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Intestinal bacteria metabolize dietary substances to produce bioactive postbiotics, among which some are recognized for their role in promoting host health. We here explored the postbiotic potential of two omega-3 α-linolenic acid-derived metabolites: trans -10- cis -15-octadecadienoic acid (t10,c15-18:2) and cis -9- cis -15-octadecadienoic acid (c9,c15-18:2). Dietary intake of lipids rich in omega-3 α-linolenic acid elevated levels of t10,c15-18:2 and c9,c15-18:2 in the serum and feces of mice, an effect dependent on the presence of intestinal bacteria. Notably, t10,c15-18:2 mitigated skin inflammation in mice that became hypersensitive after exposure to 2,4-dinitrofluorobenzene, an experimental model for allergic contact dermatitis. In particular, t10,c15-18:2-but not c9,c15-18:2-attenuated ear swelling and edema, characteristic symptoms of contact hypersensitivity. The anti-inflammatory effects of t10,c15-18:2 were due to its ability to suppress the release of vascular endothelial growth factor A from keratinocytes, thereby mitigating the enhanced vascular permeability induced by hapten stimulation. Our study identified retinoid X receptor as a functional receptor that mediates the downregulation of skin inflammation upon treatment with t10,c15-18:2. Our results suggest that t10,c15-18:2 holds promise as an omega-3 fatty acid-derived postbiotic with potential therapeutic implications for alleviating the skin edema seen in allergic contact dermatitis-induced inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Saika, Nagatake, Kishino, Kitamura, Honda, Hosomi, Tiwari, Node, Kawai, Kondo, Ishida, Kabashima, Ogawa and Kunisawa.)

Patients receiving neuraxial treatment with morphine for pain relief often experience a distressing pruritus. Neuroinflammation-mediated plasticity of sensory synapses in the spinal cord is critical for the development of pain and itch. Caspase-6, as an intracellular cysteine protease, is capable of inducing central nociceptive sensitization through regulating synaptic transmission and plasticity. Given the tight interaction between protein kinase Mζ (PKMζ) and excitatory synaptic plasticity, this pre-clinical study investigates whether caspase-6 contributes to morphine-induced itch and chronic itch via PKMζ. Intrathecal morphine and contact dermatitis were used to cause pruritus in mice. Morphine antinociception, itch-induced scratching behaviors, spinal activity of caspase-6, and phosphorylation of PKMζ and ERK were examined. Caspase-6 inhibitor Z-VEID-FMK, exogenous caspase-6 and PKMζ inhibitor ZIP were utilized to reveal the mechanisms and prevention of itch. Herein, we report that morphine induces significant scratching behaviors, which is accompanied by an increase in spinal caspase-6 cleavage and PKMζ phosphorylation (but not expression). Intrathecal injection of Z-VEID-FMK drastically reduces morphine-induced scratch bouts and spinal phosphorylation of PKMζ, without abolishing morphine analgesia. Moreover, intrathecal strategies of ZIP dose-dependently reduce morphine-induced itch-like behaviors. Spinal phosphorylation of ERK following neuraxial morphine is down-regulated by ZIP therapy. Recombinant caspase-6 directly exhibits scratching behaviors and spinal phosphorylation of ERK, which is compensated by PKMζ inhibition. Also, spinal inhibition of caspase-6 and PKMζ reduces the generation and maintenance of dermatitis-induced chronic itch. Together, these findings demonstrate that spinal caspase-6 modulation of PKMζ phosphorylation is important in the development of morphine-induced itch and dermatitis-induced itch in mice., (Copyright © 2023 IBRO. Published by Elsevier Inc. All rights reserved.)

Dermal regulatory T cells (Tregs) are essential for maintenance of skin homeostasis and control of skin inflammatory responses. In mice, Tregs in the skin are characterized by high expression of CD103, the αE integrin. Evidence indicates that CD103 promotes Treg retention within the skin, although the mechanism underlying this effect is unknown. The main ligand of CD103, E-cadherin, is predominantly expressed by cells in the epidermis. However, because Tregs are predominantly located within the dermis, the nature of the interactions between E-cadherin and CD103-expressing Tregs is unclear. In this study, we used multiphoton intravital microscopy to examine the contribution of CD103 to Treg behavior in resting and inflamed skin of mice undergoing oxazolone-induced contact hypersensitivity. Inhibition of CD103 in uninflamed skin did not alter Treg behavior, whereas 48 h after inducing contact hypersensitivity by oxazolone challenge, CD103 inhibition increased Treg migration. This coincided with E-cadherin upregulation on infiltrating myeloid leukocytes in the dermis. Using CD11c-enhanced yellow fluorescent protein (EYFP) × Foxp3-GFP dual-reporter mice, inhibition of CD103 was found to reduce Treg interactions with dermal dendritic cells. CD103 inhibition also resulted in increased recruitment of effector CD4+ T cells and IFN-γ expression in challenged skin and resulted in reduced glucocorticoid-induced TNFR-related protein expression on Tregs. These results demonstrate that CD103 controls intradermal Treg migration, but only at later stages in the inflammatory response, when E-cadherin expression in the dermis is increased, and provide evidence that CD103-mediated interactions between Tregs and dermal dendritic cells support regulation of skin inflammation., (Copyright © 2023 by The American Association of Immunologists, Inc.)

The stratum corneum is the outermost layer of the epidermis. It acts as an interface with the external environment and functions as a barrier that prevents microorganisms and allergens from penetrating the skin, while preventing bodily fluids, electrolytes and proteins from being lost in a process aimed at maintaining homeostasis. With the novel coronavirus disease 2019 (COVID-19) outbreak, there has been an increase in hygiene practice, particularly hand washing and the use of hand sanitisers. These practices have undoubtedly assisted a great deal in combatting the rate of transmission and contributed immensely to saving lives. However, repeated hand washing and the use of sanitisers have both been linked with marked skin dryness and contact dermatitis. This especially holds true when the above-mentioned practices are carried out in the absence of intermittent hand moisturiser usage.

p-phenylenediamine (PPD) is a common component of hair dye known to induce immediate allergy, even acute dermatitis and contact dermatitis. MAS-related G protein coupled receptor-X2 (MRGPRX2) in mast cells (MCs) mediates small molecular substances-induced pseudo-allergic reactions. However, the role of MRGPRX2 in PPD-induced immediate contact allergy needs further exploration. The aim of this study was to investigate whether PPD activates MCs via MRGPRX2 and induces immediate allergies that contribute to contact dermatitis. Wild-type (WT) and kit w-sh/w-sh mice (MUT) were treated with PPD to observe local inflammation and MC degranulation in vivo. The release of inflammatory mediators was measured in vitro. Histamine 1 receptor (H 1 R) -/- mice were used to analyze itch type. PPD caused immediate contact allergy in WT mice, induced scratching, and local inflammatory reactions, while exhibiting minimal effects on MUT mice. PPD did not induce histamine release, but induced significant tryptase release in vivo and in vitro. PPD activated MRGPRX2 to induce MC degranulation in vitro. PPD caused immediate contact allergy in WT mice, induced scratching and local inflammatory reactions, while exhibited minimal effect on MUT mice. PPD did not induce histamine release, while induced significant tryptase release in vivo and in vitro. PPD induced immediate contact allergy by MCs activation via MRGPRX2 and lead to tryptase release. The scratching times showed no significant difference in WT mice or H 1 R -/- mice, which indicated PPD caused non-histaminergic itch. The results showed that PPD activated MCs via MRGPRX2 and induced immediate contact allergy, leading to the release of tryptase without monoamine release, which might induce non-histaminergic itch., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with excessive inflammation and defective skin barrier function. Activated protein C (APC) is a natural anticoagulant with anti-inflammatory and barrier protective functions. However, the effect of APC on AD and its engagement with protease activated receptor (PAR)1 and PAR2 are unknown. Methods: Contact hypersensitivity (CHS), a model for human AD, was induced in PAR1 knockout (KO), PAR2KO and matched wild type (WT) mice using 2,4-dinitrofluorobenzene (DNFB). Recombinant human APC was administered into these mice as preventative or therapeutic treatment. The effect of APC and PAR1KO or PARKO on CHS was assessed via measurement of ear thickness, skin histologic changes, inflammatory cytokine levels, Th cell phenotypes and keratinocyte function. Results: Compared to WT, PAR2KO but not PAR1KO mice displayed less severe CHS when assessed by ear thickness; PAR1KO CHS skin had less mast cells, lower levels of IFN-γ, IL-4, IL-17 and IL-22, and higher levels of IL-1β, IL-6 and TGF-β1, whereas PAR2KO CHS skin only contained lower levels of IL-22 and IgE. Both PAR1KO and PAR2KO spleen cells had less Th1/Th17/Th22/Treg cells. In normal skin, PAR1 was present at the stratum granulosum and spinosum, whereas PAR2 at the upper layers of the epidermis. In CHS, however, the expression of PAR1 and PAR2 were increased and spread to the whole epidermis. In vitro, compared to WT cells, PAR1KO keratinocytes grew much slower, had a lower survival rate and higher para permeability, while PAR2KO cells grew faster, were resistant to apoptosis and para permeability. APC inhibited CHS as a therapeutic but not as a preventative treatment only in WT and PAR1KO mice. APC therapy reduced skin inflammation, suppressed epidermal PAR2 expression, promoted keratinocyte growth, survival, and barrier function in both WT and PAR1KO cells, but not in PAR2KO cells. Conclusions: APC therapy can mitigate CHS. Although APC acts through both PAR1 and PAR2 to regulate Th and mast cells, suppression of clinical disease in mice is achieved mainly via inhibition of PAR2 alone. Thus, APC may confer broad therapeutic benefits as a disease-modifying treatment for AD.

