Mouse Model Of Skin Inflammation Research Articles

Rosmarinic Acid Ameliorates Dermatophagoides farinae Extract-Induced Atopic Dermatitis-like Skin Inflammation by Activating the Nrf2/HO-1 Signaling Pathway

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases. AD pathogenesis is associated with increased oxidative stress, impairment of the skin barrier, and activation of the immune response. Rosmarinic acid (RA), a caffeic acid ester, is known for its anti-inflammatory and antioxidant properties. However, the effects of RA on Dermatophagoides farinae extract (DfE)-induced AD-like skin inflammation, as well as its ability to regulate oxidative stress through the Nrf2/HO-1 pathway in TNF-α/IFN-γ-treated keratinocytes, remain unclear. We investigated RA activity in a DfE-induced AD-like skin inflammation mouse model and IFN-γ/TNF-α-stimulated keratinocytes. We found that RA attenuates DfE-induced inflammation by decreasing dermatitis scores and serum inflammatory marker levels and mast cell infiltration. Additionally, RA significantly suppressed IFN-γ/TNF-α-induced chemokine production in keratinocytes and reduced Th cytokine levels in concanavalin A-stimulated splenocytes. Importantly, RA also increased Nrf2/HO-1 expression in TNF-α/IFN-γ-treated keratinocytes. In conclusion, this study demonstrated that RA effectively alleviates DfE-induced AD-like skin lesions by reducing the levels of inflammatory cytokines and chemokines. Furthermore, RA promotes Nrf2/HO-1 signaling in keratinocytes, which may help mitigate DfE-induced oxidative stress, thereby alleviating AD-like skin inflammation. These findings highlight the potential of RA as a therapeutic agent for treating AD and other skin inflammation.

The molecular mechanism of berberine affecting psoriasis skin inflammation by regulating keratinocyte pyroptosis via the p38 MAPK/NF-κB pathway.

Berberine (BBR), a Rhizoma Coptis-sourced isoquinoline alkaloid, is an effective drug for psoriasis treatment with its therapeutic mechanism remaining unclear. We delved into the mechanism of BBR affecting psoriatic skin inflammation by regulating keratinocyte pyroptosis. A psoriasis-like skin inflammation mouse model was induced by imiquimod (IMQ) and treated with BBR and a p38 activator anisomycin. Human epidermal keratinocytes (HEKs) were stimulated with five chemokines (M5) [interleukin (IL)-17A, IL-22A, oncostatin M, tumor necrosis factor-α, IL-1α] to simulate psoriasis immune microenvironment, then treated with BBR and anisomycin. Psoriasis skin lesions, skin tissue damage, cell viability and death, and gasdermin D-N (GSDMD-N) and NOD-like receptor protein 3 (NLRP3) positive cell numbers were assessed. The p38 mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) pathway and levels of the NLRP3/GSDMD pathway-related proteins and inflammatory factors were determined. BBR alleviated M5-induced HEK pyroptosis by inactivating NLRP3 inflammasomes. BBR inhibited the p38 MAPK/NF-κB pathway, and its effects on HEKs were partly averted by activating the p38 MAPK/NF-κB pathway. BBR repressed NLRP3 inflammasome activation and pyroptosis by inhibiting the p38 MAPK/NF-κB pathway. Collectively, BBR suppressed keratinocyte NLRP3/GSDMD pathway pyroptosis by suppressing the p38 MAPK/NF-κB pathway, thereby affecting psoriasis skin inflammation.

Oroxylin A attenuates psoriasiform skin inflammation by direct targeting p62 (sequestosome 1) via suppressing M1 macrophage polarization.

