Macrophage-like THP-1 Cells Research Articles

In vitro and in vivo properties of the bovine antimicrobial peptide, Bactenecin 5.

Antimicrobial peptides (AMP), part of the innate immune system, are well studied for their ability to kill pathogenic microorganisms. However, many also possess important immunomodulatory effects, and this area has potential for the development of novel therapies to supplement traditional methods such as the use of antibiotics. Here, we characterise the microbicidal and immunomodulatory potential of the proline-rich bovine AMP, Bactenecin 5 (Bac5). We demonstrate broad antimicrobial activity, including against some mycobacterial species, which are important pathogens of fish, cattle and humans. Bac5 is able to activate macrophage-like THP-1 cells and can synergistically trigger the upregulation of tnf-α when co-stimulated with M. marinum. Furthermore, Bac5 sensitises A549 epithelial cells to stimulation with TNF-α. For the first time, we characterise the activity of Bac5 in vivo, and show it to be a potent chemokine for macrophages in the zebrafish (Danio rerio) embryo model of infection. Bac5 also supports the early recruitment of neutrophils in the presence of M. marinum. In the absence of host adaptive immunity, exogenous injected Bac5 is able to slow, although not prevent, infection of zebrafish with M. marinum.

Lysosomal oxidation of LDL alters lysosomal pH, induces senescence, and increases secretion of pro-inflammatory cytokines in human macrophages

We have shown that aggregated LDL is internalized by macrophages and oxidized in lysosomes by redox-active iron. We have now investigated to determine whether the lysosomal oxidation of LDL impairs lysosomal function and whether a lysosomotropic antioxidant can prevent these alterations. LDL aggregated by SMase (SMase-LDL) caused increased lysosomal lipid peroxidation in human monocyte-derived macrophages or THP-1 macrophage-like cells, as shown by a fluorescent probe, Foam-LPO. The pH of the lysosomes was increased considerably by lysosomal LDL oxidation as shown by LysoSensor Yellow/Blue and LysoTracker Red. SMase-LDL induced senescence-like properties in the cells as shown by β-galactosidase staining and levels of p53 and p21. Inflammation plays a key role in atherosclerosis. SMase-LDL treatment increased the lipopolysaccharide-induced secretion of TNF-α, IL-6, and MCP-1. The lysosomotropic antioxidant, cysteamine, inhibited all of the above changes. Targeting lysosomes with antioxidants, such as cysteamine, to prevent the intralysosomal oxidation of LDL might be a novel therapy for atherosclerosis.

Of mice and men: Interaction of Corynebacterium diphtheriae strains with murine and human phagocytes

ABSTRACTSeven non-toxigenic C. diphtheriae strains and one toxigenic strain were analyzed with regard to their interaction with murine macrophages (BMM) and human THP-1 macrophage-like cells. Proliferation assays with BMM and THP-1 revealed similar intracellular CFUs for C. diphtheriae strains independent of the host cell. Strain ISS4060 showed highest intracellular CFUs, while the toxigenic DSM43989 was almost not detectable. This result was confirmed by TLR 9 reporter assays, showing a low signal for DSM43989, indicating that the bacteria are not endocytosed. In contrast, the non-pathogenic C. glutamicum showed almost no intracellular CFUs independent of the host cell, but was recognized by TLR9, indicating that the bacteria were degraded immediately after endocytosis. In terms of G-CSF and IL-6 production, no significant differences between BMM and THP-1 were observed. G-CSF production was considerably higher than IL-6 for all C. diphtheriae strains and the C. glutamicum did not induce high cytokine secretion in general. Furthermore, all corynebacteria investigated in this study were able to induce NFκB signaling but only viable C. diphtheriae strains were able to cause host cell damage, whereas C. glutamicum did not. The absence of Mincle resulted in reduced G-CSF production, while no influence on the uptake of the bacteria was observed. In contrast, when MyD88 was absent, both the uptake of the bacteria and cytokine production were blocked. Consequently, phagocytosis only occurs when the TLR/MyD88 pathway is functional, which was also supported by showing that all corynebacteria used in this study interact with human TLR2.

Development of an In Vitro Model of Inflammatory Cytokine Influences on Intervertebral Disk Cells in 3D Cell Culture Using Activated Macrophage-Like THP-1 Cells.

