Keratinocyte Function Research Articles

Stress signalling and STAT1 activation characterize the keratinocytic gene expression pattern in Hidradenitis suppurativa.

The pathogenetic factors generating the innate immune signal necessary for T-cell activation, initiation and chronification of Hidradenitis suppurativa (HS, also known as Acne inversa) are still poorly understood. Emerging evidence suggests that a defective keratinocyte function critically contributes to HS disease development and progression. To elucidate the role of keratinocytes in HS lesion formation, we compared the transcriptomes of lesional and perilesional epidermis isolated from HS patients by RNA sequencing (RNA Seq). Pairwise-matched lesional and perilesional HS skin samples of five different donors were obtained and epidermal keratinocytes freshly isolated and processed for RNA extraction and RNA seq. Lesionally regulated genes were analysed by large-scale promoter analysis and functional annotation clustering to identify epidermally overrepresented transcription factor binding sites and functionally related gene groups. Results were experimentally validated with independent epidermal isolates of patient-matched lesional and perilesional HS skin employing qRT-PCR, cell culture, immunoblot and immunostaining. We show that HS is characterized by a strong epidermal stress state evident by a significant overrepresentation of an AP-1-driven gene signature and a substantial activation of the stress-activated cJun N-terminal kinase (JNK) pathway in lesional epidermis. Additionally, our data reveal a strong induction of STAT1 activation in lesional HS epidermis that likely results from IFNγ production and triggered expression of key inflammatory genes coordinating innate immune activation and the adaptive T-cell response in HS. Our data implicate a key role of stress signalling and JAK/STAT1 activation in disease progression of HS and suggest interference with JAK/STAT1 signalling as a potentially promising therapeutic approach for HS.

637 Non-redundant roles for GLUT1 and GLUT3 in melanoma and primary melanocytes

The GLUT1 transporter mediates glucose uptake in most tissues and is upregulated in both inflammatory and malignant skin diseases. Most melanomas overexpress both GLUT1 and GLUT3, but it is unclear whether the closely related transporters are functionally redundant. Consistent with its function in keratinocytes, GLUT1 knockdown in melanoma cells (UACC-62 and Mel-juso) inhibited glucose uptake and cell proliferation. Notably, GLUT3 knockdown did not impair glucose transport yet showed even more dramatic effects on cell proliferation and morphology. Fractionation revealed the preferential localization of GLUT1 to the plasma membrane while GLUT3 preferentially localized to endosomes. RNA-sequencing, Western blotting, and immunofluorescence revealed an essential role for GLUT3 in signal transducer and activator of transcription 3 (STAT3) activation, which has been implicated in melanoma growth and metastasis. Deletion of GLUT1 or GLUT3 in primary mouse melanocytes through Tyrosinase-cre expression confirmed a critical role for GLUT3 in STAT3 activation and non-redundant roles for GLUT1 and GLUT3 in pigmentation and melanocyte proliferation ex vivo. These studies identify unique functions for GLUT1 and GLUT3 in melanoma and melanocytes and highlight the GLUT3-STAT3 signaling axis as a potential therapeutic target in melanoma.

431 AMP-IBP5 improves tight junction barrier function in human keratinocytes and suppresses dermatitis-like lesions in a mouse model of atopic dermatitis

Background: A novel antimicrobial peptide derived from insulin-like growth factor-binding protein 5 (AMP-IBP5) displays both antimicrobial and immunomodulatory properties. However, the role of AMP-IBP5 in the regulation of skin barrier remains unclear. Objective: To investigate the effects of AMP-IBP5 on skin barrier function. Methods: Normal human epidermal keratinocytes were stimulated with AMP-IBP5 and Western blot was used to analyze the expression of tight junction (TJ)-related proteins. Immunofluorescence staining was used to examine the intercellular distribution of TJ-related proteins.

724 MPZL3 functions as a negative regulator of sebaceous gland size and sebocyte proliferation

The sebaceous gland (SG) engages in holocrine secretion to produce lipid-rich sebum critical for epidermal barrier function, skin and hair lubrication, and microbiome management. SG undergoes continuous self-renewal to replenish differentiated sebocytes lost during holocrine secretion, as well as hair cycle-dependent oscillations in size. Previous studies have shown that a mitochondrially localized, nuclear encoded immunoglobulin-like v-type protein, Myelin Protein Zero-like-3 (MPZL3), regulates lipid metabolism and energy expenditure, as well as epidermal keratinocyte differentiation and function.

