Cytokines TSLP Research Articles

Anti-Inflammatory Effects of Cannabigerol In Vitro and In Vivo Are Mediated Through the JAK/STAT/NFκB Signaling Pathway

Cannabinoid compounds have potential as treatments for a variety of conditions, with cannabigerol (CBG) being known for its anti-inflammatory properties. In this study, we investigated the effects of CBG in a cellular model of 1-chloro-2,4-dinitrobenzene (DNCB)-induced atopic dermatitis (AD). In the cellular model, we confirmed the cytotoxicity of CBG and downregulated the expression of inflammatory markers CCL26, IL1B, IL6, and TNF (p < 0.001). In the mouse model, clinical, histological, and immunological changes were analyzed. The results showed that CBG improved dermatitis severity score, epidermal thickness, and mast cell count and reduced inflammatory cytokines (Tslp, Il1b, Il4, Il6, Il13, Il17, Il18, Il22, and Il33) by qRT-PCR (p < 0.001). Western blot results showed modulated changes in JAK1, JAK2, TYK2, STAT1, STAT2, STAT3, p-STAT3, STAT6, and p-STAT6 (p < 0.05). Subsequently, p-IκBα, NF-κB, and p-NF-κB signaling factors were also reduced (p < 0.05)., with corresponding changes in skin barrier factors. The results of this study indicate that CBG effectively alleviates AD-like symptoms and suggest the potential of CBG as a therapeutic agent.

Synthetic small interfering RNAs selectively suppress the expression of proinflammatory cytokine genes (IL-25 and TSLP) in experiments in vitro

Allergic rhinitis (AR) is an inflammatory disease of the upper respiratory tract (nasal mucosa). AR affects up to 40% of the world’s population; in the Russian Federation, the incidence is 18% to 30%, depending on the region. Despite the fact that AR is not a severe pathology, it causes significant economic burden. Another threat associated with this disease is that in 40% of cases, patients with AR eventually develop a more severe disabling pathology – AD. Widespread prevalence and significant economic disadvantages caused by AR determine the importance of developing new ways of prevention and control of this disease, as the existing methods of therapy are insufficient. However, the search for new ways of therapy is impossible without a detailed investigation of the molecular mechanisms of AR pathogenesis. For a long time it was considered that this allergic inflammation is formed by Th2-dependent mechanism with involvement of Th2-lymphocytes, B-cells and eosinophils and pro-inflammatory cytokines: IL-4, IL-5 and IL-13. However, experimental evidence has now accumulated on the role of epithelial cells of the respiratory tract and the proinflammatory cytokines they secrete (IL-25, IL-33 and TSLP) in the pathogenesis of AR and AD. IL-25 has been shown to induce the production of IL-4, IL-5 and IL-13, directing a Th2-type immune response. At the same time, mice with inactivated IL-25 developed barely any Th2-immune response. Inactivation of IL-33 significantly reduces inflammation (mediated by eosinophils) of the respiratory tract. Mice knockout for the cytokine receptor TSLP did not develop nasal hyperreactivity in response to allergen, but the level of nasal mucosal inflammation remained high. Currently, work is actively progressing on the development of new drugs capable of specifically blocking the activity of the listed cytokines; first of all, drugs based on neutralizing monoclonal antibodies. However, there are other technologies that can be used to regulate the activity of genes, such as the technology based on the RNA interference. It can be used to suppress the expression of any gene with a known nucleotide sequence, including genes encoding pro-inflammatory cytokines.Considering the above, the aim of this work was to design synthetic miRNA molecules and study their ability to specifically block the expression of genes encoding proinflammatory cytokines IL-25 and TSLP in experiments in vitro.

Schizonepeta tenuifolia Briq-Saposhnikovia divaricata decoction alleviates atopic dermatitis via downregulating macrophage TRPV1.

