João Demétrio Martins

Autophagy and Inflammasome Interplay

Inflammation is a defensive response of the organism to manage harmful stimuli sensed by innate immune cells. The signal alarm is triggered by the recognition of pathogen-associated molecular patterns, such as microbial components, or host-derived damage-associated molecular patterns (DAMPs), namely high-mobility group box 1 protein (HMGB1) and purine metabolites, through a set of highly conserved receptors in immune cells termed pattern recognition receptors. Among these receptors, membrane-associated toll-like receptors (TLRs) and cytosolic nucleotide binding and oligomerization domain (nod)-like receptors (NLRs) assume particular relevance in the inflammatory process. Once activated, NLRs induce the assembly of multiprotein complexes called inflammasomes, leading to production of proinflammatory cytokines (e.g., interleukin-1) and induction of inflammatory cell death (pyroptosis) through the activation of caspase-1. Although these processes intend to protect the body from insults, prolonged or exacerbated inflammatory responses associated with inflammasome activation are related to a growing number of diseases. Recently, inflammasome activation and autophagy were shown to be linked and to mutually influence each other. Therefore, we aim, in this review, to discuss the recent evidences concerning the cross talk between autophagy and inflammasome activation and its potential roles in disease progression.

Oxidative stress-dependent activation of the eIF2α–ATFr unfolded protein response branch by skin sensitizer 1-fluoro-2,4-dinitrobenzene modulates dendritic-like cell maturation and inflammatory status in a biphasic manner

The pathogenesis of allergic contact dermatitis, the most common manifestation of immunotoxicity in humans, is intimately connected to hapten-induced maturation of dendritic cells (DC). The molecular mechanisms driving this maturational program are not completely known; however, initial danger signals such as the generation of reactive oxygen species (ROS) were shown to play a critical role. Recent evidence linking ROS production, endoplasmic reticulum (ER) stress, and the pathogenesis of several inflammatory diseases led us to analyze, in the present work, the ability of the skin sensitizer 1-fluoro-2,4-dinitrobenzene (DNFB) to evoke ER stress in DC-like THP-1 cells and the concomitant consequences to their immunobiology. We found that DNFB triggers a ROS-dependent activation of the PERK-eIFα-ATF4 unfolded protein response (UPR) branch conferring cytoprotection and modulating the maturation/proinflammatory cell status in a biphasic manner. Early DNFB induction of ATF4 positively modulates autophagy-related genes MAP1LC3B and ATG3 and stabilizes the transcription factor Nrf2, causing a strong induction of the HMOX1-detoxifying gene. Moreover, we observed that in a first phase, DNFB-induced ATF4 upregulates IL8 mRNA levels while blocking CD86, IL1B, IL12B, and CXL10 transcription. Later, following ATF4 decay, HMOX1 and IL8 transcription drastically decrease and CD86, IL1B, and Il12B are upregulated. Overall, our results evidence a connection between sensitizer-induced redox imbalance and the establishment of ER stress in DC-like cells and provide new insights into the role of UPR effectors such as ATF4 to the complex DC maturational program.

Toxicity assessment of the herbicide metolachlor comparative effects on bacterial and mitochondrial model systems.

Metolachlor is one of the most intensively used chloroacetamide herbicides. However, its effects on the environment and on non-target animals and humans as well as its interference at a cell/molecular level have not yet been fully elucidated. The aim of this study was: firstly, to evaluate the potential toxicity of metolachlor at a cell/subcellular level by using two in vitro biological model systems (a strain of Bacillus stearothermophilus and rat liver mitochondria); secondly, to evaluate the relative sensibility of these models to xenobiotics to reinforce their suitability for pollutant toxicity assessment. Our results show that metolachlor inhibits growth and impairs the respiratory activity of B.stearothermophilus at concentrations two to three orders of magnitude higher than those at which bacterial cells are affected by other pesticides. Also at concentrations significantly higher than those of other pesticides, metolachlor depressed the respiratory control ratio, membrane potential and respiration of rat liver mitochondria when malate/glutamate or succinate were used as respiratory substrates. Moreover, metolachlor impaired the respiratory activity of rat liver mitochondria in the same concentration range at which it inhibited bacterial respiratory system (0.4-5.0 micromol/mg of protein). In conclusion, the high concentration range at which metolachlor induces toxicity in vitro suggests that this compound is safer than other pesticides previously studied in our laboratory, using the same model systems. The good parallelism between metolachlor effects on both models and the toxicity data described in the literature, together with results obtained in our laboratory with other compounds, indicate the suitability of these systems to assess toxicity in vitro.

