Thymic Stromal Lymphopoietin mRNA Research Articles

Endoplasmic reticulum stress enhances the expression of TLR3-induced TSLP by airway epithelium.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pleiotropic cytokine that regulates T-helper 2 (Th2) immune responses in the lung and plays a major role in severe uncontrolled asthma. Emerging evidence suggests a role for endoplasmic reticulum (ER) stress in the pathogenesis of asthma. In this study, we determined if ER stress and the unfolded protein response (UPR) signaling are involved in TSLP induction in the airway epithelium. For this, we treated human bronchial epithelial basal cells and differentiated primary bronchial epithelial cells with ER stress inducers and the TSLP mRNA and protein expression was determined. A series of siRNA gene knockdown experiments were conducted to determine the ER stress-induced TSLP signaling pathways. cDNA collected from asthmatic bronchial biopsies was used to determine the gene correlation between ER stress and TSLP. Our results show that ER stress signaling induces TSLP mRNA expression via the PERK-C/EBP homologous protein (CHOP) signaling pathway. AP-1 transcription factor is important in regulating this ER stress-induced TSLP mRNA induction, though ER stress alone cannot induce TSLP protein production. However, ER stress significantly enhances TLR3-induced TSLP protein secretion in the airway epithelium. TSLP and ER stress (PERK) mRNA expression positively correlates in bronchial biopsies from participants with asthma, particularly in neutrophilic asthma. In conclusion, these results suggest that ER stress primes TSLP that is then enhanced further upon TLR3 activation, which may induce severe asthma exacerbations. Targeting ER stress using pharmacological interventions may provide novel therapeutics for severe uncontrolled asthma.NEW & NOTEWORTHY TSLP is an epithelial-derived cytokine and a key regulator in the pathogenesis of severe uncontrolled asthma. We demonstrate a novel mechanism by which endoplasmic reticulum stress signaling upregulates airway epithelial TSLP mRNA expression via the PERK-CHOP signaling pathway and enhances TLR3-mediated TSLP protein secretion.

400 308-nm excimer lamp ameliorates MC-903 induced atopic dermatitis with a reduction of thymic stromal lymphopoietin mRNA levels

Purpose Narrow band-ultraviolet B (NB-UVB) is efficacious for atopic dermatitis (AD). However, its detailed mechanism remains to be elucidated. The purpose of this study is to explore an in vivo-effect of NB-UVB on AD, utilizing MC903-induced AD model mice. Method To induce AD-like dermatitis, MC903, a vitamin D3 analogue, was applied on the shaved dorsal skin of C57BL/6 mice once a day for twelve days. Mice were irradiated with a 308-nm excimer lamp at 100 mJ/cm2 once every other day from days 0 to 10.

Inhibition of thymic stromal lymphopoietin production by FK3453

To prevent the onset and aggravation of allergic diseases, it is necessary to modulate excessive Th2-type immune responses. It is well accepted that thymic stromal lymphopoietin (TSLP) plays important roles in the change of Th1/Th2 balance to Th2 dominance and would be a druggable target. In this study, using a drug repositioning strategy, we identified 6-(2-amino-4-phenylpyrimidine-5-yl)-2-isopropylpyridazin-3(2H)-one (FK3453) as a novel inhibitor of TSLP production. FK3453 inhibited constitutive production of TSLP in the KCMH-1 mouse keratinocyte cell line and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced one in PAM212 cells. FK3453 also inhibited TSLP mRNA expression induced by a mixture of tumor necrosis factor alpha (TNF-α), interleukin (IL)-4, fibroblast-stimulation lipopeptide-1, and protease activated-receptor agonist and TPA in normal human epidermal keratinocytes (NHEKs). Although FK3453 inhibited TPA-induced IL-33 expression in NHEKs in addition to TSLP, it did not inhibit TNF-α and IL-6 production. In addition, FK3453 did not inhibit MAP kinase (ERK) phosphorylation. We have confirmed that topical treatment with FK3453 inhibited TSLP production in the lipopolysaccharide-induced air pouch-type inflammation model. FK3453 could be a lead compound for a novel type of medicine which prevents the onset and aggravation of allergic diseases.

