Saadia Kerdine-Römer

HMOX1 and NQO1 Genes are Upregulated in Response to Contact Sensitizers in Dendritic Cells and THP-1 Cell Line: Role of the Keap1/Nrf2 Pathway

Electrophilicity is one of the most common features of skin contact sensitizers and is necessary for protein haptenation. The Keap1 (Kelch-like ECH-associated protein 1)/Nrf2 -signaling pathway is dedicated to the detection of electrophilic stress in cells leading to the upregulation of genes involved in protection or neutralization of chemical reactive species. Signals provided by chemical stress could play an important role in dendritic cell activation and the aim of this work was to test whether contact sensitizers were specific activators of the Keap1/Nrf2 pathway. CD34-derived dendritic cells (CD34-DC) and the THP-1 myeloid cell line were treated by a panel of sensitizers (Ni, 1-chloro 2,4-dinitrobenzene, cinnamaldehyde, 7-hydroxycitronellal, 1,4-dihydroquinone, alpha-methyl-trans-cinnamaldehyde, 2-4-tert-(butylbenzyl)propionaldehyde or Lilial, and 1,4-phenylenediamine), irritants (sodium dodecyl sulfate, benzalkonium chloride), and a nonsensitizer molecule (chlorobenzene). Three well-known Nrf2 activators (tert-butylhydroquinone, lipoic acid, sulforaphane) were also tested. Expression of hmox1 and nqo1 was measured using real-time PCR and cellular accumulation of Nrf2 was assessed by Western blot. Our results showed an increased expression at early time points of hmox1 and nqo1 mRNAs in response to sensitizers but not to irritants. Accumulation of the Nrf2 protein was also observed only with chemical sensitizers. A significant inhibition of the expression of hmox1 and nqo1 mRNAs and CD86 expression was found in 1-chloro 2,4-dinitrobenzene-treated THP-1 cells preincubated with N-acetyl cysteine, a glutathione precursor. Altogether, these data suggested that the Keap1/Nrf2-signaling pathway was activated by electrophilic molecules including sensitizers in dendritic cells and in the THP-1 cell line. Monitoring of this pathway may provide new biomarkers (e.g., Nrf2, hmox1) for the detection of the sensitization potential of chemicals.

TLR7 and TLR8 agonists trigger different signaling pathways for human dendritic cell maturation.

Dendritic cells (DCs) play an important role in bridging innate and adaptive immunity. These APCs have the ability to recognize specific molecular signatures of pathogens through TLRs. In particular, the intracellular TLR7 and TLR8, mediating the recognition of ssRNA by DCs, play a major role in the immune response during viral infection. Although differences have been identified between TLR7 and TLR8, in terms of cellular expression and functions, the signaling pathways that lead to DC maturation following TLR7 or TLR8 engagement are largely unknown. We compared the signaling pathways involved in human CD34‐DC maturation induced by agonists selective for TLR7 (imiquimod) or TLR8 (3M002). TLR7 and TLR8 activation up‐regulated CCR7, CD40, CD86, and CD83 expression and IL‐6 and IL‐12p40 production. However, only TLR8 activation led to IL‐12p70 production and il‐12p35 mRNA expression. We found that upon TLR7 and TLR8 activation, JNK and NF‐κB positively regulated the expression of CCR7, CD86, CD83, and CD40 and the production of IL‐6 and IL‐12p40. However, although p38MAPK participated in the up‐regulation of maturation markers in response to TLR7 activation, this kinase exerted an inhibitory effect on CD40 expression and IL‐12 production in TLR8‐stimulated DCs. We also showed that the Jak/STAT signaling pathway was involved in CD40 expression and cytokine production in TLR7‐stimulated DCs but negatively regulated CD83 expression and cytokine secretion in DCs activated through TLR8. This study showed that TLR7 and TLR8 activate similar signaling pathways that play different roles in DC maturation, depending on which TLR is triggered.

