A method for biomarker measurements in peripheral blood mononuclear cells isolated from anxious and depressed mice: β-arrestin 1 protein levels in depression and treatment

A limited number of biomarkers in the central and peripheral systems which are known may be useful for diagnosing major depressive disorders and predicting the effectiveness of antidepressant (AD) treatments. Since 60% of depressed patients do not respond adequately to medication or are resistant to ADs, it is imperative to delineate more accurate biomarkers. Recent clinical studies suggest that β-arrestin 1 levels in human mononuclear leukocytes may be an efficient biomarker. If potential biomarkers such as β-arrestin 1 could be assessed from a source such as peripheral blood cells, then they could be easily monitored and used to predict therapeutic responses. However, no previous studies have measured β-arrestin 1 levels in peripheral blood mononuclear cells (PBMCs) in anxious/depressive rodents. This study aimed to develop a method to detect β-arrestin protein levels through immunoblot analyses of mouse PBMCs isolated from whole blood. In order to validate the approach, β-arrestin levels were then compared in naïve, anxious/depressed mice, and anxious/depressed mice treated with a selective serotonin reuptake inhibitor (fluoxetine, 18 mg/kg/day in the drinking water). The results demonstrated that mouse whole blood collected by submandibular bleeding permitted isolation of enough PBMCs to assess circulating proteins such as β-arrestin 1. β-Arrestin 1 levels were successfully measured in healthy human subject and naïve mouse PBMCs. Interestingly, PBMCs from anxious/depressed mice showed significantly reduced β-arrestin 1 levels. These decreased β-arrestin 1 expression levels were restored to normal levels with chronic fluoxetine treatment. The results suggest that isolation of PBMCs from mice by submandibular bleeding is a useful technique to screen putative biomarkers of the pathophysiology of mood disorders and the response to ADs. In addition, these results confirm that β-arrestin 1 is a potential biomarker for depression

The Nrf2-Antioxidant Response Element Signaling Pathway Controls Fibrosis and Autoimmunity in Scleroderma

Systemic sclerosis (SSc) is an autoimmune disease with fibrosis of the skin and internal organs and vascular alterations. Dysregulations in the oxidant/antioxidant balance are known to be a major factor in the pathogenesis of the disease. Indeed, reactive oxygen species (ROS) trigger neoepitopes leading to a breach of immune tolerance and autoimmune responses, activate fibroblasts to proliferate and to produce excess of type I collagen. ROS also alter endothelial cells leading to vascular dysfunction. Glutathione (GSH) is the most potent antioxidant system in eukaryotic cells. Numerous studies have reported a defect in GSH in SSc animal models and humans, but the origin of this defect remains unknown. The transcription factor NRF2 is a key player in the antioxidant defense, as it can induce the transcription of antioxidant and cytoprotective genes, including GSH, through its interaction with the antioxidant response elements. In this work, we investigated whether NRF2 could be implicated in the pathogenesis of SSc, and if this pathway could represent a new therapeutic target in this orphan disease with no curative medicine. Skin biopsies from 11 patients and 10 controls were harvested, and skin fibroblasts were extracted. Experimental SSc was induced both in BALB/c and in nrf2−/− mice by daily intradermal injections of hypochloric acid. In addition, diseased BALB/c mice were treated with an nrf2 agonist, dimethyl fumarate, or placebo. A drop in nrf2 and target genes mRNA levels was observed in skin fibroblasts of SSc patients compared to controls. Moreover, the nrf2 pathway is also downregulated in skins and lungs of SSc mice. In addition, we observed that nrf2−/− mice have a more severe form of SSc with increased fibrosis and inflammation compared to wild-type SSc mice. Diseased mice treated with the nrf2 agonist dimethyl fumarate (DMF) exhibited reduced fibrosis and immune activation compared to untreated mice. The ex vivo treatment of skin fibroblasts from SSc mice with DMF restores GSH intracellular content, decreases ROS production and cell proliferation. These results suggest that the nrf2 pathway is highly dysregulated in human and SSc mice with deleterious consequences on fibrosis and inflammation and that Nrf2 modulation represents a therapeutic target in SSc

Immune-competent in vitro co-culture models as an approach for skin sensitisation assessment

