Redox-activity and in vitro effects of regional atmospheric aerosol pollution: Seasonal differences and correlation between oxidative potential and in vitro toxicity of PM1.

Particulate Matter (PM) is a complex and heterogeneous mixture of atmospheric particles recognized as a threat to human health. Oxidative Potential (OP) measurement is a promising and integrative method for estimating PM-induced health impacts since it is recognized as more closely associated with adverse health effects than ordinarily used PM mass concentrations. OP measurements could be introduced in the air quality monitoring, along with the parameters currently evaluated. PM deposition in the lungs induces oxidative stress, inflammation, and DNA damage. The study aimed to compare the OP measurements with toxicological effects on BEAS-2B and THP-1 cells of winter and summer PM1 collected in the Po Valley (Italy) during 2021. PM1 was extracted in deionized water by mechanical agitation and tested for OP and, in parallel, used to treat cells. Cytotoxicity, genotoxicity, oxidative stress, and inflammatory responses were assessed by MTT test, DCFH-DA assay, micronucleus, γ-H2AX, comet assay modified with endonucleases, ELISA, and Real-Time PCR. The evaluation of OP was performed by applying three different assays: dithiothreitol (OPDTT), ascorbic acid (OPAA), and 2',7'-dichlorofluorescein (OPDCFH), in addition, the reducing potential was also analysed (RPDPPH). Seasonal differences were detected in all the parameters investigated. The amount of DNA damage detected with the Comet assay and ROS formation highlights the presence of oxidative damage both in winter and in summer samples, while DNA damage (micronucleus) and genes regulation were mainly detected in winter samples. A positive correlation with OPDCFH (Spearman's analysis, p < 0.05) was detected for IL-8 secretion and γ-H2AX. These results provide a biological support to the implementation in air quality monitoring of OP measurements as a useful proxy to estimate PM-induced cellular toxicological responses. In addition, these results provide new insights for the assessment of the ability of secondary aerosol in the background atmosphere to induce oxidative stress and health effects.

Impact of endocrine disruptors on peripheral blood mononuclear cells in vitro: role of gender.

Humans can be exposed to endocrine disruptors (EDs) in numerous ways. EDs can interfere with endogenous hormones at different levels, resulting in numerous adverse human health outcomes, including immunotoxicity. In this regard, this study aimed to investigate in vitro the possible effects of EDs on immune cells and possible gender differences. Peripheral blood mononuclear cells from healthy humans, both males and females, were exposed to 6 different EDs, namely atrazine (herbicide), cypermethrin (insecticide), diethyl phthalate (plasticizer), 17α-ethynylestradiol (contraceptive drug), perfluorooctanesulfonic acid (persistent organic pollutant), and vinclozolin (fungicide). We evaluated the effect of EDs on RACK1 (receptor for activated C kinase 1) expression, considering it as a bridge between the endocrine and the immune system, and putatively used as screening tool of immunotoxic effects of EDs. The exposure to EDs resulted at different extent in alteration in RACK1 expression, pro-inflammatory activity, natural killer lytic ability, and lymphocyte differentiation, with sex-related differences. In particular, diethyl phthalate and perfluorooctanesulfonic acid resulted the most active EDs tested, with gender differences in terms of effects and magnitude. The results from our study evidenced the ability of EDs to directly affect immune cells.

Role of miR-24-3p and miR-146a-5p in dendritic cells' maturation process induced by contact sensitizers.

