Neuroprotective protein ADNP-dependent histone remodeling complex promotes T helper 2 immune cell differentiation.

Type 2 immune responses are critical in tissue homeostasis, anti-helminth immunity, and allergy. T helper 2 (Th2) cells produce interleukin-4 (IL-4), IL-5, and IL-13 from the type 2 gene cluster under regulation by transcription factors (TFs) including GATA3. To better understand transcriptional regulation of Th2 cell differentiation, we performed CRISPR-Cas9 screens targeting 1,131 TFs. We discovered that activity-dependent neuroprotector homeobox protein (ADNP) was indispensable for immune reactions to allergen. Mechanistically, ADNP performed a previously unappreciated role in gene activation, forming a critical bridge in the transition from pioneer TFs to chromatin remodeling by recruiting the helicase CHD4 and ATPase BRG1. Although GATA3 and AP-1 bound the type 2 cytokine locus in the absence of ADNP, they were unable to initiate histone acetylation or DNA accessibility, resulting in highly impaired type 2 cytokine expression. Our results demonstrate an important role for ADNP in promoting immune cell specialization.

Sensory Neurons Co-opt Classical Immune Signaling Pathways to Mediate Chronic Itch.

Mammals have evolved neurophysiologic reflexes, such as coughing and scratching, to expel invading pathogens and noxious environmental stimuli. It is well established that these responses are also associated with chronic inflammatory diseases, including asthma and atopic dermatitis. However, the mechanisms by which inflammatory pathways promote sensations such as itch remain poorly understood. Here, we show that type 2 cytokines directly activate sensory neurons in both mice and humans. Further, we demonstrate that chronic itch is dependent on neuronal IL-4Rα and JAK1 signaling. We also observe that patients with recalcitrant chronic itch that failed other immunosuppressive therapies markedly improve when treated with JAK inhibitors. Thus, signaling mechanisms previously ascribed to the immune system may represent novel therapeutic targets within the nervous system. Collectively, this study reveals an evolutionarily conserved paradigm in which the sensory nervous system employs classical immune signaling pathways to influence mammalian behavior.

Innate type 2 immunity controls hair follicle commensalism by Demodex mites.

Demodex mites are commensal parasites of hair follicles (HFs). Normally asymptomatic, inflammatory outgrowth of mites can accompany malnutrition, immune dysfunction, and aging, but mechanisms restricting Demodex outgrowth are not defined. Here, we show that control of mite HF colonization in mice required group 2 innate lymphoid cells (ILC2s), interleukin-13 (IL-13), and its receptor, IL-4Ra-IL-13Ra1. HF-associated ILC2s elaborated IL-13 that attenuated HFs and epithelial proliferation at anagen onset; in their absence, Demodex colonization led to increased epithelial proliferation and replacement of gene programs for repair by aberrant inflammation, leading to the loss of barrier function and HF exhaustion. Humans with rhinophymatous acne rosacea, an inflammatory condition associated with Demodex, had increased HF inflammation with decreased type 2 cytokines, consistent with the inverse relationship seen in mice. Our studies uncover a key role for skin ILC2s and IL-13, which comprise an immune checkpoint that sustains cutaneous integrity and restricts pathologic infestation by colonizing HF mites.

Paneth Cell-Derived Lysozyme Defines the Composition of Mucolytic Microbiota and the Inflammatory Tone of the Intestine.

Paneth cells are the primary source of C-type lysozyme, a ß-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1-/- hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.

Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion.