Nuclear Factor Erythroid 2-Related Factor 2 Nuclear Translocation Induces Myofibroblastic Dedifferentiation in Idiopathic Pulmonary Fibrosis

AIMSnOxidants have been implicated in the pathophysiology of idiopathic pulmonary fibrosis (IPF), especially in myofibroblastic differentiation. We aimed at testing the hypothesis that nuclear factor erythroid 2-related factor 2 (Nrf2), the main regulator of endogenous antioxidant enzymes, is involved in fibrogenesis via myofibroblastic differentiation. Fibroblasts were cultured from the lungs of eight controls and eight IPF patients. Oxidants-antioxidants balance, nuclear Nrf2 expression, and fibroblast phenotype (α-smooth muscle actin and collagen I expression, proliferation, migration, and contraction) were studied under basal conditions and after Nrf2 knockdown or activation by Nrf2 or Keap1 siRNA transfection. The effects of sulforaphane (SFN), an Nrf2 activator, on the fibroblast phenotype were tested under basal and pro-fibrosis conditions (transforming growth factor β [TGF-β]).nnnRESULTSnDecreased Nrf2 expression was associated with a myofibroblast phenotype in IPF compared with control fibroblasts. Nrf2 knockdown induced oxidative stress and myofibroblastic differentiation in control fibroblasts. Conversely, Nrf2 activation increased antioxidant defences and myofibroblastic dedifferentation in IPF fibroblasts. SFN treatment decreased oxidants, and induced Nrf2 expression, antioxidants, and myofibroblastic dedifferentiation in IPF fibroblasts. SFN inhibited TGF-β profibrotic deleterious effects in IPF and control fibroblasts and restored antioxidant defences. Nrf2 knockdown abolished SFN antifibrosis effects, suggesting that they were Nrf2 mediated.nnnINNOVATION AND CONCLUSIONnOur findings confirm that decreased nuclear Nrf2 plays a role in myofibroblastic differentiation and that SFN induces human pulmonary fibroblast dedifferentiation in vitro via Nrf2 activation. Thus, Nrf2 could be a novel therapeutic target in IPF.

Rapid Anxiolytic Effects of a 5-HT 4 Receptor Agonist Are Mediated by a Neurogenesis-Independent Mechanism

Selective serotonin reuptake inhibitors (SSRIs) display a delayed onset of action of several weeks. Past work in naive rats showed that 5-HT4 receptor agonists had rapid effects on depression-related behaviors and on hippocampal neurogenesis. We decided to investigate whether 5-HT4 receptor stimulation was necessary for the effects of SSRIs in a mouse model of anxiety/depression, and whether hippocampal neurogenesis contributed to these effects. Using the mouse corticosterone model of anxiety/depression, we assessed whether chronic treatment with a 5-HT4 receptor agonist (RS67333, 1.5u2009mg/kg/day) had effects on anxiety- and depression-related behaviors, as well as on hippocampal neurogenesis in comparison with chronic fluoxetine treatment (18u2009mg/kg/day). Then, using our anxiety/depression model combined with ablation of hippocampal neurogenesis, we investigated whether neurogenesis was necessary for the behavioral effects of subchronic (7 days) or chronic (28 days) RS67333 treatment. We also assessed whether a 5-HT4 receptor antagonist (GR125487, 1u2009mg/kg/day) could prevent the behavioral and neurogenic effects of fluoxetine. Chronic treatment with RS67333, similar to fluoxetine, induced anxiolytic/antidepressant-like activity and stimulated adult hippocampal neurogenesis, specifically facilitating maturation of newborn neurons. However, unlike fluoxetine, anxiolytic effects of RS67333 were already present after 7 days and did not require hippocampal neurogenesis. Chronic treatment with GR125487 prevented both anxiolytic/antidepressant-like and neurogenic effects of fluoxetine, indicating that 5-HT4 receptor activation is necessary for these effects of SSRIs. 5-HT4 receptor stimulation could represent an innovative and rapid onset therapeutic approach to treat depression with comorbid anxiety.