Psoriasis results from the interplay of innate and adaptive immunity in the skin. Oroxylin A (OA) has shown anti-inflammatory effects in various disorders. This study explores oroxylin A potential in treating psoriasis, particularly its impact on type I macrophage (Mφ1) polarization. Oroxylin A-mediated therapeutic effects were evaluated using imiquimod-induced or IL-23-injected psoriatic mice models, followed by proteomics assays to predict potential signalling and targeting proteins. Immunofluorescence and immunoblot assays verified that oroxylin A suppresses NF-kB signalling in M1 macrophages. Co-immunoprecipitation and microscale thermophoresis (MST) assays further demonstrated that p62 (sequestosome 1) is the target protein for oroxylin A in macrophages. Oroxylin A-p62-mediated suppression of psoriasis was validated in an imiquimod-induced p62 conditional knockout (cKO) mice model. Oroxylin A demonstrated therapeutic efficacy in murine models induced by imiquimod or IL-23 by attenuating cutaneous inflammation and mitigating Mφ1 polarization via NF-κB signalling. Proteomics analysis suggested SQSTM1/p62 as a key target, confirmed to interact directly with oroxylin A. Oroxylin A disrupted the p62-PKCζ interaction by binding to PB1 domain of p62. Its anti-inflammatory effects were significantly reduced in macrophages from p62 cKO mice compared to the wild-type (WT) mice in psoriasis model, supporting oroxylin A role in suppressing Mφ1 polarization through its interaction with p62. Our findings demonstrated oroxylin A suppressed psoriasiform skin inflammation in mouse models by blocking the PKCζ-p62 interaction, subsequently inhibiting the activation of NF-κB p65 phosphorylation in macrophages.

Diminished γδ T Cells during Murine Allergic Skin Inflammation Is Mediated by IL-4 Signaling in Keratinocytes.

Atopic dermatitis results in diminished barrier function and altered production of antimicrobial peptides. Dendritic epidermal T cells (DETCs) play an important role in the wound repair and inflammation process. Our previous work identified an IL-4-dependent loss of DETCs in Stat6VT mice and in the MC903-induced skin inflammation mouse model. However, the mechanisms through which IL-4 mediates the loss of DETCs are unclear. In this study, we show that IL-4Rα germline knockout mice (Il4ra-/-) have increased DETCs, faster wound healing, and increased epidermal differentiation complex gene and fibronectin expression. The absence of IL-4Rα minimized the MC903-induced loss of DETCs, and reciprocal bone marrow chimera experiments in Il4ra-/- and wild-type mice demonstrated structural nonhematopoietic IL-4-responsive cell-mediated DETC homeostasis. Skin keratinocyte-derived IL-15 decreased dramatically in the MC903 model, while injection of IL-15 rescued DETC loss by promoting DETC proliferation and limiting apoptosis. Conditional deletion of IL-4Rα from keratinocytes using Il4rafl/fl K14-Cre mice showed an increase of DETCs, increased IL-15 production, and diminished skin inflammation following wounding. These results suggest that IL-4-dependent effects on DETCs in allergic skin inflammation are mediated by the IL-4Rα receptor of keratinocytes.

Foxp3+ Treg control allergic skin inflammation by restricting IFN-γ-driven neutrophilic infiltration and NETosis

BackgroundAtopic dermatitis (AD), a chronic inflammatory skin disease with T cell activation as a key feature, in which Th2 cell–mediated responses play a pivotal role. Regulatory T cells (Treg) are central immune cells that restrict autoimmunity and inflammation in the body. Patients with immune dysregulation, polyendocrinopathy, or enteropathy X-linked syndrome, an immune disease characterized by a deficiency in Treg, develop skin inflammation and allergic disorders, indicating that Treg play a crucial role in the development of allergic skin inflammation. Objectivewe investigated the underlying mechanisms by which Treg control cutaneous allergic inflammation. MethodsAn allergic skin inflammation mouse model was constructed using MC903, and Treg-depleted mouse model was constructed using diphtheria toxin. Neutralization of IFN-γ was constructed using anti-mouse-IFN-γ mouse antibody. Neutrophil infiltration was analyzed by flow cytometry and immunohistochemistry. Neutrophil extracellular traps (NETs), a process called NETosis, were detected using immunofluorescence. In vitro neutrophil stimulation and immunocytochemistry was conducted to demonstrate the effect of IFN-γ on NETosis. ResultsThe depletion of Foxp3+ Treg led to significantly exacerbated AD-like skin inflammation, including increased recruitment of neutrophils and expression of Th1 cytokine IFN-γ. Neutrophil infiltrating in skin of Treg-depleted mice released more NETs than wild type. Neutralization of IFN-γ abolished neutrophil infiltration and NETosis in Treg-depleted mice. Neutrophils stimulated with IFN-γ were more prone to release NETs in vitro. Finally, Foxp3+ Treg control cutaneous allergic inflammation by regulating IFN-γ-driven neutrophilic infiltration and NETosis. ConclusionOur results highlight the previously underestimated Treg-IFN-γ-neutrophil inflammatory axis.