699 An in-vitro keratinocyte model for the evaluation of skin damage induced by 5G radiation

With the rapid spread of next-generation cellular communication technology, wireless bands between 3 – 100 GHz (5G), are increasingly in use in major population centers. The electromagnetic radiation involved remains non-ionizing, however it is of higher energy compared to existing cellular or wi-fi bands; and, this technology’s range is shorter – so that more emitter stations will be needed, placed closer to one another, and thus also closer to the user. This will result in higher net EM energy exposure. There is no clear consensus on the risks inherent in this exposure yet, but there is concern among the public and the scientific community, based on indications that this radiation can affect immune function, trigger inflammatory responses, and influence gene expression linked to protein folding, oxidative stress, tissue / ECM matrix turnover, and more. Further, some skin appendages have been shown to act as antennae for this radiation, which may lead to localized damage. To better understand the effects of this radiation on skin, we irradiated a normal human epidermal keratinocyte culture using a 6 GHz signal generator. Biological effects were evaluated by ELISA or immunostaining. After a remarkably short 1-h exposure time, clear effects were observable in several indicators of keratinocyte function, including: an increase in key inflammatory cytokine IL1-alpha; a reduction in collagenase inhibitor TIMP1; an increase in wound healing and epidermal differentiation facilitator ANGPLT4; and an increase in SA100A9, involved in immune recruitment during injury. These results indicate a clear influence of 5G irradiation on the keratinocytes, suggesting induced changes in skin homeostasis which may be consistent with a state of injury and damage response. In upcoming studies, we hope to be able to show these effects in more depth, and propose some possible solutions for daily users of 5G technology.

403 Glyoxal induces senescence in in vitro skin models

Senescence is a well-known cellular event characterized by specific markers like a permanent cell proliferation arrest and the secretion of messenger molecules forming the Senescence-Associated Secretory Phenotype (SASP). The SASP composition depends on many factors such as the cell type and the nature of the stress that induced senescence. Since the skin constitutes a barrier with the external environment, it is particularly subjected to different types of stresses and thus to premature cellular aging. In this study, we have demonstrated that glyoxal provokes senescence in our skin in vitro models. Indeed, application of glyoxal on keratinocytes during the reconstruction induced a senescent phenotype of epidermis with a thinning of epidermal thickness (H&E), an increase of senescent markers (p16), an increase of inflammatory cytokine (IL1a), and a decrease of terminal differentiation epidermal protein expression (filaggrin, loricrin). Our study provides evidence that glyoxal can affect keratinocyte function and act as a driver of human skin aging. Hence, senotherapeutics aimed at modulating glyoxal-associated senescent phenotype, could be relevant to prevent the aging process in skin. These innovative in vitro models have been used to develop and screen ingredients. Results allowed to select ingredients with high potential to fight against consequences of glyoxal stress.

A Systematic Review of Keratinocyte Secretions: A Regenerative Perspective.

Cell regenerative therapy is a modern solution for difficult-to-heal wounds. Keratinocytes, the most common cell type in the skin, are difficult to obtain without the creation of another wound. Stem cell differentiation towards keratinocytes is a challenging process, and it is difficult to reproduce in chemically defined media. Nevertheless, a co-culture of keratinocytes with stem cells usually achieves efficient differentiation. This systematic review aims to identify the secretions of normal human keratinocytes reported in the literature and correlate them with the differentiation process. An online search revealed 338 references, of which 100 met the selection criteria. A total of 80 different keratinocyte secretions were reported, which can be grouped mainly into cytokines, growth factors, and antimicrobial peptides. The growth-factor group mostly affects stem cell differentiation into keratinocytes, especially epidermal growth factor and members of the transforming growth factor family. Nevertheless, the reported secretions reflected the nature of the involved studies, as most of them focused on keratinocyte interaction with inflammation. This review highlights the secretory function of keratinocytes, as well as the need for intense investigation to characterize these secretions and evaluate their regenerative capacities.

