Photoaging of polystyrene-based microplastics amplifies inflammatory response in macrophages.

The continuous release of municipal and industrial products into the environment poses a growing concern for public health. Among environmental pollutants, polystyrene (PS) stands out as a primary constituent of environmental plastic waste, given its widespread use and high production rates owing to its durability and user-friendly properties. The detection of polystyrene microparticles (PS-MPs) in various living organisms has been well-documented, posing a serious threat due to their potential passage into the human ecosystem. In this manuscript, we aimed to study the toxicological effects of low concentrations of pristine and photoaged PS-MPs in a murine macrophage cell line. To this purpose, PS-MPs were photoaged by indoor exposure to visible light to simulate environmental weathering due to solar irradiation (PS-MPs3h). Physical characterization revealed that the irradiation treatment results in particle degradation and the possible release of nanoparticles. Monocultures of the RAW264.7 cell line were then exposed to PS-MPs and PS-MPs3h at concentrations comparable to experimental measurements from biological samples, to assess cytotoxicity, intracellular oxidative stress, primary genotoxicity, and inflammatory effects. Significant toxicity-related outcomes were observed in cells treated with both pristine PS-MPs and PS-MPs3h even at low concentrations (0,10 µg/ml and 1 µg/ml). PS-MPs3h exhibited greater adverse effects compared to PS-MPs, including reduced cell viability, increased ROS production, elevated DNA damage, and upregulation of IL-6 and NOS2 gene expression. Therefore, we can conclude that changes induced by environmental aging in the physicochemical composition of PS microplastics play a crucial role in the adverse health outcomes associated with microplastic exposure.

Chemical Characterization, Free Radical Scavenging, and Cellular Antioxidant Properties of the Egadi Island Endemic Brassica macrocarpa Guss Leaf Extract.

The genus Brassica is an important source of food in the Mediterranean diet with documented nutritional and medicinal properties. However, few studies have investigated the phytochemical composition and the biological activity of wild Sicilian taxa. Thus, we aimed to study the chemical profile and the antioxidant potential, in vitro and in LPS-stimulated RAW 264.7 cells, of a methanolic extract of leaves of wild Brassica macrocarpa Guss (B. macrocarpa) (Egadi Islands; Sicily-Italy). B. macrocarpa methanolic extract showed a large amount of glucosinolates and different phenolic compounds. It exhibited antioxidant activity in the DPPH assay and in LPS-stimulated RAW 264.7 cells, being able to reduce NO and ROS levels and NOS2 mRNA expression. Our study demonstrated that Sicilian B. macrocarpa methanolic extract, in LPS-stimulated macrophages, efficiently counteracts oxidative stress and displays radical scavenging activity. Future studies are required to identify the contribution of the single phytocomponents, to characterize the action mechanism, and to reveal possible applications in human health.

Extracellular Vesicles and Immunity: At the Crossroads of Cell Communication.

Extracellular vesicles (EVs), comprising exosomes and microvesicles, are small membranous structures secreted by nearly all cell types. They have emerged as crucial mediators in intercellular communication, playing pivotal roles in diverse physiological and pathological processes, notably within the realm of immunity. These roles go beyond mere cellular interactions, as extracellular vesicles stand as versatile and dynamic components of immune regulation, impacting both innate and adaptive immunity. Their multifaceted involvement includes immune cell activation, antigen presentation, and immunomodulation, emphasising their significance in maintaining immune homeostasis and contributing to the pathogenesis of immune-related disorders. Extracellular vesicles participate in immunomodulation by delivering a wide array of bioactive molecules, including proteins, lipids, and nucleic acids, thereby influencing gene expression in target cells. This manuscript presents a comprehensive review that encompasses in vitro and in vivo studies aimed at elucidating the mechanisms through which EVs modulate human immunity. Understanding the intricate interplay between extracellular vesicles and immunity is imperative for unveiling novel therapeutic targets and diagnostic tools applicable to various immunological disorders, including autoimmune diseases, infectious diseases, and cancer. Furthermore, recognising the potential of EVs as versatile drug delivery vehicles holds significant promise for the future of immunotherapies.