Abstract 1405: Development of a humanized anti-IL-22 antibody for cancer and inflammation therapy

Abstract Interleukin-22 (IL-22) is an inflammatory cytokine involved in the pathology of autoimmune diseases such as psoriasis, atopic dermatitis, and ulcerative colitis. IL-22 has also been shown to promote epithelial cell proliferation, stemness and tumorigenesis in cancer, and is associated with a more aggressive phenotype in a variety of cancers including inflammatory colon cancer models. IL-22 plays a central role in this pathogenic process by driving activation of STAT3 and other signaling cascades. Recent studies demonstrated that neutralization of IL-22 reduced dysplasia and tumor development in preclinical models. However, there has not been a clear application for anti-IL-22 therapy for cancer and autoimmune diseases, likely due to identifying the right disease indication and their low potency and efficacy. Thus, there remains an opportunity to explore the potential of more potent anti-IL-22 therapeutics. We have developed humanized anti-IL-22 antibodies with high affinities to human, cynomolgus and mouse IL-22 using BioAtla’s proprietary antibody discovery and engineering platforms. In vitro data demonstrated that our anti-IL-22 antibodies inhibited IL-22-induced p-STAT3 activities. In addition, our antibodies showed a 10-fold increased binding activity to human IL-22 compared to Fezakinumab, which was previously developed by Pfizer for the treatment of autoimmune diseases. To test the functions of our antibodies in vivo, we established an inflammation-driven sporadic colitis-associated colorectal (CAC) cancer mouse model. Our humanized anti-IL-22 antibody, a potent, species cross-reactive anti-IL-22 blocking antibody, significantly reduced tumor progression in CAC model. These data suggest that targeting IL-22 reduces tumor occurrence by reducing inflammation-induced tumorigenesis. In addition, we evaluated our anti-IL22 antibodies in an imiquimod-induced psoriasiform skin inflammation mouse model. The mice treated with anti-IL22 antibodies had significantly reduced skin lesions compared to isotype antibody-treated mice. Q-PCR analysis of the mouse skin demonstrated that treatment with our anti-IL-22 antibody led to significantly decreased RNA levels of the imiquimod-induced inflammatory marker CXCL3. In conclusion, the development of a potent, species cross-reactive anti-IL-22 antibody using BioAtla’s antibody discovery and engineering platforms addresses the past challenges of anti-IL-22 therapeutics and allows for translational studies in relevant animal efficacy and safety models. Citation Format: Jian Chen, Cathy Chang, Gerhard Frey, Haizhen Liu, Jing Wang, William J. Boyle, Jay M. Short. Development of a humanized anti-IL-22 antibody for cancer and inflammation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1405.

Matrix metalloproteinase landscape in the imiquimod-induced skin inflammation mouse model

Inflammation and autoimmunity are known as central processes in many skin diseases, including psoriasis. It is therefore important to develop pre-clinical models that describe disease-related aspects to enable testing of pharmaceutical drug candidates and formulations. A widely accepted pre-clinical model of psoriasis is the imiquimod (IMQ)-induced skin inflammation mouse model, where topically applied IMQ provokes local skin inflammation. In this study, we investigated the abundance of a subset of matrix metalloproteinases (MMPs) in skin from mice with IMQ-induced skin inflammation and skin from naïve mice using targeted proteomics. Our findings reveal a significant increase in the abundance of MMP-2, MMP-7, MMP-8, and MMP-13 after treatment with IMQ compared to the control skin, while MMP-3, MMP-9, and MMP-10 were exclusively detected in the IMQ-treated skin. The increased abundance and broader representation of MMPs in the IMQ-treated skin provide valuable insight into the pathophysiology of skin inflammation in the IMQ model, adding to previous studies on cytokine levels using conventional immunochemical methods. Specifically, the changes in the MMP profiles observed in the IMQ-treated skin resemble the MMP patterns found in skin lesions of individuals with psoriasis. Ultimately, the differences in MMP abundance under IMQ-induced inflammation as compared to non-inflamed control skin can be exploited as a model to investigate drug efficacy or performance of drug delivery systems.