We developed a new model for evaluation of the influence of proinflammatory cytokines on intervertebral disc cells in a 3D culture based on co-culturing of these cells with activated macrophage-like THP-1 cells. The levels of TNFα, IL-1β, IL-6, IL-8, IL-10, and IL-12p70 production were assessed by flow cytofluorometry using microspheres. Considerable differences in the level of spontaneous cytokine secretion by normal and degenerated intervertebral disc cells were revealed. A significant increase in the level of IL-1β and IL-8 was observed during co-culturing, which confirms consistency of the developed model.

Nano-TiO2 stability in medium and size as important factors of toxicity in macrophage-like cells

TiO2 along with nano-TiO2 are commonly found in consumer products. In vivo studies have observed an accumulation of nano-TiO2 in macrophages. However, characteristics of nano-TiO2 determining toxicity remain unclear. In our study, the cytotoxic effects of 14 diverse nano-TiO2 on THP-1 macrophage-like cells were measured by 3 cytotoxicity assays (MTS, WST-1 and LDH). Total averaged cytotoxicity was calculated using principal component analysis. Characteristics of all 14 nano-TiO2 included hydrodynamic diameter, zeta potential, shape, polydispersity index (PDI) and concentration; moreover, crystal form, specific surface area and crystallite size were measured for 10 nano-TiO2.The variables affecting cytotoxicity were chosen using LASSO (least absolute shrinkage and selection operator). Except for concentration, PDI in media measured within 1 h after preparation of the nanomaterial dispersion was selected as a variable affecting cytotoxicity: stable dispersion resulted in higher cytotoxic effects. Crystallite size has been shown to have nonlinear effects (particles of sizes between 20 and 60 nm were cytotoxic while smaller and larger ones were not) and thus it has been excluded from LASSO. The shape (particles/fibre) and crystal form did not affect the cytotoxicity. PDI and the nonlinear effect of size could be an explanation for the inconsistencies of the cytotoxicity of nano-TiO2 in various studies.

Whole-genome expression analysis in THP-1 macrophage-like cells exposed to diverse metal-containing nanomaterials

Whole-genome expression analysis in THP-1 macrophage-like cells exposed to diverse metal-containing nanomaterials

Fumonisin production and toxic capacity in airborne black Aspergilli

This study presents the distribution of fumonisin (FB)-producing and non-producing airborne Aspergilli (Nigri) in apartments (AP), basements (BS) and a grain mill (GM) in Croatia, and their cytotoxic, immunomodulation and genotoxic potency in comparison with FB1 and FB2. Concentration of black Aspergilli was 260-fold higher in GM than in living environment with domination of A. tubingensis and A. welwitschiae. FB2- but not FB1- was confirmed via HPLC-MS and detection of fum1 and fum8 genes for one isolate of A. niger (0.015 μg/mL) and 8/15 isolates of A. welwitschiae (0.128–13.467 μg/mL). After 24 h, both FB1 and FB2 were weakly cytotoxic (MTT assay) to human lung A549 cells and THP-1 macrophage-like cells. In THP-1 cells FB1 but not FB2 provoked a higher increase of IL-8, TNF-α and IL-1β (ELISA). In A549 cells DNA damaging effect of FB1 was slightly higher than that of FB2 (Comet assay). In THP-1 macrophage-like cells A. tubingensis and A. piperis evoked immunomodulatory effects which corresponded to their cytotoxicity (IC50 = 0.228–0.323 mg/mL); A. welwitschiae, A. tubingensis and A. piperis exerted cytotoxic (IC50 = 0.214–0.460 mg/mL) and genotoxic effects in A549 cells. Presumably, secondary metabolites other than FB2 may have contributed to the toxicity of black Aspergilli.