The environmental pollutant BDE-47 modulates immune responses in invitro and in vivo murine models.

2,2',4,4'-tetra-bromodiphenyl ether (BDE-47) is widespread in the environment and biological samples. Its association with health risks is an increasing concern, yet information on BDE-47 immunotoxicity remains limited. This study investigated the impact of BDE-47 on innate and adaptive immune responses through in vitro and in vivo approaches. BDE-47's capacity to directly induce cell responses and modulate responses induced by known stimuli was studied in vitro using the RAW 264.7 murine macrophage cell line and spleen-derived lymphocytes, and in vivo using keyhole limpet hemocyanin (KLH)-immunized BALB/c mice orally administered (28 d) at dose levels (7.5, 15.0 and 30 mg/kg/bw/d) derived from relevant toxicokinetic data from rodent models. RAW 264.7 cells stimulated with lipopolysaccharide (LPS) and exposed to BDE-47 exhibited unchanged cell viability but decreased release of interleukin (IL)-6. Primary splenocytes from naïve mice stimulated with anti-CD3/anti-CD28 antibodies and exposed to BDE-47 showed a significant decrease of IL-17 A and IFNγ production. In vivo data showed that BDE-47 significantly reduced the KLH-specific antibody response. A generally decreasing trend of IFNγ, IL-10 and IL-5 production was observed after in vitro antigen-specific restimulation of spleen cells. Histopathological effects on liver, spleen, small intestine and thyroid were detected at the highest dose in the absence of general toxicity. In addition, the expression of Mm_mir155 and Mm_let7a was induced in livers of exposed mice. The data obtained in this study suggest that exposure to BDE-47 may perturb innate and adaptive immune responses, thus possibly decreasing resistance to bacterial and viral infections.

Extracellular vesicles from PBDE-47 treated M(LPS) THP-1 macrophages modulate the expression of markers of epithelial integrity, EMT, inflammation and muco-secretion in ALI culture of airway epithelium.

AIMS: The lung epithelial cells form a physical barrier to the external environment acting as the first line of defence against potentially harmful environmental stimuli. These cells interact with several other cellular components, of which macrophages are some of the most relevant. We analysed the effects of the PBDE-47 on the microRNA cargo of THP-1 macrophage like derived small Extracellular Vesicles (sEVs) and the effects on A549 lung epithelial cells. MAIN METHODS: sEVs from M(LPS) THP-1 macrophage-like cells after PBDE-47 treatment (sEVsPBDE+LPS) were characterized by nanoparticle tracking analysis and their microRNA cargo studied by qPCR. Confocal microscopy was applied to study sEVs cellular uptake by A549 cells. The expression of tight junctions (TJs), adhesion molecules, inflammation markers and mucus production in A549 cultured in air liquid interface (ALI) conditions were studied by Real Time PCR and confocal microscopy. KEY FINDINGS: sEVsPBDE+LPS microRNA cargo analysis showed that the PBDE-47 modulated the expression of the miR-15a-5p, miR29a-3p, miR-143-3p and miR-122-5p. Furthermore, ALI cultured A549 cells incubated with sEVsPBDE+LPS showed that zonula occludens-1 (p ≤ 0.04), claudin (p ≤ 0.02), E-cadherin (p ≤ 0.006) and Vimentin (p ≤ 0.0008) mRNAs were increased in A549 cells after sEVsPBDE+LPS treatment. Indeed, Interleukin (IL)-8 (p ≤ 0.008) and mucin (MUC5AC and MUC5B) (p ≤ 0.03 and p ≤ 0.0001) mRNA expression were up- and down-regulated, respectively. SIGNIFICANCE: PBDE-47 treated macrophages secrete sEVs with altered microRNA cargo that affect the mRNA expression of TJs, adhesion molecules, cytokines and EMT markers damaging the normal function of the lung epithelium, potentially contributing to the development of lung diseases.