Schizonepeta tenuifolia -Saposhnikovia divaricata (Jingjie-Fangfeng, JF) has been used for years to treat allergic inflammatory skin diseases like atopic dermatitis, but the specific effects and mechanisms of JF are still unclear. We aim to investigate the therapeutic effect and mechanism of JF in MC903-induced atopic dermatitis-like model. JF decoction was subjected to rigorous HPLC and GC analysis. The JF decoction was then freshly prepared and administered to MC903-induced atopic dermatitis -like mice models to investigate its therapeutic effects. Our evaluation focused on several markers of inflammation including the TEWL index, ear thickness, swelling, and specific inflammation indicators such as TSLP, IL33, IgE, and immune cell presence at the lesion sites. We measured Transient Receptor Potential Vanilloid 1 (TRPV1) expression levels through immunofluorescent staining in skin tissue from both atopic dermatitis patients and the MC903-treated mice. Furthermore, TRPV1 expression and macrophage activation markers were measured in LPS/IFN-γ-stimulated Raw264.7 and THP-1 cell models in vitro. Additionally, we developed cell lines that overexpress TRPV1 and investigated how JF treatment affects NF-κB p65 phosphorylation in these cells to understand better the role of TRPV1 in atopic dermatitis. The JF decoction met the standards outlined in the Chinese pharmacopeia. The JF decoction significantly alleviated inflammatory skin symptoms and helped restore skin barrier function. Additionally, it reduced the levels of IgE and pro-inflammatory cytokines TSLP, IL-33, and IL-4. There was also a noticeable decrease in mast cell infiltration and degranulation. Notably, JF decoction reduced infiltrated macrophages with limited affection on T cell infiltration. It also decreased F4/80+/TRPV1+ cells in atopic dermatitis mice and TRPV1 expression in LPS/IFNγ-stimulated microphages. Additionally, we observed that CD68+/TRPV1+ cells increased in human atopic dermatitis tissue. Further studies showed that JF water extract (JF-WE) suppressed TRPV1 expression in macrophages, potentially by affecting NF-κB p65 phosphorylation rather than the JAK-STAT6 pathway. This study offers initial evidence of the effectiveness of JF-WE in suppressing inflammation in atopic dermatitis. The therapeutic effect might stems from its ability to downregulate TRPV1 expression and subsequent NF-κB p65 phosphorylation in macrophages.

Skin Care Function of Lactoferrin Was Characterized Using Recombinant Human Epidermal Model

The effect of lactoferrin on skin was simulated using a recombinant human epidermal model. The anti-inflammatory and soothing effect of lactoferrin was verified using IL-1α and TSLP Elisa assay. The effects of lactoferrin on the expression of related genes and proteins were detected using qPCR and immunofluorescence staining. The results showed that lactoferrin can effectively enhance the Transepidermal Electrical Resistance (TEER) and inhibit the secretion of inflammatory cytokine IL-1α and TSLP. In addition, it was confirmed using qPCR that lactoferrin had high expression levels on AQP3, FLG, IVL, CLDN1 and HAS1 genes. Immunofluorescence staining confirmed that lactoferrin had high fluorescence intensity and expression in AQP3, Filaggrin and Involucrin. The results showed that lactoferrin improved the skin barrier at higher than 1.5 mg/mL. At the same time, it can have anti-inflammatory and moisturizing effects. This study provides a strong basis for the application of lactoferrin in cosmetics and daily chemical products.

Immune stimulus exposure as a trigger for the development of chronic pruritus and circulating blood type 2 inflammation

BackgroundChronic pruritus (CP) is a poorly characterized condition associated with intense pruritus without a primary skin eruption. This condition tends to emerge more commonly in older adults, and there is limited research on triggering factors. ObjectiveTo explore the clinical characteristics and pathophysiology of chronic pruritus following exposure to an immune stimulus. MethodsClinical characteristics and plasma samples were collected from 15 patients who developed CP following an immune stimulus such as checkpoint inhibitors or vaccination. A multiplex panel was used to analyze plasma cytokine concentrations within these patients. ResultsMost immunotherapy-treated patients experienced CP during treatment or after 21 to 60 days of receiving treatment, while vaccine-stimulated patients developed pruritus within a week of vaccination. Plasma cytokine analysis revealed elevated levels of twelve cytokines in patients with immune-stimulated CP compared to healthy controls. Notably, Th2-related cytokines IL-5 (FC 2.65; q<0.25) and TSLP (FC 1.61 q<0.25) were upregulated. LimitationsLimitations of this study include limited sample size, particularly in the plasma cytokine assay. Conclusions and RelevanceThis study reveals triggers of CP development and describes alterations in blood Th2 markers in CP patients, including IgE, increased blood eosinophils, and cytokines IL-5 and TSLP.