MiRNAs are non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Although allergic contact dermatitis has been studied extensively, few studies addressed miRNA expression and their role in dendritic cell activation. The main aim of this work was to investigate the role of miRNAs in the underlying mechanism of dendritic cell maturation induced by contact sensitizers of different potency. Experiments were conducted using THP-1-derived immature DCs (iDCs). Contact allergens of different potency were used: p-benzoquinone, Bandrowski's base, and 2,4-dinitrochlorobenzene as extreme; nickel sulfate hexahydrate, diethyl maleate and 2-mercaptobenzothiazole as moderate; and α-hexyl cinnamaldehyde, eugenol, and imidazolidinyl urea as weak. Selective inhibitor and mimic miRNAs were then used and several cell surface markers was evaluated as targets. Also, patients patch tested with nickel were analyzed to determine miRNAs expression. Results indicate an important role of miR-24-3p and miR-146a-5p in DCs activation. miR-24-3p was up-regulated by extreme and weak contact allergens, while miR-146a-5p was up-regulated by weak and moderate contact allergens and down-regulated only by the extreme ones. Also, the involvement of PKCß in contact allergen-induced miR-24-3p and miR-146a-5p expression was demonstrated. Furthermore, the expression of the two miRNAs maintains the same trend of expression in both in vitro and in human conditions after nickel exposure. Results obtained suggest the involvement of miR-24 and miR-146a in DCs maturation process in the proposed in vitro model, supported also by human evidences.

An integrated in silico-in vitro investigation to assess the skin sensitization potential of 4-Octylphenol.

One of the major challenges in chemical toxicity testing is the possibility to protect human health against adverse effects with non-animal methods. In this paper, 4-Octylphenol (OP) was tested for skin sensitization and immunomodulatory effects using an integrated in silico-in vitro test approach. In silico tools (QSAR TOOLBOX 4.5, ToxTree and VEGA) were used together with several in vitro tests including HaCaT cells (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA and expression of genes TNF, IL1A, IL6 and IL8 by RT- qPCR), RHE model (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA) and THP-1 activation assay (CD86/CD54 expression and IL-8 release). Additionally, the immunomodulatory effect of OP was investigated using lncRNAs MALAT1 and NEAT1 expression and LPS-induced THP-1 activation (CD86/CD54 expression and IL-8 release). The in silico tools predicted OP as a sensitizer. In vitro tests are also concordant with the in silico prediction. OP increased IL-6 expression (HaCaT cells); IL-18 and IL-8 expressions (RHE model). An irritant potential was also shown by a great expression of IL-1α (RHE model); and increased expression of CD54 marker and IL-8 in THP-1 cells. Immunomodulatory effects of OP were demonstrated by the downregulation of NEAT1, MALAT1 (epigenetic markers), IL6 and IL8; and an increase in LPS-induced CD54 and IL-8 expressions. Overall, results indicate that OP is a skin sensitizer, being positive in three key events of the AOP for skin sensitization, also showing immunomodulatory effects.

New approach methodologies in immunotoxicology: challenges and opportunities.

To maintain the integrity of an organism, a well-functioning immune system is essential. Immunity is dynamic, with constant surveillance needed to determine whether to initiate an immune response or to not respond. Both inappropriate immunostimulation and decreased immune response can be harmful to the host. A reduced immune response can lead to high susceptibility to cancer or infections, whereas an increased immune response can be related to autoimmunity or hypersensitivity reactions. Animal testing has been the gold standard for hazard assessment in immunotoxicity but a lot of efforts are ongoing to develop non-animal-based test systems, and important successes have been achieved. The term "new approach methodologies" (NAMs) refer to the approaches which are not based on animal models. They are applied in hazard and risk assessment of chemicals and include approaches such as defined approaches for data interpretation and integrated approaches to testing and assessment. This review aims to summarize the available NAMs for immunotoxicity assessment, taking into consideration both inappropriate immunostimulation and immunosuppression, including implication for cancer development.

In Vitro Effects of Bisphenol Analogs on Immune Cells Activation and Th Differentiation.