Gastro-intestinal helminth infections trigger the release of interleukin-33 (IL-33), which induces type-2 helper T cells (Th2 cells) at the site of infection to produce IL-13, thereby contributing to host resistance in a T cell receptor (TCR)-independent manner. Here, we show that, as a prerequisite for IL-33-induced IL-13 secretion, Th2 cells required the expression of the epidermal growth factor receptor (EGFR) and of its ligand, amphiregulin, for the formation of a signaling complex between T1/ST2 (the IL-33R) and EGFR. This shared signaling complex allowed IL-33 to induce the EGFR-mediated activation of the MAP-kinase signaling pathway and consequently the expression of IL-13. Lack of EGFR expression on T cells abrogated IL-13 expression in infected tissues and impaired host resistance. EGFR expression on Th2 cells was TCR-signaling dependent, and therefore, our data reveal a mechanism by which antigen presentation controls the innate effector function of Th2 cells at the site of inflammation.

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13.

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host­virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13­treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.

Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy.

IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)-based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.

IL-33 potentiates histaminergic itch.

BACKGROUND: Itch is a common symptom that can greatly diminish quality of life. Histamine is a potent endogenous pruritogen, and while antihistamines are often the first-line treatment for itch, in conditions like chronic spontaneous urticaria (CSU), many patients remain symptomatic while receiving maximal doses. Mechanisms that drive resistance to antihistamines are poorly defined. OBJECTIVES: Signaling of the alarmin cytokine IL-33 in sensory neurons is postulated to drive chronic itch by inducing neuronal sensitization to pruritogens. Thus, we sought to determine if IL-33 can augment histamine-induced (histaminergic) itch. METHODS: Itch behavior was assessed in response to histamine after IL-33 or saline administration. Various stimuli and conditional and global knockout mice were utilized to dissect cellular mechanisms. Multiple existing transcriptomic data sets were evaluated, including single-cell RNA sequencing of human and mouse skin, microarrays of isolated mouse mast cells at steady state and after stimulation with IL-33, and microarrays of skin biopsy samples from subjects with CSU and healthy controls. RESULTS: IL-33 amplifies histaminergic itch independent of IL-33 signaling in sensory neurons. Mast cells are the top expressors of the IL-33 receptor in both human and mouse skin. When stimulated by IL-33, mouse mast cells significantly increase IL-13 levels. Enhancement of histaminergic itch by IL-33 relies on a mast cell- and IL-13-dependent mechanism. IL-33 receptor expression is increased in lesional skin of subjects with CSU compared to healthy controls. CONCLUSIONS: Our findings suggest that IL-33 signaling may be a key driver of histaminergic itch in mast cell-associated pruritic conditions such as CSU.

Thymic stromal lymphopoietin induces IL-4/IL-13 from T cells to promote sebum secretion and adipose loss.

BACKGROUND: The cytokine TSLP promotes type 2 immune responses and can induce adipose loss by stimulating lipid loss from the skin through sebum secretion by sebaceous glands, which enhances the skin barrier. However, the mechanism by which TSLP upregulates sebaceous gland function is unknown. OBJECTIVES: This study investigated the mechanism by which TSLP stimulates sebum secretion and adipose loss. METHODS: RNA-sequencing analysis was performed on sebaceous glands isolated by laser capture microdissection and single-cell RNA-sequencing analysis was performed on sorted skin T cells. Sebocyte function was analyzed by histological analysis and sebum secretion in vivo and by measuring lipogenesis and proliferation in vitro. RESULTS: This study found that TSLP sequentially stimulated the expression of lipogenesis genes followed by cell death genes in sebaceous glands to induce holocrine secretion of sebum. TSLP did not affect sebaceous gland activity directly. Rather, single-cell RNA-sequencing revealed that TSLP recruited distinct T-cell clusters that produce IL-4 and IL-13, which were necessary for TSLP-induced adipose loss and sebum secretion. Moreover, IL-13 was sufficient to cause sebum secretion and adipose loss in vivo and to induce lipogenesis and proliferation of a human sebocyte cell line in vitro. CONCLUSIONS: This study proposes that TSLP stimulates T cells to deliver IL-4 and IL-13 to sebaceous glands, which enhances sebaceous gland function, turnover, and subsequent adipose loss.

Cholinergic sensing of allergen exposure by airway epithelium promotes type 2 immunity in the lungs.