Hypertension is a chronic disease associated with chronic inflammation involving activated macrophages. Antihypertensive drugs (for example, angiotensin-converting enzyme inhibitors-ACEIs) used in the treatment of hypertension have immunomodulatory properties. On the other hand, the immunological effect of diuretics and combined drugs (diuretics + ACEI) is unclear. Therefore, we examined the influence of diuretics and combination drugs (ACEI + diuretic) on cellular response (contact hypersensitivity), production of reactive oxygen intermediates (ROIs), and nitric oxide (NO), and the secretion of interleukin-12 (IL-12). CBA mice were administered i.p. captopril (5 mg/kg) with or without hydrochlorothiazide (10 mg/kg) or furosemide (5 mg/kg) for 8 days. On the third day, the mice were administered i.p. mineral oil, and macrophages were collected 5 days later. In the presented results, we show that diuretics administered alone or with captopril increase the generation of ROIs and reduce the formation of NO by macrophages. Moreover, tested drugs inhibit the secretion of IL-12. Diuretics and combined drugs reduce the activity of contact hypersensitivity (both activation and induction phases). Our research shows that the tested drugs modulate the cellular response by influencing the function of macrophages, which is important in assessing the safety of antihypertensive therapy.

As a natural dietary ingredient, berberine possesses multiple biological activities including anti-inflammatory effects. In this work, glucocorticoid receptor (GR)-mediated alleviation of inflammation by berberine was investigated by a combination of in vitro , in silico , and in vivo approaches. The fluorescence polarization assay showed that berberine bound to GR with an IC 50 value of 9.14 ± 0.16 pM. Molecular docking and molecular dynamics simulation suggested that berberine bound stably to the active site of GR via hydrogen bonding and hydrophobic interactions. Berberine induced GR nuclear translocation but did not activate the glucocorticoid response element in HeLa cells. Furthermore, both gene and protein expressions of PEPCK were significantly attenuated by berberine in HepG2 cells. Interestingly, berberine downregulated CBG mRNA and protein levels without up-regulating TAT mRNA and protein levels in HepG2 cells, demonstrating its dissociated characteristics that could separate transrepression from transactivation. In addition, the in vitro and in vivo anti-inflammatory effects of berberine were confirmed in lipopolysaccharide-induced RAW 264.7 cells and in a mouse model of allergic contact dermatitis, respectively. In conclusion, berberine might serve as a potential selective GR modulator.

Although allergic contact dermatitis (ACD) is the most common T cell-mediated inflammatory responses against an allergen in the skin, the pathogenesis of ACD remains incompletely understood. In the sensitization phase in ACD, hapten-bearing dermal dendritic cells (DCs) play a pivotal role in the transport of an antigen to the lymph nodes (LNs), where they present the antigen to naïve T cells. Here we report that Allergin-1, an inhibitory immunoreceptor containing immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic region, is highly expressed on dermal DCs. Mice deficient in Allergin-1 exhibited exacerbated fluorescein isothiocyanate (FITC)-induced type 2 contact hypersensitivity (CHS) such as ear swelling and skin eosinophilia. Allergin-1-deficient mice also showed larger numbers of CD4 + T cells and FITC-bearing DCs and greater expressions of type 2 cytokines, including IL-5, IL-10 and IL-13, in the draining LNs than did wild type mice. In sharp contrast, Allergin-1-deficient mice showed comparable level of type 1 CHS induced by 2,4-dinitrofluorobenzene (DNFB). These results suggest that Allergin-1 on dermal DC inhibits type 2, but not type 1, immune responses in the sensitization phase of CHS., Competing Interests: Declaration of competing interest This research was funded in part by Ono Pharmaceutical Co., Ltd. S. Shibayama is an employee of Ono Pharmaceutical Co., Ltd. The sponsor had no control over the interpretation, writing, or publication of this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: The flower buds of Sophora japonica L. are a major traditional medicine in China, Japan, and Korea and are used to stop bleeding and 'cool the blood'. Accordingly, they are used to treat bleeding haemorrhoids, hypertension, and pyoderma. In addition, it was recently found that the flower buds of S. japonica (SJ) have cosmetic whitening properties., Materials and Methods: Compounds in SJ and their targets and related diseases were investigated using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform. Target gene information was obtained from the UniProt database. Network construction was carried out using Cytoscape 3.72. Contact dermatitis (CD)-related gene searching was performed using the Cytoscape string App. Docking analysis was conducted using AutoDock Vina. Six-week-old Balb/c male mice with DNFB (1-fluoro-2,4-dinitrofluorobenzene)-induced CD were treated with a methanol extract of the flower buds of S. japonica (MESJ), and its effects on skin colour, lesions, and immune cell infiltration, and on histopathological abnormalities such as epidermal hyperplasia were investigated., Results: Eleven compounds targeted 13 CD-related genes, that is, serum albumin (ALB), prostaglandin G/H synthase (COX) 2, C-X-C motif chemokine (CXCL) 2, CXCL10, ICAM1, IFN-γ, IL-10, IL-1α, IL-1β, IL-2, IL-6, E-selectin, and TNF. In the murine DNFB model, MESJ significantly suppressed scaling, erythema, and skin thickening as compared with DNFB controls and epithelial hyperplasia and immune cell infiltrations induced by repeated DNFB application., Conclusions: Our animal study showed that the mode of action of MESJ was closely related to the prevention of epithelial hyperplasia and immune cell infiltration. The results obtained demonstrated that the flower buds of S. japonica offer a potential means of treating CD, and suggest that the therapeutic mechanism of CD is explained by relations between 11 major components of SJ, including kaempferol and quercetin, and 13 CD-related genes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The present study evaluated the anti-inflammatory effect of nanoencapsulated curcuminoid preparations of poly(vinyl pyrrolidone) (Nano-cur) and free curcuminoids (Cur) in an experimental model of croton oil-induced cutaneous inflammation. Male Swiss mice, weighing 25-30 g, received oral treatment by gavage 1 h before CO application or topical treatment immediately after CO application (200 μg diluted in 70% acetone) with a single dose of Cur and Nano-cur. After 6 h, the animals were anesthetized and euthanized. The ears were sectioned into disks (6.0 mm diameter) and used to determine edema, myeloperoxidase (MPO) activity, and oxidative stress. Photoacoustic spectroscopy (PAS) was used to evaluate the percutaneous penetration of Cur and Nano-cur. Topical treatment with both preparations had a similar inhibitory effect on the development of edema, MPO activity, and the oxidative response. The PAS technique showed that the percutaneous permeation of both topically applied preparations was similar. Oral Nano-cur administration exerted a higher anti-inflammatory effect than Cur. Topical Cur and Nano-cur application at the same dose similarly inhibited the inflammatory and oxidative responses. Oral Nano-cur administration inhibited such responses at doses that were eight times lower than Cur, suggesting the better bioavailability of Nano-cur compared with Cur.Graphical abstract.

