Stimulation Of Keratinocytes Research Articles

Type 2 Cytokine–Dependent Skin Barrier Regulation in Personalized 2-Dimensional and 3-Dimensional Skin Models of Atopic Dermatitis: A Pilot Study

Keratinocytes from healthy donors stimulated with type 2 cytokines are often used to experimentally study atopic dermatitis (AD) inflammatory responses. Due to potential intrinsic alterations, it seems favorable to use keratinocytes from AD patients. Keratinocytes isolated from hair follicles offer a non-invasive approach to investigate AD-derived keratinocytes. To evaluate whether such AD-derived keratinocytes are suitable to mimic AD inflammatory responses we compared hair follicle-derived keratinocytes from healthy donors and AD patients in a type 2 cytokine environment. Stimulation of AD-derived keratinocytes with IL-4 and IL-13 induced higher expression changes of AD-associated markers as compared to healthy keratinocytes. The combination of IL-4 and IL-13 generally induced highest expression changes, but IL-13 alone also induced significant changes of AD-specific markers. Similar to the 2D cultures, IL-4/IL-13 stimulation of 3D skin models generated with AD-derived keratinocytes modulated the expression of several AD-relevant factors. Whole transcriptome analysis revealed that IL-4 and IL-13 acted similarly on these 3D skin models. Histologically, IL-13 alone and in combination with IL-4 increased epidermal spongiosis, a histological hallmark of AD skin. Taken together, our pilot study suggests that hair follicle-derived keratinocytes from AD patients represent a useful model system to study AD-related inflammation in a personalized in vitro model.

Targeting IL-1 controls refractory pityriasis rubra pilaris.

Pityriasis rubra pilaris (PRP) is a rare inflammatory skin disease with a poorly understood pathogenesis. Through a molecularly driven precision medicine approach and an extensive mechanistic pathway analysis in PRP skin samples, compared to psoriasis, atopic dermatitis, healed PRP, and healthy controls, we identified IL-1β as a key mediator, orchestrating an NF-κB-mediated IL-1β-CCL20 axis, including activation of CARD14 and NOD2. Treatment of three patients with the IL-1 antagonists anakinra and canakinumab resulted in rapid clinical improvement and reversal of the PRP-associated molecular signature with a 50% improvement in skin lesions after 2 to 3 weeks. This transcriptional signature was consistent with in vitro stimulation of keratinocytes with IL-1β. With the central role of IL-1β underscoring its potential as a therapeutic target, our findings propose a redefinition of PRP as an autoinflammatory keratinization disorder. Further clinical trials are needed to validate the efficacy of IL-1β antagonists in PRP.

Breast Cancer–Related Lymphedema Results in Impaired Epidermal Differentiation and Tight Junction Dysfunction

Breast cancer-related lymphedema (BCRL) is characterized by skin changes, swelling, fibrosis, and recurrent skin infections. Clinical studies have suggested that lymphedema results in skin barrier defects; however, the underlying cellular mechanisms and the effects of bacterial contamination on skin barrier function remain unknown. In matched biopsies from patients with unilateral BCRL, we observed decreased expression of filaggrin and the tight junction protein zona occludens-1 (ZO-1) in skin affected by moderate lymphedema, or by subclinical lymphedema in which dermal backflow of lymph was identified by indocyanine green lymphography, relative to controls (areas without backflow and from the unaffected arm). In vitro stimulation of keratinocytes with lymph fluid obtained from patients undergoing lymphedema surgery led to the same changes, as well as increased expression of keratin 14, a marker of immature keratinocytes. Finally, using mouse models of lymphedema, we showed that like the clinical scenario, the expression of skin barrier proteins was decreased relative to normal skin and that colonization with S. epidermidis bacteria amplified this effect, as well as lymphedema severity. Taken together, our findings suggest that lymphatic fluid stasis contributes to skin barrier dysfunction in lymphedema.