Candida tropicalis induces pro-inflammatory cytokine production, NF-κB and MAPKs pathways regulation, and dectin-1 activation

The prevalence of Candida infection induced by non-albicans Candida (NAC) species is increasing. However, as a common NAC species, C. tropicalis has received much less study in terms of host immunity than C. albicans has. In this study, we evaluated the pro-inflammatory cytokine responses evoked by C. tropicalis and determined whether dectin-1 and downstream NF-κB and mitogen-activated protein kinases (MAPKs) signaling pathways played roles in inflammation in human peripheral blood mononuclear cells (PBMCs) and THP-1 macrophage-like cells. Exposure of PBMCs and THP-1 macrophage-like cells to C. tropicalis led to the enhanced gene expression and secretion of TNF-α and IL-6 in a time- and dose-dependent manner. THP-1 macrophage-like cells being challenged by C. tropicalis resulted in the activation of the NF-κB, p38, and ERK1/2 MAPK signaling pathways. We also found that the expression of dectin-1 was increased with C. tropicalis treatment. These data reveal that dectin-1 may play a role in sensing the inflammation response induced by C. tropicalis and that NF-κB and MAPK are involved in the downstream signaling pathways in macrophages.

Exosome-encapsulated miR-6089 regulates inflammatory response via targeting TLR4.

Exosome-encapsulated microRNAs (miRNAs) have been identified as potential biomarkers in autoimmune diseases. However, little is known about the role of exosome-delivered miRNAs in rheumatoid arthritis (RA). In this study, we investigated the profile of specific exosomal miRNAs by microarray analysis of serum exosomes from three patients with RA and three healthy controls.Quantitative real-time PCR (qRT-PCR) was performed to validate the aberrantly expressed exosomal miRNAs. A total of 20 exosome-encapsulated miRNAs were identified to be differently expressed in the serum of patients with RA compared with controls. Interestingly, we found that exosome-encapsulated miR-6089 was significantly decreased after validation by qRT-PCR in serum exosomes from 76 patients with RA and 20 controls. Besides, miR-6089 could inhibit lipopolysaccharide (LPS)-induced cell proliferation and activation of macrophage-like THP-1 cells. TLR4 was a direct target for miR-6089. MiR-6089 regulated the generation of IL-6, IL-29, and TNF-α by targetedly controlling TLR4 signaling. In conclusion, exosome-encapsulated miR-6089 regulates LPS/TLR4-mediated inflammatory response, which may serve as a novel, promising biomarker in RA.

Decidual stromal cell-derived PGE2 regulates macrophage responses to microbial threat.

Bacterial chorioamnionitis causes adverse pregnancy outcomes, yet host-microbial interactions are not well characterized within gestational membranes. The decidua, the outermost region of the membranes, is a potential point of entry for bacteria ascending from the vagina to cause chorioamnionitis. We sought to determine whether paracrine communication between decidual stromal cells and macrophages shaped immune responses to microbial sensing. Decidual cell-macrophage interactions were modeled in vitro utilizing decidualized, telomerase-immortalized human endometrial stromal cells (dTHESCs) and phorbol ester-differentiated THP-1 macrophage-like cells. The production of inflammatory mediators in response to LPS was monitored by ELISA for both cell types, while phagocytosis of bacterial pathogens (Escherichia coli and Group B Streptococcus (GBS)) was measured in THP-1 cells or primary human placental macrophages. Diclofenac, a non-selective cyclooxygenase inhibitor, and prostaglandin E2 (PGE2 ) were utilized to interrogate prostaglandins as decidual cell-derived paracrine immunomodulators. A mouse model of ascending chorioamnionitis caused by GBS was utilized to assess the colocalization of bacteria and macrophages in vivo and assess PGE2 production. In response to LPS, dTHESC and THP-1 coculture demonstrated enhancement of most inflammatory mediators, but a potent suppression of macrophage TNF-α generation was observed. This appeared to reflect a paracrine-mediated effect of decidual cell-derived PGE2 . In mice with GBS chorioamnionitis, macrophages accumulated at sites of bacterial invasion with increased PGE2 in amniotic fluid, suggesting such paracrine effects might hold relevance in vivo. These data suggest key roles for decidual stromal cells in modulating tissue responses to microbial threat through release of PGE2 .

