Ethylhexadecyldimethylammonium bromide, a quaternary ammonium compound, controls inflammatory response through NRF2 pathway in a human immortalized keratinocyte cell line.

Many everyday products contain quaternary ammonium compounds (QAC) and some of them are known to be skin irritants such as benzalkonium chloride. Others, such as didecyldimethylammonium chloride, have been shown to cause allergic contact dermatitis. Ethylhexadecyldimethylammonium bromide (EHD) is a QAC for which sensitization potential is not clearly known. Therefore, we have studied its mechanism in human keratinocytes (KC), the main cells of the epidermis. We used the well-described human KC cell line KERTr exposed to EHD, cinnamaldehyde (CinA), a well-known skin sensitizer, and a mixture of both. Since 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, protein or mRNA expression of NRF2 pathway-related enzymes and pro-inflammatory cytokines were investigated by Western blot and RT-qPCR. The activity of the NRF2 pathway on inflammation was studied by RT-qPCR in NRF2-invalidated KERTr cells. We showed that EHD cannot induce the NRF2 pathway, unlike contact sensitizers like CinA. EHD triggers an inflammatory response by inducing the mRNA expression of pro-inflammatory cytokines such as IL-1ß or IL-6. Moreover, mixing EHD and CinA inhibits the effect of CinA on NRF2 expression and mitigates the inflammatory response induced by EHD alone. EHD treatment of KERTr cells in which NRF2 has been invalidated showed an exacerbation of the inflammatory response at the transcriptional level. Hence, EHD may elicit an inflammatory response in KC via the NF-κB pathway, which could lead to irritation when applied to the skin. This inflammation is negatively controlled by the basal activity of the NRF2 pathway.

Photobiomodulation Controls Keratinocytes Inflammatory Response through Nrf2 and Reduces Langerhans Cells Activation.

Photobiomodulation (PBM) is rapidly gaining traction as a valuable tool in dermatology for treating many inflammatory skin conditions using low levels of visible light or near-infrared radiation. However, the physiological regulatory pathways responsible for the anti-inflammatory effect of PBM have not been well defined. Since previous studies showed that nuclear factor-erythroid 2 like 2 (Nrf2) is a master regulator of the skin inflammatory response, we have addressed its role in controlling inflammation by PBM. Primary human keratinocytes (KCs) stimulated with 2,4-dinitrochlorobenzene (DNCB) to mimic pro-inflammatory stress were illuminated with two wavelengths: 660 nm or 520 nm. Both lights significantly reduced the mRNA expression of the DNCB-triggered TNF-α, IL-6, and IL-8 cytokines in KCs, while they enhanced Nrf2 pathway activation. PBM-induced Nrf2 is a key regulator of the inflammatory response in KCs since its absence abolished the regulatory effect of light on cytokines production. Further investigations of the mechanisms contributing to the immunoregulatory effect of PBM in inflamed human skin explants showed that 660 nm light prevented Langerhans cells migration into the dermis, preserving their dendricity, and decreased pro-inflammatory cytokine production compared to the DNCB-treated group. This study is the first to report that the PBM-mediated anti-inflammatory response in KCs is Nrf2-dependent and further support the role of PBM in skin immunomodulation. Therefore, PBM should be considered a promising alternative or complementary therapeutic approach for treating skin-related inflammatory diseases.

Quaternary ammonium compounds in hypersensitivity reactions.