Ethnopharmacological Relevance: Numerous epidemiological and clinical studies have demonstrated the protective role of dietary isoflavones against development of several chronic diseases. ISO-1, one fraction of isoflavone powders derived from soybean cake, is reported to attenuate inflammation and photodamage., Aim of the Study: Contact dermatitis is a common inflammatory skin disease, which accounts for most occupational skin disorders. Instead of oral administration, we aimed to explore the effects of topical ISO-1 application on contact dermatitis by using 2,4-dinitrochlorobenzene (DNCB)-stimulated HaCaT keratinocytes and DNCB-induced mouse dermatitis as models., Materials and Methods: In the in vitro study, we first evaluated the biologic effects of DNCB on HaCaT keratinocytes. HaCaT keratinocytes were treated with 2,4-dinitrochlorobenzene (DNCB), and cell viability was measured by MTT assay. Then, we detect the prominent induction of IL-8 mRNA expression after DNCB and ISO-1 treatment by reverse transcription polymerase chain reaction (RT-PCR), and release of IL-8 from HaCaT keratinocytes was measured by ELISA assay. HaCaT keratinocytes were pretreated with ISO-1 and then treated with DNCB, phosphorylation of JNK, p38, ERK and IκBα was analyzed by western blot. In the in vivo study, the hairless mice were used for an induced contact dermatitis model. The surface changes in the dorsal skin after DNCB and ISO-1 treatment were recorded using photography, and TEWL, erythema were measured using an MPA-580 cutometer. Blood was also collected from mice for measurement of white blood cell counts., Results: Results showed ISO-1 inhibited DNCB-induced IL-8 production and also suppressed DNCB-induced phosphorylation of JNK and p38, and IκBα in HaCaT. In the animal model of DNCB-induced contact dermatitis, topical ISO-1 treatment significantly decreased DNCB-induced erythema and transepidermal water loss (TEWL) in mouse skin. ISO-1 also reduced DNCB-induced skin thickening and increase of white blood cell count., Conclusions: ISO-1 is promising for improvement of DNCB-induced inflammation and skin barrier impairment, suggesting the potential application of topical ISO-1 for inflammatory dermatoses., (Copyright © 2020 Elsevier B.V. All rights reserved.)

CX3CL1 can function as both an adhesion molecule and a chemokine for CX3CR1 + cells, such as T cells, monocytes, and NK cells. Recent studies have demonstrated that CX3CL1-CX3CR1 interaction is associated with the development of various inflammatory skin diseases. In this study, we examined CX3CR1 involvement in 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity using CX3CR1 -/- mice. Ear swelling and dermal edema were attenuated after DNFB challenge in CX3CR1 -/- mice. Expression of TNF-α, IL-6, and M1 macrophage markers was decreased in the ears of CX3CR1 -/- mice, whereas expression of M2 macrophage markers including arginase-1 was increased. Decreased TNF-α and IL-6 expression and increased arginase-1 expression were found in peritoneal macrophages from CX3CR1 -/- mice. Furthermore, ear swelling was attenuated by depleting dermal macrophages in wild-type mice to a similar level to CX3CR1 -/- mice. These results suggest that CX3CR1 deficiency could induce skewed polarization towards M2 phenotype in macrophages, resulting in attenuation of contact hypersensitivity response.

The hair cycle consists of three different phases: anagen (growth), catagen (regression), and telogen (resting). During the anagen phase, hair follicle stem cells (HFSCs) in the bulge and the secondary hair germ proliferate and generate the outer and inner root sheath cells and the hair shafts. We previously identified NG2-immunoreactive (NG2+) cells as HFSCs in both regions of the hair follicles. Recently, the interaction between the hair cycle and the cutaneous immune system has been re-examined under physiological and pathological conditions. However, the roles of NG2+ HFSCs in the skin's immune system remain completely elucidated. In the present study, we investigated whether the elimination of NG2+ HFSCs affects the induction of allergic contact dermatitis, using a herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene system. When the GCV solution was applied to the skin of NG2-HSVtk transgenic (Tg) rats during the depilation-induced anagen phase, NG2+ HFSCs in the Tg rat skin induced apoptotic cell death. Under exposure of a hapten, the selective ablation of NG2+ HFSCs during the anagen phase aggravated the sensitization phase of allergic contact dermatitis. These findings suggest that NG2+ HFSCs and their progeny have immunosuppressive abilities during the anagen phase.

Suprabasin (SBSN) is expressed not only in epidermis but also in epithelial cells of the upper digestive tract where metals such as nickel are absorbed. We have recently shown that SBSN level is decreased in the stratum corneum and serum of atopic dermatitis (AD) patients, especially in intrinsic AD, which is characterized by metal allergy. By using SBSN-null (Sbsn -/- ) mice, this study was conducted to investigate the outcome of SBSN deficiency in relation to AD. Sbsn -/- mice exhibited skin barrier dysfunction on embryonic day 16.5, but after birth, their barrier function was not perturbed despite the presence of ultrastructural changes in stratum corneum and keratohyalin granules. Sbsn -/- mice showed a comparable ovalbumin-specific skin immune response to wild type (WT) mice and rather lower contact hypersensitivity (CHS) responses to haptens than did WT mice. The blood nickel level after oral feeding of nickel was significantly higher in Sbsn -/- mice than in WT mice, and CHS to nickel was elevated in Sbsn -/- mice under nickel-loading condition. Our study suggests that the completely SBSN deficient mice retain normal barrier function, but harbor abnormal upper digestive tract epithelium that promotes nickel absorption and high CHS to nickel, sharing the features of intrinsic AD.

Objective: To investigate the anti-inflflammatory effects of Sanguisorbae Radix on contact dermatitis (CD)., Methods: Mice were sensitized by painting 30 µL of 1-fluoro-2,4-dinitrofluorobenzene (DNFB) onto each ear for 3 days. Four days later, mice were challenged by painting with 50 µL of DNFB onto the shaved dorsum every 2 days. Sanguisorbae Radix methanol extract (MESR) was applied onto the shaved dorsum every 2 days. The effects of MESR on skin thickness, skin weights, histopathological changes, skin lesions and cytokine production in DNFB-induced CD mice were investigated, as well as its effects on body weights and spleen/body weight ratio., Results: Topical application of MESR effectively inhibited enlargement of skin thickness and weight (P<0.05). MESR treatment also inhibited hyperplasia, spongiosis and immune cell infiltration induced by DNFB in inflamed tissues and improved lesions on dorsum skin in CD mice. Moreover, treatment with MESR suppressed the increase in the levels of tumor necrosis factor α (TNF-α,P<0.01) and interferon γ (IFN-γ,P<0.05), respectively. Finally, MESR had no effect on body weight gain or spleen/body weight ratio., Conclusion: These data suggest that MESR acts as an anti-inflflammatory agent that decreases the production of TNF-α and IFN-γ, resulting in reductions of skin lesions and histopathological changes in inflamed skin tissues.

Contact hypersensitivity (CHS) is the most common occupational dermatological disease. Dendritic cells (DCs) mediate the sensitization stage of CHS, while T-cells facilitate the effector mechanisms that drive CHS. Black raspberry ( Rubus occidentalis , BRB) and BRB phytochemicals possess immunomodulatory properties, but their dietary effects on CHS are unknown. We examined the effects of diets containing BRB and protocatechuic acid (PCA, a constituent of BRB and an anthocyanin metabolite produced largely by gut microbes), on CHS, using a model induced by 2,4-dinitrofluorobenze (DNFB). Mice were fed control diet or diets supplemented with BRB or PCA. In vitro bone-marrow derived DCs and RAW264.7 macrophages were treated with BRB extract and PCA. Mice fed BRB or PCA supplemented diets displayed decreased DNFB-induced ear swelling, marked by decreased splenic DC accumulation. BRB extract diminished DC maturation associated with reduced Cd80 expression and Interleukin (IL)-12 secretion, and PCA reduced IL-12. Dietary supplementation with BRB and PCA induced differential decreases in IL-12-driven CHS mediators, including Interferon (IFN)-γ and IL-17 production by T-cells. BRB extracts and PCA directly attenuated CHS-promoting macrophage activity mediated by nitric oxide and IL-12. Our results demonstrate that BRB and PCA mitigate CHS pathology, providing a rationale for CHS alleviation via dietary supplementation with BRB or BRB derived anthocyanins.