TSLP disease-associated genetic variants combined with airway TSLP expression influence asthma risk

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine important in initiation of allergic inflammation. Single nucleotide polymorphisms (SNPs) in TSLP are associated with asthma, yet studies have shown inconsistent associations between circulating TSLP and asthma. Studies that integrate the combined effects of TSLP genotype, TSLP mRNA, circulating TSLP levels, and asthma outcome are lacking. This study sought to recruit a novel cohort based on asthma-relevant TSLP SNPs and determine their impact on TSLP mRNA expression and TSLP circulating protein levels, and their individual and combined effects on asthma. This study developed an algorithm to prioritize TSLP SNPs and recruited 51 carriers and noncarriers based on TSLP genotypes. TSLP mRNA was quantified in nasal epithelial cells and circulating TSLP levels in plasma. This study determined the associations of defined TSLP risk genotypes and/or TSLP mRNA and protein levels with asthma. TSLP mRNA expression, but not circulating TSLP, was significantly increased in people who are asthmatic compared with in people who are nonasthmatic (P= .007; odds ratio, 1.44). Notably, 90% of children with the defined TSLP risk genotypes and high nasal TSLP mRNA expression (top tertile) had asthma compared with 40% of subjects without risk genotypes and with low TSLP expression (bottom tertile) (P= .024). No association between circulating TSLP and asthma was observed. Collectively, these data suggest childhood asthma is modified by the combined effects of TSLP genotype and TSLP expression in the nasal epithelium. The increased asthma risk likely manifests when genetic variation enables expression quantitative trait loci in the TSLP locus to elevate TSLP. It is important to consider both biomarkers when factoring asthma risk.

Effects of Resveratrol on Thymic Stromal Lymphopoietin Expression in Mast Cells.

Background and objectives: Cytokine thymic stromal lymphopoietin (TSLP) plays a pivotal role in the pathogenesis of atopic diseases such as atopic dermatitis, allergic rhinitis, and asthma. Resveratrol (RSV) exerts various pharmacological effects such as antioxidant, anti-inflammatory, neuroprotective, and anticancer. Although, it has been verified the beneficial effects of RSV on various subjects, the effect of RSV on thymic stromal lymphopoietin (TSLP) regulation has not been elucidated. Materials and Methods: Here, we examined how RSV regulates TSLP in HMC-1 cells. Enzyme-linked immunosorbent assay, real-time polymerase chain reaction, Western blotting, and calcium assay were performed to evaluate the effect of RSV. Results: TSLP production and mRNA expression were reduced by RSV. RSV down-regulated nuclear factor-κB activation, IκBα phosphorylation as well as activation of receptor-interacting protein2 and caspase-1 in HMC-1 cells. In addition, RSV treatment decreased the up-regulation of intracellular calcium in HMC-1 cells. Conclusions: These results suggest that RSV might be useful for the treatment of atopic diseases through blocking of TSLP.

The Expressions of TSLP, IL-33, and IL-17A in Monocyte Derived Dendritic Cells from Asthma and COPD Patients are Related to Epithelial-Macrophage Interactions.

Background. The cross-talk between the external and internal environment in the respiratory tract involves macrophage/dendritic cell (DC) transepithelial network. Epithelium triggers dendritic cell-mediated inflammation by producing thymic stromal lymphopoietin (TSLP), IL-33, and IL-17A. The study aimed to evaluate the expression of TSLP, IL-33, and IL-17A in human monocyte derived dendritic cells (moDCs) co-cultured with respiratory epithelium and monocyte derived macrophages (moMφs) in asthma, chronic obstructive pulmonary disease (COPD) and healthy controls. Methods. The study used a triple-cell co-culture model, utilizing nasal epithelial cells, along with moMφs and moDCs. Cells were cultured in mono-, di-, and triple-co-cultures for 24 h. Results. Co-culture with epithelium and moMφs significantly increased TSLP in asthma and did not change IL-33 and IL-17A mRNA expression in moDCs. moDCs from asthmatics were characterized by the highest TSLP mRNA expression and the richest population of TSLPR, ST2, and IL17RA expressed cells. A high number of positive correlations between the assessed cytokines and CHI3L1, IL-12p40, IL-1β, IL-6, IL-8, TNF in moDCs was observed in asthma and COPD. Conclusion. TSLP, IL-33, and IL-17A expression in moDCs are differently regulated by epithelium in asthma, COPD, and healthy subjects. These complex cell–cell interactions may impact airway inflammation and be an important factor in the pathobiology of asthma and COPD.