Quaternary ammonium compounds (QAC) are commonly used disinfectants, antiseptics, preservatives, and detergents due to their antibacterial property and represent the first used biocides before phenolic or nitrogen products. Their common structure consists of one or more quaternary ammonium bound with four lateral substituents. Their amphiphilic structure allows them to intercalate into microorganism surfaces which induces an unstable and porous membrane that explains their antimicrobial activity towards bacteria, fungi, and viruses. QAC are thus found in many areas, such as household products, medicines, hygiene products, cosmetics, agriculture, or industrial products but are also used in medical practice as disinfectants and antiseptics and in health care facilities where they are used for cleaning floors and walls. QAC exposure has already been involved in occupational asthma in healthcare workers or professional cleaners by many authors. They also have been suggested to play a role in contact dermatitis (CD) and urticaria in workers using cosmetics such as hairdressers or healthcare workers, inciting reglementary agencies to make recommendations regarding those products. However, distinguishing the irritant or sensitizing properties of chemicals is complex and as a result, the sensitizing property of QAC is still controverted. Moreover, the precise mechanisms underlying the possible sensitization effect are still under investigation, and to date, only a few studies have documented an immunological mechanism. Besides, QAC have been suggested to be responsible for neuromuscular blocking agents (NMBA) sensitization by cross-reactivity. This hypothesis is supported by a higher prevalence of quaternary ammonium (QA)-specific IgE in the professionally exposed populations, such as hairdressers, cleaners, or healthcare workers, suggesting that the sensitization happens with structurally similar compounds present in the environment. This review summarizes the newest knowledge about QAC and their role in hypersensitivities. After describing the different QAC, their structure and use, the most relevant studies about the effects of QAC on the immune system will be reviewed and discussed.

Models of Dendritic Cells to Assess Skin Sensitization.

Allergic contact dermatitis (ACD) is a complex skin pathology occurring in reaction against environmental substances found in the workplace (cement, hair dyes, textile dyes), in the private environment (e.g., household products, cosmetic ingredients), or following skin exposure to drugs. Many cells are involved in the initiation of ACD during the sensitization phase. The four key events (KE) of skin sensitization AOP are covalent binding to skin proteins (KE1), keratinocyte activation (KE2), activation of DCs (KE3), and T-cell activation and proliferation (KE4), leading to the adverse outcome of ACD. Dendritic cells (DCs) are thus playing a key role in ACD pathophysiology. Indeed, in the presence of chemical sensitizers, DCs migrate from the skin to the draining lymph nodes and present peptide-chemical conjugates to T cells, leading to their activation and proliferation. In vitro methods have been actively developed to assess the activation of DCs by chemicals to establish a reliable in vitro sensitization test. Therefore, this review will detail the most used methods and protocols to develop DC models in vitro. Three different models of DCs will be addressed: 1) DCs derived from Cord Blood (CD34-DCs), 2) DCs derived from Monocytes (Mo-DCs), and 3) DCs derived from mice Bone-Marrow (BM-DCs). In addition, a model of exposition to contact sensitizers to assess KE3 of skin sensitization will be detailed for each of the models presented.

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-1ß, 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-1ß 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.

Regulation of the immune response to contact sensitizers by Nrf2.

The skin is frequently exposed to chemical stress by organic chemicals or metal ions that can directly or indirectly challenge its immune components and may lead to T-cell-mediated delayed-type hypersensitivity reactions. The disruption of the skin's homeostasis by exposure to contact sensitizers (CSs) can trigger an inflammatory immune response that results in eczema and allergic contact dermatitis. The recognition of these chemicals depends on the expression of pattern recognition receptors on sentinel skin cells, mainly the innate resident immune cells orchestrating the skin's immune response and involving both oxidative and inflammatory pathways. The main driver of these both pathways is the Nrf2/Keap1 pathway, a major ubiquitous regulator of cellular oxidative and electrophilic stress, activated in various innate immune cells of the skin, including keratinocytes and epidermal Langerhans cells in the epidermis and dermal dendritic cells in the dermis. Nrf2 also shows a strong protective capacity by downregulating inflammatory pathways. In this review, the important role of Nrf2 in the regulation of the immune response to CSs will be discussed and highlighted.

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

[This corrects the article DOI: 10.3389/fimmu.2018.01896.].

Phloretin suppresses neuroinflammation by autophagy-mediated Nrf2 activation in macrophages.