Impact of the flame retardant 2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) in THP-1 macrophage-like cell function via small extracellular vesicles.

2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) is one of the most widespread environmental brominated flame-retardant congeners which has also been detected in animal and human tissues. Several studies have reported the effects of PBDEs on different health issues, including neurobehavioral and developmental disorders, reproductive health, and alterations of thyroid function. Much less is known about its immunotoxicity. The aim of our study was to investigate the effects that treatment of THP-1 macrophage-like cells with PBDE-47 could have on the content of small extracellular vesicles' (sEVs) microRNA (miRNA) cargo and their downstream effects on bystander macrophages. To achieve this, we purified sEVs from PBDE-47 treated M(LPS) THP-1 macrophage-like cells (sEVsPBDE+LPS) by means of ultra-centrifugation and characterized their miRNA cargo by microarray analysis detecting the modulation of 18 miRNAs. Furthermore, resting THP-1 derived M(0) macrophage-like cells were cultured with sEVsPBDE+LPS, showing that the treatment reshaped the miRNA profiles of 12 intracellular miRNAs. This dataset was studied in silico, identifying the biological pathways affected by these target genes. This analysis identified 12 pathways all involved in the maturation and polarization of macrophages. Therefore, to evaluate whether sEVsPBDE+LPS can have some immunomodulatory activity, naïve M(0) THP-1 macrophage-like cells cultured with purified sEVsPBDE+LPS were studied for IL-6, TNF-α and TGF-ß mRNAs expression and immune stained with the HLA-DR, CD80, CCR7, CD38 and CD209 antigens and analyzed by flow cytometry. This analysis showed that the PBDE-47 treatment does not induce the expression of specific M1 and M2 cytokine markers of differentiation and may have impaired the ability to make immunological synapses and present antigens, down-regulating the expression of HLA-DR and CD209 antigens. Overall, our study supports the model that perturbation of miRNA cargo by PBDE-47 treatment contributes to the rewiring of cellular regulatory pathways capable of inducing perturbation of differentiation markers on naïve resting M(0) THP-1 macrophage-like cells.

2,2'4,4'-Tetrabromodiphenyl Ether (PBDE-47) Modulates the Intracellular miRNA Profile, sEV Biogenesis and Their miRNA Cargo Exacerbating the LPS-Induced Pro-Inflammatory Response in THP-1 Macrophages.

The 2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) is one of the most prominent PBDE congeners detected in the environment and in animal and human tissues. Animal model experiments suggested the occurrence of PBDE-induced immunotoxicity leading to different outcomes and recently we demonstrated that this substance can impair macrophage and basophil activities. In this manuscript, we decided to further examine the effects induced by PBDE-47 treatment on innate immune response by looking at the intracellular expression profile of miRNAs as well as the biogenesis, cargo content and activity of human M(LPS) macrophage cell-derived small extracellular vesicles (sEVs). Microarray and in silico analysis demonstrated that PBDE-47 can induce some epigenetic effects in M(LPS) THP-1 cells modulating the expression of a set of intracellular miRNAs involved in biological pathways regulating the expression of estrogen-mediated signaling and immune responses with particular reference to M1/M2 differentiation. In addition to the cell-intrinsic modulation of intracellular miRNAs, we demonstrated that PBDE-47 could also interfere with the biogenesis of sEVs increasing their number and selecting a de novo population of sEVs. Moreover, PBDE-47 induced the overload of specific immune related miRNAs in PBDE-47 derived sEVs. Finally, culture experiments with naïve M(LPS) macrophages demonstrated that purified PBDE-47 derived sEVs can modulate macrophage immune response exacerbating the LPS-induced pro-inflammatory response inducing the overexpression of the IL-6 and the MMP9 genes. Data from this study demonstrated that PBDE-47 can perturb the innate immune response at different levels modulating the intracellular expression of miRNAs but also interfering with the biogenesis, cargo content and functional activity of M(LPS) macrophage cell-derived sEVs.