Abnormal thymic stromal lymphopoietin expression in the gastrointestinal mucosa of patients with eosinophilic gastroenteritis

ObjectiveTo investigate the differential expression of the thymic stromal lymphopoietin isoforms, short and long, and discern their biological implications under eosinophilic gastroenteritis. MethodsThe expression of thymic stromal lymphopoietin and its two isoforms in tissues was assessed by quantitative RT‐PCR in healthy controls (n=24) and patients with eosinophilic gastroenteritis (n=17). ResultsThymic stromal lymphopoietin mRNA was significantly reduced in eosinophilic gastroenteritis when compared with healthy controls (p <0.0001). A significantly lower amount of short thymic stromal lymphopoietin mRNA was observed in eosinophilic gastroenteritis when compared with controls (p <0.05), while a significantly higher amount of long thymic stromal lymphopoietin mRNA was observed in eosinophilic gastroenteritis when compared with controls (p <0.05). Peak eosinophilic count is significantly positively correlated with the expression of long thymic stromal lymphopoietin mRNA in the gastrointestinal mucosal of patients with eosinophilic gastroenteritis (rs=0.623,p <0.005), while peak eosinophilic count is significantly negatively correlated with the expression of short thymic stromal lymphopoietin mRNA in the gastrointestinal mucosal of patients with eosinophilic gastroenteritis (rs=‐0.4474, p <0.05). ConclusionsAbnormal mucosal thymic stromal lymphopoietin expression may contribute to gastrointestinal mucosa damage in eosinophilic gastroenteritis.

Regulation of Intestinal Epithelial Thymic Stromal Lymphopoietin Expression by Retinoic Acid Receptor Alpha

We have previously targeted the retinoic acid receptor alpha (RARα) isoform in murine intestinal epithelial cells (IECs) to explore the role of epithelial intrinsic retinoid signaling in intestinal immune homeostasis. We noted a significant decrease in the CD11c+ and CD103+ dendritic cell (DC) subpopulations in the intestines of intestinal‐epithelial specific RARα‐deficient (RARαvillin) mice. In the present study, we identified Thymic Stromal Lymphopoietin (TSLP) as a contributing factor that influences intrinsic RARα signaling within IECs as this epithelial‐derived cytokine preferentially stimulates CD103+ DCs to a more tolerogenic phenotype in the GI tract, induces Tregs and drives TH2 polarization. TSLP also influences immune homeostasis by modulating the activation of myeloid cells, inhibition of proinflammatory DCs, and production of IL‐12. Notably, TSLP levels are decreased in Crohn’s disease (CD) where inflammation is driven by IL‐12.We hypothesize that RA intrinsic signaling within IECs modulates TSLP expression via activation of RARα. We sought to generate a RARα−/− murine intestinal epithelial cell line utilizing the CRISPR/CAS9 system to examine the effects of RARα ablation. Second, we aim to establish RARα’s role in regulating intestinal epithelial TSLP expression in both the RARαvillin mice and the CRISPR‐generated RARα−/− cell line.Exon 5 in the open reading frame of the RARα gene in Mode‐K murine IECs was targeted using CRISPR/CAS9. CRISPR cleavage and knockdown of RARα activity was confirmed via surveyor/luciferase assays. The cell lines were phenotyped with regards to their morphology, proliferative ability, and viability. Next, we examined TSLP mRNA expression in the colons of RARαvillin mice and the CRISPR RARα−/− knockout cell line via qRT‐PCR under baseline and stimulated conditions. Wild‐type (WT) and RARα−/− cells were stimulated with RARα‐selective agonist, BMS753, and muramyl dipeptide (MDP), a NOD2‐selective agonist.TSLP expression in surface colonic epithelium was elevated 2.3‐fold in RARαvillin mice compared to WT litter mates. TSLP expression was elevated 10‐fold in the Mode‐K RARα−/− cells versus parent cells at baseline. BMS753 stimulation resulted in a 15‐fold increase in TSLP expression in the RARα−/− cells compared to baseline. When stimulated with MDP, a NOD2‐selective agonist and known stimulus of TSLP production, TSLP expression was elevated 60‐fold in the RARα−/− cells, but significantly impaired compared to the WT cells (400‐fold), suggesting a different regulatory mechanism under stimulated conditions.Identifying a link between dietary factors, such as RA, its receptors and the TSLP molecular pathway can facilitate our understanding of how environmental components contribute to the pathogenesis of inflammatory bowel disease. TSLP expression is controlled by RARα in colonic IECs where it may act as a repressor of TSLP promoter transactivation under baseline conditions. This suggests an important role for RA on myeloid and T cell function via effects on intestinal epithelial TSLP expression.Support or Funding InformationUniversity of Calgary, Department of Medicine and Crohn's Colitis Canada