BACKGROUND: Macrophages play a dual role in neuroinflammatory disorders such as multiple sclerosis (MS). They are involved in lesion onset and progression but can also promote the resolution of inflammation and repair of damaged tissue. In this study, we investigate if and how phloretin, a flavonoid abundantly present in apples and strawberries, lowers the inflammatory phenotype of macrophages and suppresses neuroinflammation. METHODS: Transcriptional changes in mouse bone marrow-derived macrophages upon phloretin exposure were assessed by bulk RNA sequencing. Underlying pathways related to inflammation, oxidative stress response and autophagy were validated by quantitative PCR, fluorescent and absorbance assays, nuclear factor erythroid 2-related factor 2 (Nrf2) knockout mice, western blot, and immunofluorescence. The experimental autoimmune encephalomyelitis (EAE) model was used to study the impact of phloretin on neuroinflammation in vivo and confirm underlying mechanisms. RESULTS: We show that phloretin reduces the inflammatory phenotype of macrophages and markedly suppresses neuroinflammation in EAE. Phloretin mediates its effect by activating the Nrf2 signaling pathway. Nrf2 activation was attributed to 5' AMP-activated protein kinase (AMPK)-dependent activation of autophagy and subsequent kelch-like ECH-associated protein 1 (Keap1) degradation. CONCLUSIONS: This study opens future perspectives for phloretin as a therapeutic strategy for neuroinflammatory disorders such as MS. TRIAL REGISTRATION: Not applicable.

CD36-mediated uptake of myelin debris by macrophages and microglia reduces neuroinflammation.

BACKGROUND: The presence of foamy macrophages and microglia containing intracellular myelin remnants is a pathological hallmark of neurodegenerative disorders such as multiple sclerosis (MS). Despite the importance of myelin internalization in affecting both central nervous system repair and neuroinflammation, the receptors involved in myelin clearance and their impact on the phagocyte phenotype and lesion progression remain to be clarified. METHODS: Flow cytometry, quantitative PCR, and immunohistochemistry were used to define the mRNA and protein abundance of CD36 in myelin-containing phagocytes. The impact of CD36 and nuclear factor erythroid 2-related factor 2 (NRF2) on the phagocytic and inflammatory features of macrophages and microglia was assessed using a pharmacological CD36 inhibitor (sulfo-N-succinimidyl oleate) and Nrf2-/- bone marrow-derived macrophages. Finally, the experimental autoimmune encephalomyelitis (EAE) model was used to establish the impact of CD36 inhibition on neuroinflammation and myelin phagocytosis in vivo. RESULTS: Here, we show that the fatty acid translocase CD36 is required for the uptake of myelin debris by macrophages and microglia, and that myelin internalization increased CD36 expression through NRF2. Pharmacological inhibition of CD36 promoted the inflammatory properties of myelin-containing macrophages and microglia in vitro, which was paralleled by a reduced activity of the anti-inflammatory lipid-sensing liver X receptors and peroxisome proliferator-activated receptors. By using the EAE model, we provide evidence that CD36 is essential for myelin debris clearance in vivo. Importantly, CD36 inhibition markedly increased the neuroinflammatory burden and disease severity in the EAE model. CONCLUSION: Altogether, we show for the first time that CD36 is crucial for clearing myelin debris and suppressing neuroinflammation in demyelinating disorders such as MS.

Tau accumulates in Crohn's disease gut.

A sizeable body of evidence has recently emerged to suggest that gastrointestinal (GI) inflammation might be involved in the development of Parkinson's disease (PD). There is now strong epidemiological and genetical evidence linking PD to inflammatory bowel diseases and we recently demonstrated that the neuronal protein alpha-synuclein, which is critically involved in PD pathophysiology, is upregulated in inflamed segments of Crohn's colon. The microtubule associated protein tau is another neuronal protein critically involved in neurodegenerative disorders but, in contrast to alpha-synuclein, no data are available about its expression and phosphorylation patterns in inflammatory bowel diseases. Here, we examined the expression levels of tau isoforms, their phosphorylation profile and truncation in colon biopsy specimens from 16 Crohn's disease (CD) and 6 ulcerative colitis (UC) patients and compared them to samples from 16 controls. Additional experiments were performed in full thickness segments of colon of five CD and five control subjects, in primary cultures of rat enteric neurons and in nuclear factor erythroid 2-related factor (Nrf2) knockout mice. Our results show the upregulation of two main human tau isoforms in the enteric nervous system (ENS) in CD but not in UC. This upregulation was not transcriptionally regulated but instead likely resulted from a decrease in protein clearance via an Nrf2 pathway. Our findings, which provide the first detailed characterization of tau in CD, suggest that the key proteins involved in neurodegenerative disorders such as alpha-synuclein and tau, might also play a role in CD.