Selective inhibition of overactive warmth-sensitive Ca2+-permeable TRPV3 channels by antispasmodic agent flopropione for alleviation of skin inflammation

The temperature-sensitive Ca2+-permeable TRPV3 ion channel is robustly expressed in the skin keratinocytes, and its gain-of-function mutations are involved in the pathology of skin lesions. Here, we report the identification of an antispasmodic agent flopropione that alleviates skin inflammation by selective inhibition of TRPV3. In whole-cell patch clamp recordings, flopropione selectively inhibits macroscopic TRPV3 currents in a concentration-dependent manner with an IC50 value of 17.8± 3.5μM. At the single-channel level, flopropione inhibits TRPV3 channel open probability without alteration of its unitary conductance. In an invivo mouse model of skin inflammation induced by the skin sensitizer DNFB, flopropione also alleviates dorsal skin lesions and ear skin swelling. Further molecular docking combined with site-directed mutagenesis reveals that two residues E501 and I505 in the channel S2-helix are critical for flopropione-mediated inhibition of TRPV3. Taken together, our findings demonstrate that the spasmolytic drug flopropione as a selective inhibitor of TRPV3 channel not only provides a valuable tool molecule for understanding of TRPV3 channel pharmacology but also holds repurposing potential for therapy of skin disorders, such as dermatitis and pruritus.

CREB1-driven CXCR4hi neutrophils promote skin inflammation in mouse models and human patients

Neutrophils have a pathogenic function in inflammation via releasing pro-inflammatory mediators or neutrophil extracellular traps (NETs). However, their heterogeneity and pro-inflammatory mechanisms remain unclear. Here, we demonstrate that CXCR4hi neutrophils accumulate in the blood and inflamed skin in human psoriasis, and correlate with disease severity. Compared to CXCR4lo neutrophils, CXCR4hi neutrophils have enhanced NETs formation, phagocytic function, neutrophil degranulation, and overexpression of pro-inflammatory cytokines and chemokines in vitro. This is accompanied by a metabolic shift in CXCR4hi neutrophils toward glycolysis and lactate release, thereby promoting vascular permeability and remodeling. CXCR4 expression in neutrophils is dependent on CREB1, a transcription factor activated by TNF and CXCL12, and regulated by de novo synthesis. In vivo, CXCR4hi neutrophil infiltration amplifies skin inflammation, whereas blockade of CXCR4hi neutrophils through CXCR4 or CXCL12 inhibition leads to suppression of immune responses. In this work, our study identifies CREB1 as a critical regulator of CXCR4hi neutrophil development and characterizes the contribution of CXCR4hi neutrophils to vascular remodeling and inflammatory responses in skin.

Kaempferol therapy improved MC903 induced-atopic dermatitis in a mouse by suppressing TSLP, oxidative stress, and type 2 inflammation

BackgroundAtopic dermatitis is a common skin disease caused by genetic susceptibility, environmental factors, immune response, and skin barrier dysfunction. Kaempferol is a natural flavonoid widely found in tea, vegetables, and fruits and has been reported to have excellent anti-inflammation activity. However, the therapeutic effect of kaempferol on atopic dermatitis is unclear. ObjectiveThis study aimed to elucidate the effect of kaempferol on skin inflammation in atopic dermatitis. MethodsThe suppressive effect of kaempferol administration on skin inflammation was examined using MC903-induced atopic dermatitis-like skin inflammation mouse model. Quantification of skin dermatitis and transepidermal water loss was performed. A histopathological study was performed to examine thymic stromal lymphopoietin expression, cornified envelope proteins such as filaggrin, loricrin, and involucrin, and the numbers of infiltrating inflammatory cells, including lymphocytes, macrophages, and mast cells in the dermatitis area. The expressions of IL-4 and IL-13 were investigated by qPCR and flow cytometry analysis using skin tissues. The expression of HO-1 was investigated by western blot and qPCR. ResultsKaempferol therapy significantly suppressed MC903-induced dermatitis, TEWL, TSLP, and HO-1 expression, and infiltration of inflammatory cells. Kaempferol therapy improved the decreased expressions of filaggrin, loricrin, and involucrin in MC903-induced dermatitis skin site. The expressions of IL-4, and IL-13 were partially decreased in kaempferol-treated mice. ConclusionKaempferol might improve MC903-induced dermatitis via suppression of type 2 inflammation and improvement of barrier dysfunction by inhibition of TSLP expression and oxidative stress. Kaempferol might have the potential to be a new treatment for atopic dermatitis.