Particulate matter stimulates the NADPH oxidase system via AhR-mediated epigenetic modifications

Stimulation of human keratinocytes with particulate matter 2.5 (PM2.5) elicits complex signaling events, including a rise in the generation of reactive oxygen species (ROS). However, the mechanisms underlying PM2.5-induced ROS production remain unknown. Here, we show that PM2.5-induced ROS production in human keratinocytes is mediated via the NADPH oxidase (NOXs) system and the Ca2+ signaling pathway. PM2.5 treatment increased the expression of NOX1, NOX4, and a calcium-sensitive NOX, dual oxidase 1 (DUOX1), in human epidermal keratinocyte cell line. PM2.5 bound to aryl hydrocarbon receptor (AhR), and this complex bound to promoter regions of NOX1 and DUOX1, suggesting that AhR acted as a transcription factor of NOX1 and DUOX1. PM2.5 increased the transcription of DUOX1 via epigenetic modification. Moreover, a link between DNA demethylase and histone methyltransferase with the promoter regions of DUOX1 led to an elevation in the expression of DUOX1 mRNA. Interestingly, PM2.5 increased NOX4 expression and promoted the interaction of NOX4 and Ca2+ channels within the cytoplasmic membrane or endoplasmic reticulum, leading to Ca2+ release. The increase in intracellular Ca2+ concentration activated DUOX1, responsible for ROS production. Our findings provide evidence for a PM2.5-mediated ROS-generating system network, in which increased NOX1, NOX4, and DUOX1 expression serves as a ROS signal through AhR and Ca2+ activation.

Correlational analysis of PLIN1 with inflammation in diabetic foot ulcer wounds

BackgroundThe persistent presence of inflammation is a recognized pathogenic mechanisms of diabetic foot ulcers (DFUs). We aimed to investigate the expression of PLIN1 in tissues from DFU patients and assess its potential association with inflammation-induced damage. MethodsWe performed transcriptome sequencing and correlation analysis of the foot skin from patients with or without DFUs. Additionally, we examined the correlation between PLIN1 and related inflammatory indicators by analyzing PLIN1 expression in tissue and serum samples and through high-glucose stimulation of keratinocytes (HaCaT cells). ResultsPLIN1 is upregulated in the tissue and serum from DFU patients. Additionally, PLIN1 shows a positive correlation with leukocytes, neutrophils, monocytes, C-reactive protein, and procalcitonin in the serum, as well as IL-1β and TNF-α in the tissues. Experiments with Cells demonstrated that reduced expression of PLIN1 leads to significantly decreased expression of iNOS, IL-1β, IL-6, IL-18, and TNF-α. PLIN1 may mediate wound inflammatory damage through the NF-κB signaling pathway. ConclusionOur findings suggest that PLIN1 mediates the inflammatory damage in DFU, offering new prospects for the treatment of DFU.

Metabolic re-programming of keratinocytes in response to contact allergens.

Allergic contact dermatitis (ACD) is a common skin disease caused by the recognition of haptens by the immune system. Keratinocytes play an important role in the initiation and facilitation of inflammatory responses in ACD. Immune responses are associated with major changes in metabolism. However, metabolic re-programming is not well studied in ACD; specifically, knowledge of metabolic alterations in structural cells is lacking. Metabolic re-programming in ACD was studied using publicly available transcriptome datasets. Primary pooled keratinocytes and a keratinocyte cell line (HaCaT) were stimulated with contact allergens, and inflammatory responses and expression of metabolic markers were measured by qPCR and flow cytometry, respectively. ACD is characterized by metabolic re-programming with a metabolic profile similar to atopic dermatitis. Exposure to contact allergens causes a wide array of metabolic alterations. Stimulation of keratinocytes with contact allergens induced inflammatory responses typical for ACD and was associated with an up-regulation of proteins representative for glucose uptake, fatty acid metabolism, oxidative phosphorylation and to some extent arginine biosynthesis. Changes in these metabolic pathways were also observed when comparing lesional with non-lesional contact dermatitis skin. ACD is, similarly to other inflammatory skin diseases, characterized by metabolic re-programming. Contact allergen exposure induces expression of a wide array of metabolic pathways, which is at least in part mediated through metabolic re-programming of keratinocytes.

KRT17 from skin cells with high glucose stimulation promotes keratinocytes proliferation and migration.