Roles of Dectin-1 in phagocytosis of Candida albicans by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1

Objective To investigate the roles of Dectin-1 in phagocytosis of Candida albicans (C. albicans) by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1. Methods THP-1 macrophage-like cells served as the target cells, and were transfected with small interfering RNA (siRNA) targeting Dectin-1 to down-regulate the expression of Dectin-1 receptor (siRNA-Dectin-1 group) . THP-1 macrophage-like cells transfected with nonsense siRNA (siRNA-NC) served as a negative control group. After transfection, the THP-1 macrophage-like cells in the above 2 groups were co-cultured with heat-killed C. albicans separately. And then, fluorescence microscopy was performed to count THP-1 macrophage-like cells phagocytosing C. albicans, and flow cytometry was used to determine the mean fluorescence intensity (MFI) of dihydrorhodamine (DHR) -123 fluorescent cells. Statistical analysis was done by one-way analysis of variance (ANOVA) and t test with the SPSS19.0 software. Results After transfection with siRNA-Dectin-1, the mRNA and protein expression of Dectin-1 significantly decreased in THP-1 macrophage-like cells (t= 26.163, P < 0.001) . After 1-, 2-, 4-hour co-culture of THP-1 macrophage-like cells with C. albicans, fluorescence microscopy showed that the phagocytosis rates of C. albicans by THP-1 macrophage-like cells were significantly lower in the siRNA-Dectin-1 group than in the negative control group (17.5% vs. 22.1%, 18.6% vs. 24.3%, 39.2% vs. 59.1%, respectively, all P < 0.05) , so were the percentage of THP-1 macrophage-like cells phagocytosing more than 3 C. albicans cells (2.2% vs. 4.7%, 2.5% vs. 5.4%, 5.1% vs. 8.3%, respectively, all P < 0.05) . After 30-minute, 1-, 2- and 4-hour co-culture of THP-1 macrophage-like cells with DHR-123-labelled C. albicans, flow cytometry showed that the MFI of C. albicans-phagocytosing cells was significantly lower in the siRNA-Dectin-1 group than in the negative control group (36.8 vs. 45.7, 54.3 vs. 62.4, 72.1 vs. 84.9, 93.6 vs. 116.7, respectively, all P < 0.05) . Conclusion Dectin-1 receptor plays an important role in the phagocytosis of C. albicans by macrophages. Key words: Candida albicans; Macrophages; Phagocytosis; Leukemia, monocytic, acute; Dectin-1

Lipid A Has Significance for Optimal Growth of Coxiella burnetii in Macrophage-Like THP-1 Cells and to a Lesser Extent in Axenic Media and Non-phagocytic Cells.

Lipid A is an essential basal component of lipopolysaccharide of most Gram-negative bacteria. Inhibitors targeting LpxC, a conserved enzyme in lipid A biosynthesis, are antibiotic candidates against Gram-negative pathogens. Here we report the characterization of the role of lipid A in Coxiella burnetii growth in axenic media, monkey kidney cells (BGMK and Vero), and macrophage-like THP-1 cells by using a potent LpxC inhibitor -LPC-011. We first determined the susceptibility of C. burnetii LpxC to LPC-011 in a surrogate E. coli model. In E. coli, the minimum inhibitory concentration (MIC) of LPC-011 against C. burnetii LpxC is < 0.05 μg/mL, a value lower than the inhibitor's MIC against E. coli LpxC. Considering the inhibitor's problematic pharmacokinetic properties in vivo and Coxiella's culturing time up to 7 days, the stability of LPC-011 in cell cultures was assessed. We found that regularly changing inhibitor-containing media was required for sustained inhibition of C. burnetii LpxC in cells. Under inhibitor treatment, Coxiella has reduced growth yields in axenic media and during replication in non-phagocytic cells, and has a reduced number of productive vacuoles in such cells. Inhibiting lipid A biosynthesis in C. burnetii by the inhibitor was shown in a phase II strain transformed with chlamydial kdtA. This exogenous KdtA enzyme modifies Coxiella lipid A with an α-Kdo-(2 → 8)-α-Kdo epitope that can be detected by anti-chlamydia genus antibodies. In inhibitor-treated THP-1 cells, Coxiella shows severe growth defects characterized by poor vacuole formation and low growth yields. Coxiella progenies prepared from inhibitor-treated cells retain the capability of normally infecting all tested cells in the absence of the inhibitor, which suggests a dispensable role of lipid A for infection and early vacuole development. In conclusion, our data suggest that lipid A has significance for optimal development of Coxiella-containing vacuoles, and for robust multiplication of C. burnetii in macrophage-like THP-1 cells. Unlike many bacteria, C. burnetii replication in axenic media and non-phagocytic cells was less dependent on normal lipid A biosynthesis.