Diminished γδ T Cells during allergic skin inflammation is mediated by IL-4 signaling in keratinocytes

Abstract Atopic dermatitis results in profound changes in the function of the skin that include diminished barrier function and altered production of antimicrobial peptides. Dendritic epidermal γδ T cells (DETC) are a subset of γδ T cells that play an important role in the wound repair and inflammation process. Our previous work identified a loss of DETC in Stat6VT mice and in MC903 induced skin inflammation mouse model that was dependent on IL-4. However, the mechanisms through which IL-4 mediates this loss of DETCs are still not clear. In this study, we show that IL-4Rα germline knockout mice (Il4ra−/−) have an increased DETC population in skin that correlates with a higher rate of wound closure and increased EDC gene and fibronectin 1 expression. In Il4ra−/− mice, MC903 induced loss of DETCs was alleviated. Bone marrow chimeras of Il4ra−/− and WT bone marrow into Il4ra−/− mice retained the increased DETCs in the skin of Il4ra−/− host mice. Importantly, IL-15 secretion from skin keratinocytes was decreased dramatically in the MC903 induced skin inflammation model and injection of IL-15 rescued the loss of DETCs. Furthermore, conditional knock out of IL-4Rα from Keratinocytes of Il4ra fl/fl K14cre mice also showed an increase of this DETC populations in skin, increased IL-15 expression and less skin inflammation. Together, these results identify an IL-4-dependent circuit that regulates DETCs in the skin by repressing IL-15 expression in keratinocytes. This provides further insights into the ability of IL-4 to promote the development of atopic lesions and impair wound healing. Supported by grants from NIH (R01AI095282)

Abstract 6335: Developing anti-IL-22 therapeutics for inflammation and cancer

Abstract Chronic inflammation facilitates the development of cancer, age-related, inflammatory autoimmune and other diseases. Proinflammatory cytokines play a central role in this pathogenic process by driving activation of NF-ƙB, STAT3 and other signaling cascades. IL-22 is an inflammatory cytokine involved in the pathology of autoimmune diseases such as psoriasis, atopic dermatitis, and ulcerative colitis. IL-22 has also been shown to promote epithelial cell proliferation, stemness and tumorigenesis in cancer. Recent studies demonstrated that neutralization of IL-22 reduced dysplasia and tumor development in preclinical models. To date, there has not been a clear application for anti-IL-22 therapy for autoimmune diseases and cancer, likely due to identifying the right disease indication and to their low potency and low efficacy. Thus, there remains an opportunity to explore the potential of more potent anti-IL-22 therapeutics. Using BioAtla’s proprietary antibody discovery and engineering platforms, we have generated humanized anti-IL-22 antibodies with high affinities to human, cynomolgus and mouse IL-22. In vitro data demonstrated that our anti-IL-22 antibodies inhibited IL-22-induced p-STAT3, CXCL-1 and IL-10 activities. In addition, our antibodies showed a 10-fold increased binding activity to human IL-22 compared to Fezakinumab, which was previously developed by Pfizer for the treatment of autoimmune diseases. BioAtla’s high affinity anti-IL-22 antibodies were shown to be more potent relative to Fezakinumab using multiple in vitro assays. To test the functions of our antibodies in vivo, we also established mouse models to determine anti-IL22 efficacy. More specifically, to study whether targeting IL-22 in an inflammation-induced tumor microenvironment reduces tumor occurrence, we developed an inflammation-driven sporadic colitis-associated colorectal cancer mouse model for ongoing cancer studies that will be discussed. In addition, we evaluated our anti-IL22 antibodies in an imiquimod-induced psoriasiform skin inflammation mouse model. The mice treated with potent anti-IL-22 antibodies had significantly reduced skin lesions, including erythema, scales, and skin thickness compared to isotype antibody-treated mice. Q-PCR analysis of the mouse skin demonstrated that treatment with our anti-IL-22 antibody led to significantly decreased RNA levels of the imiquimod-induced inflammatory marker CXCL3. Thus, the enhanced anti-IL-22 therapy is a promising strategy for targeting anti-inflammation in autoimmune diseases, such as psoriasis and atopic dermatitis, and inflammatory cancers, including colorectal cancer. In conclusion, the development of a potent, species cross reactive anti-IL-22 antibody using BioAtla’s antibody discovery and engineering platforms addresses the challenges of anti-IL-22 therapeutics and allows for translational studies in relevant animal efficacy and safety models. Citation Format: Christina Wheeler, Jian Chen, Cathy Chang, Gerhard Frey, Haizhen Liu, Jing Wang, Kathryn Woodard, Solmarie Joyner, Wei Zhou, William J. Boyle, Jay M. Short. Developing anti-IL-22 therapeutics for inflammation and cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6335.