Improvements in Human Keratinocytes and Antimicrobial Effect Mediated by Cell-Free Supernatants Derived from Probiotics

The skin acts as a physical and physiological barrier, thereby protecting the body from various environmental components and stimuli. Cell-free supernatants (CFS) derived from probiotics can improve skin functions and retain moisture. In this study, to assess the efficacy of CFS derived from Ligilactobacillus salivarius and Limosilactobacillus fermentum, we investigated the barrier strengthening and moisturizing effects of CFS in keratinocytes along with their antibacterial effects. We also determined the adhesive effects of probiotics on colorectal cells. To confirm improvements in moisturization and barrier function mediated by CFS in keratinocytes, hyaluronic acid (HA) production, and mRNA expression of HA synthases (HAS)2, HAS3, and FLG were measured. The results showed that CFS from L. salivarius MG242 and L. fermentum MG901 increased the expression of these genes along with the production of HA (2.40- and 1.95-fold of control). Additionally, CFS derived from L. salivarius MG242 and L. fermentum MG901 inhibited the growth of S. aureus and E. coli, thereby demonstrating inhibitory effects against harmful pathogens observed on the skin. These results indicate that the use of CFS derived from L. salivarius MG242 and L. fermentum MG901 may increase moisturization in the skin and improve barrier function of keratinocytes along with elimination of potential pathogens.

Lrig1- and Wnt-dependent niches dictate segregation of resident immune cells and melanocytes in murine tail epidermis.

ABSTRACTThe barrier-forming, self-renewing mammalian epidermis comprises keratinocytes, pigment-producing melanocytes and resident immune cells as first-line host defense. In murine tail skin, interfollicular epidermis patterns into pigmented ‘scale’ and hypopigmented ‘interscale’ epidermis. Why and how mature melanocytes accumulate in scale epidermis is unresolved. Here, we delineate a cellular hierarchy among epidermal cell types that determines skin patterning. Already during postnatal development, melanocytes co-segregate with newly forming scale compartments. Intriguingly, this process coincides with partitioning of both Langerhans cells and dendritic epidermal T cells to interscale epidermis, suggesting functional segregation of pigmentation and immune surveillance. Analysis of non-pigmented mice and of mice lacking melanocytes or resident immune cells revealed that immunocyte patterning is melanocyte and melanin independent and, vice versa, immune cells do not control melanocyte localization. Instead, genetically enforced progressive scale fusion upon Lrig1 deletion showed that melanocytes and immune cells dynamically follow epithelial scale:interscale patterns. Importantly, disrupting Wnt-Lef1 function in keratinocytes caused melanocyte mislocalization to interscale epidermis, implicating canonical Wnt signaling in organizing the pigmentation pattern. Together, this work uncovers cellular and molecular principles underlying the compartmentalization of tissue functions in skin.

Whey Derivatives and Galactooligosaccharides Stimulate the Wound Healing and the Function of Human Keratinocytes through the NF-kB and FOXO-1 Signaling Pathways.

Skin repair requires the activation of keratinocytes and is mediated by controlled inflammation and cell migration and proliferation, ending with the regeneration of well-differentiated cell layers. Whey derivatives contain galactooligosaccharides (GOS), which have potential beneficial effects on wound healing due to their activity as toll-like receptor ligands, although their direct nonprebiotic effects in the skin have not yet been described. In this study, we investigated the effects of different whey-derived products and purified GOS on a human keratinocyte cell line. We found that the inflammatory cytokine interleukin-8 (IL-8) was upregulated by nuclear factor kappa B (NF-kB) signaling triggered by whey derivatives and GOS and that wound healing was accelerated by promoting cell migration and the loss of E-cadherin in the absence of epithelial–mesenchymal transition. Interestingly, the treatments enhanced the mitochondrial function in association with the translocation of the Forkhead Box O1 (FOXO-1) transcription factor. Finally, we detected the increased expression of the differentiation markers induced by GOS and whey derivatives. All together, our results show that GOS-containing products can promote wound closure and skin health by direct activity on keratinocyte functions. Among the preparations tested, the fermented compound produced by autochthonous microorganisms was the most active in modulating keratinocyte activity, supporting the biological value of whey derivatives for health.

Zinc supplementation prevents arsenic-induced dysregulation of ZRANB2 splice function

Environmentally relevant (100 nM) inorganic arsenic (iAs) exposure displaces zinc from zinc fingers of upstream splice regulator ZRANB2 disrupting the splicing of its target TRA2B. Excess zinc displaced iAs from ZRANB2 zinc fingers in cell free system. Thus, the hypothesis that zinc supplementation could prevent iAs-mediated disruption of ZRANB2 splice function in human keratinocytes was tested. The data show that zinc supplementation prevented iAs-induced dysregulation of TRA2B splicing by ZRANB2 as well as the induction of ZRANB2 protein expression. These results provide additional support for the hypothesis that zinc supplementation could prevent iAs-mediated disease in iAs-exposed populations.