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

There 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 downregulates zymosan-induced neutrophil activation and modulates migration.

Polymorphonuclear neutrophils (PMNs) are the first line of defense against pathogens and their activation needs to be tightly regulated in order to limit deleterious effects. Nrf2 (Nuclear factor (erythroïd-derived 2)-like 2) transcription factor regulates oxidative stress and/or represses inflammation in various cells such as dendritic cells or macrophages. However, its involvement in PMN biology is still unclear. Using Nrf2 KO mice, we thus aimed to investigate the protective role of Nrf2 in various PMN functions such as oxidative burst, netosis, migration, cytokine production and phagocytosis, mainly in response to zymosan. We found that zymosan induced Nrf2 accumulation in PMNs leading to the upregulation of some target genes including Hmox-1, Nqo1 and Cat. Nrf2 was able to decrease zymosan-induced PMN oxidative burst; sulforaphane-induced Nrf2 hyperexpression confirmed its implication. Tnfα, Ccl3 and Cxcl2 gene transcription was decreased in zymosan-stimulated Nrf2 KO PMNs, suggesting a role for Nrf2 in the regulation of proinflammatory cytokine production. However, Nrf2 was not involved in phagocytosis. Finally, spontaneous migration of Nrf2 KO PMNs was lower than that of WT PMNs. Moreover, in response to low concentrations of CXCL2 or CXCL12, Nrf2 KO PMN migration was decreased despite similar CXCR2 and CXCR4 expression and ATP levels in PMNs from both genotypes. Nrf2 thus seems to be required for an optimal migration. Altogether these results suggest that Nrf2 has a protective role in several PMN functions. In particular, it downregulates their activation in response to zymosan and is required for an adequate migration.

Nrf2 Involvement in Chemical-Induced Skin Innate Immunity.

Exposure to certain chemicals disturbs skin homeostasis. In particular, protein-reactive chemical contact sensitizers trigger an inflammatory immune response resulting in eczema and allergic contact dermatitis. Chemical sensitizers activate innate immune cells which orchestrate the skin immune response. This involves oxidative and inflammatory pathways. In parallel, the Nrf2/Keap1 pathway, a major ubiquitous regulator of cellular oxidative and electrophilic stress is activated in the different skin innate immune cells including epidermal Langerhans cells and dermal dendritic cells, but also in keratinocytes. In this context, Nrf2 shows a strong protective capacity through the downregulation of both the oxidative stress and inflammatory pathways. In this review we highlight the important role of Nrf2 in the control of the innate immune response of the skin to chemical sensitizers.

Cutting Edge: Nrf2 Regulates Neutrophil Recruitment and Accumulation in Skin during Contact Hypersensitivity.

Neutrophils are essential during contact hypersensitivity (CHS), a common skin allergic disease. NF-E2-related factor-2 (Nrf2) is a key regulator of redox balance and skin homeostasis playing a protective role in CHS. In this study, we investigated Nrf2 role in neutrophil recruitment during the sensitization phase of CHS. Comparing wild-type and Nrf2 knockout mice, we demonstrated that Nrf2 regulated dinitrochlorobenzene-induced xenoinflammation, notably neutrophil recruitment to sensitized skin. Nrf2 protective role was associated with high expression of antioxidant genes (ho-1, gclc, nqo1…) and decreased chemokine production (CCL2, CCL4, CCL11). Interestingly, skin sensitization induced CD36 upregulation in skin-resident macrophages. In vitro results confirmed that the transcription of cd36 gene in macrophages was dependent on Nrf2 and led to an improved capacity to phagocyte-damaged neutrophils by efferocytosis. Nrf2 emerges as a critical target in the sensitization phase of CHS regulating neutrophil recruitment and accumulation in the skin through antioxidant-dependent and -independent mechanisms.

The THP-1 cell toolbox: a new concept integrating the key events of skin sensitization.