Discovery and multi-parametric optimization of a high-affinity antibody against interleukin-25 with neutralizing activity in a mouse model of skin inflammation.

BackgroundInterleukin (IL)25 has been implicated in tissue homeostasis at barrier surfaces and the initiation of type two inflammatory signaling in response to infection and cell injury across multiple organs. We sought to discover and engineer a high affinity neutralizing antibody and evaluate the antibody functional activity in vitro and in vivo.MethodsIn this study, we generated a novel anti-IL25 antibody (22C7) and investigated the antibody’s therapeutic potential for targeting IL25 in inflammation.ResultsA novel anti-IL25 antibody (22C7) was generated with equivalent in vitro affinity and potency against the human and mouse orthologs of the cytokine. This translated into in vivo potency in an IL25-induced air pouch model where 22C7 inhibited the recruitment of monocytes, macrophages, neutrophils and eosinophils. Furthermore, 22C7 significantly reduced ear swelling, acanthosis and disease severity in the Aldara mouse model of psoriasiform skin inflammation. Given the therapeutic potential of IL25 targeting in inflammatory conditions, 22C7 was further engineered to generate a highly developable, fully human antibody while maintaining the affinity and potency of the parental molecule.ConclusionsThe generation of 22C7, an anti-IL25 antibody with efficacy in a preclinical model of skin inflammation, raises the therapeutic potential for 22C7 use in the spectrum of IL25-mediated diseases.

Chemokines form nanoparticles with DNA and can superinduce TLR-driven immune inflammation.

Chemokines control the migratory patterns and positioning of immune cells to organize immune responses to pathogens. However, many chemokines have been associated with systemic autoimmune diseases that have chronic IFN signatures. We report that a series of chemokines, including CXCL4, CXCL10, CXCL12, and CCL5, can superinduce type I IFN (IFN-I) by TLR9-activated plasmacytoid DCs (pDCs), independently of their respective known chemokine receptors. Mechanistically, we show that chemokines such as CXCL4 mediate transcriptional and epigenetic changes in pDCs, mostly targeted to the IFN-I pathways. We describe that chemokines physically interact with DNA to form nanoparticles that promote clathrin-mediated cellular uptake and delivery of DNA in the early endosomes of pDCs. Using two separate mouse models of skin inflammation, we observed the presence of CXCL4 associated with DNA in vivo. These data reveal a noncanonical role for chemokines to serve as nucleic acid delivery vectors to modulate TLR signaling, with implications for the chronic presence of IFN-I by pDCs in autoimmune diseases.

Effects of conditioned medium obtained from human adipose-derived stem cells on skin inflammation.