A58 REGULATION OF INTESTINAL EPITHELIAL THYMIC STROMAL LYMPHOPOIETIN GENE EXPRESSION BY RETINOIC ACID RECEPTOR ALPHA

Abstract Background We have targeted the retinoic acid receptor alpha (RARα) isoform in murine intestinal epithelial cells (IECs) to explore the role of epithelial intrinsic retinoid signaling in intestinal immune homeostasis. We noted a significant decrease in the CD11c+ and CD103+ dendritic cell (DC) subpopulations in the intestines of intestinal-epithelial specific RARα-deficient (RARα villin) mice. In the present study, we identified Thymic Stromal Lymphopoietin (TSLP) as a contributing factor that influences intrinsic RARα signaling within IECs. This cytokine preferentially stimulates CD103+ DCs to a more tolerogenic phenotype in the GI tract, induces Tregs and drives TH2 polarization. TSLP also influences immune homeostasis by modulating the activation of myeloid cells, inhibition of proinflamamtory DCs, and production of IL-12. Notably, TSLP levels are decreased in Crohn’s disease (CD) where inflammation is driven by IL-12. Aims We hypothesize that RA intrinsic signaling within IECs modulates TSLP expression via activation of RARα. We sought to generate a RARα -/- murine intestinal epithelial cell line utilizing the CRISPR/CAS9 system to examine the effects of RARa ablation. Second, we aim to establish RARα’s role in regulating intestinal epithelial TSLP expression in both the RARα villin mice and the CRISPR-generated RARα -/- cell line. Methods Exon 5 in the open reading frame of the RARα gene in Mode-K murine IECs was targeted using CRISPR/CAS9. CRISPR cleavage and knockdown of RARα activity was confirmed via surveyor/luciferase assays. The cell lines were phenotyped with regards to their morphology, proliferative ability, and viability. Next, we examined TSLP mRNA expression in the colons of RARα villin mice and the CRISPR RARα -/- knockout cell line via qRT-PCR under baseline and stimulated conditions. Wild-type (WT) and RARα-/- cells were stimulated with RARα-selective agonist, BMS753, and muramyl dipeptide (MDP), a NOD2-selective agonist. Results TSLP expression in surface colonic epithelium was elevated 2.3-fold in RARα villin mice compared to WT litter mates. TSLP expression was elevated 10-fold in the Mode-K RARα-/- cells versus parent cells at baseline. BMS753 stimulation resulted in a 15-fold increase in TSLP expression in the RARα-/- cells compared to baseline. When stimulated with MDP, a NOD2-selective agonist known to stimulate TSLP expression, TSLP expression was elevated 60-fold in the RARα-/- cells, but significantly impaired compared to the WT cells (400-fold). Conclusions TSLP expression is controlled by RARα in colonic IECs where it may act as a repressor of TSLP promoter transactivation under baseline conditions. This suggests an important role for RA on myeloid and T cell function via effects on intestinal epithelial TSLP expression. Funding Agencies CCCUniversity of Calgary, Department of Medicine

Activation of Transient Receptor Potential Melastatin Family Member 8 (TRPM8) Receptors Induces Proinflammatory Cytokine Expressions in Bronchial Epithelial Cells.

PurposeCold air is a major environmental factor that exacerbates asthma. Transient receptor potential melastatin family member 8 (TRPM8) is a cold-sensing channel expressed in the airway epithelium. However, its role in airway inflammation remains unknown. We investigated the role of TRPM8 in innate immune responses in bronchial epithelial cells and asthmatic subjects.MethodsThe TRPM8 mRNA and protein expression on BEAS2B human bronchial epithelial cells was examined by real-time polymerase chain reaction (PCR), immunofluorescence staining and western blotting. Additionally, interleukin (IL)-4, IL-6, IL-8, IL-13, IL-25 and thymic stromal lymphopoietin (TSLP) levels before and after menthol, dexamethasone and N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl) piperazine-1-carboxamide (BCTC) treatments were measured via real-time PCR. TRPM8 protein levels in the supernatants of induced sputum from asthmatic subjects and normal control subjects were measured using enzyme-linked immunosorbent assay, and mRNA levels in sputum cell lysates were measured using real-time PCR.ResultsTreatment with up to 2 mM menthol dose-dependently increased TRPM8 mRNA and protein in BEAS2B cells compared to untreated cells (P < 0.001) and concomitantly increased IL-25 and TSLP mRNA (P < 0.05), but not IL-33 mRNA. BCTC (10 μM) significantly abolished menthol-induced up-regulation of TRPM8 mRNA and protein and IL-25 and TSLP mRNA (P < 0.01). TRPM8 protein levels were higher in the supernatants of induced sputum from asthmatic subjects (n = 107) than in those from healthy controls (n = 19) (P < 0.001), and IL-25, TSLP and IL-33 mRNA levels were concomitantly increased (P < 0.001). Additionally, TRPM8 mRNA levels correlated strongly with those of IL-25 and TSLP (P < 0.001), and TRPM8 protein levels were significantly higher in bronchodilator-responsive asthmatic subjects than in nonresponders.ConclusionsTRPM8 may be involved in the airway epithelial cell innate immune response and a molecular target for the treatment of asthma.