AIMS: Investigate the immunomodulatory effects of bisphenols in the THP-1 cell line and peripheral blood mononuclear cells in response to lipopolysaccharide (LPS) activation or to phorbol 12-myristate 13-acetate (PMA) and ionomycin. BACKGROUND: We have previously demonstrated the usefulness of the evaluation of RACK1 expression as a link between endocrine disrupting activity and the immunotoxic effect of xenobiotics. We demonstrated that while BPA and BPAF reduced RACK1 expression, BPS was able to increase it. OBJECTIVE: Bisphenol A (BPA) is one of the most commonly used chemicals in the manufacturing of polycarbonate plastics and plastic consumer products. Its endocrine disrupting (ED) potential and changes in European regulations have led to replacing BPA in many uses with structurally similar chemicals, like bisphenol AF (BPAF) and bisphenol S (BPS). However, emerging data indicated that bisphenol analogues may not be safer than BPA both in toxic effects and ED potential. METHODS: THP-1 cell line and peripheral blood mononuclear cells were activated with lipopolysaccharide (LPS) or with phorbol 12-myristate 13-acetate (PMA) and ionomycin. RESULTS: BPA and BPAF decreased LPS-induced expression of surface markers and the release of pro-inflammatory cytokines, while BPS increased LPS-induced expression of CD86 and cytokines. BPA, BPAF, and BPS affected PMA/ionomycin-induced T helper differentiation and cytokine release with gender-related alterations in some parameters investigated. CONCLUSION: Data confirm that bisphenols can modulate immune cell differentiation and activation, further supporting their immunotoxic effects.

In Vitro Effects of Cypermethrin and Glyphosate on LPS-Induced Immune Cell Activation.

(1) Background: The insecticide cypermethrin (Cypm) and the herbicide glyphosate (Glyp) are among the most widely used pesticides. While the two pesticides have been considered to have low toxicity in mammals, some indication of potential immunotoxicity has emerged. The aim of this work was to investigate in vitro the effects of Cypm and Glyp on bacteria lipopolysaccharide (LPS)-induced immune cell activation and of Cypm on 2-mercaptobenzothiazole (MBT)-induced maturation of dendritic cells (DCs). (2) Methods: The release of the inflammatory cytokines TNF-α and IL-8, the expression of the surface markers CD54 and CD86 in human primary peripheral blood mononuclear cells (PBMC), and THP-1 cells were investigated together with CD83, HLA-DR, IL-6, and IL-18 in DCs. (3) Results: While no significant modulation on LPS-induced immune cell activation was observed following Glyp exposure, with only a trend toward an increase at the highest concentration tested, Cypm reduced the responses to LPS and to MBT, supporting a direct immunosuppressive effect. Overall, the present study contributes to our understanding of pesticide-induced immunotoxicity, and the results obtained support evidence showing the immunosuppressive effects of Cypm.

Air Pollution: Possible Interaction between the Immune and Nervous System?

Exposure to environmental pollutants is a serious and common public health concern associated with growing morbidity and mortality worldwide, as well as economic burden. In recent years, the toxic effects associated with air pollution have been intensively studied, with a particular focus on the lung and cardiovascular system, mainly associated with particulate matter exposure. However, epidemiological and mechanistic studies suggest that air pollution can also influence skin integrity and may have a significant adverse impact on the immune and nervous system. Air pollution exposure already starts in utero before birth, potentially causing delayed chronic diseases arising later in life. There are, indeed, time windows during the life of individuals who are more susceptible to air pollution exposure, which may result in more severe outcomes. In this review paper, we provide an overview of findings that have established the effects of air pollutants on the immune and nervous system, and speculate on the possible interaction between them, based on mechanistic data.

Computational modelling and simulation for immunotoxicity prediction induced by skin sensitisers.

In many domains regulating chemicals and chemical products, there is a legal requirement to determine skin sensitivity to allergens. While many in vitro assays to detect contact hypersensitivity have been developed as alternatives to animal testing over the past ten years and significant progress has been made in this area, there is still a need for continued investment in the creation of techniques and strategies that will allow accurate identification of potential contact allergens and their potency in vitro. In silico models are promising tools in this regard. However, none of the state-of-the-art systems seems to function well enough to serve as a stand-alone hazard identification tool, especially in evaluating the possible allergenicity effects in humans. The Universal Immune System Simulator, a mechanistic computational platform that simulates the human immune system response to a specific insult, provides a means of predicting the immunotoxicity induced by skin sensitisers, enriching the collection of computational models for the assessment of skin sensitization. Here, we present a specific disease layer implementation of the Universal Immune System Simulator for the prediction of allergic contact dermatitis induced by specific skin sensitizers.