Use of the microorganism Bacillus stearothermophilus as a model to evaluate toxicity of the lipophilic environmental pollutant endosulfan

Microorganisms are very powerful tools for the supply of information about the toxic effects of lipophilic compounds, since an impairment of cell growth usually occurs as a result of perturbations related, in most cases, with the partition of toxicants in membranes. The thermophilic eubacterium Bacillus stearothermophilus has been used as a model system to identify alpha- and beta-endosulfan interactions with the membrane possibly related with the insecticide toxicity. Two approaches have been pursued: (a) bacterial growth is followed and the effects of endosulfan isomers determined; (b) biophysical studies with the fluorescent fluidity probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to assess the effects of alpha- and beta-endosulfan on the organization of the membrane lipid bilayer. The effects on growth were quantitatively evaluated by determination of growth parameters, namely the lag phase, the specific growth rate and the cell density reached by cultures in the stationary phase. Growth inhibition by alpha and beta-endosulfan dependent on the concentration is diminished or removed by the addition of 2.5 mM Ca2+ to bacterial cultures. Fluorescence DPH polarization consistently showed opposite effects of Ca2+ and alpha- and beta-endosulfan on the physical state of bacterial polar lipid dispersions.

Development of an in Vitro Dendritic Cell-Based Test for Skin Sensitizer Identification

The sensitizing potential of chemicals is currently assessed using animal models. However, ethical and economic concerns and the recent European legislative framework triggered intensive research efforts in the development and validation of alternative methods. Therefore, the aim of this study was to develop an in vitro predictive test based on the analysis and integration of gene expression and intracellular signaling profiles of chemical-exposed skin-derived dendritic cells. Cells were treated with four known sensitizers and two nonsensitizers, and the effects on the expression of 20 candidate genes and the activation of MAPK, PI3K/Akt, and NF-κB signaling pathways were analyzed by real-time reverse transcription polymerase chain reaction and Western blotting, respectively. Genes Trxr1, Hmox1, Nqo1, and Cxcl10 and the p38 MAPK and JNK signaling pathways were identified as good predictor variables and used to construct a dichotomous classifier. For validation of the model, 12 new chemicals were then analyzed in a blind assay, and from these, 11 were correctly classified. Considering the total of 18 compounds tested here, 17 were correctly classified, representing a concordance of 94%, with a sensitivity of 92% (12 of 13 sensitizers identified) and a specificity of 100% (5 of 5 nonsensitizers identified). Additionally, we tested the ability of our model to discriminate sensitizers from nonallergenic but immunogenic compounds such as lipopolysaccharide (LPS). LPS was correctly classified as a nonsensitizer. Overall, our results indicate that the analysis of proposed gene and signaling pathway signatures in a mouse fetal skin-derived dendritic cell line represents a valuable model to be integrated in a future in vitro test platform.

Molecular mechanisms of the metabolite 4-hydroxytamoxifen of the anticancer drug tamoxifen: use of a model microorganism

A strain of the thermophilic eubacterium Bacillus stearothermophilus was used as a model system to identify membrane mediated cytotoxic effects of 4-hydroxytamoxifen, following previous studies with tamoxifen. With this experimental approach we attempted to further clarify tamoxifen and 4-hydroxytamoxifen membrane interactions often evoked as responsible for their multiple cellular effects. Bacterial growth and the oxygen consumption rate provided quantitative data of the cytotoxic action of hydroxytamoxifen. The effects of hydroxytamoxifen on the physical properties of bacterial lipid membrane preparations were also evaluated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Cultures of B. stearothermophilus grown in a complex medium containing hydroxytamoxifen in the concentration range of 1 to 7 microM exhibited progressively longer lag adapting periods, decreased specific growth rates and lower growth yields, as compared to control cultures. Hydroxytamoxifen also affected the electron redox flow of B. stearothermophilus protoplasts and induced significant perturbation of the structural order of bacterial lipid dispersions. We concluded that the bacterial model provides useful information about the nature and repercussion of membrane physical interactions of this lipophilic drug, on the basis of an easy and economic methodology.

Phospholipidomic Profile Variation on THP-1 Cells Exposed to Skin or Respiratory Sensitizers and Respiratory Irritant.