Toxicity of surface-modified PLGA nanoparticles toward lung alveolar epithelial cells

In vitro cytotoxicity and inflammatory response following exposure to nanoparticles (NPs) made of poly(lactide-co-glycolide) (PLGA) have been investigated on A549 human lung epithelial cells. Three different PLGA NPs (230 nm) were obtained using different stabilizers (polyvinyl alcohol, chitosan, or Pluronic(®) F68) to form respectively neutral, positively or negatively charged NPs. Polystyrene NPs were used as polymeric but non-biodegradable NPs, and titanium dioxide (anatase and rutile) as inorganic NPs, for comparison. Cytotoxicity was evaluated through mitochondrial activity as well as membrane integrity (lactate dehydrogenase release, trypan blue exclusion, propidium iodide staining). The cytotoxicity of PLGA-based and polystyrene NPs was lower or equivalent to the one observed after exposure to titanium dioxide NPs. The inflammatory response, evaluated through the release of the IL-6, IL-8, MCP-1, TNF-α cytokines, was low for all NPs. However, some differences were observed, especially for negative PLGA NPs that led to a higher inflammatory response, which can be correlated to a higher uptake of these NPs. Taken together, these results show that both coating of PLGA NPs and the nature of the core play a key role in cell response.

Biodegradable Nanoparticles Meet the Bronchial Airway Barrier: How Surface Properties Affect Their Interaction with Mucus and Epithelial Cells

Despite the wide interest raised by lung administration of nanoparticles (NPs) for the treatment of various diseases, little information is available on their effect toward the airway epithelial barrier function. In this study, the potential damage of the pulmonary epithelium upon exposure to poly(lactide-co-glycolide) (PLGA) NPs has been assessed in vitro using a Calu-3-based model of the bronchial epithelial barrier. Positively and negatively charged as well as neutral PLGA NPs were obtained by coating their surface with chitosan (CS), poloxamer (PF68), or poly(vinyl alcohol) (PVA). The role of NP surface chemistry and charge on the epithelial resistance and mucus turnover, using MUC5AC as a marker, was investigated. The interaction with mucin reduced the penetration of CS- and PVA-coated NPs, while the hydrophilic PF68-coated NPs diffused across the mucus barrier leading to a higher intracellular accumulation. Only CS-coated NPs caused a transient but reversible decrease of the trans-epithelial electrical resistance (TEER). None of the NP formulations increased MUC5AC mRNA expression or the protein levels. These in vitro results highlight the safety of PLGA NPs toward the integrity and function of the bronchial airway barrier and demonstrate the crucial role of NP surface properties to achieve a controlled and sustained delivery of drugs via the pulmonary route.

Allergic Skin Inflammation Induced by Chemical Sensitizers Is Controlled by the Transcription Factor Nrf2

Allergic contact dermatitis (ACD) is induced by low-molecular weight electrophilic chemicals and metal ions. Chemical contact sensitizers trigger reactive oxygen species production and provoke electrophilic stress, leading to the accumulation of the transcription factor nuclear-related factor 2 (Nrf2) in innate immune cell types. The objective of this work was to identify the role of Nrf2 in the regulation of ACD. We used the local lymph node assay (LLNA) and the mouse ear swelling test (MEST) to study the role of Nrf2 in both the sensitization and elicitation phase in nrf2 knockout (nrf2(-/-)) and wild-type (nrf2(+/+)) mice. Five chemicals were used: two compounds known to react with cysteine residues, 2,4-dinitrochlorobenzene (DNCB) and cinnamaldehyde (CinA); one sensitizer known to exhibit mixed reactivity to cysteine and lysine residues, isophorone diisocyanate; and one reacting specifically with lysine residues, trimellitic anhydride and croton oil, a well-known irritant. In the MEST assay, DNCB (1 and 2%) induced a significant increase in ear thickness in nrf2(-/-) compared with nrf2(+/+) mice, suggesting a role for Nrf2 in the control of the inflammatory process. When DNCB was used at 0.25 and 0.5% or when mice were treated with CinA, inflammation was found only in nrf2(-/-) mice. In the LLNA, all chemical sensitizers induced an increase of lymphocyte proliferation in nrf2(-/-) compared with nrf2(+/+) mice for the same chemical concentration. These results reveal an important role for Nrf2 in controlling ACD and lymphocyte proliferation in response to sensitizers.