LGI3 promotes human keratinocyte migration in high-glucose environments by increasing the expression of β-catenin.

The leucine-rich repeat LGI family member 3 (LGI3) has been reported to regulate various functions in epidermal keratinocytes. In this study, we investigated the effects of LGI3 on keratinocyte migration in environments with different glucose concentrations. Our results showed that cell migration is markedly impaired in high-glucose environments compared to in low-glucose environments (control). Nevertheless, the use of LGI3 in high-glucose environments restores cell migration to the normal level. Therefore, we performed LGI3 knockdown to identify the role of LGI3 in cell migration. It was observed that transfecting LGI3 siRNA into HaCaT cells reduces the expression of LGI3 and inhibits wound closure. These results indicate that LGI3 is deeply involved in wound healing in high-glucose environments. Western blot analysis showed that in high-glucose environments, LGI3 increases the phosphorylation of Akt, forkhead box protein O1, and focal adhesion kinase. However, no change was observed in the levels of glycogen synthase kinase 3β, c-Jun N-terminal kinase, extracellular signal-regulated kinase, or p38 mitogen-activated protein kinase. Further results showed that LY294002, a specific inhibitor of phosphatidylinositol 3-kinase, reduced LGI3-induced cell migration. It is generally known that Akt activation leads to the accumulation of β-catenin, an important mediator of keratinocyte migration. LGI3 greatly increased the expression of β-catenin in high-glucose environments comparison to that in the low-glucose environments. Taken together, these data indicate that LGI3 induces keratinocyte migration in high-glucose environments as a result of β-catenin accumulation via Akt phosphorylation. Therefore, LGI3 can be considered a new treatment option for diabetic wound healing.

The mechanism on Prevotella melaninogenica promoting the inflammatory progression of oral lichen planus.

Oral lichen planus (OLP) is a common chronic inflammatory disease occurring in the oral mucosa. Bacteria are a key driver of mucosal immune responses and can induce changes in gene expression and function of epithelial keratinocytes. IL-36γ can induce the expression of antimicrobial peptides, cytokines, and chemokines, and is widely involved in many chronic inflammatory diseases. Our aim is to explore the role of IL-36γ in the pathological process of OLP when Prevotella melaninogenica (P. melaninogenica) invades the oral mucosa. The expression of IL-36γ in OLP lesions and mice was detected by immunohistochemistry. Recombinant human IL-36Gamma (rhIL-36γ) was used to treat oral keratinocytes and the expression levels of inflammatory cytokines were detected by qRT-PCR and ELISA. The expression of IL-36γ and TRPV1 was detected by western blotting following co-culturing P. melaninogenica with oral keratinocytes. The mRNA expression of IL-36γ was detected by qRT-PCR. From our results, IL-36γ was upregulated in OLP lesions. Exogenous rhIL-36γ promoted the expression of pro-inflammatory cytokines and antibacterial peptides in oral keratinocytes. The expression of IL-36γ was significantly increased following the stimulation of P. melaninogenica in oral keratinocytes and mice. TRPV1 activation was induced by P. melaninogenica and its activation enhanced the expression of IL-36γ. IL-36Ra could reduce the inflammation in OLP in vitro. In summary, overexpression of IL-36γ in OLP lesions could promote its pathogenesis by inducing inflammation. P. melaninogenica invasion of oral keratinocytes could induce the expression of IL-36γ by the activation of TRPV1, thereby regulating the interaction between bacteria and oral epithelial cells.

Expression of Retinal G Protein-Coupled Receptor, a Member of the Opsin Family, in Human Skin Cells and Its Mediation of the Cellular Functions of Keratinocytes.