Impaired diabetic wound healing is an important issue in diabetic complications. Proliferation and migration of keratinocytes are major processes of skin wound repair after injury. However, hyperkeratosis can affect the speed of wound healing. Based on the results of preliminary experiments on increased KRT17 expression after high glucose stimulation of human skin tissue cells, a cell model of human immortalized keratinocyte (HaCaT) stimulation with different concentrations of KRT17 was established in vitro, and the promotion in cell proliferation and migration were discovered. KRT17 silencing promoted diabetic wound healing in the db/db diabetic wound model. Transcriptome sequencing (RNA-seq) was performed on HaCaT cells after KRT17 stimulation, and analysis showed significant enrichment in the PI3K-AKT signaling pathway, in which the regulation of cell c-MYB mRNA, a key molecule regulating cell proliferation and migration, was significantly upregulated. In vitro assays showed increased c-MYB expression and enhanced pAKT activity after HaCaT cell stimulation by KRT17. We speculate that KRT17 is upregulated under high glucose and promotes keratinocyte proliferation and migration caused hyperkeratosis, through the c-MYB/PI3K-AKT pathway, contributing to delayed wound healing.

Decreased epidermal AXL expression and increased infiltration of AXL‐expressing dendritic cells in psoriasis

AbstractBackgroundPsoriasis is a chronic skin inflammatory disease characterized by epidermal proliferation and inflammatory cell infiltration. AXL is a tyrosine kinase receptor that promotes cell proliferation and invasion in cancer cells, and its expression is elevated in multiple tumors. However, less is known about its expression and function in inflammatory diseases.ObjectivesThe aim of this study is to examine AXL expression in psoriasis and investigate the biological function of AXL under psoriatic conditions.ResultsAXL mRNA expression was decreased in psoriatic skin compared to healthy skin, and an inverse correlation with neutrophil‐to‐lymphocyte ratio and platelet‐to‐lymphocyte ratio was observed. Immunohistochemical staining of psoriatic skin revealed decreased AXL expression of the epidermis and an increased number of AXL‐positive dendritic cells in the dermis. Stimulation of epidermal keratinocytes with antimicrobial peptide LL37, but not with IL‐17A, resulted in decreased AXL expression. Knockdown of AXL in epidermal keratinocytes did not affect cell proliferation or expression of psoriasis‐associated cytokines and chemokines.ConclusionDecreased epidermal AXL expression and increased infiltration of AXL‐expressing dendritic cells were revealed in psoriasis.

Antimicrobial Peptides for Diabetic Wound Healing: Preclinical and Clinical Studies.

The purpose of this review is to provide verified data on the current knowledge acquired from preclinical and clinical studies regarding topically used antimicrobial peptides (AMPs) with diabetic wound healing activity. The electronic databases were searched for articles published from 2012 to 2022. The 20 articles comparing topically used AMPs in diabetic wound healing treatment versus control treatments (placebo or active therapy) were selected. AMPs have several unique advantages in diabetic wound healing, such as a broad spectrum of antimicrobial activity even against antibiotic-resistant strains, and the capability to modulate the host's immune response and affect wound healing processes through various mechanisms of action. AMPs through antioxidant activity, stimulation of angiogenesis, keratinocytes, and fibroblast migration and proliferation may be considered an important support during conventional therapy used for diabetic wound treatment.

Inflammatory stimulation of osteoblasts and keratinocytes from a SAPHO patient for implant risk evaluation