The linear ubiquitin chain assembly complex (LUBAC), which consists of HOIP, SHARPIN and HOIL-1L, promotes NF-κB activation and protects against cell death by generating linear ubiquitin chains. LUBAC contains two RING-IBR-RING (RBR) ubiquitin ligases (E3), and the HOIP RBR is responsible for catalysing linear ubiquitination. We found that HOIL-1L RBR plays a crucial role in regulating LUBAC. HOIL-1L RBR conjugates monoubiquitin onto all LUBAC subunits, followed by HOIP-mediated conjugation of linear chains onto monoubiquitin, and these linear chains attenuate the functions of LUBAC. The introduction of E3-defective HOIL-1L mutants into cells augmented linear ubiquitination, which protected the cells against Salmonella infection and cured dermatitis caused by reduced LUBAC levels due to SHARPIN loss. Our results reveal a regulatory mode of E3 ligases in which the accessory E3 in LUBAC downregulates the main E3 by providing preferred substrates for autolinear ubiquitination. Thus, inhibition of HOIL-1L E3 represents a promising strategy for treating severe infections or immunodeficiency.

Ethnopharmacological Relevance: Popularly used in India and sub-Hymalaian region, Moringa oleifera (Moringaceae) is associated with healing properties demonstrated in its use as treatment of acute and chronic skin diseases. Our study aimed at investigating the effects of M. oleifera seed oil (MOSO) in animal models for inflammatory and hyperproliferative skin conditions., Materials and Methods: MOSO was analyzed using gas chromatography/mass spectrometry. The anti-inflammatory and anti-hyperproliferative effects of treatment with either MOSO or oleic acid (OA), its main constituent, was evaluated. Acute and chronic inflammation was induced by applying 12-O-Tetradecanoylphorbol-13-acetate (TPA) and acute inflammation with either Arachidonic Acid (AA) or Phenol onto the ear of Swiss mice. Systemic activity and the influence of glucocorticoid receptors (GC) was also evaluated., Results: Topical application of MOSO and OA inhibited ear edema caused by TPA, and Phenol. Only MOSO inhibited ear edema induced by AA. Neutrophil migration was also inhibited by treatment with MOSO. Topical application of MOSO, but not OA, significantly reduced chronic skin inflammation and epidermal hypertrophy induced by multiple TPA applications. Pre-treatment with GC antagonist mifepristone reversed the anti-inflammatory effect of MOSO and OA on the TPA model. Repeated administration of MOSO show a similar effect to dexamethasone on thymus weight, though MOSO did not present any influence on skin thickness, as well as in the weight of the spleen, adrenal gland and lymph node., Conclusion: The results suggest that MOSO is effective as a treatment for skin diseases that rely on keratinocyte hyperproliferation. OA is also effective in acute inflammation. Both MOSO and OA depend on GC activation for anti-inflammatory effect but do not exhibit the same adverse effects seen in topical treatment with dexamethasone. We hereby evidence the use of MOSO as a topical anti-inflammatory agent in inflammatory skin diseases, thus, expanding its therapeutic potential., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

To evaluate the medicinal properties of a glycyrrhizin (GL)-deficient strain of Glycyrrhiza uralensis, we investigated the anti-allergic effect of the hot water extract obtained from its roots on contact hypersensitivity in mice, and compared it with that of the hot water extract of a commercial crude drug, Glycyrrhiza Radix. The hot water root extract of the GL-deficient strain contained glucoglycyrrhizin (GGL) and rhaoglucoglycyrrhizin (RGL) instead of GL, and it showed anti-allergic activity against contact hypersensitivity in a fashion similar to that of the crude drug extract. We further confirmed the presence of glycyrrhetinic acid (GA), a major metabolite of GL, in mice serum after oral administration of the hot water root extract of a GL-deficient strain. We demonstrated that GGL underwent hydrolysis by intestinal microflora of mice to form GA. These results suggest that a GL-deficient strain of G. uralensis is a useful medicinal resource since the glycosides of GA work in a fashion similar to that of GL when orally administered.

Mast cells are involved in many physiological reactions in which their functions can be very diverse. Models of allergic skin inflammation and systemic anaphylactic reactions in mice are validated methods in which the role of mast cells is well established. In this chapter, we will therefore present protocols for passive cutaneous anaphylaxis and contact hypersensitivity, i.e., models which can be used to identify and characterize the role of mast cells as well as mast cell mediators and receptors in allergic IgE-dependent and IgE-independent skin inflammation, and for passive systemic anaphylaxis, a model ideally suited to characterize the systemic effects of mast cell-derived mediators and mast cell receptors.

To elucidate the roles of epidermal keratinocytes in the toxicological outcomes of chemically induced contact dermatitis, genome-scale transcriptional analyses were performed using normal human keratinocytes (NHKCs) treated with 10 μM sodium lauryl sulfate (SLS) or 5 μM urushiol. In Gene Ontology (GO) enrichment analyses, SLS- and urushiol-induced upregulated DEGs are commonly associated with the regulation of pro-inflammatory responses and epidermal differentiation processes in NHKCs whereas cellular protein metabolic process was also identified as a commonly downregulated DEG signature. Among the downregulated DEGs, CXCL14 was investigated as a potential biomarker for a new in vitro skin sensitization test using OECD TG429 reference chemicals. CXCL14 was significantly downregulated in NHKCs in response to 62.5% of the OECD TG429 sensitizers in a concentration-dependent manner. When the sensitizer-induced upregulation of chemokine CXCL8 was included in the analysis, 87.5% of the OECD TG429 reference sensitizing chemicals significantly induced either CXCL8 upregulation or CXCL14 downregulation in NHKCs. Only one OECD TG429 reference non-sensitizer changed the constitutive CXCL14 expression in NHKCs whereas five out of six non-sensitizers altered CXCL8 production. The reference irritating non-sensitizer SLS caused a false-positive outcome. The downregulation of constitutively expressed CXCL14 was regulated by both the MAPK/ERK and JAK3/STAT6 pathways in NHKCs. CXCL14 can be used as a mechanism-based biomarker in the development of in vitro skin sensitization tests and may help improve the distinction between allergenic sensitizers and non-sensitizers., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Chronic itch is one of the disturbing symptoms of inflammatory skin diseases. Kappa opioid receptor agonists are effective in suppressing scratching in mice against different pruritogens. Nalbuphine, a nonscheduled kappa opioid receptor agonist and mu opioid receptor antagonist, has been in clinical use for post-operative pain management since the 1980s and recently has been in clinical trials for chronic itch of prurigo nodularis (https://www.trevitherapeutics.com/nalbuphine). We studied whether nalbuphine is effective against chronic scratching induced by rostral neck application of 1-fluoro-2,4-dinitrobenzene (DNFB), an accepted mouse model of contact dermatitis to study pruritoceptive itch. Mice were treated once a week with either saline or nalbuphine 20 min before the third, fifth, seventh, and ninth sensitizations with DNFB and the number of scratching bouts was counted for 30 min. Skin samples from the neck of mice at week 4 were used to measure protein levels and mRNA expressions of chemokines and cytokines. Different sets of mice were used to study sedation and anhedonic-like behavior of nalbuphine. We found that: nalbuphine (a) antagonized scratching in a dose- and time-dependent manner without affecting locomotion, b) decreased IL-31, and increased anti-inflammatory IL-10, and c) induced more elevations in the levels of CCL2, CCL3, CCL12, CXCL1, CXCL2, CXCL9, CXCL10, IL-1β, IL-16, TIMP-1, M-CSF, TREM-1 and M1-type macrophages compared to saline. Increases in chemokines and cytokines and M1 macrophages by nalbuphine suggest an inflammatory phase of healing in damaged skin due to scratching. Our data indicate that nalbuphine is an effective antipruritic in murine model of pruritoceptive itch., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Irritant Contact Dermatitis (ICD) is characterized by epidermal hyperplasia and inflammatory cytokine release. IL-6 has been shown to be involved in the pathogenesis of ICD; however, the involvement of the IL-22/IL-22R α axis and its relation to IL-6 in the inflammatory response following irritant exposure are unknown. Using a chemical model of ICD, it was observed that mice with a keratinocyte-specific knockout of IL-6R α (IL-6R α Δker ) presented with increased inflammation and IL-22R α and IL-22 protein expression relative to WT following irritant exposure, indicating that IL-6R α deficiency in epidermal keratinocytes leads to the upregulation of IL-22R α and its ligand during ICD. Furthermore, it was shown that IL-6 negatively regulates the expression of IL-22R α on epidermal keratinocytes. This effect is functional as the effects of IL-22 on keratinocyte proliferation and differentiation were markedly reduced when keratinocytes were pretreated with IL-6 prior to IL-22 treatment. These results show that IL-6 modulates the IL-22/IL-22R α axis in the skin and suggest that this occurrence may be associated with the increased epidermal hyperplasia and exacerbated inflammatory response observed in IL-6R α Δker mice during ICD., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 Benjamin Frempah et al.)