Potential of Bioactive Compounds for Atopic Dermatitis

Atopic Dermatitis (AD) is a complicated condition that places tremendous physiological and psychological strain on individuals. Natural products have long been used to cure diseases such as cancer, asthma, gastrointestinal disorders, neurological disorders, and infections. The study findings reveal that natural compounds, particularly quercetin, gallic acid, and ginsenosides, have promised preventive effects against atopic dermatitis. The study addresses the medicinal properties of bioactive compounds and emphasizes their ability to exert anti-inflammatory action. These compounds exhibit anti-inflammatory properties by reducing the quantity and functionality of various inflammatory cells such as cytokines neutrophils, monocytes, lymphocytes, Langerhans cells, interleukins (ILs, such as IL-1 IL-5, and IL-4, IL-13, and IL-31), TNF-α, TSLP, and IgE, etc. The studies would pave the way for the development of natural compounds specifically designed to treat atopic dermatitis in humans. Atopic dermatitis is routinely treated using bioactive and phytoconstituents derived from them. As a result, the review emphasizes recent advances in understanding the clinical characteristics, etiology, pathogenesis, treatment with bioactive compounds, and management of atopic dermatitis.

Role of G-protein-coupled estrogen receptor in the pathogenesis of chronic asthma

Background and AimG protein-coupled estrogen receptor (GPER) is an estrogen receptor located on the plasma membrane. We previously reported that the administration of G-1, a GPER-specific agonist, suppressed development of acute ovalbumin (OVA)-induced asthma in a mouse model. Herein, we evaluate the involvement of GPER in a mouse model of chronic OVA asthma. MethodsG-1 or saline was administered subcutaneously to BALB/c mice with chronic OVA asthma, and pathological and immunological evaluation was performed. In addition, Foxp3-expressing CD4-positive T-cells in the spleen and ILC2 in the lungs were measured using flow cytometry. ResultsWe observed a significant decrease in the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) in the G-1 treated group. In the airways, inflammatory cell accumulation, Th2 cytokines (IL-4, IL-5, IL-13, and eotaxin) and epithelial cytokine TSLP were suppressed, while in the BALF, anti-inflammatory cytokines (IL-10 and TGF-β) were increased. Furthermore, in splenic mononuclear cells, Foxp3-expressing CD4-positive T-cells were increased in the G-1 group, whereas treatment with G-1 did not change the percentage of ILC2 in the lungs. ConclusionG-1 administration suppressed allergic airway inflammation in mice with chronic OVA asthma. GPER may be a potential therapeutic target for chronic allergic asthma.

Anti-Atopic Dermatitis Effect of TPS240, a Novel Therapeutic Peptide, via Suppression of NF-κB and STAT3 Activation

Atopic dermatitis (AD) is a relapsing skin disease with persistent inflammation as a causal factor for symptoms and disease progression. Current therapies provide only temporary relief and require long-term usage accompanied by side effects due to persistent relapses. A short peptide, TPS240, has been tested for its potential to subside AD. In this study, we confirmed the anti-atopic effect of TPS240 in vivo and in vitro using a DNCB-induced AD mouse model and TNF-α/IFN-γ-stimulated HaCaT cells. In the AD mouse model, topical treatment with TPS240 diminished AD-like skin lesions and symptoms such as epidermal thickening and mast cell infiltration induced by DNCB, similar to the existing treatment, dexamethasone (Dex). Furthermore, skin atrophy, weight loss, and abnormal organ weight changes observed in the Dex-treated group were not detected in the TPS240-treated group. In TNF-α/IFN-γ-stimulated HaCaT cells, TPS240 reduced the expression of the inflammatory chemokines CCL17 and CCL22 and the pruritic cytokines TSLP and IL-31 by inhibiting NF-κB and STAT3 activation. These results suggest that TPS240 has an anti-atopic effect through immunomodulation of AD-specific cytokines and chemokines and can be used as a candidate drug for the prevention and treatment of AD that can solve the safety problems of existing treatments.