The present report exemplifies a translational method, which could assist the clinical pre-evaluation of patients at risk before surgical interventions. In this study, a presurgical implant decision in a case of SAPHO (synovitis, acne, palmoplantar pustulosis, hyperostosis, osteitis) is described. Since the etiology of this syndrome is likely to involve genetic, infectious and immunological components, lipopolysaccharides (LPS) may conceptually trigger cytokine production leading to a specific chronic inflammation and immunological host response. This may hamper proper healing or accentuate the destruction of periodontal host tissues. In our approach, we examined the ex vivo cell viability and immune responses of primary osteoblasts and keratinocytes under sterile inflammation induced by P. gingivalis LPS. Keratinocytes and osteoblasts were obtained from biopsies of the keratinized gingiva and alveolar bone tissues of a SAPHO human subject. Enzymatically dissociated cells were thus cultured and incubated to LPS at different concentrations (50ng/ml, 200ng/ml, 500ng/ml and 1µg/ml) for 24 h in order to test inflammatory cytokine response (quantitative real time PCR) and toxicity (cell viability). Healthy primary keratinocytes and osteoblasts were used as control cells. The highest concentration of LPS (1µg/ml) significantly reduced cell viability (p < 0.05). Meanwhile, all tested LPS concentrations similarly enhanced the mRNA expressions of selected inflammatory cytokines (TNFalpha, IL-6, IL-8, IL-1beta and IL-1alpha) up to =~3.5-fold, when compared to the healthy cell controls (p < 0.05). This study demonstrated a valuable inflammatory risk evaluation before implant placement, which was successfully performed based on the presented laboratory diagnostic/prognostic approach.
 Accepted for publication: December 12, 2022
 Published online: January, 2022

Leser–Trelat sign in cosmetologist’s practice

LeserTrelat syndrome (eruptive seborrheic keratosis) is an unusial paraneoplastic dermatosis characterized by sudden appearance of seborrheic keratomas on the skin and a progressive increase of their number. This condition was first described in 1901 by the German surgeon E. Leser and the French surgeon U. Trelat. It occurs with the same frequency in both men and women aged 40+. The etiology and pathogenesis have not been studied in detail, however, there is evidence that the development of the syndrome may be associated with stimulation of the epidermal growth factor, which leads to stimulation of keratinocytes. The manifestation of the syndrome is usually start at the same time with cancer development, mostly it can be combained with adenocarcinoma of the gastrointestinal tract (47.7%), with intestinal carcinoma (32%), with lymphoproliferative diseases (21%), less often with malignant neoplasms of the lungs, breast, prostate.
 The clinical picture of the LeserTrelat sign is characterized by the sudden appearance оf seborrheic keratomas, which have typical clinical and histological signs. The most typical localization of seborrheic keratosis are back and chest (76%), limbs (18%), face (21%), abdomen (15%), neck (13%), armpits (6%), inguinal folds (3%). Keratosis can appear rapidly, over several months or even weeks. The rapid appearance of multiple seborheic keratomas may precede or develop with the oncological process in the internal organs.
 Treatment is carried out at the same time with the establishment and treatment of the underlying disease and consists in removing the largest keratomas by destructive methods (surgical excision, radiowave method, cryodestruction, electrocoagulation).
 The prognosis is favorable if paraneoplastic process was early detected.
 The article describes clinical cases of multiple seborrheic keratosis and the tactics of examining this group of patients.

Bioelectronic microfluidic wound healing: a platform for investigating direct current stimulation of injured cell collectives.

Upon cutaneous injury, the human body naturally forms an electric field (EF) that acts as a guidance cue for relevant cellular and tissue repair and reorganization. However, the direct current (DC) flow imparted by this EF can be impacted by a variety of diseases. This work delves into the impact of DC stimulation on both healthy and diabetic in vitro wound healing models of human keratinocytes, the most prevalent cell type of the skin. The culmination of non-metal electrode materials and prudent microfluidic design allowed us to create a compact bioelectronic platform to study the effects of different sustained (12 hours galvanostatic DC) EF configurations on wound closure dynamics. Specifically, we compared if electrotactically closing a wound's gap from one wound edge (i.e., uni-directional EF) is as effective as compared to alternatingly polarizing both the wound's edges (i.e., pseudo-converging EF) as both of these spatial stimulation strategies are fundamental to the eventual translational electrode design and strategy. We found that uni-directional electric guidance cues were superior in group keratinocyte healing dynamics by enhancing the wound closure rate nearly three-fold for both healthy and diabetic-like keratinocyte collectives, compared to their non-stimulated respective controls. The motility-inhibited and diabetic-like keratinocytes regained wound closure rates with uni-directional electrical stimulation (increase from 1.0 to 2.8% h-1) comparable to their healthy non-stimulated keratinocyte counterparts (3.5% h-1). Our results bring hope that electrical stimulation delivered in a controlled manner can be a viable pathway to accelerate wound repair, and also by providing a baseline for other researchers trying to find an optimal electrode blueprint for in vivo DC stimulation.