Effects of endocrine active contaminating pesticides on RACK1 expression and immunological consequences in THP-1 cells.

We have previously demonstrated that RACK1, which expression is under steroid hormone control, plays an important role in the activation of immune cells and its expression can be useful to evaluate the immunotoxic profile of endocrine disrupting chemicals (EDCs). Hence, we investigated the effects of three contaminating and persistent pesticides: the fungicide vinclozolin (VIN), the herbicide atrazine (ATR) and the insecticide cypermethrin (CYP) on RACK1 expression and on innate immune response. VIN resulted in modest alteration of RACK1 while ATR and CYP reduced in a dose dependent manner RACK1 expression, ultimately leading to the decrease in lipopolysaccharide-induced IL-8 and TNF-α release and CD86 and CD54 surface marker expression. Moreover, our data indicate that, after exposure to EDCs, alterations of RACK1 expression can also occur with mechanisms not directly mediated by an interaction with a nuclear or membrane steroid receptors. Therefore, RACK1 could represent a useful EDCs screening tool to evaluate their immunotoxic potential and to dissect their mechanisms of action.

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

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

Direct Effects of Glyphosate on In Vitro T Helper Cell Differentiation and Cytokine Production.

Glyphosate (G) is the active ingredient of the most used herbicides worldwide. Its use is currently very debated, as several studies indicating its hazard and toxicity are emerging. Among them, there is evidence of adverse effects on the immune system. The aim of this work was to investigate if G could directly affect immune cells. Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were used as experimental model. PBMC were expose to G and stimulated with PMA/ionomycin, T helper (Th) cell differentiation and cytokine production were assessed by flow cytometry and enzyme-linked immunosorbent assay, respectively. A reduction of Th1/Th2 ratio, mainly due to a decrease in Th1 cells, was observed following G exposure. Results show an enhancement of IL-4 and IL-17A production, and a reduction of IFN-γ. Based on literature evidence that suggest G being an endocrine disruptor, we investigated the role of nuclear estrogen receptors (ER). ERα/ERß inhibition by ICI 182,780 abolished the effects of G on IFN-γ and IL-4 release, suggesting a role of ER in the observed effects. To further characterize the mechanism of action of G, miRNAs, both in exosome and intracellular, were investigated. A statistically significant increase in miR-500a-5p was observed following G treatment. The blockage of miR-500a-5p, using a specific antagomir, prevented G-induced reduction of IFN-γ production. Finally a relationship between miR-500a-5p up-regulation and ER was observed. Overall, these results suggest that G can directly act on T cells, altering T cell differentiation and cytokines production.

The Modified THP-1 Activation Assay for the In Vitro Identification of Drug-Inducing Systemic Hypersensitivity.

The development of new low molecular weight drugs has many chances of failure and is an expensive process. Currently, there are no screening methods and/or models to assess the hazard of hypersensitivity reactions to drugs (DHRs) in the preclinical phase. DHRs represent 6-15% of adverse drug reactions. Although rare, DHRs represent a serious health problem for predisposed individuals, resulting, in some cases, in life-threatening pathologies. To date, there are no in vitro or in vivo sensitive models able to predict the sensitizing potential of drugs in the preclinical tests, and these reactions are highlighted only after the drug has been placed on the market, affecting both population and public health. This article describes a novel approach methodology for the study of the sensitizing potential of drugs based on the use of the human promyelocytic cell line THP-1 as a surrogate for dendritic cells. The method is based on the upregulation of specific surface markers (CD86 and CD54) and on the production of IL-8. In our experience, the THP-1 activation assay allowed the correct identification of drugs known to induce systemic hypersensitivity in humans, including the one associated with specific HLAs. This method may help to discover possible systemic hypersensitivity reactions early in the preclinical phase of drug development.

Human keratinocytes and monocytes co-culture cell system: An important contribution for the study of moderate and weak sensitizers.