BACKGROUND: Nonneuronal cells, including epithelial cells, can produce acetylcholine (ACh). Muscarinic ACh receptor antagonists are used clinically to treat asthma and other medical conditions; however, knowledge regarding the roles of ACh in type 2 immunity is limited. OBJECTIVE: Our aim was to investigate the roles of epithelial ACh in allergic immune responses. METHODS: Human bronchial epithelial (HBE) cells were cultured with allergen extracts, and their ACh production and IL-33 secretion were studied in vitro. To investigate immune responses in vivo, naive BALB/c mice were treated intranasally with different muscarinic ACh receptor antagonists and then exposed intranasally to allergens. RESULTS: At steady state, HBE cells expressed cellular components necessary for ACh production, including choline acetyltransferase and organic cation transporters. Exposure to allergens caused HBE cells to rapidly release ACh into the extracellular medium. Pharmacologic or small-interfering RNA-based blocking of ACh production or autocrine action through the M3 muscarinic ACh receptors in HBE cells suppressed allergen-induced ATP release, calcium mobilization, and extracellular secretion of IL-33. When naive mice were exposed to allergens, ACh was quickly released into the airway lumen. A series of clinical M3 muscarinic ACh receptor antagonists inhibited allergen-induced IL-33 secretion and innate type 2 immune response in the mouse airways. In a preclinical murine model of asthma, an ACh receptor antagonist suppressed allergen-induced airway inflammation and airway hyperreactivity. CONCLUSIONS: ACh is released quickly by airway epithelial cells on allergen exposure, and it plays an important role in type 2 immunity. The epithelial ACh system can be considered a therapeutic target in allergic airway diseases.

MiR-155-5p Differentially Regulates IL-13Rα1 and IL-13Rα2 Expression and Signalling, Driving Abnormal Lung Epithelial Cell Phenotype in Severe Asthma.

MiR-155-5p is known to increase in innate and adaptive immune cells in response to IL-13 and is associated with asthma severity. However, little is known about its role in airway structural cells. BECs isolated from healthy donors and severe asthma patients were stimulated with IL-13. MiR-155-5p expression and release were measured by RT-PCR in BECs and in their derived exosomes. Modulation of miR-155-5p in BECs was performed using transfection of miR-155-5p inhibitor and mimic. IL-13Rα1, IL-13Rα2, MUC5AC, IL-8 and Eotaxin-1 expression were measured by RT-PCR and western blot. BECs repair process was assessed by wound healing assay. IL-13Rα1 and IL-13Rα2 expression and downstream pathways were evaluated by western blot. Dual Luciferase assay was used to determine miR-155-5p target genes associated to IL-13 receptors signaling. BECs from severe asthma showed an increased expression and exosomal release of miR-155-5p at baseline that was amplified by IL-13 stimulation. BECs from asthmatics expressed more IL-13Rα1 and less IL-13Rα2 than healthy donors and IL-13Rα1 but not IL-13Rα2 induced miR-155-5p expression under IL-13 stimulation. MiR-155-5p overexpression favored MUC5AC, IL-8 and Eotaxin-1 through IL-13Rα1/SOCS1/STAT6 pathway to the detriment of a delayed repair process with a downregulated IL-13Rα2/MAPK14/c-Jun/c-Fos signaling. Dual Luciferase assay confirmed that miR-155-5p modulates both IL-13 receptors pathways by directly targeting SOCS1, c-Fos and MAPK14. MiR-155-5p is overexpressed in severe asthma BECs and regulates IL-13Rα1 and IL-13Rα2 expression and signaling, favoring expression of mucin and eosinophils related genes to detriment of airway repair. These results show that miR-155-5p may contribute to airway epithelial cell dysfunction in severe asthma.

Ex Vivo Infection of Human Skin Models with Herpes Simplex Virus 1: Accessibility of the Receptor Nectin-1 during Formation or Impairment of Epidermal Barriers Is Restricted by Tight Junctions.