Stimulation with THBS4 activates pathways that regulate proliferation, migration and inflammation in primary human keratinocytes

As in other mammalian tissues, the extracellular matrix (ECM) of skin functions as mechanical support and regulative environment that guides the behavior of the cells. ECM is a gel-like structure that is primarily composed of structural and nonstructural proteins. While the content of structural proteins is stable, the level of nonstructural ECM proteins, such as thrombospondin-4 (THBS4), is dynamically regulated. In a previous work we demonstrated that THBS4 stimulated cutaneous wound healing. In this work we discovered that in addition to proliferation, THBS4 stimulated the migration of primary keratinocytes in 3D. By using a proteotransciptomic approach we found that stimulation of keratinocytes with THBS4 regulated the activity of signaling pathways linked to proliferation, migration, inflammation and differentiation. Interestingly, some of the regulated genes (eg IL37, TSLP) have been associated with the pathogenesis of atopic dermatitis (AD). We concluded that THBS4 is a promising candidate for novel wound healing therapies and suggest that there is a potential convergence of pathways that stimulate cutaneous wound healing with those active in the pathogenesis of inflammatory skin diseases.

ОЦЕНКА ЦИТОКИНОВОГО СТАТУСА РОТОВОЙ ЖИДКОСТИ У ДЕТЕЙ С РЕЦИДИВИРУЮЩИМ АФТОЗНЫМ СТОМАТИТОМ

A special place in the pathogenesis of recurrent aphthous stomatitis is occupied by the state of local immunity, which can affect the clinical course and prognosis of chronic diseases of the oral mucosa. The inflammatory process in recurrent aphthous stomatitis is initiated by stimulation of keratinocytes of the oral mucosa by a currently unknown antigen, which leads to stimulation of T-lymphocytes and the release of cytokines and various interleukins.
 Cytokines are the main nonspecific humoral factor of immunity, providing the initiation and development of an inflammatory response during the development of a protective immune response. A huge number of works are devoted to the study of cytokine status in recurrent aphthous stomatitis, which plays one of the key roles in the pathogenesis of the disease. However, most of the studies were conducted in patients over the age of 18, in addition, the results obtained are contradictory. This determined the purpose of this study – to assess the cytokine status of oral fluid in children with recurrent aphthous stomatitis. The study examined 70 children who were divided into 2 groups. The main group consisted of 45 children aged 7 to 14 years, patients with recurrent aphthous stomatitis, during the relapse of the disease. The main group was divided into 2 subgroups: A – 20 children with the duration of recurrent aphthous stomatitis up to 5 years, group B – 25 children with the duration of recurrent aphthous stomatitis more than 5 years. All patients in the main group had concomitant diseases, in most cases, diseases of the gastrointestinal tract. The control group consisted of 25 practically healthy children without recurrent aphthous stomatitis. In children with recurrent aphthous stomatitis in the oral fluid, a significant increase in the concentration of the anti-inflammatory cytokine TNF-α and a decrease in the concentration of IL-10, an imbalance in the level of IL-2 depending on the duration of the disease, as evidenced by a pronounced inflammatory process of the oral mucosa with epithelial destruction.

347 Chronic Inflammation of Keratinocytes Leads to Autophagy Impairment in Psoriasis

Psoriasis is an inflammatory disorder which leads to the improper stratification of epidermal keratinocytes. Autophagic flux analysis of our established cell lines has shown that autophagy is functionally active in healthy keratinocytes but is impaired in psoriatic keratinocytes. To mirror psoriasis in vitro we custom-made a cytokine cocktail that induced IL-1β release in keratinocyte cultures. Stimulation of healthy keratinocytes by this chronic inflammatory cocktail similarly causes autophagy block and accumulation of key autophagy proteins.

Stimulation of alpha-1 adrenoceptors may intensify cutaneous inflammation in complex regional pain syndrome.