IntroductionCell therapy using adipose-derived mesenchymal stem cells (ASCs) is a promising avenue of regenerative medicine for the treatment of various diseases. It has been considered that ASCs exert their therapeutic effects through the secretion of multiple factors that are critical for tissue remodeling or the suppression of inflammation. Recently, conditioned medium (CM) from ASCs that contains a complex of secreted factors has received attention as a cost-effective alternative to cell therapy.MethodsWe investigated the effects of CM obtained from ASCs (ASCs-CM) using human dermal fibroblasts (hDFs) and human epidermal keratinocytes with or without interleukin (IL)-1β and examined mRNA levels of marker genes. We also examined alterations in cell proliferation and morphology of hDFs following treatment with ASCs-CM. We further investigated the effects of ASCs-CM treatment on prevention of skin inflammation using a mouse model.ResultsIn hDFs and human epidermal keratinocytes, the ASCs-CM treatment suppressed pro-inflammatory factors and enhanced regenerative and remodeling factors with or without interleukin (IL)-1β exposure. The ASCs-CM treatment also enhanced cell proliferation of hDFs and prevented morphological changes in response to IL-1β exposure. Furthermore, in a mouse model of skin inflammation, treatment with ASCs-CM reduced the inflammatory reactions, including redness and thickness.ConclusionsCM from ASCs may represent a potential alternative to ASC therapy for the treatment of inflammatory skin conditions.

Characterization of a novel non-steroidal glucocorticoid receptor agonist optimized for topical treatment

Glucocorticoids (GCs) are commonly used topical treatments for skin diseases but are associated with both local and systemic side effects. In this study, we describe a selective non-steroidal glucocorticoid receptor (GR) agonist for topical use, LEO 134310, which is rapidly deactivated in the blood resulting in low systemic exposure and a higher therapeutic index in the TPA-induced skin inflammation mouse model compared with betamethasone valerate (BMV) and clobetasol propionate (CP). Selectivity of LEO 134310 for GR was confirmed within a panel of nuclear receptors, including the mineralocorticoid receptor (MR), which has been associated with induction of skin atrophy. Topical treatment with LEO 134310 in minipigs did not result in any significant reduction in epidermal thickness in contrast to significant epidermal thinning induced by treatment with BMV and CP. Thus, the profile of LEO 134310 may potentially provide an effective and safer treatment option for skin diseases compared with currently used glucocorticoids.

Protocol to Induce Follicular T Helper Cells, Germinal Centers, and Skin Lesions in Mouse Models for Skin Blistering Diseases.

Autoreactive T cells in autoantibody-mediated autoimmune diseases can be divided into two major subsets: (i) follicular T helper cells (Tfh) that provide T cell help in germinal centers (GC) and (ii) effector T (Teff) cells that immigrate into peripheral tissue sites such as the skin and mediate local inflammation. To study the sequence of events leading to the loss of tolerance in autoantibody-mediated autoimmune diseases it is required to investigate both T cell subsets simultaneously. This approach is hampered mainly because the appearance of skin inflammation in mouse models is a random process, which makes it difficult to define the location of inflammation at the right time point. To overcome this problem, we developed a scratching technique for ear skins that leads to the establishment of chronic autoimmune wounds in the mouse model for the pemphigoid-like disease epidermolysis bullosa acquisita. By defining the exact place where the skin wounds should form, this protocol enables a detailed analysis of skin-immigrating Teff cells. Of note, this protocol induces GC in draining lymph nodes in parallel so that Tfh cells in GC can be investigated concurrently. This protocol is not restricted to T cells and can be used for any other skin-immigrating inflammatory cells.

Epidermal Fatty Acid‒Binding Protein Mediates Depilatory-Induced Acute Skin Inflammation