Herpes simplex virus 1 (HSV-1) must overcome epidermal barriers to reach its receptors on keratinocytes and initiate infection in human skin. The cell-adhesion molecule nectin-1, which is expressed in human epidermis, acts as an efficient receptor for HSV-1 but is not within reach of the virus upon exposure of human skin under nonpathological conditions. Atopic dermatitis skin, however, can provide an entry portal for HSV-1 emphasizing the role of impaired barrier functions. Here, we explored how epidermal barriers impact HSV-1 invasion in human epidermis and influence the accessibility of nectin-1 for the virus. Using human epidermal equivalents, we observed a correlation of the number of infected cells with tight-junction formation, suggesting that mature tight junctions prior to formation of the stratum corneum prevent viral access to nectin-1. Consequently, impaired epidermal barriers driven by Th2-inflammatory cytokines interleukin 4 (IL-4) and IL-13 as well as the genetic predisposition of nonlesional atopic dermatitis keratinocytes correlated with enhanced infection supporting the impact of functional tight junctions for preventing infection in human epidermis. Comparable to E-cadherin, nectin-1 was distributed throughout the epidermal layers and localized just underneath the tight-junctions. While nectin-1 was evenly distributed on primary human keratinocytes in culture, the receptor was enriched at lateral surfaces of basal and suprabasal cells during differentiation. Nectin-1 showed no major redistribution in the thickened atopic dermatitis and IL-4/IL-13-treated human epidermis in which HSV-1 can invade. However, nectin-1 localization toward tight junction components changed, suggesting that defective tight-junction barriers make nectin-1 accessible for HSV-1 which enables facilitated viral penetration. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a widely distributed human pathogen which productively infects epithelia. The open question is which barriers of the highly protected epithelia must the virus overcome to reach its receptor nectin-1. Here, we used human epidermal equivalents to understand how physical barrier formation and nectin-1 distribution contribute to successful viral invasion. Inflammation-induced barrier defects led to facilitated viral penetration strengthening the role of functional tight-junctions in hindering viral access to nectin-1 that is localized just underneath tight junctions and distributed throughout all layers. We also found nectin-1 ubiquitously localized in the epidermis of atopic dermatitis and IL-4/IL-13-treated human skin implying that impaired tight-junctions in combination with a defective cornified layer allow the accessibility of nectin-1 to HSV-1. Our results support that successful invasion of HSV-1 in human skin relies on defective epidermal barriers, which not only include a dysfunctional cornified layer but also depend on impaired tight junctions.

Chronic skin damage induces small intestinal damage via IL-13-induced apoptosis.

The gut-skin axis has recently been widely recognized, and both the gut and skin have been found to affect each other through a bidirectional connection; however, the precise mechanisms remain to be elucidated. Therefore, we aimed to investigate the effects of chronic skin damage (CSD) on mouse intestines. Following the CSD model, 4% sodium dodecyl sulfate was applied to the back-shaved murine skin six times for 2 weeks after tape stripping. The small and large intestines were analyzed histologically and immunologically, respectively. Intestinal permeability was measured using fluorescein isothiocyanate-conjugated-dextran. The role of interleukin-13 (IL-13) in the ileum was investigated using an anti-IL-13 antibody. Apoptotic intestinal cells were analyzed using TUNEL staining. Villus atrophy was observed in the small intestine in the CSD model, along with increased permeability. Mast cells, but not T cells, eosinophils, or innate lymph cell-2, were increased in the intestinal mucosa. However, no significant changes were observed in the large intestine. mRNA expression of IL-13 was increased only in the ileum of the CSD model. Apoptotic intestinal epithelial cells were significantly increased in the ileum of the CSD model. Administration of an anti-IL-13 antibody ameliorated the intestinal damage caused by CSD, along with decreased apoptotic cells and mast cell infiltration. Skin damage causes morphological changes in the small intestine, accompanied by increased intestinal permeability, possibly through the IL-13-induced apoptosis of mast cells in the epithelium. Surfactant-mediated mechanical skin damage can cause a leaky gut.

