Thymic Stromal Lymphopoietin Production Research Articles

Co-Stimulation with TWEAK and TGF-β1 Induces Steroid-Insensitive TSLP and CCL5 Production in BEAS-2B Human Bronchial Epithelial Cells.

Steroid-resistant asthma is a common cause of refractory asthma. Type 2 inflammation is the main inflammatory response in asthma, and the mechanism underlying the steroid-resistance of type 2 inflammation has not been completely elucidated. Tumor-necrosis-factor-like apoptosis-inducing factor (TWEAK) and transforming growth factor (TGF)-β1 are involved in epithelial-mesenchymal transition (EMT) and the production of thymic stromal lymphopoietin (TSLP) and C-C motif chemokine ligand 5 (CCL5). We herein hypothesize that the combined exposure to TWEAK and TGF-β1 may result in the development of steroid resistance in bronchial epithelial cells. The bronchial epithelial cell line BEAS-2B was cultured with or without TGF-β1 or TWEAK, in the presence or absence of dexamethasone (DEX). The roles of Smad-independent pathways and MAP kinase phosphatase 1 (MKP-1) were also explored. Co-stimulation of TWEAK and TGF-β1 induced E-cadherin reduction, N-cadherin upregulation, and TSLP and CCL5 production, which were not suppressed by DEX. Inhibition of the nuclear factor kappa beta (NF-κB) and mitogen-activated protein kinase pathways downregulated steroid-unresponsive TSLP and CCL5 production, whereas knockdown of MKP-1 improved steroid-unresponsive TSLP production, induced by co-stimulation with TWEAK and TGF-β1. Therefore, co-stimulation with TWEAK and TGF-β1 can induce the steroid-insensitive production of TSLP and CCL5 in the bronchial epithelium and may contribute to airway inflammation.

MERS-CoV Infection and Its Impact on the Expression of TSLP Cytokine and IgG Antibodies: An In Vivo and In Vitro Study.

Thymic stromal lymphopoietin (TSLP) is a proinflammatory cytokine produced by epithelial cells that is involved in the activation of allergic disorders. To date, no study has examined TSLP induction during Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Herein, we aimed to study the effects of the recombinant spike protein of MERS-CoV on TSLP production. Additionally, the effects of recombinant human TSLP (rhTSLP) on B cell survival and antibody production were investigated. B cells were separated using the Human B Cell Enrichment Kit, and B cell survival was measured using the WST-1 Assay Kit. Enzyme-linked immunosorbent assay (ELISA) was used to measure TSLP levels in the sera of both MERS-CoV-infected (n=4; median age, 53 years) and healthy individuals (n=5; median age, 35 years). We showed that the group of infected patients had significantly higher levels of TSLP than healthy controls (37.6 pg/mL vs 19.8 pg/mL, *p<0.05). The levels of TSLP in A549 cells were remarkably increased after 48h of stimulation with recombinant full-length spike protein (rSP) (32.2 pg/mL, p=0.01). B cell survival was greatly enhanced by rhTSLP alone or in combination with rSP (0.02 vs 0.046, and 0.045; **p<0.01, respectively). Our data also showed a significant synergistic effect of rhTSLP and rSP on the augmented response of IgG antibodies against the spike protein of MERS-CoV compared with unstimulated cells (0.156 vs 0.22; *p<0.05). TSLP production is induced in vivo after MERS-CoV infection and in vitro after treatment with the rSP of MERS-CoV, which has a significant effect on the survival of B cells. Our data suggest that TSLP can be used as a strong mucosal adjuvant for vaccine development against MERS-CoV infection. However, further investigation is required to study the functional role of TSLP in MERS-CoV infection.

Baicalin Attenuates Type 2 Immune Responses in a Mouse Allergic Asthma Model through Inhibiting the Production of Thymic Stromal Lymphopoietin

Plain Language SummaryBaicalin is a flavonoid chemical extracted and purified from Scutellaria baicalensis Georgi, which possesses broad pharmacological properties. Our work objected to explore the protective effect of baicalin on allergic asthma and the possible mechanisms on regulating type 2 immune response. Mice were stimulated with OVA and administrated with baicalin. We found that eosinophils infiltrate in BALF were reduced remarkably in baicalin-treated asthmatic mice. Baicalin decreased OVA-induced inflammatory cytokines and serum immunoglobulin E secretion significantly. Moreover, baicalin alleviated the asthmatic pathological changes and substantially suppressed TSLP expression in the lung tissues. Our study indicates that baicalin attenuates OVA-induced allergic asthma in mice effectively by suppressing type 2 immune responses, which may provide a new mechanistic insight into the anti-asthmatic activity of baicalin.

Antipruritic effect of ursolic acid through MRGPRX2/MrgprB2-dependent inhibition of mast cell degranulation and reduced TSLP production

Ursolic acid (UA), a pentacyclic triterpene, exhibits diverse pharmacological effects, including potential treatment for allergic diseases. It downregulates thymic stromal lymphopoietin (TSLP) and disrupts mast cell signaling pathways. However, the exact molecular mechanism by which UA interferes with mast cell action remains unclear. Therefore, the current study aimed to uncover molecular entities underlying the effect of UA on mast cells and its potential antipruritic effect, specifically investigating its modulation of key molecules such as TRPV4, PAR2, and MRGPRX2, which are involved in TSLP regulation and sensation.Calcium imaging experiments revealed that UA pretreatment significantly suppressed MRGPRX2 activation (and its mouse orthologue MrgprB2), a G protein-coupled receptor predominantly expressed in mast cells. Molecular docking predictions suggested potential interactions between UA and MRGPRX2/MrgprB2. UA pretreatment also reduced mast cell degranulation through MRGPRX2 and MrgprB2-dependent mechanisms. In a dry skin mouse model, UA administration decreased tryptase and TSLP production in the skin, and diminished TSLP response in the sensory neurons. While PAR2 and TRPV4 activation enhances TSLP production, UA did not inhibit their activity. Notably, UA attenuated compound 48/80-induced scratching behaviors in mice and suppressed spontaneous scratching in a dry skin model.The present study confirms the effective inhibition of UA on MRGPRX2/MrgprB2, leading to reduced mast cell degranulation and suppressed scratching behaviors. These findings highlight the potential of UA as an antipruritic agent for managing various allergy- or itch-related conditions.