Mechanisms of IL-12 Synthesis by Human Dendritic Cells Treated with the Chemical Sensitizer NiSO4

Allergic contact dermatitis, caused by metallic ions, is a T cell-mediated inflammatory skin disease. IL-12 is a 70-kDa heterodimeric protein composed of IL-12p40 and IL-12p35, playing a major role in the generation of allergen-specific T cell responses. Dendritic cells (DCs) are APCs involved in the induction of primary immune responses, as they possess the ability to stimulate naive T cells. In this study, we address the question whether the sensitizer nickel sulfate (NiSO4) itself or in synergy with other signals can induce the secretion of IL-12p70 in human monocyte-derived DCs (Mo-DCs). We found that IL-12p40 was produced by Mo-DC in response to NiSO4 stimulation. Addition of IFN-γ concomitantly to NiSO4 leads to IL-12p70 synthesis. NiSO4 treatment leads to the activation of MAPK, NF-κB pathways, and IFN regulatory factor 1 (IRF-1). We investigated the role of these signaling pathways in IL-12 production using known pharmacological inhibitors of MAPK and NF-κB pathways and RNA interference-mediated silencing of IRF-1. Our results showed that p38 MAPK, NF-κB, and IRF-1 were involved in IL-12p40 production induced by NiSO4. Moreover, IRF-1 silencing nearly totally abrogated IL-12p40 and IL-12p70 production provoked by NiSO4 and IFN-γ. In response to NiSO4, we observed that STAT-1 was phosphorylated on both serine and tyrosine residues and participated to NiSO4-induced IRF-1 activation. N-acetylcysteine abolished STAT-1 phosphorylation, suggesting that STAT-1 activation may be dependent on NiSO4-induced alteration of the redox status of the cell. These results indicate that p38 MAPK, NF-κB, and IRF-1 are activated by NiSO4 in Mo-DC and cooperate for IL-12 production.

Nrf2-signaling and BDNF: A new target for the antidepressant-like activity of chronic fluoxetine treatment in a mouse model of anxiety/depression

Several studies have shown that Nrf2, a major redox-sensitive transcription factor involved in the cellular defense against oxidative stress, increases susceptibility to depressive-like behavior. However, little is known about the influence of antidepressant drugs on Nrf2 signaling and expression of its target genes (GCLC, NQO1, HO-1) in the brain. We found that chronic treatment of a mouse model of anxiety/depression (CORT model) with a selective serotonin reuptake inhibitor (SSRI, fluoxetine, 18mg/kg/day) reversed CORT-induced anxiety/depression-like behavior in mice. Chronic fluoxetine treatment restored CORT-induced decreases in Nrf2 protein levels and its target genes in the cortex and hippocampus. Furthermore, we found that chronic fluoxetine also increased brain derived neurotrophic factor (BDNF) protein levels in cortex and hippocampus of CORT-treated Nrf2 knockout mice (KO, Nrf2(-/-)). Taken together, these data suggest that Nrf2 signaling contributes to fluoxetine-induced neuroprotection via an unexpected mechanism involving 5-HT transporter SERT blockade, and not through enhancement of BDNF expression.

Surface coating mediates the toxicity of polymeric nanoparticles towards human-like macrophages

The purpose of this study was to investigate the toxicity of a series of poly(lactide-co-glycolic) (PLGA) nanoparticles on human-like THP-1 macrophages. Positively-, negatively-charged and neutral nanoparticles (200 nm) were prepared using chitosan (CS), poloxamer 188 (PF68) and poly(vinyl alcohol) (PVA) as stabilizer. Stabilizer-free PLGA nanoparticles were obtained as well. When used at therapeutically relevant concentrations (up to 0.1 mg/mL in vitro), all tested nanoparticles showed no or scarce signs of toxicity, as assessed by cell mitochondrial activity, induction of apoptosis and necrosis, production of intracellular reactive oxygen species (ROS) and secretion of pro-inflammatory cytokines. At high concentrations (above 1mg/mL), cytotoxicity was found to be induced by the presence of stabilizers, whatever the toxicological pattern of the stabilizer itself. While stabilizer-free PLGA nanoparticles exerted no cytotoxicity, the slightly cytotoxic CS polymer conferred PLGA nanoparticles significant cytotoxicity when used as nanoparticle stabilizer; more surprisingly, the otherwise innocuous PVA and PF68 polymers also conferred a significant cytotoxicity to PLGA nanoparticles. These results unveiled the critical toxicological contribution played by stabilizers used for the formulation of PLGA nanoparticles when used at high concentrations, which may have implications for local toxicities of PLGA-based nanomedicine, and provided additional insight in cytotoxic effects of internalized nanoparticles.