Ursolic acid downregulates thymic stromal lymphopoietin through the blockade of intracellular calcium/caspase‑1/NF‑κB signaling cascade in HMC‑1 cells.

Thymic stromal lymphopoietin (TSLP) plays an important role in allergic disorders, including atopic dermatitis and asthma. Ursolic acid (UA) has various pharmacological properties, such as antioxidant, anti‑inflammatory and anticancer. However, the effect of UA on TSLP regulation has not been fully elucidated. The aim of the present study was to analyze how UA regulates the production of TSLP in the human mast cell line HMC‑1. Enzyme‑linked immunosorbent assay, quantitative polymerase chain reaction analysis, western blotting, caspase‑1 assay and fluorescent measurements of intracellular calcium levels were conducted to analyze the regulatory effects of UA. The results revealed that UA inhibited TSLP production and mRNA expression. In addition, UA reduced the activation of nuclear factor‑κB and degradation of IκBα. Caspase‑1 activity was increased by exposure to phorbol myristate acetate plus calcium ionophore, whereas it was reduced by UA. Finally, UA treatment prevented an increase in intracellular calcium levels. These results indicated that UA may be a useful agent for the treatment and/or prevention of atopic and inflammatory diseases, and its effects are likely mediated by TSLP downregulation.

Abnormal thymic stromal lymphopoietin expression in the gastrointestinal mucosa of patients with eosinophilic gastroenteritis

ObjectiveTo investigate the differential expression of the thymic stromal lymphopoietin isoforms, short and long, and discern their biological implications under eosinophilic gastroenteritis. MethodsThe expression of thymic stromal lymphopoietin and its two isoforms in tissues was assessed by quantitative RT-PCR in healthy controls (n=24) and patients with eosinophilic gastroenteritis (n=17). ResultsThymic stromal lymphopoietin mRNA was significantly reduced in eosinophilic gastroenteritis when compared with healthy controls (p<0.0001). A significantly lower amount of short thymic stromal lymphopoietin mRNA was observed in eosinophilic gastroenteritis when compared with controls (p<0.05), while a significantly higher amount of long thymic stromal lymphopoietin mRNA was observed in eosinophilic gastroenteritis when compared with controls (p<0.05). Peak eosinophilic count is significantly positively correlated with the expression of long thymic stromal lymphopoietin mRNA in the gastrointestinal mucosal of patients with eosinophilic gastroenteritis (rs=0.623, p<0.005), while peak eosinophilic count is significantly negatively correlated with the expression of short thymic stromal lymphopoietin mRNA in the gastrointestinal mucosal of patients with eosinophilic gastroenteritis (rs=−0.4474, p<0.05). ConclusionsAbnormal mucosal thymic stromal lymphopoietin expression may contribute to gastrointestinal mucosa damage in eosinophilic gastroenteritis.

A steroid alkaloid derivative 02F04 upregulates thymic stromal lymphopoietin expression slowly and continuously through a novel Gq/11-ROCK-ERK1/2 signaling pathway in mouse keratinocytes