Identification of an LPS-Induced Chemo-Attractive Peptide from Ciona robusta.

BACKGROUND: Previously published work has demonstrated that the LPS injection of Ciona robusta leads to the overexpression of a truncated form of an immune-related mRNA (C8short) by means of Ciona robusta (CR) alternative polyadenylation (APA) (CR-APA). METHODS: The 3D structure of the C8short-derived Ciona robusta chemo-attractive peptide (CrCP) was evaluated by homology modeling. The biological activity of the CrCP was studied in vitro using a primary human dermal cell line (HuDe). Real-Time PCR was used to investigate the expression levels of genes involved in cell motility. NF-κB signaling was studied by western blotting. RESULTS: In silico modeling showed that CrCP displayed structural characteristics already reported for a short domain of the vertebrate CRK gene, suggesting its possible involvement in cell migration mechanisms. In vitro assays demonstrated that CrCP was capable of inducing the motility of HuDe cells in both wound healing and chemo-attractive experiments. qPCR demonstrated the capability of CrCP to modulate the expression of the matrix metalloproteinase-7 (MMP-7) and E-cadherin genes. Finally, western blot analysis demonstrated that treatment with CrCP induced activation of the NF-κB signaling pathway. CONCLUSION: Our results describe the characterization of the 3D structure and chemo-attractive activity of an LPS-induced CrCP peptide from Ciona robusta.

In vitro exposure to 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) impairs innate inflammatory response.

Polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants that are added to numerous products to prevent accidental fires. PBDEs are present in the environment and they bio-accumulate in human and animal tissues. Recently, their presence has been correlated to several pathologies but little is known about their effect on the human innate immune system activity. In this study we investigated the effect of the congener 2,2',4,4'-Tetrabromodiphenyl ether (PBDE-47) on the functional activity of the THP-1 human macrophages cell line and on ex vivo freshly isolated human basophils. Cytotoxicity and genotoxicity studies showed that PBDE-47 was able to induce toxic effects on the THP-1 cell line viability at concentrations ≥25 µM. Immune function of THP-1 was studied after stimulation with bacterial lipopolysaccharide (LPS) and PBDE-47 exposure at concentrations granting macrophage viability. Two dimensional electrophoresis showed modification of the proteome in the 3 µM PBDE-47 treated sample and Real Time PCR and ELISA demonstrated a statistically significant reduction in the expression of IL-1ß, IL-6 and TNF-α cytokines. Furthermore, PBDE-47 was able to perturbate genes involved in cell motility upregulating CDH-1 and downregulating MMP-12 expressions. Finally, basophil activation assay showed reduced CD63 activation in PBDE-47 treated samples. In conclusion, our study demonstrated that PBDE-47 may perturb the activities of cells involved in innate immunity dampening the expression of macrophage pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) and genes involved in cell motility (MMP-12 and E-cadherin) and interfering with basophil activation suggesting that this compound can impair innate immune response.

Relationship between domestic smoking and metals and rare earth elements concentration in indoor PM2.5.

Cigarette smoke is the main source of indoor chemical and toxic elements. Cadmium (Cd), Thallium (Tl), Lead (Pb) and Antimony (Sb) are important contributors to smoke-related health risks. Data on the association between Rare Earth Elements (REE) Cerium (Ce) and Lanthanum (La) and domestic smoking are scanty. To evaluate the relationship between cigarette smoke, indoor levels of PM2.5 and heavy metals, 73 children were investigated by parental questionnaire and skin prick tests. The houses of residence of 41 "cases" and 32 "controls" (children with and without respiratory symptoms, respectively) were evaluated by 48-h PM2.5 indoor/outdoor monitoring. PM2.5 mass concentration was determined by gravimetry; the extracted and mineralized fractions of elements (As, Cd, Ce, La, Mn, Pb, Sb, Sr, Tl) were evaluated by ICP-MS. PM2.5 and Ce, La, Cd, and Tl indoor concentrations were higher in smoker dwellings. When corrected for confounding factors, PM2.5, Ce, La, Cd, and Tl were associated with more likely presence of respiratory symptoms in adolescents. We found that: i) indoor smoking is associated with increased levels of PM2.5, Ce, La, Cd, and Tl and ii) the latter with increased presence of respiratory symptoms in children.