Background: Photoreceptive proteins play critical physiological roles in human skin cells. The retinal G protein-coupled receptor (RGR) is a photoisomerase in the human retina, but its expression and cellular functions in human skin cells have not been reported. Objectives: We aimed to detect RGR expression in various skin cells and evaluate its regulation of the cellular functions of keratinocytes. Methods: The expression, distribution, and subcellular location of the RGR in normal human epidermal keratinocytes and cells with pathological conditions including psoriasis, seborrheic keratosis, and squamous cell carcinoma were determined using microscopic tools (immunohistochemical staining, immunofluorescence staining, and immunoelectron microscopy) and Western blotting (WB). The protein levels of the RGR in primary human melanocytes, keratinocytes, and fibroblasts isolated from the neonatal foreskin were measured by WB. The expression and subcellular localization of the RGR in these cells were detected by immunofluorescence staining under a fluorescence microscope and laser scanning confocal microscope. Additionally, the levels of RGR expression in normal keratinocytes exposed to ultraviolet (UV)-A or total ultraviolet radiation (UVR) in the presence or absence of all-trans-retinal were measured by WB. Furthermore, the effects of the RGR on human keratinocyte functions including proliferation, migration, and apoptosis were evaluated using the Cell Counting Kit 8, wound healing, and Transwell assays after reducing the RGR mRNA level in keratinocytes using small interfering RNA technology. Results: The RGR was primarily located in the epidermal basal and spinous layers and skin appendages. Its expression increased in psoriatic lesions, seborrheic keratosis, and squamous cell carcinoma. Confocal microscopy showed that the RGR was located in the cell membrane and nucleus of keratinocytes, melanocytes, and fibroblasts. Keratinocytes had a higher expression of the RGR than melanocytes and fibroblasts, as well as nuclear expression, according to nuclear/cytoplasmic fractionation. Colloidal gold immunoelectron microscopy technology further confirmed that the RGR is mainly located in the nucleoplasm and mitochondria and is scattered in the cytoplasm and other organelles in the epidermal keratinocytes. Notably, RGR knockdown in keratinocytes led to the inhibition of cell proliferation and migration, augmenting cell apoptosis. Conclusions: This study is the first to demonstrate the presence of RGR in the human skin. Our findings indicate that the RGR may play a critical role in the physiological function of epidermal keratinocytes.

Ginsenoside Rb1 selectively improved keratinocyte functions in vitro without affecting tissue regeneration in zebrafish larvae tail regrowth.

Ginsenoside Rb1 selectively improved keratinocyte functions in vitro without affecting tissue regeneration in zebrafish larvae tail regrowth.

Immune-enhancing effect of water-soluble beta-glucan derived from enzymatic hydrolysis of yeast glucan

Immunostimulants play an important role in the treatment of immunodeficiency. Macrophages are the first line in our immune defense system and play a critical role in the immune response. Therefore, finding new and better substances to induce an immune response by activating macrophages is an attractive research topic, especially in the fields of immunopharmacology and cancer prevention. Keratinocytes actively crosstalk with immune cells during wound repair, so enhancing the function of keratinocytes is also an important part of improving immunity. Beta-glucans are naturally occurring polysaccharides, consisting of d-glucose monomers linked by beta-glycosidic bonds. Several studies have investigated the immunomodulatory effects of beta-glucan, such as its anti-inflammatory and antibacterial properties. However, the use of yeast cell wall glucan has been limited because it is not soluble in water. In this study, we produced low-molecular-weight water-soluble yeast glucan (WSY glucan) and confirmed various aspects of its immune-enhancing effect. The structure of the beta-(1→3) and (1→6) bonds of WSY glucan were confirmed by nuclear magnetic resonance spectroscopy (1H-NMR) analysis. Our results showed that treatment with WSY glucan significantly and dose-dependently induced the production of inflammatory mediators (prostaglandin E2 (PGE2) and nitric oxide (NO)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-6) in macrophages. In addition, WSY glucan treatment showed changes in the morphological structure of the macrophages and promoted phagocytic activity of the macrophages and wound healing in keratinocytes. Based on these results, WSY glucan is considered as a potential candidate for the treatment of diseases related to the weakening of the immune system without the limitation of insolubility.

Unraveling the Role of Sex Hormones on Keratinocyte Functions in Human Inflammatory Skin Diseases.