Thymic stromal lymphopoietin (TSLP), a master switch of allergic inflammation, plays an important role in the pathogenesis of allergic diseases. Although many compounds upregulate TSLP expression in vivo or in vitro, most of them are pollutants or toxicants. In the previous study, for the first time, we found that a steroid alkaloid derivative 02F04, which has a unique skeletal structure compared with other TSLP-inducing chemicals, significantly induced TSLP production in mouse keratinocytes. However, it is not investigated thoroughly that how 02F04 produces TSLP and why. In this study, we did a detailed investigation on the inducible effect and underlying molecular mechanism of 02F04 on TSLP production. We found that the peak time of TSLP mRNA level induced by 02F04 at 48 h led to a slow and continuous TSLP production in PAM212 cells. Besides, 02F04-induced TSLP production was significantly suppressed by inhibitors of Rho-associated protein kinase (ROCK), guanine nucleotide-binding protein subunit alpha q/11 (Gq/11) and extracellular signal-regulated kinase 1/2 (ERK1/2) at not only protein but also mRNA levels, and by siRNA-mediated knockdown of Gq or G11. This suggested that ROCK, Gq/11 and ERK1/2 signaling pathways were involved in 02F04-induced TSLP production. Increase in the level of p-ERK1/2 induced by 02F04 was suppressed by both inhibitors of ROCK and Gq/11, indicating that ROCK and Gq/11 molecules were located at the upstream of ERK1/2 to regulate 02F04-induced TSLP production. Gq/11 was located at the upstream of ROCK because the specific Gq/11 inhibitor of YM-254890 significantly reduced 02F04-induced actin stress fiber formation. Taken together, 02F04 upregulates a slow and continuous TSLP production through a novel Gq/11-ROCK-ERK1/2 signaling pathway. The thorough understanding the effect and mechanism of 02F04 on TSLP production is expected to supply it as a novel TSLP-regulating compound and a potential new tool for investigating the role of TSLP in allergic disorders.

Thymic Stromal Lymphopoietin-Related Allergic Pathway in Patients With Vernal Keratoconjunctivitis.

To investigate the expression of thymic stromal lymphopoietin (TSLP) and the relevant signaling pathways in the giant papillae obtained from patients with vernal keratoconjunctivitis (VKC) and to study the potential functional role and molecular mechanism of TSLP. Giant papillae from VKC patients and control samples were used to perform immunohistochemical staining and analyze the mRNA expression of TSLP and related pathway by real-time polymerase chain reaction. TSLP was markedly expressed in the epithelial cells and some inflammatory cells of giant papillae, but not in the control conjunctival tissue. TSLP mRNA expression in the giant papillae of VKC was increased by 9.63 ± 0.99 (mean ± SD) fold compared with controls (P < 0.01). CD11c and OX40L immunoreactive cells largely infiltrated the giant papillae as observed by immunohistochemical staining. CD4Th2 cell infiltration was observed through high immunoreactivity of CD4. Th2 cytokines (IL-4, IL-5 and IL-13) and OX40 in the VKC specimens showed increased expression. Augmented gene expression levels of CD4 (6.88 ± 1.84), OX40L (7.60 ± 1.79), OX40 (7.25 ± 1.38), IL-4 (6.89 ± 1.46), IL-5 (8.42 ± 1.55), and IL-13 (9.69 ± 1.94) were significantly different from controls (P < 0.05). Our observations provide strong evidence that TSLP may be a crucial factor that contributes to the development and progression of allergic conjunctivitis. The results also demonstrated that TSLP activates dendritic cells to prime CD4T cells to differentiate into Th2 type and triggers Th2-dominant allergic inflammation through the TSLP/OX40L/OX40 signaling as part of immunopathogenesis of VKC.

Elevated expression of IL-17RB and ST2 on myeloid dendritic cells is associated with a Th2-skewed eosinophilic inflammation in nasal polyps

BackgroundInterleukin(IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP) underlie the crosstalk between epithelial cells and dendritic cells (DCs) during the development of Th2 responses. This study aimed to measure the expressions of IL-17RB, ST2 and TSLPR, receptor of IL-25, IL-33, and TSLP respectively, on myeloid DCs in nasal polyps (NP) and evaluate their association with local Th2 inflammation and disease severity in patients with NP.MethodsSamples were collected from 30 NP patients and 16 control subjects recruited prospectively. The mRNA expression of cytokines, including TSLP, IL-25 and IL-33, as well as interferon (IFN)-γ, IL-4, IL-5, IL-13 and IL-17A in NP and control tissues was examined by qualitative polymerase chain reaction (qPCR). The expression of IL-17RB, ST2 and TSLPR as well as other surface markers on myeloid DCs (mDCs) was examined by flow cytometry.ResultsIncreased numbers of total and activated mDCs were found in NP patients. mDCs demonstrated significantly higher expression of IL-17RB, ST2 and TSLPR than those in control tissues. The activated mDCs exhibited up-regulations of OX40L and ICOSL, but down-regulation of PDL1 in NP. Moreover, the IL-17RB, ST2 and TSLPR levels on mDCs were positively correlated with IL-25, IL-33 and TSLP mRNA levels, respectively, in NP. Furthermore, IL-17RB and ST2 expressions on mDCs were correlated with the IL-5 mRNA level as well as eosinophil number in NP. Importantly, the IL-17RB expression on mDCs and the OX40L expression on activated mDCs in NP were positively correlated with CT score and total nasal symptom score.ConclusionsIncreased expressions of IL-17RB and ST2 on mDCs are associated with enhanced local Th2 inflammation in NP, suggesting that mDCs might play a role in IL-25- and IL-33-induced type 2 responses and eosinophilic inflammation in NP.