A pilot study of brisk walking in sedentary combination antiretroviral treatement (cART)- treated patients: benefit on soluble and cell inflammatory markers.

BACKGROUND: Chronic HIV infection is associated with low-level inflammation and increased risk of chronic diseases and mortality. The objective was to assess the effects of moderate intensity exercise on metabolic and inflammatory markers in HIV-infected treated persons. METHODS: This was a pilot study enrolling cART-treated, sedentary persons with metabolic complications in a 12-week protocol, consisting of three sessions per week of 60 min brisk walking with (strength-walk group) or without (walk group) 30 min circuit-training. Assessments at baseline and week 12 (W12) included body morphometrics and total body dual-energy X-ray absorptiometry; lipid and glucose blood profile; plasma level of high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), D-dimer, interleukin-18 (IL-18), soluble CD14, and CD38 and HLA-DR expression on CD4+ and CD8+ T-cells. RESULTS: Forty-nine patients were included and 35 (71%) completed the program: 21 in the walk and 14 in the strength-walk group. At W12, significant improvements were observed of body mass index, waist and hip circumference, and total cholesterol both overall and in the walk group, and of LDL cholesterol in both training groups. In the whole group, significant reductions were observed in hsCRP, IL-6, D-dimer, IL-18, and of CD8+/CD38+/HLA-DR+ cell frequencies. HsCRP and CD8+/CD38+/HLA-DR+ frequency decreased significantly in both training groups when examined separately whereas IL-6 and D-dimer in the walk group only. CONCLUSIONS: Brisk walking, with or without strength exercise, could improve lipid profile and inflammatory markers in chronic HIV infection. TRIAL REGISTRATION: ACTRN12615001258549, registered 17 November 2015, "retrospectively registered" Web address of trial: http://www.ANZCTR.org.au/ACTRN12615001258549.aspx.

LPS injection reprograms the expression and the 3' UTR of a CAP gene by alternative polyadenylation and the formation of a GAIT element in Ciona intestinalis.

The diversification of cellular functions is one of the major characteristics of multicellular organisms which allow cells to modulate their gene expression, leading to the formation of transcripts and proteins with different functions and concentrations in response to different stimuli. CAP genes represent a widespread family of proteins belonging to the cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 superfamily which, it has been proposed, play key roles in the infection process and the modulation of immune responses in host animals. The ascidian Ciona intestinalis represents a group of proto-chordates with an exclusively innate immune system that has been widely studied in the field of comparative and developmental immunology. Using this biological system, we describe the identification of a novel APA mechanism by which an intronic polyadenylation signal is activated by LPS injection, leading to the formation of a shorter CAP mRNA capable of expressing the first CAP exon plus 19 amino acid residues whose sequence is contained within the first intron of the annotated gene. Furthermore, such an APA event causes the expression of a translational controlling cis-acting GAIT element which is not present in the previously isolated CAP isoform and identified in the 3'-UTR of other immune-related genes, suggesting an intriguing scenario in which both transcriptional and post-transcriptional control mechanisms are involved in the activation of the CAP gene during inflammatory response in C. intestinalis.

JC polyomavirus mutants escape antibody-mediated neutralization.