Variation in Macrophage Phagocytosis of Streptococcus agalactiae Does Not Reflect Bacterial Capsular Serotype, Multilocus Sequence Type or Association with Invasive Infection

Background:Group B Streptococcus (GBS) is an encapsulated Gram-positive coccus that is an important cause of infections in adults with chronic medical conditions, pregnant women, and neonates. GBS causes a range of clinical syndromes, from asymptomatic colonization to deep-seated invasive and highly lethal infections. Macrophages are important sentinels of innate immunity, protecting host tissues from infection when bacteria advance beyond cutaneous or mucosal barriers. We hypothesized that the capacity for macrophages to phagocytose unopsonized GBS would vary across distinct clinical strains, and such differences would reflect serotype diversity.Methods:A high-throughput screen using the phorbol ester-differentiated THP-1 macrophage-like human cell line was used to quantify phagocytosis of a diverse group of 35 different human clinical isolates of GBS representing a wide variety of capsular serotypes. Validation studies were conducted using human primary phagocytes.Results:Phagocytosis of GBS differed widely across clinical isolates but this was not related to capsular serotype, genetic sequence type, pilus type, or clinical source of the GBS isolate (colonizing or invasive strain).Conclusions:Structural and/or biochemical differences among diverse GBS strains are reflected in a diverse capacity for macrophages to ingest them through non-opsonic phagocytosis. Mechanisms explaining these differences are not clear.

The Role of AIRE in the Immunity Against Candida Albicans in a Model of Human Macrophages.

Autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a primary immunodeficiency caused by mutations in the autoimmune regulator gene (AIRE). Patients with AIRE mutations are susceptible to Candida albicans infection and present with autoimmune disorders. We previously demonstrated that cytoplasmic AIRE regulates the Syk-dependent Dectin-1 pathway. In this study, we further evaluated direct contact with fungal elements, synapse formation, and the response of macrophage-like THP-1 cells to C. albicans hyphae to determine the role of AIRE upon Dectin receptors function and signaling. We examined the fungal synapse (FS) formation in wild-type and AIRE-knockdown THP-1 cells differentiated to macrophages, as well as monocyte-derived macrophages from APECED patients. We evaluated Dectin-2 receptor signaling, phagocytosis, and cytokine secretion upon hyphal stimulation. AIRE co-localized with Dectin-2 and Syk at the FS upon hyphal stimulation of macrophage-like THP-1 cells. AIRE-knockdown macrophage-like THP-1 cells exhibited less Dectin-1 and Dectin-2 receptors accumulation, decreased signaling pathway activity at the FS, lower C. albicans phagocytosis, and less lysosome formation. Furthermore, IL-1β, IL-6, or TNF-α secretion by AIRE-knockdown macrophage-like THP-1 cells and AIRE-deficient patient macrophages was decreased compared to control cells. Our results suggest that AIRE modulates the FS formation and hyphal recognition and help to orchestrate an effective immune response against C. albicans.

Porous 3D Prussian blue/cellulose aerogel as a decorporation agent for removal of ingested cesium from the gastrointestinal tract

In the present study, we successfully synthesized a porous three-dimensional Prussian blue-cellulose aerogel (PB-CA) composite and used it as a decorporation agent for the selective removal of ingested cesium ions (Cs+) from the gastrointestinal (GI) tract. The safety of the PB-CA composite was evaluated through an in vitro cytotoxicity study using macrophage-like THP-1 cells and Caco-2 intestinal epithelial cells. The results revealed that the PB-CA composite was not cytotoxic. An adsorption study to examine the efficiency of the decorporation agent was conducted using a simulated intestinal fluid (SIF). The adsorption isotherm was fitted to the Langmuir model with a maximum Cs+ adsorption capacity of 13.70 mg/g in SIF that followed pseudo-second-order kinetics. The PB-CA composite showed excellent stability in SIF with a maximum Cs+ removal efficiency of 99.43%. The promising safety toxicology profile, remarkable Cs+ adsorption efficacy, and excellent stability of the composite demonstrated its great potential for use as an orally administered drug for the decorporation of Cs+ from the GI tract.

CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation

ABSTRACTThe macroautophagy/autophagy and inflammasome pathways are linked through their roles in innate immunity and chronic inflammatory disease. Ceramide-1-phosphate (C1P) is a bioactive sphingolipid that regulates pro-inflammatory eicosanoid production. Whether C1P also regulates autophagy and inflammasome assembly/activation is not known. Here we show that CPTP (a protein that traffics C1P from its site of phosphorylation in the trans-Golgi to target membranes) regulates both autophagy and inflammasome activation. In human epithelial cells, knockdown of CPTP (but not GLTP [glycolipid transfer protein]) or expression of C1P binding-site point mutants, stimulated an 8- to 10-fold increase in autophagosomes and altered endogenous LC3-II and SQSTM1/p62 protein expression levels. CPTP depletion-induced autophagy elevated early markers of autophagosome formation (Golgi-derived ATG9A-vesicles, WIPI1), required key phagophore assembly and elongation factors (ATG5, ATG7, ULK1), and suppressed MTOR phosphorylation and that of its downstream target, RPS6KB1/p70S6K. Wild-type CPTP overexpression exerted a protective effect against starvation-induced autophagy. In THP-1 macrophage-like surveillance cells, CPTP knockdown induced not only autophagy but also elevated CASP1/caspase-1 levels, and strongly increased IL1B/interleukin-1β and IL18 release via a NLRP3 (but not NLRC4) inflammasome-based mechanism, while only moderately increasing inflammatory (pyroptotic) cell death. Inflammasome assembly and activation stimulated by CPTP depletion were autophagy dependent. Elevation of intracellular C1P by exogenous C1P treatment (instead of CPTP inhibition) also induced autophagy and IL1B release. Our findings identify human CPTP as an endogenous regulator of early-stage autophagosome assembly and inflammasome-driven, pro-inflammatory cytokine generation and release.

α- and β-d-Glucans from the edible mushroom Pleurotus albidus differentially regulate lipid-induced inflammation and foam cell formation in human macrophage-like THP-1 cells

Macrophages play an essential role in lipid metabolism; however, the excessive uptake of modified lipids and cholesterol crystals (CC) leads to the formation of pro-inflammatory lipid-laden macrophages called foam cells. Since the α-1,6- and β-1,3-d-glucans from the basidiome and the mycelium of the edible mushroom Pleurotus albidus have previously been shown to regulate macrophage function, these glucans were tested in macrophage-like THP-1 cells previously exposed to acetylated low-density lipoproteins (acLDL) or CC. The glucans inhibited lipid-induced inflammation, but only the β-1,3-d-glucan regulated both the NLRP3 inflammasome activation and the expression of genes involved on lipid efflux in acLDL- or CC-pretreated cells, thereby reducing foam cell formation. In contrast, the two α-1,6-glucans tested inhibited foam cell formation only in acLDL-pretreated cells and had no effect on the expression of the peroxisome proliferator-activated receptor gamma and liver X receptor alpha genes, suggesting that these glucans regulate lipid influx rather than lipid efflux. Thus, α- and β-d-glucans differentially regulate lipid-induced inflammation and foam cell formation in macrophage-like cells. Furthermore, results emphasize that P. albidus has potential to be used as a functional food or as a source for the extraction of biologically-active glucans.

Expression of immune-related molecules in THP-1 macrophage-like cells exposed to nanoparticles

Expression of immune-related molecules in THP-1 macrophage-like cells exposed to nanoparticles

Effects of the yeast form of Sporothrix schenckii on activation of p38MAPK and expression of interleukin-6 in human THP-1 macrophage-like cells