Cutaneous expression of thymic stromal lymphopoietin (TSLP) in vitiligo patients: a case-control study.

Thymic stromal lymphopoietin (TSLP) is a major pro-allergic cytokine promoting T helper-2 responses. Our aim was to study and verify the hypothesis of the role of TSLP in the pathogenesis of vitiligo. This case-control study was conducted on 25 patients with generalized non-segmental vitiligo (recruited from the Dermatology outpatient clinic, Kasr El Ainy, Faculty of Medicine, Cairo University) and 25 healthy controls fulfilling the inclusion criteria over a period of 7 months (January 2017-July 2017). Patients underwent complete medical history, detailed assessment of vitiligo, and photographic documentation. Skin biopsies were taken from the back from both patients' vitiliginous skin and from normal skin of controls for which relative TSLP messenger RNA (mRNA) tissue expression levels were measured using quantitative real-time polymerase chain reaction technique. There was a statistically significant difference between the TSLP mRNA expression levels in patients and controls (P < 0.001) with lower levels in the former group. This study revealed lower TSLP mRNA expression levels in vitiliginous skin than in normal skin suggesting an imminent role of TSLP in the pathogenesis of vitiligo.

IL-17A-associated IKK-\u03b1 signaling induced TSLP production in epithelial cells of COPD patients

Thymic stromal lymphopoietin (TSLP) is a cytokine expressed in the epithelium, involved in the pathogenesis of chronic disease. IL-17A regulates airway inflammation, oxidative stress, and reduction of steroid sensitivity in chronic obstructive pulmonary disease (COPD). TSLP and IL-17A were measured in induced sputum supernatants (ISs) from healthy controls (HC), healthy smokers (HS), and COPD patients by enzyme-linked immunosorbent assay. Human bronchial epithelial cell line (16HBE) and normal bronchial epithelial cells were stimulated with rhIL-17A or ISs from COPD patients to evaluate TSLP protein and mRNA expression. The effects of the depletion of IL-17A in ISs, an anticholinergic drug, and the silencing of inhibitor kappa kinase alpha (IKKα) on TSLP production were evaluated in 16HBE cells. Coimmunoprecipitation of acetyl-histone H3(Lys14)/IKKα was evaluated in 16HBE cells treated with rhIL-17A and in the presence of the drug. TSLP and IL-17A levels were higher in ISs from COPD patients and HS compared with HC. TSLP protein and mRNA increased in 16HBE cells and in normal bronchial epithelial cells stimulated with ISs from COPD patients compared with ISs from HC and untreated cells. IKKα silencing reduced TSLP production in 16HBE cells stimulated with rhIL-17A and ISs from COPD patients. RhIL-17A increased the IKKα/acetyl-histone H3 immunoprecipitation in 16HBE cells. The anticholinergic drug affects TSLP protein and mRNA levels in bronchial epithelial cells treated with rhIL-17A or with ISs from COPD patients, and IKKα mediated acetyl-histone H3(Lys14). IL-17A/IKKα signaling induced the mechanism of chromatin remodeling associated with acetyl-histone H3(Lys14) and TSLP production in bronchial epithelial cells. Anticholinergic drugs might target TSLP derived from epithelial cells during the treatment of COPD.

Thymic stromal lymphopoietin in human pancreatic ductal adenocarcinoma: expression and prognostic significance.