Regulation and function of adiponectin in the intestinal epithelial cells in response to Trichinella spiralis infection

Besides metabolic homeostasis regulation, adipokines are recently emerged as important players in regulating immunity and inflammation. Helminth infection has known to modulate circulating adipokine secretion; however, the regulation and function of adipokines in response to helminth infection is still unclear. Here, we investigated the regulation and function of adiponectin during T. spiralis infection. While there was no change in circulating level of adiponectin, we found an increased adiponectin, but not leptin expression in the small intestine. Interestingly, the intestinal adiponectin expression was strongly associated with the expression of epithelial cell-derived cytokines IL-25, IL-33, and TSLP following infection. Indeed, mice deficiency of IL-25 receptor exhibited no intestinal adiponectin induction upon helminth infection. Interestingly, IL-25-induced adiponectin modulated intestinal epithelial cell responses by enhancing occludin and CCL17 expression. Using LPS-induced intestinal epithelial barrier dysfunctions in a Caco-2 cell monolayer model, adiponectin pretreatment enhanced a Transepithelial electrical resistance (TEER) and occludin expression. More importantly, adiponectin pretreatment of Caco2 cells prevented T. spiralis larval invasion in vitro and its administration during infection enhanced intestinal IL-13 secretion and worm expulsion in vivo. Altogether, our data suggest that intestinal adiponectin expression induced by helminth infection through the regulation of IL-25 promotes worm clearance and intestinal barrier function.

In vitro and in vivo anti-eczema effect of Artemisia annua aqueous extract and its component profiling

Ethnopharmacological relevanceArtemisia annua L. belongs to the Asteraceae family and has a long history of clinical application in China. It has been widely used for centuries to treat fever, malaria, jaundice and some skin diseases (such as scabies and sores). Modern pharmacological studies have shown that it has anti-inflammatory, immunomodulatory, antimalarial and antibacterial effects. Aim of studyThis study aimed to investigate the anti-eczema effect of A. annua aqueous extract (AAE), profile its potential bioactive components and try to explore its possible underlying mechanisms. Materials and methodsThe MTT assay was employed to assess the cytotoxicity of AAE. The anti-eczema effect of AAE was evaluated using both an in vitro 3D epidermal inflammation model and an in vivo guinea pig itching model. The bioactive components of AAE were characterized by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry coupled with the UNIFI platform. ResultsIn this study, we found that AAE is safe for primary human skin keratinocytes at concentrations ranging from 31.3 μg/mL to 250 μg/mL. Further investigations indicate that AAE can increase the itching threshold, inhibit the expression of the inflammatory cytokine TSLP, and promote the expression of FLG mRNA. Additionally, the utilization of UPLC-QTOF/MS and UNIFI platform enabled us to identify 61 potential bioactive components of AAE, with sesquiterpenes and phenolic acids being the most abundant components. ConclusionsIn this study, the anti-inflammatory and anti-itch effects of the A. annua extract were revealed, along with sesquiterpenes and phenolic acids were identified as potential bioactive components according to literature. The AAE extract holds potential for utilization in the treatment of eczema.

Topical Calcipotriol Plus Imiquimod Immunotherapy for Nonkeratinocyte Skin Cancers

Nonkeratinocyte cutaneous malignancies, including breast cancer cutaneous metastasis and melanoma in situ, are often poor surgical candidates. Imiquimod (IMQ), a toll-like receptor 7 agonist that activates innate immunity in the skin, is used to treat these cutaneous malignancies. However, IMQ's modest effect on the activation of adaptive immunity limits its efficacy as a monotherapy. In this study, we demonstrate that topical TSLP cytokine inducers-calcipotriol and retinoic acid-synergize with IMQ to activate CD4+ T-cell immunity against nonkeratinocyte cutaneous malignancies. Topical calcipotriol plus IMQ treatment reduced breast tumor growth compared with calcipotriol or IMQ alone (P < 0.0001). Calcipotriol plus IMQ-mediated tumor suppression was associated with significant infiltration of CD4+ effector T cells in the tumor microenvironment. Notably, topical calcipotriol plus IMQ immunotherapy enabled immune checkpoint blockade therapy to effectively control immunologically cold breast tumors, which was associated with induction of CD4+ T-cell immunity. Topical treatment with calcipotriol plus IMQ and retinoic acid plus IMQ also blocked subcutaneous melanoma growth. These findings highlight the synergistic effect of topical TSLP induction in combination with innate immune cell activation as an effective immunotherapy for malignancies affecting the skin.