Alpha-1 adrenoceptors are overexpressed in the epidermis of a subgroup of patients with complex regional pain syndrome (CRPS). Activating α 1 -adrenoceptors in epidermal cells increases production of the proinflammatory cytokine interleukin-6 (IL-6), a mediator of inflammation. To investigate whether this might exacerbate inflammation in CRPS, primary keratinocytes or dermal fibroblasts were cultured from skin biopsies obtained from the affected limb of 25 patients and a similar site in 28 controls. The fundamental proinflammatory cytokine, tumor necrosis factor alpha, was administered for 24 hours to initiate inflammation. After this, cells were incubated for 6 hours with the α 1 -adrenoceptor agonist phenylephrine. Exposure to tumor necrosis factor alpha induced proinflammatory cytokine mRNA production and protein secretion in keratinocytes and fibroblasts and enhanced α 1B -adrenoceptor mRNA expression in keratinocytes. Additional stimulation of α 1 adrenoceptors with phenylephrine increased the production of IL-6 mRNA and protein secretion in both cell types. Under all conditions, gene and protein α 1 -adrenoceptor levels and cytokine gene expression and protein secretion were similar, overall, in patients and controls, except for abnormally high α 1 -adrenoceptor protein levels in the keratinocytes of 3 of 17 patients. These findings suggest that persistent inflammation in CRPS is not due to dysfunction of skin cells but is a normal response to extrinsic signals. After α 1 -adrenoceptor stimulation of keratinocytes, increases in IL-6 mRNA but not protein were proportional to basal α 1 -adrenoceptor protein levels. Skin cells play an important role in persistent inflammation in CRPS. Potentially, a positive feedback loop between α 1 -adrenoceptors and IL-6 production in skin cells contributes to this inflammatory state.

Equine keratinocytes in the pathogenesis of insect bite hypersensitivity: Just another brick in the wall?

Equine insect bite hypersensitivity (IBH) is the most common skin disease affecting horses. It is described as an IgE-mediated, Type I hypersensitivity reaction to salivary gland proteins of Culicoides insects. Together with Th2 cells, epithelial barrier cells play an important role in development of Type I hypersensitivities. In order to elucidate the role of equine keratinocytes in development of IBH, we stimulated keratinocytes derived from IBH-affected (IBH-KER) (n = 9) and healthy horses (H-KER) (n = 9) with Culicoides recombinant allergens and extract, allergic cytokine milieu (ACM) and a Toll like receptor ligand 1/2 (TLR-1/2-L) and investigated their transcriptomes. Stimulation of keratinocytes with Culicoides allergens did not induce transcriptional changes. However, when stimulated with allergic cytokine milieu, their gene expression significantly changed. We found upregulation of genes encoding for CCL5, -11, -20, -27 and interleukins such as IL31. We also found a strong downregulation of genes such as SCEL and KRT16 involved in the formation of epithelial barrier. Following stimulation with TLR-1/2-L, keratinocytes significantly upregulated expression of genes affecting Toll like receptor and NOD-receptor signaling pathway as well as NF-kappa B signaling pathway, among others. The transcriptomes of IBH-KER and H-KER were very similar: without stimulations they only differed in one gene (CTSL); following stimulation with allergic cytokine milieu we found only 23 differentially expressed genes (e.g. CXCL10 and 11) and following stimulation with TLR-1/2-L they only differed by expression of seven genes. Our data suggests that keratinocytes contribute to the innate immune response and are able to elicit responses to different stimuli, possibly playing a role in the pathogenesis of IBH.

Antiviral Effect of hBD-3 and LL-37 during Human Primary Keratinocyte Infection with West Nile Virus.

West Nile virus (WNV) is an emerging flavivirus transmitted through mosquito bites and responsible for a wide range of clinical manifestations. Following their inoculation within the skin, flaviviruses replicate in keratinocytes of the epidermis, inducing an innate immune response including the production of antimicrobial peptides (AMPs). Among them, the cathelicidin LL-37 and the human beta-defensin (hBD)-3 are known for their antimicrobial and immunomodulatory properties. We assessed their role during WNV infection of human primary keratinocytes. LL-37 reduced the viral load in the supernatant of infected keratinocytes and of the titer of a viral inoculum incubated in the presence of the peptide, suggesting a direct antiviral effect of this AMP. Conversely, WNV replication was not inhibited by hBD-3. The two peptides then demonstrated immunomodulatory properties whether in the context of keratinocyte stimulation by poly(I:C) or infection by WNV, but not alone. This study demonstrates the immunostimulatory properties of these two skin AMPs at the initial site of WNV replication and the ability of LL-37 to directly inactivate West Nile viral infectious particles. The results provide new information on the multiple functions of these two peptides and underline the potential of AMPs as new antiviral strategies in the fight against flaviviral infections.