IL-4 and IL-13 are not involved in IL-31-induced itch-associated scratching behaviour in mice.

Itchy skin or pruritus is a common cutaneous symptom that causes an urge to scratch, and the role of interleukins (IL) in itchy skin has been widely studied. IL-4 and IL-13 are known to induce chronic itch. Similarly, the direct role of IL-31 in inducing itch has been demonstrated in clinical situations such as atopic dermatitis and prurigo nodularis. Moreover, IL-4 receptor α antibodies (dupilumab) and IL-31 receptor A antibodies (nemolizumab) inhibit pruritus. However, the interplay between these ILs in pruritus remains unclear. Therefore, we investigated the reciprocal effects of these cytokines on pruritus in mice. The intradermal administration of IL-31 induced itch-associated scratching behaviour in a dose-dependent manner. Interestingly, the amount of IL-31 and IL-4/IL-13, co-administration or 30 min pre-administration of IL-4/IL-13 and intradermal or intravenous pre-administration of IL-4 did not affect IL-31-induced itch-associated scratching behaviour when it was observed for 30 min, 2 h, 24 h or 48 h. Pre-administration of neutralising antibodies against IL-4 and IL-13 also did not affect IL-31-induced itch-associated scratching behaviour. These results suggest that IL-31 can induce itching independently of IL-4 and IL-13 in vivo.

The stimulation of TH2 cells results in increased IL-5 and IL-13 production via the H4 receptor.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease resulting in decreased quality of life. Histamine and specifically the H4 receptor play a key role in the inflammatory process in AD and serve as targets for novel therapeutic approaches. OBJECTIVE: In the present study we aimed to elucidate the immunopathological mechanisms with which the H4 receptor impacts TH2 cells and contributes to AD pathophysiology. METHODS: Total CD4+ T cells obtained from healthy or AD individuals and in vitro differentiated TH2 cells were cultured under different conditions and the mRNA expression or protein production of target molecules were determined using quantitative real-time PCR and ELISA. RESULTS: H4 receptor mRNA expression was upregulated concentration dependent upon IL-4 stimulation in in vitro differentiated TH2 cells progressively during the differentiation. Transcriptomic analysis of in vitro differentiated TH2 versus TH1 cells revealed that the H4 receptor among other genes represents one of the highly upregulated genes in TH2 cells. Most importantly, increased amounts of IL-5 and IL-13 mRNA expression were detected in in vitro differentiated TH2 cells as well as protein secretion in the presence of histamine or of the H4 receptor-selective-agonist when compared to the untreated control. CONCLUSION: We show for the first time an H4 receptor dependent upregulation of the pro-inflammatory cytokines IL-5 and IL-13 in human TH2 cells by histamine. This suggests that the blockade of the H4 receptor may lead to downregulation of these cytokines and amelioration of AD symptoms as reported in first clinical studies.

NOTCH4 potentiates the IL-13 induced genetic program in M2 alternative macrophages through the AP1 and IRF4-JMJD3 axis.

IL-13 signaling polarizes macrophages to an M2 alternatively activated phenotype, which regulates tissue repair and anti-inflammatory responses. However, an excessive activation of this pathway leads to severe pathologies, such as allergic airway inflammation and asthma. In this work, we identified NOTCH4 receptor as an important modulator of M2 macrophage activation. We show that the expression of NOTCH4 is induced by IL-13, mediated by Janus kinases and AP1 activity, probably mediated by the IL-13Rα1 and IL-13Rα2 signaling pathway. Furthermore, we demonstrate an important role for NOTCH4 signaling in the IL-13 induced gene expression program in macrophages, including various genes that contribute to pathogenesis of the airways in asthma, such as ARG1, YM1, CCL24, IL-10, or CD-163. We also demonstrate that NOTCH4 signaling modulates IL-13-induced gene expression by increasing IRF4 activity, mediated, at least in part, by the expression of the histone H3K27me3 demethylase JMJD3, and by increasing AP1-dependent transcription. In summary, our results provide evidence for an important role of NOTCH4 signaling in alternative activation of macrophages by IL-13 and suggest that NOTCH4 may contribute to the increased severity of lesions in M2 inflammatory responses, such as allergic asthma, which points to NOTCH4 as a potential new target for the treatment of these pathologies.