Occupational exposure to trichloroethylene (TCE) has been associated with severe, generalized contact hypersensitivity (CHS) skin disorder, which is considered a delayed-type hypersensitivity reaction mediated by antigen-specific T cells. Transforming growth factor-β activated kinase-1 (TAK1) is essential for regulating the development and effector function of T cells. We hypothesized that disrupting TAK1 activity might inhibit TCE-induced CHS response. In this study, a local lymph node assay was employed to build a CHS model induced by TCE combined with the inducible-TAK1 deletion system to study the effect of TAK1 on it. It was observed that TAK1 deficiency ameliorated the TCE-induced CHS response and was associated with defective T cell expansion and activation and IFN-γ production in vivo. Furthermore, we investigated the effects of TCE and its metabolites trichloroacetic acid (TCA) and dichloroacetic acid (DCA) on CD4 + T cell function and the effect of TAK1 on it in vitro. The results showed that TCE, TCA and DCA augmented the proliferation, activation and differentiation of CD4 + T cells through Jnk MAPK and NF-κB pathways. TAK1 deletion significantly attenuated these effects induced by TCE, TCA or DCA on CD4 + T cells. In conclusion, it is suggested that TAK1 plays a critical role both in TCE-induced CHS response in vivo and in TCE and its metabolite-induced CD4 + T cell activation in vitro. Local inhibition of TAK1 might offer a promising alternative feasible strategy for TCE-induced CHS., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Irritant contact dermatitis (ICD) is characterized by epidermal hyperplasia, infiltration of leucocytes into lesional skin and inflammatory cytokine release. The cellular infiltrate during ICD comprises primarily cells of the myeloid lineage. Our group has previously shown that the cytokine IL-6 confers a protective effect to lesional skin during ICD. How IL-6Rα function in myeloid cells is involved in the inflammatory response during ICD is, however, unknown. In the present study, utilizing a chemical model of ICD, it is shown that mice with a myeloid-specific knockout of the IL-6Rα (IL-6Rα Δmyeloid ) display an exaggerated inflammatory response to benzalkonium chloride (BKC) and Jet propellant-8 (JP8) fuel, two well-characterized irritants relative to littermate control. Results from immunohistochemical and flow cytometric analyses revealed that IL-6Rα Δmyeloid mouse skin displayed increased epidermal hyperplasia and inflammatory monocyte influx into lesional skin but lower numbers of resident macrophages relative to littermate controls after irritant exposure. Multiplex immunoassay revealed significantly higher levels of pro-inflammatory cytokines IL-1α and TNF-α, but reduced expression of chemokine proteins including CCL2-5, CCL7, CCL11, CXCL1 and CXCL10 in IL-6Rα Δmyeloid mouse skin relative to littermate control following irritant exposure. These results highlight a previously unknown role of IL-6Rα function in myeloid cells in modulating the inflammatory response and myeloid population dynamics during ICD., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Coconut oil is used as a dietary oil worldwide, and its healthy effects are recognized by the fact that coconut oil is easy to digest, helps in weight management, increases healthy cholesterol, and provides instant energy. Although topical application of coconut oil is known to reduce skin infection and inflammation, whether dietary coconut oil has any role in decreasing skin inflammation is unknown. In this study, we showed the impact of dietary coconut oil in allergic skin inflammation by using a mouse model of contact hypersensitivity (CHS). Mice maintained on coconut oil showed amelioration of skin inflammation and increased levels of cis-5, 8, 11-eicosatrienoic acid (mead acid) in serum. Intraperitoneal injection of mead acid inhibited CHS and reduced the number of neutrophils infiltrating to the skin. Detailed mechanistic studies unveiled that mead acid inhibited the directional migration of neutrophils by inhibiting the filamentous actin polymerization and leukotriene B 4 production required for secondary recruitment of neutrophils. Our findings provide valuable insights into the preventive roles of coconut oil and mead acid against skin inflammation, thereby offering attractive therapeutic possibilities., (© 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

The function of regulatory immune cells in peripheral tissues is crucial to the onset and severity of various diseases. Interleukin-10 (IL-10)-producing regulatory B (IL-10 + B reg ) cells are known to suppress various inflammatory diseases. However, evidence for the mechanism by which IL-10 + B reg cells are generated and maintained is still very limited. Here, we found that IL-10 + B reg cells suppress the activation of IL-13-producing type 2 innate lymphoid cells (IL-13 + ILC2s) in an IL-10-dependent manner in mice with oxazolone-induced severe contact hypersensitivity (CHS). Mast cell (MC) IL-5 was important for maintaining the population of IL-10 + B reg cells in peripheral lymphoid tissues. Overall, these results uncover a previously unknown mechanism of MCs as a type of immunoregulatory cell and elucidate the cross-talk among MCs, IL-10 + B reg cells, and IL-13 + ILC2s in CHS.

Background: Tinea is an infectious disease by dermatophytes, of which Trichophyton species accounts for the overwhelming majority of case. Tinea often causes itching with inflammation. In terms of pruritus by fungal infection, however, tinea has not been investigated sufficiently to date., Aim: To evaluate itch caused by Trichophyton infection and the effect of antifungal agents on the infection, by measuring scratch behaviour and profiles of inflammatory cytokines and chemokines., Methods: We used a previously established mouse model of contact hypersensitivity induced by trichophytin, a crude extract from Trichophyton mentagrophytes. Scratching behaviour was recorded using a counting device that measured an electric current induced in a coil by movement of magnets that had been inserted into the hind paws of each animal. We investigated expression of various genes in lesional skin of mice and in normal human epidermal keratinocytes. We also investigated the antipruritic effects of the corticosteroid dexamethasone (DEX) and three antifungal agents: ketoconazole (KCZ), terbinafine (TBF) and liranaftate (LNF)., Results: Biphasic peaks of scratching were observed at 1 h and at 6-7 h during an observation period of 14 h after trichophytin induction. For lesional skin, RNA was extracted 24 h after trichophytin challenge, and increased expression was seen in the genes for interleukin (IL)-17A, interferon-γ, tumour necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-2 and Dectin-1, whereas there was no obvious change in the genes for IL-31 and prostaglandin (PG)E2. Furthermore, KCZ inhibited histidine decarboxylase (HDC) expression in vitro and in vivo, and inhibited scratching in the very early phase. LNF inhibited expression of thymic stromal lymphopoietin (TSLP) and IL-8 in vitro, and TSLP, TNF-α, IL-1α and MIP2 in vivo, and also scratching in the early phase. TBF did not induce any significant alterations in either gene expression or scratching. DEX suppressed expression of all the chemical mediators except HDC in vitro and in vivo, and inhibited scratching., Conclusion: Antifungals can inhibit itching induced by fungal infection through different mechanisms., (© 2018 British Association of Dermatologists.)

Contact hypersensitivity (CHS) is frequently used as an animal model for human allergic contact dermatitis (ACD). Diets of pomegranate polyphenols (PPs) or soy isoflavones (SIs) each alleviated CHS symptoms; however, the effect of diets containing a mixture of PPs and SIs on CHS is unclear. We investigated the CHS-inhibitory effects of diets supplemented with a mixture of PPs and SIs at human physiologically relevant doses. Consuming the mixture of PPs and SIs attenuated ear swelling and reduced infiltration of Gr-1-positive cells. Ear swelling decreased in the PP and SI-treated mice compared to the SI-treated mice. The auricle tissues of the PP and SI-fed mice exhibited decreased production of CXCL2 and MCP-5 compared to the SI- and PP-treated mice, respectively. These results suggest that dietary supplementation with a mixture of PPs and SIs may have ACD-preventive effects and may prove more beneficial than supplementation with PPs or SIs alone.