Thymic stromal lymphopoietin (TSLP) has emerged as an important, but contradictory, player conditioning tumor growth. In certain contexts, by driving T helper (h) 2 responses via tumor-associated OX40 Ligand (OX40L)+ dendritic cells (DCs), TSLP may play a pro-tumorigenic role. The study elucidates the importance of TSPL in pancreatic ductal adenocarcinoma (PDAC), by analyzing: i) TSLP levels in PDAC cell-line supernatants and plasma from patients with locally-advanced/metastatic PDAC, pre- and post-treatment with different chemotherapeutic protocols, in comparison with healthy donors; ii) TSLP and OX40L expression in PDAC and normal pancreatic tissues, by immunohistochemistry; iii) OX40L expression on ex vivo-generated normal DCs in the presence of tumor-derived TSLP, by flow cytometry; iv) clinical relevance in terms of diagnostic and prognostic value and influence on treatment modality and response.Some PDAC cell lines, such as BxPC-3, expressed both TSLP mRNA and protein. Normal DCs, generated ex vivo in the presence of TSLP-rich-cell supernatants, displayed increased expression of OX40L, reduced by the addition of a neutralizing anti-TSLP polyclonal antibody. OX40L+ cells were detected in pancreatic tumor inflammatory infiltrates. Abnormally elevated TSLP levels were detected in situ in tumor cells and, systemically, in locally-advanced/metastatic PDAC patients. Of the chemotherapeutic protocols applied, gemcitabine plus oxaliplatin (GEMOX) significantly increased circulating TSLP levels. Elevated plasma TSLP concentration was associated with shorter overall survival and increased risk of poor outcome. Plasma TSLP measurement successfully discriminated PDAC patients from healthy controls.These data show that TSLP secreted by pancreatic cancer cells may directly impact PDAC biology and patient outcome.

Interleukin-32 induced thymic stromal lymphopoietin plays a critical role in the inflammatory response in human corneal epithelium

Interleukin (IL)-32, a novel cytokine, participates in a variety of inflammatory disorders. Thymic stromal lymphopoietin (TSLP) plays important roles in mucosal epithelial cells, especially in allergy-induced inflammation, through the TSLP-TSLPR (thymic stromal lymphopoietin receptor) signalling pathway. However, the association of IL-32 with TSLP on the ocular surface remains unclear. The present work aimed to assess the functional association of IL-32 with TSLP in the control of pro-inflammatory cytokine levels in the corneal epithelium. Human corneal tissue specimens and human corneal epithelial cells (HCECs) were administered different concentrations of IL-32 in the presence or absence of various inhibitors to assess TSLP levels and localization, as well as the molecular pathways that control pro-inflammatory cytokine production. TSLP mRNA levels were determined by real time RT- PCR, while protein levels were quantitated by ELISA and immunohistochemical staining. TSLP protein expression was examined in donor corneal epithelium samples. IL-32 significantly upregulated TSLP and pro-inflammatory cytokines (TNFα and IL-6) in HCECs at the gene and protein levels. The production of pro-inflammatory molecules by IL-32 was increased by recombinant TSLP. Interestingly, both NF-κB (quinazoline) and caspase-1 (VX-765) inhibitors suppressed the IL-32-related upregulation of pro-inflammatory cytokines (TNFα and IL-6). These findings demonstrate that IL-32 and IL-32-induced-TSLP are critical cytokines that participate in inflammatory responses through the caspase-1 and NF-κB signalling pathways in the corneal epithelium, suggesting new molecular targets for inflammatory diseases of the ocular surface. The effects of IL-32 on cell proliferation and apoptosis were investigated by MTT assays and RT-PCR,respectively. The results demonstrated that IL-32 inhibits cells apoptosis in HCECs.

Thyroid-Stimulating Hormone-Stimulated Human Adipocytes Express Thymic Stromal Lymphopoietin.

When recombinant human (rh) thyroid-stimulating hormone (TSH) is administered to thyroid cancer survivors, an acute extra-thyroidal effect raises pro-inflammatory cytokines and activates platelets. Thymic stromal lymphopoietin (TSLP) is a cytokine recently implicated in platelet activation. Our aim was to measure platelet microparticle levels after rhTSH stimulation in vivo, and to investigate TSLP expression in TSH-stimulated human adipocytes in culture. Blood samples for total and platelet microparticle analysis were obtained from thyroid cancer survivors before (day 1) and after rhTSH administration (day 5). Adipocytes, differentiated from stromal preadipocytes isolated from adipose tissue from surgical patients, were stimulated with TSH. TSLP mRNA expression, protein expression, and protein release into the adipocyte medium were measured. The level of platelet microparticles in thyroid cancer patients rose 5-fold after rhTSH stimulation. TSH upregulated TSLP mRNA expression in adipocytes in culture through a pathway that was inhibited by 66% by H89, a protein kinase A inhibitor. TSLP protein expression rose in response to TSH, and TSH-stimulated TSLP release into the medium was completely blocked by dexamethasone. In conclusion, TSLP is a novel TSH-responsive adipokine. Future studies will be needed to address the potential role of adipocyte-derived TSLP and whether it is linked to TSH-dependent platelet activation.