International audienceThere is a complex interplay between numerous cell types that act at different steps of the mechanism leading to allergic contact dermatitis. The validated in vitro methods for skin sensitisation assessment provide good statistical correspondence to local lymph node assay (LLNA) or to human data but, for the most part, poorly represent the actual in vivo situation as they generally involve single cell type culture in 2D. Significant progress has been made over the past decades to develop new technologies of data generation concurrently with novel approaches to improve the models especially by the use of co-culture. The importance of heterotypic cell-cell interactions in the in vitro assessment of skin sensitisation should not be overlooked. This review addresses the technical aspects to take into consideration when co-culturing depending on the desired objective and describes the different keratinocytes and dendritic cells co-cultures developed in 2D and 3D. To date, from a regulatory point of view, no alternative method to animal testing for skin sensitisation potential assessment using a keratinocytes and dendritic cells co-culture model is yet proposed. This review also presents several directions of further development

Nrf2 expression and activity in human T lymphocytes: stimulation by T cell receptor activation and priming by inorganic arsenic and tert-butylhydroquinone.

International audienceThe transcription factor nuclear factor-erythroid 2-related-2 (Nrf2) controls cellular redox homeostasis and displays immunomodulatory properties. Nrf2 alters cytokine expression in murine T cells, but its effects in human T lymphocytes are unknown. This study investigated the expression and activity of Nrf2 in human activated CD4(+) T helper lymphocytes (Th cells) that mediate the adaptive immune response. Th cells were isolated from peripheral blood mononuclear cells and activated with antibodies against CD3 and CD28, mimicking physiologic Th cell stimulation by dendritic cells. Nrf2 is hardly detectable in unstimulated Th cells. Activation of Th cells rapidly and strongly increases the levels of Nrf2 protein by increasing NRF2 gene transcription. Th cell activation also enhances mRNA and protein levels of Nrf2 target genes encoding antioxidant enzymes. Blocking Nrf2 expression using chemical inhibitors or siRNAs prevents these gene inductions. Pretreatment with inorganic arsenic, a Nrf2 inducer that does not alter NRF2 gene expression, increases protein level and transcriptional activity of Nrf2 induced by Th cell stimulation. Inorganic arsenic enhances nuclear translocation of Nrf2, its interaction with the coactivator protein p300, and its DNA binding activity. Inhibition of Nrf2 expression abrogates the effects of inorganic arsenic on mRNA levels of antioxidant genes, but does not alter the expression of IL-2, TNF-α, interferon-γ, or IL-17 in Th cells activated in the absence or presence of the metalloid. In conclusion, this study demonstrates for the first time that stimulation of human Th cells increases transcription of the NRF2 gene and activity of the Nrf2 protein. However, modulation of Nrf2 levels does not modify the secretion of inflammatory cytokines from these T lymphocytes

Nrf2-dependent repression of interleukin-12 expression in human dendritic cells exposed to inorganic arsenic

International audienc

The Inflammatory Response in Human Keratinocytes Exposed to Cinnamaldehyde Is Regulated by Nrf2

Keratinocytes (KC) play a crucial role in epidermal barrier function, notably through their metabolic activity and the detection of danger signals. Chemical sensitizers are known to activate the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), leading to cellular detoxification and suppressed proinflammatory cytokines such as IL-1β, a key cytokine in skin allergy. We investigated the role of Nrf2 in the control of the proinflammatory response in human KC following treatment with Cinnamaldehyde (CinA), a well-known skin sensitizer. We used the well-described human KC cell line KERTr exposed to CinA. Our results showed that 250 μM of CinA did not induce any Nrf2 accumulation but increased the expression of proinflammatory cytokines. In contrast, 100 μM of CinA induced a rapid accumulation of Nrf2, inhibited IL-1β transcription, and downregulated the zymosan-induced proinflammatory response. Moreover, Nrf2 knockdown KERTr cells (KERTr ko) showed an increase in proinflammatory cytokines. Since the inhibition of Nrf2 has been shown to alter cellular metabolism, we performed metabolomic and seahorse analyses. The results showed a decrease in mitochondrial metabolism following KERTr ko exposure to CinA 100 µM. In conclusion, the fate of Nrf2 controls proinflammatory cytokine production in KCs that could be linked to its capacity to preserve mitochondrial metabolism upon chemical sensitizer exposure