Foxp3 and Bcl6 deficiency synergistically induces spontaneous development of atopic dermatitis-like skin disease.

Atopic dermatitis (AD) is a common chronic skin disease caused by immune dysfunction, specifically the hyperactivation of Th2 immunity. AD is a complex disease with multiple factors contributing to its development; however, the interaction between these factors is not fully understood. In this study, we demonstrated that the conditional deletion of both the forkhead box p3 (Foxp3) and B-cell lymphoma 6 (Bcl6) genes induced the spontaneous development of AD-like skin inflammation with hyperactivation of type 2 immunity, skin barrier dysfunction, and pruritus, which were not induced by the single deletion of each gene. Furthermore, the development of AD-like skin inflammation was largely dependent on IL-4/13 signaling but not on immunoglobulin E (IgE). Interestingly, we found that the loss of Bcl6 alone increased the expression of thymic stromal lymphopoietin (TSLP) and interleukin (IL)-33 in the skin, suggesting that Bcl6 controls Th2 responses by suppressing TSLP and IL-33 expression in epithelial cells. Our results suggest that Foxp3 and Bcl6 cooperatively suppress the pathogenesis of AD. Furthermore, these results revealed an unexpected role of Bcl6 in suppressing Th2 responses in the skin.

STAT6 suppression prevents bleomycin-induced dermal fibrosis.

Fibrosis of the skin and internal organs is a hallmark of systemic sclerosis (SSc). Although the pathogenesis of SSc is poorly understood, increasing evidence suggests that interleukins (IL)-4 and - 13 contribute to the pathogenesis of skin fibrosis by promoting collagen production and myofibroblast differentiation. Signal transducers and activators of transcription 6 (STAT6) is one of the most important downstream transcription factors activated by both IL-4 and IL-13. However, it is not completely understood whether STAT6 plays a role during the pathogenesis of skin fibrosis in SSc. In this study, we observed increased STAT6 phosphorylation in fibrotic skin samples collected from SSc patients as well as bleomycin-injected murine mice. Knockout of Stat6 in mice significantly (1) suppressed the expression of fibrotic cytokines including Il13, Il17, Il22, Ccl2, and the alternatively activated macrophage marker Cd206; (2) reduced the production of collagen and fibronectin, and (3) attenuated late-stage skin fibrosis and inflammation induced by bleomycin. Consistently, mice treated with STAT6 inhibitor AS1517499 also attenuated skin fibrosis on day 28. In addition, a co-culture experiment demonstrated that skin epithelial cells with STAT6 knockdown had reduced cytokine expression in response to IL-4/IL-13, and subsequently attenuated fibrotic protein expression in skin fibroblasts. On the other side, STAT6 depletion in skin fibroblasts attenuated IL-4/IL-13-induced cytokine and fibrotic marker expression, and reduced CXCL2 expression in co-cultured keratinocytes. In summary, our study highlighted an important yet not fully understood role of STAT6 in skin fibrosis by driving innate inflammation and differentiation of alternatively activated macrophages in response to injury.

IL13Rα2 as a crucial receptor for Chi3l1 in osteoclast differentiation and bone resorption through the MAPK/AKT pathway.

