilitate the design of safer functional 2D materials.