According to the current scientific consensus, one in vitro test is insufficient to cover the key events (KE) defined by the adverse outcome pathway (AOP) for skin sensitization. To address this issue we combined different end points in the same cell line to cover all KEs defined by the skin sensitization AOP. Since dendritic cells (DC) play a key role in the sensitization phase leading to the development of allergic contact dermatitis (ACD), we used THP-1 cells as a surrogate for DC. We measured ROS production and GSH depletion for KE1 (binding to proteins), Nrf2 activation pathway and gene expressions for KE2 (keratinocyte response), phenotype modifications using cell-surface markers and cytokine production for KE3 (DC activation), and T-cell proliferation for KE4 (T-cell activation). These measurements were performed using the THP-1 cell line and an original THP-1/T-cell co-culture system following exposure to a variety of chemicals, including irritant, non-sensitizers, and chemicals sensitizers (pro/prehaptens). Results showed that treatment with sensitizers such as cinnamaldehyde (100 µM) or methylisothiazolinone (150 µM) was able to trigger the three main key events (KE1, KE2, and KE3) of the sensitization phase of ACD in THP-1 cells. In addition, all sensitizers were able to induce T lymphocyte proliferation (KE4), while non-sensitizers and irritants did not. Our study shows for the first time that addressing the four main KE of skin sensitization AOP in a single cell line is an achievable task.

Impairment of glyoxalase-1, an advanced glycation end-product detoxifying enzyme, induced by inflammation in age-related osteoarthritis.

BACKGROUND: Accumulation of advanced glycation end-products (AGEs) is involved in age-related osteoarthritis (OA). Glyoxalase (Glo)-1 is the main enzyme involved in the removal of AGE precursors, especially carboxymethyl-lysine (CML). We aimed to investigate the expression of several AGEs and Glo-1 in human OA cartilage and to study chondrocytic Glo-1 regulation by inflammation, mediated by interleukin (IL)-1ß. METHODS: Ex vivo, we quantified AGEs (pentosidine, CML, methylglyoxal-hydroimidazolone-1) in knee cartilage from 30 OA patients. Explants were also incubated with and without IL-1ß, and we assessed Glo-1 protein expression and enzymatic activity. In vitro, primary cultured murine chondrocytes were stimulated with increasing concentrations of IL-1ß to assess Glo-1 enzymatic activity and expression. To investigate the role of oxidative stress in the IL-1ß effect, cells were also treated with inhibitors of mitochondrial oxidative stress or nitric oxide synthase. RESULTS: Ex vivo, only the human cartilage CML content was correlated with patient age (r = 0.78, p = 0.0031). No statistically significant correlation was found between Glo-1 protein expression and enzymatic activity in human cartilage and patient age. We observed that cartilage explant stimulation with IL-1ß decreased Glo-1 protein expression and enzymatic activity. In vitro, we observed a dose-dependent decrease in Glo-1 mRNA, protein quantity, and enzymatic activity in response to IL-1ß in murine chondrocytes. Inhibitors of oxidative stress blunted this downregulation. CONCLUSION: Glo-1 is impaired by inflammation mediated by IL-1ß in chondrocytes through oxidative stress pathways and may explain age-dependent accumulation of the AGE CML in OA cartilage.

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.

Induction of brain Nrf2-HO-1 pathway and antinociception after different physical training paradigms in mice.

AIM: Activation of the Nrf2-antioxidant response element signaling pathway is a major mechanism in the cellular defense against oxidative or electrophilic stress through conjugative reactions and by enhancing cellular antioxidant capacity. Although exercise training up-regulates antioxidant defenses system, while information regarding the intensity levels of physical exercise that acts on the cellular protection systems is limited. MAIN METHODS: The present study evaluated the effects of different durations and intensities of physical exercise on the hippocampus, cortex and hypothalamus Nrf2 and HO-1 gene expression and related protein content and the nociception thresholds in adult C57Bl male mice. Exercise training consisted of daily running on a 10-lane rodent motor-driven treadmill for either 3 or 7 weeks at three different intensities. Pain responses were evaluated after exercise and in untrained mice by Von Frey hair test and cold plate test. KEY FINDINGS: This study confirmed that only vigorous and longer duration aerobic exercise increased Nrf2 protein level in the hippocampus and HO-1 protein level in the cortex and reduced pain perception. Mechanical and thermal hypoalgesia were only observed in exercise groups after 7 weeks of physical training. SIGNIFICANCE: The overall findings in this study confirm that only the long duration intensive forced exercise reduced inflammatory pain by induction of Nrf2/HO-1 antioxidant signaling pathway.