Activation of Keratinocyte Gq-linked G-Protein Coupled Receptors Remodels Dorsal Root Ganglion Neurons Single-Cell Transcriptomic Profile

Keratinocytes are closely juxtaposed to cutaneous nerve terminals, enabling communication between keratinocytes and cutaneous nerves. We investigated potential mechanisms that mediate this communication. We genetically expressed stimulatory DREADDs (hM3Dq) into K14 basal keratinocytes (K14) in mice as a tool for mimicking the activation of Gq-linked G protein-coupled receptors (GPCRs) in K14 expressing cells. We have shown that activation of these DREADDs reduced innervation of the epidermis indicating that activation of K-14 Gq-linked GPCRs can regulate nerve fiber degeneration in the epidermis. We used bulk and single-cell RNA (scRNA-seq) sequencing from the skin and Dorsal Root Ganglion (DRG) taken from mice expressing hM3Dq into K14 keratinocytes comparing control mice receiving saline and mice receiving CNO to activate K14 keratinocytes expressing hM3Dq. Bulk RNA sequencing of the epidermis revealed changes in a set of genes associated with neurite outgrowth, including class III semaphorins, potentially impacting cutaneous nerve degeneration. Using RNAScope we found dramatic changes in the expression of semaphorins and their receptors in distinct DRG neurons subtypes. Using single-cell RNA-seq from lumbar DRG we profiled the transcriptome of 10,000 individual DRG cells and found that hM3Dq mediated stimulation of keratinocytes induced changes in DRG single-cell gene expression profile resulting in the identification of a novel DRG neuronal subtype among the nonpeptidergic nociceptors (NP1.2). Our findings indicate that activation of basal keratinocytes Gq-linked GPCRs can result in profound changes in DRG neuronal transcriptomic profile resulting in a phenotypical switch (plasticity) of DRG neurons. Furthermore, our results suggest possible therapeutic effects of activating or blocking specific keratinocyte Gq-linked GPCRs for promoting axon regeneration in small fiber neuropathy. Grant support from 1R01AR77691-01.

Enhanced Migratory Ability of Neutrophils Toward Epidermis Contributes to the Development of Psoriasis via Crosstalk With Keratinocytes by Releasing IL-17A.

Microabscess of neutrophils in epidermis is one of the histological hallmarks of psoriasis. The axis of neutrophil–keratinocyte has been thought to play a critical role in the pathogenesis of psoriasis. However, the features and mechanism of interaction between the two cell types remain largely unknown. Herein, we found that blood neutrophils were increased in psoriasis patients, positively correlated with disease severity and highly expressed CD66b, but not CD11b and CD62L compared to healthy controls. Keratinocytes expressed high levels of psoriasis-related inflammatory mediators by direct and indirect interaction with neutrophils isolated from psoriasis patients and healthy controls. The capacity of neutrophils in provoking keratinocytes inflammatory response was comparable between the two groups and is dependent on IL-17A produced by itself. Neutrophils isolated from psoriasis patients displayed more transcriptome changes related to integrin and increased migration capacity toward keratinocytes with high CD11b expression on cell surface. Of interest, neutrophils were more susceptible to keratinocyte stimulation than to fibroblasts and human umbilical vein endothelial cells (HUVECs) in terms of CD11b expression and the production of ROS and NETs. In conclusion, neutrophils from psoriasis patients gain a strong capacity of IL-17A production and integrins expression that possibly facilitates their abilities to promote production of psoriasis-related inflammatory mediators and migration, a phenomenon likely induced by their interaction with keratinocytes but not with fibroblasts. These findings provide a proof-of-concept that development of new drugs targeting migration of neutrophils could be a more specific and safe solution to treat psoriasis.