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Sensitization of wild-type (WT) mice induces hapten-reactive effector CD8 T cells producing IFN-γ and IL-17- and IL-4-producing CD4 T cells that cannot mediate CHS. Although CXCR2-dependent Ly6G + (neutrophil) cell recruitment into hapten-challenged skin is required to direct effector CD8 T cell infiltration into the challenge site to elicit CHS, 2,4-dinitrofluorobenezene (DNFB) sensitization of CXCR2 -/- mice and neutrophil-depleted WT mice induced both hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17. CD4 T cell-mediated CHS responses were not generated during DNFB sensitization of neutrophil-depleted WT mice treated with anti-IL-12 mAb or neutrophil-depleted IL-12 -/- mice. Neutrophil depletion during DNFB sensitization of WT mice markedly increased IL-12-producing hapten-primed dendritic cell numbers in the skin-draining lymph nodes. Sensitization of mice lacking the neutrophil serine protease cathepsin G (CG)-induced hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 with elevated and elongated CHS responses to DNFB challenge. Induction of CHS effector CD4 T cells producing IFN-γ in neutrophil-depleted WT mice was eliminated by s.c. injection of active, but not inactivated, CG during sensitization. Thus, hapten skin sensitization induces neutrophil release of CG that systemically inhibits hapten-presenting dendritic cell production of IL-12 and the development of hapten-reactive CD4 T cells to IFN-γ-producing CHS effector cells., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Ethnopharmacological Relevance: Tabernaemontana catharinensis, popularly known as snakeskin, is used in traditional medicine to treat skin inflammatory disorders. To confirm the topical anti-inflammatory effect of T. catharinensis leaves, we evaluated the therapeutic effect of crude extract (TcE) and its different fractions on irritant contact dermatitis model in mice and verified its anti-inflammatory action mechanism., Materials and Methods: The qualitative phytochemical analysis of TcE and its dichloromethane, n-butanol and ethyl acetate fractions was performed by UHPLC-ESI-HRMS. The gel accelerated stability was performed to ensure the effectiveness formulation. We investigated the TcE' inhibitory effect, its fractions and a gel formulation containing TcE in irritant contact dermatitis models induced by unique (1000 μg/ear) and multiple (400 μg/ear) croton oil application, evaluated by the ear edema formation, inflammatory cell infiltration (MPO activity measurement and histological procedure) and pro-inflammatory cytokines levels. The action glucocorticoid-like of TcE was investigated using a glucocorticoid receptor antagonist (mifepristone; 50 mg/kg, s.c.)., Results: The treatments (10 μg/ear) reduced the ear edema and MPO activity by 100% and 94 ± 3% (TcE) 85 ± 4% and 88 ± 3% (dichloromethane fraction), 83 ± 6% and 73 ± 11% (n-butanol fraction) and 86 ± 6% and 93 ± 4% (ethyl acetate fraction) and 100% (dexamethasone solution), respectively to the acute ICD model. The TcE and dexamethasone gel (15 mg/ear) also reduced by 66 ± 6% and 70 ± 5% the ear edema and by 58 ± 14% and 84 ± 4% the MPO activity, respectively. To the chronic ICD model, the TcE and dexamethasone (10 μg/ear) also reduced the ear edema (66 ± 6% and 70 ± 5%) and the MPO activity (58 ± 14% and 84 ± 4%); on the 9th day of the experiment. TcE and dexamethasone also reduced the pro-inflammatory cytokines (MIP-2, IL-1β and TNF-α) levels in acute ICD model induced by croton oil. Besides, mifepristone prevented the topical anti-edematogenic effect of TcE' and dexamethasone' solutions by 97 ± 9% to TcE and 75 ± 15% to dexamethasone. The accelerated stability study of T.catharinensis gels showed no relevant changes at low temperatures. The dereplication of the TcE and fractions revealed the presence of indole alkaloids, triterpenes, and flavonoids by UHPLC-ESI-HRMS. These classes of compounds are known in the literature for present potential anti-inflammatory action, supporting the results obtained., Conclusion: The results confirm the topical popular use ofT.catharinensis leaves in the treatment of skin inflammation and demonstrate the TcE' potential for the development of a promising topical anti-inflammatory agent to treat inflammatory disorders., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: The placenta is a reservoir enriched with growth factors, hormones, cytokines and minerals. While several beneficial effects of placenta extracts on wound healing, anti-aging and anti-inflammatory responses have been reported, relatively limited mechanistic exploration has been conducted to date. Here, we provide compelling evidence of anti-inflammatory and anti-oxidative activities of porcine placenta extracts (PPE) against contact dermatitis in vivo., Methods: A contact dermatitis mouse model was established by sensitizing the dorsal skin of BALB/c mice using the contact allergen, 2,4-dinitrochlorobenzene (DNCB), and molecular consequences of topical application of PPE were investigated. PPEs were pre-sterilized via γ-irradiation, which is a milder but more effective way of sterilizing biomolecules relative to the conventional autoclaving method., Results: DNCB-induced skin lesions displayed clear contact dermatitis-like symptoms and topical application of PPE dramatically alleviated both local and systemic inflammatory responses. Inflammatory epidermal thickening was completely abrogated and allergen-specific serum IgE levels significantly reduced in the presence of PPE. Moreover, anti-oxidative activities of PPE were observed both in vitro and in vivo, which may lead to attenuation of inflammatory responses. Prolonged treatment with PPE strongly inhibited production of DNCB-induced reactive oxygen species (ROS) and subsequently prevented oxidative degradation of hyaluronic acid (HA), which triggers innate inflammatory responses., Conclusion: Our findings supply valuable insights into the mechanisms underlying the anti-inflammatory effects of PPE and provide a functional basis for the clinical application of PPE in inflammatory diseases.

Contact hypersensitivity (CHS) to preservatives is receiving increased attention. Parabens are widely used in foods, pharmaceutics and cosmetics as preservatives. The skin sensitizing activity of parabens remains controversial but a few investigations have been made as to whether parabens could facilitate sensitization to other chemicals. We have shown that di-n-butyl phthalate (DBP), a phthalate ester, has an adjuvant effect in a fluorescein isothiocyanate (FITC)-induced CHS mouse model. We have also demonstrated that DBP activates transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. Comparative studies of phthalate esters revealed that TRPA1 agonistic activity and the adjuvant effect on FITC-CHS coincide. Here we focused on two commonly used parabens, butyl paraben (BP) and ethyl paraben (EP), as to their adjuvant effects. BALB/c mice were epicutneously sensitized with FITC in acetone in the presence or absence of a paraben. Sensitization to FITC was evaluated as the ear-swelling response after FITC challenge. BP but not EP enhanced skin sensitization to FITC, but the effect of BP was much weaker than that of DBP. Mechanistically, BP enhanced the trafficking of FITC-presenting CD11c + dendritic cells (DCs) from the skin to draining lymph nodes as well as cytokine production by draining lymph nodes. When the TRPA1 agonistic activity was measured with a cell line expressing TRPA1, BP exhibited higher activity than EP. The present study provides direct in vivo evidence that BP causes sensitization to other chemicals by means of a mouse FITC-CHS model.