Depilatory creams are widely used to remove unwanted body hair, but people with sensitive skin are subject to depilatory-induced skin burn/inflammation. It remains unknown what makes their skin more sensitive than others. In this study, we show that epidermal fatty acid‒binding protein (E-FABP) expressed in the skin plays a critical role in promoting depilatory-induced acute skin inflammation in mouse models. Although a depilatory cream removed hair by breaking down keratin disulfide bonds, it activated cytosolic phospholipase A2, leading to activation of the arachidonic acid/E-FABP/peroxisome proliferator-activated receptor β signaling pathway in keratinocytes. Specifically, peroxisome proliferator-activated receptor β activation induced downstream targets (e.g., cyclooxygenase 2) and chemokine (e.g., CXCL1) production, which systemically mobilized neutrophils and recruited them to localize in the skin for acute inflammatory responses. Importantly, E-FABP deletion by CRISPR-Cas9 reduced cytosolic phospholipase A2/peroxisome proliferator-activated receptor β activation in keratinocytes, and genetic deletion of E-FABP protected mice from depilatory cream-induced neutrophil recruitment and skin inflammation. Our findings suggest E-FABP as a molecular sensor for sensitive skin by triggering depilatory-induced, lipid-mediated skin inflammatory responses.

Astragalus mongholicus Bunge Water Extract Exhibits Anti-inflammatory Effects in Human Neutrophils and Alleviates Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice.

Neutrophils are the primary immune cells in innate immunity, which are related to various inflammatory diseases. Astragalus mongholicus Bunge is a Chinese medicinal herb used to treat various oxidative stress-related inflammatory diseases. However, there are limited studies that elucidate the effects of Astragalus mongholicus Bunge in human neutrophils. In this study, we used isolated human neutrophils activated by various stimulants to investigate the anti-inflammatory effects of Astragalus mongholicus Bunge water extract (AWE). Cell-free assays were used to examine free radicals scavenging capabilities on superoxide anion, reactive oxygen species (ROS), and nitrogen-centered radicals. Imiquimod (IMQ) induced psoriasis-like skin inflammation mouse model was used for investigating anti-psoriatic effects. We found that AWE inhibited superoxide anion production, ROS generation, and elastase release in human neutrophils, which exhibiting a direct anti-neutrophil effect. Moreover, AWE exerted a ROS scavenging ability in the 2,2’-Azobis (2-amidinopropane) dihydrochloride assay, but not superoxide anion in the xanthine/xanthine oxidase assay, suggesting that AWE exhibited anti-oxidation and anti-inflammatory capabilities by both scavenging ROS and by directly inhibiting neutrophil activation. AWE also reduced CD11b expression and adhesion to endothelial cells in activated human neutrophils. Meanwhile, in mice with psoriasis-like skin inflammation, administration of topical AWE reduced both the affected area and the severity index score. It inhibited neutrophil infiltration, myeloperoxidase release, ROS-induced damage, and skin proliferation. In summary, AWE exhibited direct anti-inflammatory effects by inhibiting neutrophil activation and anti-psoriatic effects in mice with IMQ-induced psoriasis-like skin inflammation. Therefore, AWE could potentially be a pharmaceutical Chinese herbal medicine to inhibit neutrophilic inflammation for anti-psoriasis.

Keratinocyte-intrinsic BCL10/MALT1 activity initiates and amplifies psoriasiform skin inflammation.

Psoriasis is a chronic inflammatory skin disease arising from poorly defined pathological cross-talk between keratinocytes and the immune system. BCL10 (B cell lymphoma/leukemia 10) and MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) are ubiquitously expressed inflammatory signaling proteins that can interact with the psoriasis susceptibility factor CARD14, but their functions in psoriasis are insufficiently understood. We report that although keratinocyte-intrinsic BCL10/MALT1 deletions completely rescue inflammatory skin pathology triggered by germline Card14 gain-of-function mutation in mice, the BCL10/MALT1 signalosome is unexpectedly not involved in the CARD14-dependent interleukin-17 receptor (IL-17R) proximal pathway. Instead, it plays a more pleiotropic role by amplifying keratinocyte responses to a series of inflammatory cytokines, including IL-17A, IL-1β, and TNF. Moreover, selective keratinocyte-intrinsic activation of BCL10/MALT1 signaling with an artificial engager molecule is sufficient to initiate lymphocyte-mediated psoriasiform skin inflammation, and aberrant BCL10/MALT1 activity is frequently detected in the skin of human sporadic psoriasis. Together, these results establish that BCL10/MALT1 signalosomes can act as initiators and crucial amplifiers of psoriatic skin inflammation and indicate a critical function for this complex in sporadic psoriasis.