M2 Macrophages promote IL-33 expression, ILC2 expansion and mucous metaplasia in response to early life rhinovirus infections.

Wheezing-associated rhinovirus (RV) infections are associated with asthma development. We have shown that infection of immature mice with RV induces type 2 cytokine production and mucous metaplasia which is dependent on IL-33 and type 2 innate lymphoid cells (ILC2s) and intensified by a second heterologous RV infection. We hypothesize that M2a macrophages are required for the exaggerated inflammation and mucous metaplasia in response to heterologous RV infection. Wild-type C57Bl/6J mice and LysMCre IL4Rα KO mice lacking M2a macrophages were treated as follows: (1) sham infection on day 6 of life plus sham on day 13 of life, (2) RV-A1B on day 6 plus sham on day 13, (3) sham on day 6 and RV-A2 on day 13, or (4) RV-A1B on day 6 and RV-A2 on day 13. Lungs were harvested one or seven days after the second infection. Wild-type mice infected with RV-A1B at day 6 showed an increased number of Arg1- and Retnla-expressing lung macrophages, indicative of M2a polarization. Compared to wild-type mice infected with RV on day 6 and 13 of life, the lungs of LysMCre IL4Rα KO mice undergoing heterologous RV infection showed decreased protein abundance of the epithelial-derived innate cytokines IL-33, IL-25 and TSLP, decreased ILC2s, decreased mRNA expression of IL-13 and IL-5, and decreased PAS staining. Finally, mRNA analysis and immunofluorescence microscopy of double-infected LysMCre IL4Rα KO mice showed reduced airway epithelial cell IL-33 expression, and treatment with IL-33 restored the exaggerated muco-inflammatory phenotype.ConclusionEarly-life RV infection alters the macrophage response to subsequent heterologous infection, permitting enhanced IL-33 expression, ILC2 expansion and intensified airway inflammation and mucous metaplasia.

High plasma levels of the TSLP cytokine in Saudi patients with chronic stable asthma

Allergic asthma is an inflammatory bronchial disease associated with the IgE response, allergic inflammation of the airways, recruitment of infiltrated inflammatory cells, increased mucus production, constriction of airways, and difficulty breathing. TSLP cytokine is derived mainly from airway epithelial cells and have been shown to play an essential role in the pathogenesis of asthma. This study was designed to determine the plasma protein concentrations of total IgE and TSLP cytokines in patients with chronic stable asthma. Furthermore, we examined TSLP-r in B cells and determined whether the expression of TSLP is correlated with increased total IgE. Thirty-one patients with chronic stable asthma and nineteen normal controls were enrolled in the study. Blood samples were separated using the Ficoll gradient method, and plasma and lymphocyte cells were collected. Plasma levels of total IgE and TSLP were quantified in patients with asthma and normal controls by specific ELISAs. Moreover, the surface expression of TSLP-r on B cells was analyzed using flow cytometry. Total IgE protein concentrations in the plasma of patients with chronic stable asthma (344 IU/ml, P < 0.002) were considerably higher than those in normal controls (mean 130 IU/ml). TSLP levels were significantly higher in the asthma group (78 Pg/ml, P < 0.02) than those in the normal control group (40 pg/ml). Additionally, the expression of TSLP-r was markedly higher in asthma patients (430 × 103 MFI, P < 0.0065) than in normal controls (280 × 103). Collectively, the findings indicate that measuring plasma levels of TSLP in patients with stable asthma can be used to assess asthma symptoms and possibly evaluate the efficacy of corticosteroid therapy.

Biomarkers in asthma in the context of atopic dermatitis in young children.