Background: To recruit leucocytes to an inflammatory site, chemokine binding to glycosaminoglycans (GAGs) is critical. Therefore, strategies to interfere with this interaction, aiming at the production of anti-inflammatory agents, were developed. These include production of modified chemokines without affinity for G protein-coupled receptors but with enhanced affinity for GAGs. Such modified chemokines compete with functional chemokines for GAG binding, prevent chemokine immobilization and presentation, and inhibit leucocyte migration. In addition to modified chemokines, a GAG-binding peptide consisting of the 30 COOH-terminal residues of CXCL9, that is CXCL9(74-103), inhibited CXCL8- and monosodium urate crystal-induced neutrophil migration., Objective: We wanted to explore whether interference with chemokine-GAG interactions by CXCL9(74-103) reduces inflammation in neutrophil-dependent dinitrofluorobenzene-induced contact hypersensitivity., Methods: For this study, we evaluated several inflammatory parameters, including ear swelling and the levels of chemokines, cytokines, proteases and neutrophils in the ears of dinitrofluorobenzene-induced mice treated with CXCL9(74-103) or buffer., Results: One intravenous injection of CXCL9(74-103), just before painting with dinitrofluorobenzene on the ear, did not affect protein levels of the major murine neutrophil attractant, that is CXCL6, in this contact hypersensitivity model. However, IL-6, CXCL1, CCL2 and matrix metalloproteinase-9 (MMP-9) protein concentrations and peroxidase activity in challenged ears were reduced. In addition, intravenous injection of the CXCL9-derived peptide led to a reduced ear swelling response, indicating that the locally produced chemokines were hindered to attract leucocytes. The inhibiting potential of CXCL9(74-103) was explained by its competition for GAG binding with CXCL1, CXCL6 and CCL3 and inhibition of transendothelial migration of neutrophils to CXCL6., Conclusions and Clinical Relevance: The CXCL9(74-103) peptide inhibited dinitrofluorobenzene-induced infiltration of neutrophils and neutrophil-dependent inflammation in ears. Therefore, CXCL9(74-103) may be a lead molecule for the development of therapeutic peptides or peptide derivatives that compete with functional chemokines for GAG binding., (© 2018 John Wiley & Sons Ltd.)

Contact hypersensitivity (CHS) is a common T cell-mediated skin disease induced by epicutaneous sensitization to haptens. Mast cells (MCs) are widely deployed in the skin and can be activated during CHS responses to secrete diverse products, including some with pro-inflammatory and anti-inflammatory functions. Conflicting results have been obtained regarding pathogenic versus protective roles of MCs in CHS, and this has been attributed in part to the limitations of certain models for studying MC functions in vivo . This review discusses recent advances in the development and analysis of mouse models to investigate the roles of MCs and MC-associated products in vivo . Notably, fluorescent avidin-based two-photon imaging approaches enable in vivo selective labeling and simultaneous tracking of MC secretory granules (e.g., during MC degranulation) and MC gene activation by real-time longitudinal intravital microscopy in living mice. The combination of such genetic and imaging tools has shed new light on the controversial role played by MCs in mouse models of CHS. On the one hand, they can amplify CHS responses of mild severity while, on the other hand, can limit the inflammation and tissue injury associated with more severe or chronic models, in part by representing an initial source of the anti-inflammatory cytokine IL-10.

Background: A standardized human model to study early pathogenic events in patients with psoriasis is missing. Activation of Toll-like receptor 7/8 by means of topical application of imiquimod is the most commonly used mouse model of psoriasis., Objective: We sought to investigate the potential of a human imiquimod patch test model to resemble human psoriasis., Methods: Imiquimod (Aldara 5% cream; 3M Pharmaceuticals, St Paul, Minn) was applied twice a week to the backs of volunteers (n = 18), and development of skin lesions was monitored over a period of 4 weeks. Consecutive biopsy specimens were taken for whole-genome expression analysis, histology, and T-cell isolation. Plasmacytoid dendritic cells (pDCs) were isolated from whole blood, stimulated with Toll-like receptor 7 agonist, and analyzed by means of extracellular flux analysis and real-time PCR., Results: We demonstrate that imiquimod induces a monomorphic and self-limited inflammatory response in healthy subjects, as well as patients with psoriasis or eczema. The clinical and histologic phenotype, as well as the transcriptome, of imiquimod-induced inflammation in human skin resembles acute contact dermatitis rather than psoriasis. Nevertheless, the imiquimod model mimics the hallmarks of psoriasis. In contrast to classical contact dermatitis, in which myeloid dendritic cells sense haptens, pDCs are primary sensors of imiquimod. They respond with production of proinflammatory and T H 17-skewing cytokines, resulting in a T H 17 immune response with IL-23 as a key driver. In a proof-of-concept setting systemic treatment with ustekinumab diminished imiquimod-induced inflammation., Conclusion: In human subjects imiquimod induces contact dermatitis with the distinctive feature that pDCs are the primary sensors, leading to an IL-23/T H 17 deviation. Despite these shortcomings, the human imiquimod model might be useful to investigate early pathogenic events and prove molecular concepts in patients with psoriasis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Endothelial upregulation of adhesion molecules serves to recruit leukocytes to inflammatory sites and appears to be promoted by NOTCH1; however, current models based on interactions between active NOTCH1 and NF-κB components cannot explain the transcriptional selectivity exerted by NOTCH1 in this context., Approach and Results: Observing that Cre/Lox-induced conditional mutations of endothelial Notch modulated inflammation in murine contact hypersensitivity, we found that IL (interleukin)-1β stimulation induced rapid recruitment of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) to genomic sites occupied by NOTCH1-RBPJ (recombination signal-binding protein for immunoglobulin kappa J region) and that NOTCH1 knockdown reduced histone H3K27 acetylation at a subset of NF-κB-directed inflammatory enhancers., Conclusions: Our findings reveal that NOTCH1 signaling supports the expression of a subset of inflammatory genes at the enhancer level and demonstrate how key signaling pathways converge on chromatin to coordinate the transition to an infla mmatory endothelial phenotype., (© 2018 American Heart Association, Inc.)

Little attention has been paid to chemicals that can enhance hypersensitivity caused by other chemicals. We have demonstrated that phthalate esters with short chain alcohols enhance fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) in a mouse model. Furthermore, phthalate esters with such an enhancing effect were found to activate transient receptor potential ankyrin 1 (TRPA1) cation channels, which are expressed on a part of sensory neurons, using a TRPA1-expressing cell line. In this study, we examined these activities of esters comprising glycerol and a short chain fatty acid, i.e. dibutyrin and tributyrin. We carried out chemical synthesis of dibutyrin isomers. Each dibutyrin isomer weakly activated TRPA1 and slightly enhanced skin sensitization to FITC. Unexpectedly, TRPA1 activation and enhancement of FITC-CHS were much more evident in the presence of tributyrin. Mechanistically, tributyrin induced increased dendritic cell trafficking from the skin to draining lymph nodes. Tributyrin enhanced interferon-γ (IFN-γ) production by draining lymph nodes, while its effect on interleukin-4 (IL-4) production was relatively less prominent. These results suggested that tributyrin concomitantly caused TRPA1 activation and an adjuvant effect on FITC-CHS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Interleukin (IL)-19 is a member of the IL-10 family of interleukins and is an immuno-modulatory cytokine produced by the main macrophages. The gastrointestinal tissues of IL-19 knockout mice show exacerbated experimental colitis mediated by the innate immune system and T cells. There is an increasing focus on the interaction and relationship of IL-19 with the function of T cells. Contact hypersensitivity (CHS) is T cell-mediated cutaneous inflammation. Therefore, we asked whether IL-19 causes CHS. We investigated the immunological role of IL-19 in CHS induced by 1-fluoro-2,4-dinitrofluorobenzene as a hapten. IL-19 was highly expressed in skin exposed to the hapten, and ear swelling was increased in IL-19 knockout mice. The exacerbation of the CHS response in IL-19 knockout mice correlated with increased levels of IL-17 and IL-6, but no alterations were noted in the production of interferon (IFN)γ and IL-4 in the T cells of the lymph nodes. In addition to the effect on T cell response, IL-19 knockout mice increased production of inflammatory cytokines. These results show that IL-19 suppressed hapten-dependent skin inflammation in the elicitation phase of CHS.

Contact dermatitis is a form of delayed-type hypersensitivity characterized by localized thickening, papules, redness and vesicles of the skin. A model of contact dermatitis involving repeated challenge of a hapten is adapted to assess dermatitis as characterized by skin thickening. Recently, it was reported that neutrophils have crucial roles in contact hypersensitivity. We thus examined the involvement of CXC chemokines bearing the glutamic acid-leucine-arginine (ELR) motif ("ELR + chemokines") and neutrophils in the ear swelling induced by 2,4,6-trinitrochlorobenzene (TNCB) challenges in the present study. Mice were sensitized by application of TNCB on their abdominal skin. They were then challenged thrice with TNCB to the ear. The CXCR2 antagonist SB225002 (9 mg/kg, i.p.) was administered before each TNCB challenge. Gene expressions and protein levels of the ELR + chemokines CXCL1, 2 and 5 was increased markedly in mouse ear after the final TNCB challenge. In addition, we indicated that gene expression of CXCL1 was enhanced in the epidermis and dermis upon TNCB challenge. Expression of the CXCL2 gene was enhanced in the epidermis, and that of the CXCL5 gene was enhanced in the dermis. The swelling induced by TNCB challenges was significantly attenuated by SB225002. Furthermore, the increases in myeloperoxidase activity, and expression of myeloperoxidase and neutrophil elastase induced by TNCB challenge in mouse ear were inhibited by SB225002. These data suggest that ear swelling resulting from TNCB challenges might be concerned by upregulated ELR + chemokine-induced neutrophil recruitment., (© 2017 John Wiley & Sons Australia, Ltd.)