In vitro approaches to address key events in chemical-induced skin sensitization have been developed, but there is still uncertain how they will be useful to predict the potency for an effective risk assessment. Keratinocytes (KCs) play a key role in all phases of skin sensitization. Dendritic cells (DCs) activation and maturation require the binding of cytokines produced by KC as a result of initial chemical exposure. We previously identified interleukin-18 (IL-18) as useful marker for determination of skin sensitization potential of chemicals. The aim of this paper was to mimic the interaction between KCs and DCs using a co-culture of NCTC 2544 and THP-1 cells. Three selected contact allergens of different potency (Bandrowski's base, diethyl maleate, and imidazolidinyl urea) were tested in time-course experiments (24, 48 and 72 h). Cell surface markers expression (CD80, CD86, and HLA-DR) was determined by flow cytometry analysis while IL-18 production was evaluated with specific sandwich ELISA. Results obtained from this simple in vitro co-culture system show the possibility to study the contribution of KCs in DCs activation through the analysis of HLA-DR expression. Results obtained demonstrate the ability of the KCs to favor the full maturation of the DCs in the presence of moderate and weak allergens, while the extreme allergen induced a complete maturation of DC alone without the need of KCs.

In vitro identification of drugs inducing systemic hypersensitivity reactions known in vivo to be associated with specific HLA genotypes.

Hypersensitivity drug reactions (HDRs) are common among drugs, despite this, there are no validated in vitro or in vivo methods for screening the sensitizing potential of drugs in the preclinical phase. We previously developed the THP-1 activation assay, based on CD86 upregulation and IL-8 production, for the in vitro identification of drugs able to induce selective dendritic cell activation. In this paper, we investigated the predictive capacity of the method toward drugs associated with HDRs for which a correlation with specific human leukocyte antigens (HLA) have been demonstrated. For that purpose, abacavir, carbamazepine and clozapine were used. Metformin was used as negative control. Dose- and time-course experiments were conducted. The surface markers CD86, CD54 and HLA-DR were evaluated by flow cytometry analysis, whereas IL-8 release by ELISA. Abacavir, carbamazepine and clozapine gave positive results with CD86 upregulation and/or IL-8 release, with abacavir also inducing HLA-DR. The test reveals the ability of drugs to induce dendritic cell activation (signals 1/2), that preceded the adaptive immune response, which will be manifested only in a minority of patients carrying the specific HLA genotypes. The idea is to integrate this simple method during drug development to identify the potential of drugs to induce hypersensitivity reactions in the pre-clinical phase.

Effect of estrogen-active compounds on the expression of RACK1 and immunological implications.

We previously demonstrated the existence of a balance among steroid hormones, i.e. glucocorticoids and androgens, in RACK1 (receptor for activated C kinase 1) expression and innate immunity activation, which may offer the opportunity to use RACK1 expression as marker to evaluate immunotoxicity of hormone-active substances. Because of the existence of close interconnections between the different steroid hormone receptors with overlapping ligand specificities and signaling pathways, in this study, we wanted to investigate a possible effect of estrogenic active compounds, namely 17ß-estradiol, diethylstilbestrol, and zearalenone, on RACK-1 expression and innate immune responses using THP-1 cells as experimental model. All compounds increased RACK1 transcriptional activity as evaluated by reporter luciferase activity, mRNA expression as assessed by real time-PCR and protein expression by western blot analysis, which paralleled an increase in LPS-induced IL-8, TNF-α production, and CD86 expression, which we previously demonstrated to be dependent on RACK1/PKCß activation. As the induction of RACK1 expression can be blocked by the antagonist G15, induced by the agonist G1 and by the non-cell permeable 17ß-estradiol conjugated with BSA, a role of GPER (previously named GPR30) activation in estrogen-induced RACK1 expression could be demonstrated. In addition, a role of androgen receptor (AR) in RACK1 transcription was also demonstrated by the ability of flutamide, a nonsteroidal antiandrogen, to completely prevent diethylstilbestrol-induced RACK1 transcriptional activity and protein expression. Altogether, our data suggest that RACK1 may represent an interesting target of steroid-active compounds, and its evaluation may offer the opportunity to screen the immunotoxic potential of hormone-active substances.