BackgroundDiverse pathways stemming from a history of atopic dermatitis (AD) might modulate different biomarkers associated with the development of asthma. Biomarkers associated with AD and asthma separately have been investigated, but none have characterized a combined AD+asthma phenotype. We investigated the clinical and molecular characteristics associated with an AD+asthma phenotype compared with AD, asthma and controls.MethodsFrom a prospective birth cohort and the outpatient allergy clinic, we included four groups of 6–12‐year‐old children: (1) healthy controls (2) previous, current, or present AD without asthma, (3) previous, current, or present AD and current asthma and (4) current asthma without AD. We performed clinical examinations and interviews and measured serum IgE, natural moisturizing factors (NMF), and plasma cytokine levels.ResultsWe found an increased number of IgE sensitizations in AD+asthma, prominent after stratifying for food allergens (p < .05). Pro‐Th2 cytokines CCL18, TSLP, and Eotaxin‐3 were elevated in AD+asthma, though not significantly higher than asthma, and elevated in asthma compared with controls. NMF levels were decreased in AD compared with asthma and control groups (p = .019, p < .001, respectively). NMF levels correlated negatively to sensitization (p = .026), though nonsignificant with only the patient groups.ConclusionOur results indicate that Th2 cytokines and increased number of sensitizations are associated with AD + asthma phenotypes compared with AD alone and that skin barrier impairment as well as decreased airway epithelial integrity may play a role in sensitization and immune modulation. Our findings suggest candidate biomarkers that should be further explored for their functional roles and prognostic potential.

Therapeutic Effect of Renifolin F on Airway Allergy in an Ovalbumin-Induced Asthma Mouse Model In Vivo.

Renifolin F is a prenylated chalcone isolated from Shuteria involucrata, a traditional minority ethnic medicine used to treat the respiratory diseases and asthma. Based on the effects of the original medicine plant, we established an in vivo mouse model of allergic asthma using ovalbumin (OVA) as an inducer to evaluate the therapeutic effects of Renifolin F. In the research, mice were sensitized and challenged with OVA to establish an allergic asthma model to evaluate the effects of Renifolin F on allergic asthma. The airway hyper-reactivity (AHR) to methacholine, cytokine levels, ILC2s quantity and mircoRNA-155 expression were assessed. We discovered that Renifolin F attenuated AHR and airway inflammation in the OVA-induced asthmatic mouse model by inhibiting the regulation of ILC2s in the lung, thereby, reducing the upstream inflammatory cytokines IL-25, IL-33 and TSLP; the downstream inflammatory cytokines IL-4, IL-5, IL-9 and IL-13 of ILC2s; and the co-stimulatory factors IL-2 and IL-7; as well as the expression of microRNA-155 in the lung. The findings suggest a therapeutic potential of Renifolin F on OVA-induced airway inflammation.

The Role of Airway Epithelial Cell Alarmins in Asthma.

The airway epithelium is the first line of defense for the lungs, detecting inhaled environmental threats through pattern recognition receptors expressed transmembrane or intracellularly. Activation of pattern recognition receptors triggers the release of alarmin cytokines IL-25, IL-33, and TSLP. These alarmins are important mediators of inflammation, with receptors widely expressed in structural cells as well as innate and adaptive immune cells. Many of the key effector cells in the allergic cascade also produce alarmins, thereby contributing to the airways disease by driving downstream type 2 inflammatory processes. Randomized controlled clinical trials have demonstrated benefit when blockade of TSLP and IL-33 were added to standard of care medications, suggesting these are important new targets for treatment of asthma. With genome-wide association studies demonstrating associations between single-nucleotide polymorphisms of the TSLP and IL-33 gene and risk of asthma, it will be important to understand which subsets of asthma patients will benefit most from anti-alarmin therapy.

Preclinical Testing of Dendritic Core-Multishell Nanoparticles in Inflammatory Skin Equivalents.