Occupational exposure to low molecular weight reactive chemicals often leads to development of allergic reactions such as allergic contact dermatitis and respiratory allergies. Further insights into the interaction of these chemicals with physiopathological relevant cellular models might provide the foundations for novel non‐animal approaches to safety assessment. In this work we used the human THP‐1 cell line to determine phospholipidome changes induced by the skin sensitizer 1‐fluoro‐2,4‐dinitrobenzene (DNFB), the respiratory allergen hexamethylene diisocyanate (HDI), and the irritant methyl salicylate (MESA). We detected that these chemicals differently induce lipid peroxidation and modulate THP‐1 IL‐1β, IL‐12B, IL‐8, CD86, and HMOX1 transcription. Decreased phosphatidylethanolamine content was detected in cells exposed to MESA, while profound alterations in the relative abundance of cardiolipin species were observed in cells exposed to DNFB. All chemicals tested induced a decrease in the relative abundance of plasmanyl phosphatidylcholine species PC (O‐16:0e/18:1) and phosphatidylinositol species PI (34:1), while increasing PI (38:4). An increased abundance of oleic acid was observed in the phospholipids of cells exposed to DNFB while a decreased abundance of palmitic acid was detected in cells treated with MESA or DNFB. We conclude that both specific and common alterations at phospholipidome levels are triggered by the different chemicals, while not allowing a complete distinction between them using a Canonical Analysis of Principal Coordinates (CAP). The common effects observed at phospholipids level with all the chemicals tested might be related to unspecific cell cytotoxic mechanisms that nevertheless may contribute to the elicitation of specific immune responses. J. Cell. Physiol. 231: 2639–2651, 2016.

Nature and kinetics of redox imbalance triggered by respiratory and skin chemical sensitizers on the human monocytic cell line THP-1

Low molecular weight reactive chemicals causing skin and respiratory allergies are known to activate dendritic cells (DC), an event considered to be a key step in both pathologies. Although generation of reactive oxygen species (ROS) is considered a major danger signal responsible for DC maturation, the mechanisms leading to cellular redox imbalance remain poorly understood. Therefore, the aim of this study was to unveil the origin and kinetics of redox imbalance elicited by 1-fluoro-2,4-dinitrobenzene (DNFB) and trimellitic anhydride chloride (TMAC), two golden standards of skin and chemical respiratory allergy, respectively. To track this goal, we addressed the time course modifications of ROS production and cellular antioxidant defenses as well as the modulation of MAPKs signaling pathways and transcription of pathophysiological relevant genes in THP-1 cells. Our data shows that the thiol-reactive sensitizer DNFB directly reacts with cytoplasmic glutathione (GSH) causing its rapid and marked depletion which results in a general increase in ROS accumulation. In turn, TMAC, which preferentially reacts with amine groups, induces a delayed GSH depletion as a consequence of increased mitochondrial ROS production. These divergences in ROS production seem to be correlated with the different extension of intracellular signaling pathways activation and, by consequence, with distinct transcription kinetics of genes such as HMOX1, IL8, IL1B and CD86. Ultimately, our observations may help explain the distinct DC phenotype and T-cell polarizing profile triggered by skin and respiratory sensitizers.

In Vitro Dendritic Cell-Based Test for Skin Sensitizers Identification and Potency Estimation

Ethical and economic concerns and the recent European legislative framework triggered intensive research efforts towards the development and validation of alternative methods. We developed an in vitro predictive test to assess the skin sensitizing potential of chemicals. The test was based on the analysis and integration of intracellular signalling pathways evoked by chemicals concomitantly with gene expression modulation in skin-derived dendritic cells. In a first approach, cells were treated with four known sensitizers and two non-sensitizers, and the effects on the expression of 20 candidate genes and on the activation of several signalling pathways were analysed by real-time RT-PCR and Western blot, respectively. The genes Trxr1, Hmox1, Nqo1 and Cxcl10 and the signalling pathways p38 MAPK and JNK were identified as good predictor variables and used to construct a dichotomous classifier. For validation of the model, a large number of chemicals were afterwards analysed in a blinded assay. From the total 18 compounds tested, 17 were correctly classified, representing a concordance of 94%, with a sensitivity of 92% and a specificity of 100%. Additionally, we also analysed the feasibility to predict the sensitizer’s potency using in vitro-generated data and several in silico-calculated descriptors. A strong correlation with LLNA EC3 values was obtained (Pearson correlation coefficient r = 0.85, p < 0.001, n = 12). Overall, our results indicate that the analysis of proposed gene and signalling pathway signatures in a mouse skin-derived dendritic cell line represents a valuable model to be integrated in a future in vitro test platform.