JC polyomavirus (JCV) persistently infects the urinary tract of most adults. Under conditions of immune impairment, JCV causes an opportunistic brain disease, progressive multifocal leukoencephalopathy (PML). JCV strains found in the cerebrospinal fluid of PML patients contain distinctive mutations in surface loops of the major capsid protein, VP1. We hypothesized that VP1 mutations might allow the virus to evade antibody-mediated neutralization. Consistent with this hypothesis, neutralization serology revealed that plasma samples from PML patients neutralized wild-type JCV strains but failed to neutralize patient-cognate PML-mutant JCV strains. This contrasted with serological results for healthy individuals, most of whom robustly cross-neutralized all tested JCV variants. Mice administered a JCV virus-like particle (VLP) vaccine initially showed neutralizing "blind spots" (akin to those observed in PML patients) that closed after booster immunization. A PML patient administered an experimental JCV VLP vaccine likewise showed markedly increased neutralizing titer against her cognate PML-mutant JCV. The results indicate that deficient humoral immunity is a common aspect of PML pathogenesis and that vaccination may overcome this humoral deficiency. Thus, vaccination with JCV VLPs might prevent the development of PML.

Isolation of a novel LPS-induced component of the ML superfamily in Ciona intestinalis.

ML superfamily represents a group of proteins playing important roles in lipid metabolism and innate immune response. In this study, we report the identification of the first component of the ML superfamily in the invertebrate Ciona intestinalis by means of a subtractive hybridization strategy. Sequence homology and phylogenetic analysis showed that this protein forms a specific clade with vertebrate components of the Niemann-Pick type C2 protein and, for this reason, it has been named Ci-NPC2. The putative Ci-NPC2 is a 150 amino acids long protein with a short signal peptide, seven cysteine residues, three putative lipid binding site and a three-dimensional model showing a characteristic ß-strand structure. Gene expression analysis demonstrated that the Ci-NPC2 protein is positively upregulated after LPS inoculum with a peak of expression 1 h after challenge. Finally, in-situ hybridization demonstrated that the Ci-NPC2 protein is preferentially expressed in hemocytes inside the vessel lumen.

Cloning and expression of a novel component of the CAP superfamily enhanced in the inflammatory response to LPS of the ascidian Ciona intestinalis.

The CAP superfamily is a group of proteins that have been linked to several biological functions such as reproduction, cancer, and immune defense. A differential screening between lipopolysaccharide (LPS)-challenged and naive Ciona intestinalis has been performed to identify LPS-induced genes. This strategy has allowed the isolation of a full-length 1471-bp cDNA encoding for a 413-amino-acid protein (CiCAP). In silico analysis has shown that this polypeptide displays a modular structure with similarities to vertebrate CAP-superfamily proteins and to a collagen-binding adhesin of Streptococcus mutans. Domain organization analysis and alignment of CiCAP to other vertebrate CAP proteins have revealed a novel structure suggesting that this protein originated from a common ancestor gene that gave rise to many subfamilies of mosaic proteins with novel functions. Quantitative mRNA expression performed by real-time polymerase chain reaction analysis has demonstrated that this gene is rapidly activated in the pharynx of C. intestinalis a few hours after LPS injection. Moreover, in situ hybridization has shown that CiCAP mRNA is highly expressed by hemocytes with large granules contained inside the pharynx vessels. Thus, CiCAP represents a protein with novel structural domains involved in ascidian immune responses.

Isolation and expression of a novel MBL-like collectin cDNA enhanced by LPS injection in the body wall of the ascidian Ciona intestinalis.

Collectins are a family of calcium-dependent lectins that are characterized by their collagen-like domains. Considerable interest has been focused on this class of proteins because of their ability to interact with components of the complement system activating a cascade of events responsible for the activation of the innate immune system. A differential screening between LPS-challenged and naïve Ciona intestinalis has been performed allowing the isolation of a full length cDNA encoding for a 221 AA protein. In silico analysis has shown that this polypeptide displays protein domains with similarities to mannose-binding lectins. A phylogenetic analysis suggested that C. intestinalis MBL has evolved early as a prototype of vertebrate MBL. Real-time PCR assay demonstrated that this gene is strongly activated after LPS injection in the tunica. In situ hybridization performed in LPS-induced animals has shown that this gene is expressed in granular amoebocytes and large granules hemocytes in the inflamed body wall tissue. Finally, an antimicrobial activity of the C. intestinalis MBL has been demonstrated.