Human skin equivalents emerged as novel tools in preclinical dermatological research. It is being claimed that they may bridge the translational gap between preclinical and clinical research, yet only a few studies have investigated their suitability for preclinical drug testing so far. Therefore, we investigated if inflammatory skin equivalents, which emulate hallmarks of atopic dermatitis (AD), are suitable to assess the anti-inflammatory effects of dexamethasone (DXM) in a cream formulation or loaded onto dendritic core-multishell nanoparticles. Topical DXM application resulted in significantly decreased expression of the proinflammatory cytokine TSLP, increased expression of the skin barrier protein involucrin, and facilitated glucocorticoid receptor translocation in a dose-dependent manner. Further, DXM treatment inhibited gene expression of extracellular matrix components, potentially indicative of the known skin atrophy-inducing side effects of glucocorticoids. Overall, we were able to successfully assess the anti-inflammatory effects of DXM and the superiority of the nanoparticle formulation. Nevertheless the identification of robust readout parameters proved challenging and requires careful study design.

The Pathogenesis of Eosinophilic Asthma: A Positive Feedback Mechanism That Promotes Th2 Immune Response via Filaggrin Deficiency.

Eosinophilic asthma (EA) is a common subtype of asthma and often progresses to severe disease. In order to understand its pathogenesis, targeted next-generation gene sequencing was performed on 77 Chinese EA patients and 431 Chinese healthy controls to obtain differential genomic variations. Among the 41 Single Nucleotide Polymorphisms (SNPs) screened for mutation sites in more than 3 patients, filaggrin gene FLG rs192116923 T>G and FLG rs75235053 C>G were newly found to be associated with EA patients with atopic dermatitis (AD) (P <0.001) and severe EA (P=0.032), respectively. Filaggrin has been shown to be mainly expressed in epithelial cells and plays an important role in formation of an effective skin barrier. Bioinformatic analysis indicated FLG rs192116923 T>G may increase the binding of Smad3 to transmit TGF-β1 signaling, and thereby inhibit filaggrin expression, and FLG rs75235053 C>G may add new splicing sites to reduce filaggrin monomers. It has been known that the level of Th2 cytokine IL-4 is increased in EA patients, and IL-4 increases airway epithelial permeability and enhances inflammatory response through some unclear mechanisms. To figure out whether filaggrin is involved in immune responses in asthma, we have treated human respiratory epithelial cell line BEAS-2B cells with IL-4 and found that the expression levels of filaggrin and E-cadherin decreased significantly in a time and dose-dependent manner, suggesting that IL-4 increased airway epithelial permeability by reducing filaggrin and adhesion molecule. In addition, in our study, IL-4 increased the expression of epithel-derived inflammatory cytokines IL-33 and TSLP which further enhanced the Th2 inflammatory response. To investigate the role of filaggrin in development of EA, knockdown filaggrin with siRNA revealed a decrease in E-cadherin levels, which were further down-regulated by IL-4 stimulation. Knockdown of filaggrin alone did not affect the levels of IL-33 and TSLP, but further exacerbated the decrease of IL-33/TSLP caused by IL-4, suggesting that filaggrin may involve in IL-4R signaling pathway to regulate the level of IL-33/TSLP. In conclusion, in the Th2 cytokine milieu of asthma, FLG deficient mutation in airway epithelial cells may increase the epithelial permeability and the expression of IL-33/TSLP which positively feedback the Th2 inflammation response.

Biologic for the Treatment of Ph-like B-Cell Acute Lymphoblastic Leukemia with Overexpression of CRLF2