Background: Atopic dermatitis (AD), psoriasis (PS), and contact dermatitis (CD) are common skin diseases, characterized by barrier disruption and systemic inflammation, with unique epidermal signatures and common inflammatory pathways identified by transcriptomic profiling. This study profiled proteomic signatures in serum from subjects with AD, PS, and CD compared with healthy controls (HC)., Objective: Identify unique proteomic signatures to distinguish between inflammatory diseases with similar epidermal disruption and overlapping epithelial inflammation., Methods: Sera from 20 subjects with moderate to severe AD, 10 subjects with CD, 12 subjects with moderate to severe PS, 10 subjects with both AD and CD, and 10 HC with no history of skin disease was analysed using high-throughput proteomic analysis that detects expression of 1129 protein targets. Protein expression was compared between disease and HC, and across diseases for statistical significance (fold change≥1.5 and false discovery rate≤0.05), to identify unique proteomic signatures for each disease., Results: Complement C5A anaphylatoxin (C5A), lipopolysaccharide binding protein (LBP), C-reactive protein (CRP), ILT-4, C-C motif ligand 18 (PARC), and sialic acid-binding Ig-like lectin 14 (SIG14) were significantly modulated in all three diseases compared with HC. We identified unique signatures for AD (Immunoglobulin E (IgE), thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC)), CD (10 proteins), and PS (kynureninase (KYNU)). Proteomic profiling in subjects with both AD and CD identified additional dysregulated proteins compared with subjects with either condition alone, indicating an exacerbated inflammation reaction., Conclusions and Clinical Relevance: Unique sera proteomic signatures may distinguish between inflammatory skin diseases despite similar epidermal barrier disruption and epithelial inflammation. This may provide insight into disease pathogenesis, diagnosis, and therapeutic intervention in difficult-to-treat subjects., (© 2017 John Wiley & Sons Ltd.)

Dibutyl phthalate (DBP) is a plasticizer used for many consumer products including cosmetics. Potential health concerns regarding DBP include reproductive and developmental toxicity, endocrine disruption and neurotoxicity. DBP is a high priority chemical as to reduction of exposure of children to it. Through reproductive toxicity studies, monobutyl phthalate (MBP) has been proposed to be the active metabolite derived from DBP. We previously demonstrated that DBP activates transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. We have also shown that DBP enhanced skin sensitization in a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse model. Through MBP formation by esterase in the skin, it is possible that MBP exerts a major effect on the biological activity we observed. To test this possibility, we directly compared DBP and MBP. A more than 40-fold higher concentration of MBP as compared with DBP was required for activation of TRPA1 in vitro. Unlike DBP, MBP did not enhance skin sensitization to FITC. These results demonstrated that DBP directly, i.e., not through its metabolite MBP, activates TRPA1 and enhances FITC-CHS. It is noteworthy that butyl benzoate, a related compound, activated TRPA1 and enhanced FITC-CHS.

Certain B cells termed as "regulatory B cells" (Bregs) can suppress the ongoing immune responses and a splenic CD5 + CD1d hi Breg subset identified earlier was shown to exert its regulatory functions through secretion of IL-10. Though FasL expression is an alternative mechanism of immune suppression used by B cells, little is known about the FasL expressing CD5 + CD1d hi Bregs. In this study, we isolated splenocytes or splenic CD19 + B cells and compared the efficiency of toll-like receptor(TLR)4 ligand (lipopolysaccharide) with TLR9 ligand (CpG), anti-CD40 and TLR9 ligand (CpG) plus anti-CD40 on the FasL expression of splenic CD5 + CD1d hi Bregs by flow cytometry. FasL expression in CD5 + CD1d hi B cells was rapidly increased after TLR4 ligation. Intriguingly, anti-CD40 and CpG plus anti-CD40 combinations failed to stimulate FasL expression in CD5 + CD1d hi B cells although the IL-10 production was up-regulated in this subset. In addition, LPS and other B10-cell inducers increased the expression of surface molecules like CD86 and CD25, which are correlated to the regulatory functions of B cells. Furthermore, NF-κB and NF-AT inhibitors decreased the TLR4-activated FasL expression in CD5 + CD1d hi B cells. Then we sorted splenic CD5 + CD1d hi Bregs using flow cytometry and found that TLR4-activated CD5 + CD1d hi Bregs suppressed the proliferation of CFSE-labeled CD4 + T cells in vitro, which was partly blocked by anti-FasL antibody. In oxazolone-sensitized mice having contact hypersensitivity, FasL expression in splenic CD5 + CD1d hi B cells was decreased compared to the control group after TLR4 ligation. Our findings suggest that the regulatory function of CD5 + CD1d hi B cells could be partly mediated by Fas-FasL pathway and this FasL expressing CD5 + CD1d hi Bregs might participate in the regulation of inflammatory diseases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mesenchymal stromal cells (MSCs) can modulate inflammation and contribute to tissue regeneration and, thus, have emerged as a promising option for cell-based therapy. However, the ability of MSCs to migrate to injured tissues still needs to be improved. In this study, we investigated whether genetically engineered MSCs could exhibit increased migratory properties and improved therapeutic efficacy. Using a mouse model of contact hypersensitivity (CHS), chemokine gene expression screening revealed that CXCL13 changed most significantly in injured tissue. Unfortunately, MSCs hardly express the corresponding receptor, CXCR5. Thus, CXCR5-overexpressing MSCs (MSC CXCR5 ) were generated that retained their abilities of proliferation, differentiation, and immunomodulation. Furthermore, MSC CXCR5 showed significantly increased migrating ability toward CXCL13. Importantly, systemic infusion of MSC CXCR5 dramatically suppressed CHS in mice, as evidenced by decreased levels of inflammatory cell infiltration and pro-inflammatory cytokine production. Numerous MSC CXCR5 migrated into inflamed ears, localized with T cells, inhibited T cell proliferation, promoted T cell apoptosis, and suppressed the production of T cell-derived pro-inflammatory factors. Collectively, these findings demonstrate that CXCR5 overexpression increases the ability of MSCs to respond to migratory stimuli and highly intensifies their immunomodulatory effects in vivo. This strategy for enhancing targeted stem/progenitor cell homing may improve the efficacy of MSC-based therapies., (Copyright © 2017. Published by Elsevier Inc.)

No standardized in vitro methods to assess potency of skin sensitizers are available. Recently, we standardized a procedure which combines the epidermal equivalent potency assay with assessment of IL-18 to provide a single test for identification and classification of skin sensitizers. This current study aimed to extend tested chemicals, and to provide a simple in vitro method for estimation of the expected sensitization induction level interpolating in vitro EC50 and IL-18 SI2 values to predict LLNA EC3 and/or human NOEL from standards curves generated using reference contact allergens. Reconstituted human epidermis was challenged with 14 chemicals not previously tested benzoquinone, chlorpromazine, chloramine T, benzyl salicylate, diethyl maleate, dihydroeugenol, 2,4-dichloronitrobenzene, benzyl cinnamate, imidazolidinyl urea, and limonene as contact sensitizers while benzyl alcohol, isopropanol, dimethyl isophthalate and 4-aminobenzoic acid as non-sensitizers in the LLNA. Where for benzyl salicylate and benzyl cinnamate no sensitization was observed in human predictive studies, positive responses to benzyl alcohol and dimethyl isophthalate were reported. The proposed method correlates better with human data, correctly predicting substances incorrectly classified by LLNA. With the exception of benzoquinone (interference with both MTT and IL-18 ELISA), and chloramine T (underestimated in the interpolation), a good estimation of LLNA EC3 and in vivo available human NOEL values was obtained., (Copyright © 2017 Elsevier B.V. All rights reserved.)