Objective To evaluate effects of the yeast form of Sporothrix schenckii on activation of p38 mitogen-activated protein kinase (p38MAPK) and expression of interleukin-6 (IL-6) in macrophage-like THP-1 cells, which were differentiated from the human acute monocytic leukemia cell line THP-1. Methods THP-1 macrophage-like cells were divided into 3 groups to be treated with the yeast form of Sporothrix schenckii at a concentration of 2 × 106 colony-forming units (CFU) /ml (yeast form group) , 100 mg/L curdlan (curdlan group) and RPMI 1640 medium (blank control group) respectively. Real-time fluorescence-based quantitative PCR was performed to measure the mRNA expression of IL-6 in THP-1 macrophage-like cells in the above 3 groups after 3-and 6-hour treatment separately, and enzyme-linked immunosorbent assay (ELISA) to detect the level of IL-6 in the culture supernatant of THP-1 macrophage-like cells after 24-hour treatment. Western blot analysis was conducted to determine the protein expression of p38MAPK and phosphorylated p38MAPK (p-p38MAPK) in the above 3 groups after 30-and 60-minute treatment separately. Other THP-1 macrophage-like cells were pretreated with 100 nmol/L dexamethasone (a p38MAPK inhibitor) for 30 minutes, and then were divided into 3 groups to be treated with the yeast form of Sporothrix schenckii, curdlan and RPMI 1640 medium respectively, and changes in the level of p-p38MAPK and mRNA expression of IL-6 were also detected. Statistical analysis was carried out with SPSS19.0 software by using one-way or multi-way analysis of variance and least significant difference (LSD) test. Results Significant differences in the mRNA expression of IL-6 in THP-1 macrophage-like cells were observed among the yeast form group, curdlan group and blank control group (F= 5 552.22, P < 0.001) after 3-hour treatment (56.81 ± 7.36, 26.69 ± 1.22 and 0.97 ± 0.05, respectively) and 6-hour treatment (378.03 ± 16.67, 276.24 ± 39.13 and 1.02 ± 0.04, respectively) . Additionally, the yeast form group showed significantly higher mRNA expression of IL-6 after 6-hour treatment than that after 3-hour treatment (q= 16.74, P < 0.001) . After 24-hour treatment, the level of IL-6 in the culture supernatant of THP-1 macrophage-like cells also significantly differed among the yeast form group, curdlan group and blank control group (59.96 ± 18.16 pg/L, 91.01 ± 17.27 pg/L, 5.50 ± 2.30 pg/L, respectively; F= 26.62, P < 0.01) , and was significantly higher in the yeast form group than in the blank control group (P < 0.01) . After 30-and 60-minute treatment, the protein expression of p-p38MAPK was significantly higher in the yeast form group than in the blank control group (both P < 0.01) . Moreover, the mRNA expression of IL-6 (4.46 ± 1.03 vs. 493.52 ± 113.87, P < 0.001) and protein expression of p-p38MAPK (2.29 ± 0.37 vs. 4.55 ± 0.46, q= 10.81, P < 0.01) were both significantly lower in the yeast form group with dexamethasone pretreatment than in that without dexamethasone pretreatment. Conclusion In vitro treatment with the yeast form of Sporothrix schenckii can enhance the expression of IL-6 in human THP-1 macrophage-like cells by activating the p38MAPK signaling pathway. Key words: Sporothrix; Macrophages; Interleukin-6; p38 Mitogen-activated protein kinases

Airborne Nanoparticle Release and Toxicological Risk from Metal-Oxide-Coated Textiles: Toward a Multiscale Safe-by-Design Approach

Nano metal oxides have been proposed as alternatives to silver (Ag) nanoparticles (NPs) for antibacterial coatings. Here, cotton and polyester-cotton fabrics were sonochemically coated with zinc oxide (ZnO) and copper oxide (CuO) NPs. By varying the reaction solvent (water or ethanol), NPs with different sizes and shapes were synthesized. The cytotoxic and pro-inflammatory effects of studied NPs were investigated in vitro in human alveolar epithelial A549 and macrophage-like THP1 cells. To understand the potential respiratory impact of the NPs, the coated textiles were subjected to the abrasion tests, and the released airborne particles were measured. A very small amount of the studied metal oxides NPs was released from abrasion of the textiles coated by the ethanol-based sonochemical process. The release from the water-based coating was comparably higher. Lung and immune cells viability decreased after 24 h of exposure only at the highest studied NPs concentration (100 μg/mL). Different from the ZnO NPs, both formulations of CuO NPs induced IL-8 release in the lung epithelial cells already at subtoxic concentrations (1-10 μg/mL) but not in immune cells. All of the studied NPs did not induce IL-6 release by the lung and immune cells. Calculations revealed that the exposures of the NPs to human lung due to the abrasion of the textiles were lower or comparable to the minimum doses in the cell viability tests (0.1 μg/mL), at which acute cytotoxicity was not observed. The results alleviate the concerns regarding the potential risk of these metal oxide NPs in their applications for the textile coating and provide insight for the safe-by-design approach.