Ph-like B-cell acute lymphoblastic leukemia (B-ALL) is associated with poor outcomes and high relapse rates. Approximately 50% of Ph-like B-ALL cases occur due to genetic alterations leading to overexpression of the cytokine receptor, CRLF2. B-ALL with overexpression of CRLF2 (CRLF2 B-ALL) occurs 5 times more often in Hispanic children than others, is prevalent in adolescents and young adults, and makes up ~1/3 of adult B-ALL. CRLF2, together with the IL-7Rα, forms a receptor complex that can be activated by circulating TSLP cytokine in patients. The human CRLF2 receptor is not activated by mouse TSLP so classic patient-derived xenografts (PDX) do not model TSLP-induced CRLF2 signaling that occurs in patients. CRLF2-mediated JAK/STAT and PI3/AKT/mTOR signals are believed to contribute to survival and proliferation of leukemia cells. We developed a novel PDX model of CRLF2 B-ALL that allows us to vary circulating levels of human TSLP (+T PDX). Primary CRLF2 B-ALL cells injected into +T PDX with circulating human TSLP (hTSLP) levels similar to pediatric leukemia patients (~4-10 pg/ml), engrafted well and showed a gene expression pattern that was more similar to the original patient sample than when injected into classic PDX. To our surprise, when +T PDX expressed physiological, but elevated levels of hTSLP (> 40 pg/ml, upper end of range reported in healthy children), CRLF2 B-ALL cells were essentially eliminated, but grew robustly in PDX without hTSLP (-T PDX). These results have been observed in 4 independent experiments for a total of 17 +T PDX and 12 -T PDX mice produced using CRLF2 B-ALL cells from two different Hispanic pediatric patients with CRLF2 B-ALL. We hypothesize that anti-leukemia effects observed at hTSLP concentrations above 40 pg/ml are mediated via TSLP-induced upregulation of the Suppressor of Cytokine Signaling (SOCS) genes. SOCS genes encode a family of proteins that regulate cytokine signaling via negative feedback through multiple mechanisms including ubiquitin-mediated degradation of JAKs and cytokine receptor components. Using the CRLF2 B-ALL cell lines, MUTZ5 and CALL-4, we found that SOCS family proteins encoded by the SOCS1 and SOCS3 genes were upregulated following culture with 15 ng/ml hTSLP, as compared to controls without hTSLP. Similarly, primary CRLF2 B-ALL cells from Hispanic pediatric patients cultured with TSLP showed upregulation of SOCS1 and SOCS3 mRNA and proteins. Next, we determined whether the upregulation of SOCS proteins was accompanied by a loss of CRLF2-mediated signals. We found that CRLF2 B-ALL cell lines and primary cells cultured with hTSLP lost the ability to upregulate STAT5 and S6 phosphorylation following hTSLP stimulation. Further, cells cultured with TSLP showed a loss of cytokine receptors as well as STAT5 phosphorylation levels that were below the baseline observed in leukemia cells never exposed to hTSLP. These data suggest that TSLP exerts anti-leukemia effects by shutting down CRLF2-mediated signals and that this shut down may occur through SOCS-mediated degradation of cytokine receptor components and/or degradation of the mutant JAK2 that provides constitutively low level STAT5 activation in these CRLF2 B-ALL cell lines and in a majority of patients with this disease. In summary, these results provide evidence that elevated hTSLP exerts a therapeutic effect on CRLF2 B-ALL and suggest that this occurs via SOCS-mediated suppression of the CRLF2 signaling pathway. These studies identify the human TSLP cytokine as a potential biologic therapy to treat CRLF2 B-ALL and reduce cancer health disparities for Hispanic children with CRLF2 B-ALL. Supported by 1R01CA209829. DisclosuresCoats:ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees. Dovat:ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees. Payne:ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees; ELF Zone Inc: Equity Ownership.

Bacteroides uniformis CECT 7771 alleviates inflammation within the gut-adipose tissue axis involving TLR5 signaling in obese mice

This study investigated the immune mechanisms whereby administration of Bacteroides uniformis CECT 7771 reduces metabolic dysfunction in obesity. C57BL/6 adult male mice were fed a standard diet or a Western diet high in fat and fructose, supplemented or not with B. uniformis CECT 7771 for 14 weeks. B. uniformis CECT 7771 reduced body weight gain, plasma cholesterol, triglyceride, glucose, and leptin levels; and improved oral glucose tolerance in obese mice. Moreover, B. uniformis CECT 7771 modulated the gut microbiota and immune alterations associated with obesity, increasing Tregs and reducing B cells, total macrophages and the M1/M2 ratio in both the gut and epididymal adipose tissue (EAT) of obese mice. B. uniformis CECT 7771 also increased the concentration of the anti-inflammatory cytokine IL-10 in the gut, EAT and peripheral blood, and protective cytokines TSLP and IL-33, involved in Treg induction and type 2 innate lymphoid cells activation, in the EAT. It also restored the obesity–reduced TLR5 expression in the ileum and EAT. The findings indicate that the administration of a human intestinal bacterium with immunoregulatory properties on the intestinal mucosa helps reverse the immuno-metabolic dysfunction caused by a Western diet acting over the gut-adipose tissue axis.