Editor's Highlight: Fragrance Allergens Linalool and Limonene Allylic Hydroperoxides in Skin Allergy: Mechanisms of Action Focusing on Transcription Factor Nrf2.

Allergic contact dermatitis is regarded as the most frequent expression of immunotoxicity in humans. Many odorant terpenes commonly used in fragrance compositions are considered as weak skin sensitizers, whereas some of their autoxidation products, allylic hydroperoxides, are classified as strong sensitizers according to the local lymph node assay. However, the mechanism of their effects on the immune system remains unclear. Since dendritic cells play a key role in allergic contact dermatitis, we studied their activation by the frequently used linalool (LINA) and limonene (LIMO), and their respective sensitizing allylic hydroperoxides (LINA-OOH, LIMO-OOH). The THP-1 cell-line was used as a surrogate for dendritic cells, the model currently employed in the validated h-CLAT in vitro test. Our data showed that allylic hydroperoxides behave differently. Both LINA-OOH and LIMO-OOH oxidized cell surface thiols 30 min after stimulation. However, the oxidative stress induced by LINA-OOH was stronger, with a higher decreased GSH/GSSG ratio and a stronger reactive species production. Moreover, LINA-OOH induced a stronger Nrf2 accumulation in correlation with nqo1 and ho-1 gene expression, 2 Nrf2 target genes. Regarding signaling pathways involved in these effects, P38 mitogen-activated protein kinase and P-ERK were activated in response to LINA-OOH but not with LIMO-OOH. CD54 and CD86 were induced 24-h postexposure. In contrast, LINA and LIMO did not modify THP-1 phenotype. This work underlies that autoxidation forming allylic hydroperoxide (ROOH) does not lead to equal chemical reactivity since LINA-OOH appears to be a stronger activator than LIMO-OOH, in regard to oxidative stress and Nrf2 pathway activation.

The nuclear factor-erythroid 2-related factor/heme oxygenase-1 axis is critical for the inflammatory features of type 2 diabetes-associated osteoarthritis.

Epidemiological findings support the hypothesis that type 2 diabetes mellitus (T2DM) is a risk factor for osteoarthritis (OA). Moreover, OA cartilage from patients with T2DM exhibits a greater response to inflammatory stress, but the molecular mechanism is unclear. To investigate whether the antioxidant defense system participates in this response, we examined here the expression of nuclear factor-erythroid 2-related factor (Nrf-2), a master antioxidant transcription factor, and of heme oxygenase-1 (HO-1), one of its main target genes, in OA cartilage from T2DM and non-T2DM patients as well as in murine chondrocytes exposed to high glucose (HG). Ex vivo experiments indicated that Nrf-2 and HO-1 expression is reduced in T2DM versus non-T2DM OA cartilage (0.57-fold Nrf-2 and 0.34-fold HO-1), and prostaglandin E2 (PGE2) release was increased in samples with low HO-1 expression. HG-exposed, IL-1ß-stimulated chondrocytes had lower Nrf-2 levels in vitro, particularly in the nuclear fraction, than chondrocytes exposed to normal glucose (NG). Accordingly, HO-1 levels were also decreased (0.49-fold) in these cells. The HO-1 inducer cobalt protoporphyrin IX more efficiently attenuated PGE2 and IL-6 release in HG+IL-1ß-treated cells than in NG+IL-1ß-treated cells. Greater reductions in HO-1 expression and increase in PGE2/IL-6 production were observed in HG+IL-1ß-stimulated chondrocytes from Nrf-2-/- mice than in chondrocytes from wild-type mice. We conclude that the Nrf-2/HO-1 axis is a critical pathway in the hyperglucidic-mediated dysregulation of chondrocytes. Impairments in this antioxidant system may explain the greater inflammatory responsiveness of OA cartilage from T2DM patients and may inform treatments of such patients.