BACKGROUND: Previous research has revealed that the 18 glycoside hydrolase gene family (GH18) member Chitinase 3-like 1 (Chi3l1) can regulate osteoclast differentiation and bone resorption. However, its downstream receptors and molecular mechanisms during osteoclastogenesis have yet to be elucidated. METHODS: Initially, we conducted a comprehensive investigation to evaluate the effects of recombinant Chi3l1 protein or Chi3l1 siRNA on osteoclast differentiation and the RANKL-induced MAPK/AKT signaling pathways. Moreover, we used immunofluorescence and immunoprecipitation assays to identify IL13Rα2 as the downstream receptor of Chi3l1. Subsequently, we investigated the impact of IL13Rα2 recombinant protein or IL13Rα2-siRNA on osteoclast differentiation and the associated signaling pathways. Finally, we performed in vivo experiments to examine the effect of recombinant IL13Rα2 protein in an LPS-induced mouse model of cranial osteolysis. RESULTS: Our findings highlight that the administration of recombinant Chi3l1 protein increased the formation of osteoclasts and bolstered the expression of several osteoclast-specific genes (TRAP, NFATC1, CTR, CTSK, V-ATPase d2, and Dc-STAMP). Additionally, Chi3l1 significantly promoted the RANKL-induced MAPK (ERK/P38/JNK) and AKT pathway activation, whereas Chi3l1 silencing inhibited this process. Next, using immunofluorescence and co-immunoprecipitation assays, we identified IL13Rα2 as the binding partner of Chi3l1 during osteoclastogenesis. IL13Rα2 recombinant protein or IL13Rα2-siRNA also inhibited osteoclast differentiation, and IL13Rα2-siRNA attenuated the RANKL-induced activation of the MAPK (ERK/P38/JNK) and AKT pathways, similar to the effects observed upon silencing of Chi3l1. Moreover, the promoting effect of recombinant Chi3l1 protein on osteoclastogenesis and the activation of the MAPK and AKT pathways was reversed by IL13Rα2 siRNA. Finally, recombinant LI13Rα2 protein significantly attenuated the LPS-induced cranial osteolysis and the number of osteoclasts in vivo. CONCLUSIONS: Our findings suggested that IL13Rα2 served as a crucial receptor for Chi3l1, enhancing RANKL-induced MAPK and AKT activation to promote osteoclast differentiation. These findings provide valuable insights into the molecular mechanisms of Chi3l1 in osteoclastogenesis, with potential therapeutic implications for osteoclast-related diseases. Video Abstract.

Analysis of intracellular communication reveals consistent gene changes associated with early-stage acne skin.

A comprehensive understanding of the intricate cellular and molecular changes governing the complex interactions between cells within acne lesions is currently lacking. Herein, we analyzed early papules from six subjects with active acne vulgaris, utilizing single-cell and high-resolution spatial RNA sequencing. We observed significant changes in signaling pathways across seven different cell types when comparing lesional skin samples (LSS) to healthy skin samples (HSS). Using CellChat, we constructed an atlas of signaling pathways for the HSS, identifying key signal distributions and cell-specific genes within individual clusters. Further, our comparative analysis revealed changes in 49 signaling pathways across all cell clusters in the LSS- 4 exhibited decreased activity, whereas 45 were upregulated, suggesting that acne significantly alters cellular dynamics. We identified ten molecules, including GRN, IL-13RA1 and SDC1 that were consistently altered in all donors. Subsequently, we focused on the function of GRN and IL-13RA1 in TREM2 macrophages and keratinocytes as these cells participate in inflammation and hyperkeratinization in the early stages of acne development. We evaluated their function in TREM2 macrophages and the HaCaT cell line. We found that GRN increased the expression of proinflammatory cytokines and chemokines, including IL-18, CCL5, and CXCL2 in TREM2 macrophages. Additionally, the activation of IL-13RA1 by IL-13 in HaCaT cells promoted the dysregulation of genes associated with hyperkeratinization, including KRT17, KRT16, and FLG. These findings suggest that modulating the GRN-SORT1 and IL-13-IL-13RA1 signaling pathways could be a promising approach for developing new acne treatments.