The skin exerts several fundamental functions that are the first physical, chemical and immune barriers to the human body. Keratinocytes, the main cell type of the epidermis, provide mechanical defense, support skin integrity and actively endorse cutaneous immune responses. Not surprisingly, considering these crucial activities, alterations in keratinocyte functions are associated with different inflammatory skin diseases. Recent findings indicate that the skin should not only be regarded as a target for hormones but that it should also be considered as an endocrine peripheral organ that is directly involved in the synthesis and metabolism of these chemical messengers. Sex hormones have multiple effects on the skin, attributed to the binding with intracellular receptors expressed by different skin cell populations, including keratinocytes, that activate downstream signaling routes that modulate specific cellular functions and activities. This review is aimed at reorganizing the current knowledge on the role exerted by sex hormones on keratinocyte function in five different inflammatory skin diseases: Hidradenitis suppurativa; Acne vulgaris; Atopic dermatitis; progesterone hypersensitivity; psoriasis. The results of our work aim to provide a deeper insight into common cellular mechanisms and molecular effectors that might constitute putative targets to address for the development of specific therapeutic interventions.

Selaginellin Inhibits Melanogenesis via the MAPK Signaling Pathway.

Hyperpigmented skin diseases such as melasma, freckles, and melanosis usually mar the appearance of patients. Traditional herbal medicines are highly accepted in inhibiting skin pigmentation, with advantages of high efficiency, low cost, and low side effects. Selaginellin (SEL), one of the active compounds of selaginella, has been proved to be exhibit antineoplastic, antioxidant, antisenescence, and antiapoptosis activities. In this study, we found that SEL can inhibit melanogenesis in vitro and in vivo. A mechanism study found that SEL inhibits melanogenesis through inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway, then down-regulating the expression of microphthalmia-associated transcription factor (MITF) and downstream genes tyrosinase (TYR) and tyrosinase-related protein 2 (TYRP2). UVB-activated paracrine function of fibroblasts and keratinocytes promotes melanogenesis of melanocytes. Interestingly, SEL antagonizes UVB-activated paracrine function of fibroblasts and keratinocytes. These findings indicate that SEL can be a potential whitening compound to inhibit melanogenesis.

Mitochondrially Localized MPZL3 Functions as a Negative Regulator of Sebaceous Gland Size and Sebocyte Proliferation

Mitochondrially localized nucleus-encoded Ig-like v-type protein, MPZL3, regulates lipid metabolism, energy expenditure, as well as epidermal keratinocyte differentiation and function (Bhaduri et al., 2015; Leiva et al., 2014; Wikramanayake et al., 2021). Previous studies have shown increased energy expenditure and insulin sensitivity (Czyzyk et al., 2013), epidermal barrier defects and a seborrheic dermatitis–like inflammatory skin phenotype (Wikramanayake et al., 2017, 2018), as well as altered hair follicle cycling (Nicu et al., 2020) in Mpzl3 global knockout (GKO) (GKO–/–) mice.

Chronic exposure to the anti-M3 muscarinic acetylcholine receptor autoantibody in pemphigus vulgaris contributes to disease pathophysiology

Pemphigus vulgaris (PV) is a potentially lethal autoimmune mucocutaneous blistering disease characterized by binding of IgG autoantibodies (AuAbs) to keratinocytes (KCs). In addition to AuAbs against adhesion molecules desmogleins 1 and 3, PV patients also produce an AuAb against the M3 muscarinic acetylcholine (ACh) receptor (M3AR) that plays an important role in regulation of vital functions of KCs upon binding endogenous ACh. This anti-M3AR AuAb is pathogenic because its adsorption eliminates the acantholytic activity of PV IgG; however, the molecular mechanism of its action is unclear. In the present study, we sought to elucidate the mode of immunopharmacologic action of the anti-M3AR AuAb in PV. Short-term exposures of cultured KCs to PV IgG or the muscarinic agonist muscarine both induced changes in the expression of keratins 5 and 10, consistent with the inhibition of proliferation and upregulated differentiation and in keeping with the biological function of M3AR. In contrast, long-term incubations induced a keratin expression pattern consistent with upregulated proliferation and decreased differentiation, in keeping with the hyperproliferative state of KCs in PV. This change could result from desensitization of the M3AR, representing the net antagonist-like effect of the AuAb. Therefore, chronic exposure of KCs to the anti-M3AR AuAb interrupts the physiological regulation of KCs by endogenous ACh, contributing to the onset of acantholysis. Since cholinergic agents have already demonstrated antiacantholytic activity in a mouse model of PV and in PV patients, our results have translational significance and can guide future development of therapies for PV patients employing cholinergic drugs.