High TSLP responses in the human infant airways are associated with pre-activated airway epithelial IFN antiviral immunity.

Thymic stromal lymphopoietin (TSLP) is a primarily epithelial-derived cytokine that drives type 2 allergic immune responses. Early life viral respiratory infections elicit high TSLP production, which leads to the development of type 2 inflammation and airway hyperreactivity. The goal of this study was to examine in vivo and in vitro the human airway epithelial responses leading to high TSLP production during viral respiratory infections in early infancy. A total of 129 infants (<1-24 m, median age 10 m) with severe viral respiratory infections were enrolled for in vivo (n = 113), and in vitro studies (n = 16). Infants were classified as 'high TSLP' or 'low TSLP' for values above or below the 50th percentile. High versus low TSLP groups were compared in terms of type I-III IFN responses and production of chemokines promoting antiviral (CXCL10), neutrophilic (CXCL1, CXCL5, CXCL8), and type 2 responses (CCL11, CCL17, CCL22). Human infant airway epithelial cell (AEC) cultures were used to define the transcriptomic (RNAseq) profile leading to high versus low TSLP responses in vitro in the absence (baseline) or presence (stimulated) of a viral mimic (poly I:C). Infants in the high TSLP group had greater in vivo type III IFN airway production (median type III IFN in high TSLP 183.2 pg/mL vs. 63.4 pg/mL in low TSLP group, p = 0.007) and increased in vitro type I-III IFN AEC responses after stimulation with a viral mimic (poly I:C). At baseline, our RNAseq data showed that infants in the high TSLP group had significant upregulation of IFN signature genes (e.g., IFIT2, IFI6, MX1) and pro-inflammatory chemokine genes before stimulation. Infants in the high TSLP group also showed a baseline AEC pro-inflammatory state characterized by increased production of all the chemokines assayed (e.g., CXCL10, CXCL8). High TSLP responses in the human infant airways are associated with pre-activated airway epithelial IFN antiviral immunity and increased baseline AEC production of pro-inflammatory chemokines. These findings present a new paradigm underlying the production of TSLP in the human infant airway epithelium following early life viral exposure and shed light on the long-term impact of viral respiratory illnesses during early infancy and beyond childhood.

Type III interferon exerts thymic stromal lymphopoietin in mediating adaptive antiviral immune response.

Previously, it was believed that type III interferon (IFN-III) has functions similar to those of type I interferon (IFN-I). However, recently, emerging findings have increasingly indicated the non-redundant role of IFN-III in innate antiviral immune responses. Still, the regulatory activity of IFN-III in adaptive immune response has not been clearly reported yet due to the low expression of IFN-III receptors on most immune cells. In the present study, we reviewed the adjuvant, antiviral, antitumor, and disease-moderating activities of IFN-III in adaptive immunity; moreover, we further elucidated the mechanisms of IFN-III in mediating the adaptive antiviral immune response in a thymic stromal lymphopoietin (TSLP)-dependent manner, a pleiotropic cytokine involved in mucosal adaptive immunity. Research has shown that IFN-III can enhance the antiviral immunogenic response in mouse species by activating germinal center B (GC B) cell responses after stimulating TSLP production by microfold (M) cells, while in human species, TSLP exerts OX40L for regulating GC B cell immune responses, which may also depend on IFN-III. In conclusion, our review highlights the unique role of the IFN-III/TSLP axis in mediating host adaptive immunity, which is mechanically different from IFN-I. Therefore, the IFN-III/TSLP axis may provide novel insights for clinical immunotherapy.

STIM/Orai-mediated calcium entry elicits spontaneous TSLP overproduction in epidermal cells of atopic dermatitis mice

Aim: Atopic dermatitis (AD) is a pruritic, chronic inflammatory skin disease. Thymic stromal lymphopoietin (TSLP) is highly expressed in the epidermis of patients with AD and induces T helper 2 (Th2) immune responses and itching. Although the mechanism underlying the stimulus-induced TSLP production in normal keratinocytes has been intensively studied, whether the production capability of TSLP is naturally enhanced in epidermal cells in AD conditions remains unclear. Previous studies demonstrated that a deficiency of polyunsaturated fatty acid (PUFA) causes AD-like pruritic skin inflammation in special diet-fed hairless mice. The aim of the study was to examine the TSLP production capability of epidermal cells isolated from diet-induced AD mouse model and its mechanism. Methods: Epidermal cells were isolated from normal and AD mice and incubated under unstimulated culture conditions to assess spontaneous TSLP production. Messenger ribonucleic acid (mRNA) and protein levels of TSLP were determined by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Results: TSLP level was markedly increased in the skin of AD mice. When epidermal cells were isolated from AD mice and cultured without stimulation, Tslp gene expression was upregulated, and a large amount of TSLP protein was extracellularly released. Such TSLP overproduction was not observed in the epidermal cells of normal mice. TSLP overproduction in AD epidermal cells was almost completely inhibited by extracellular calcium chelation, interference with plasma membrane interaction of stromal interaction molecule 1 (STIM1), blockade of the calcium release-activated calcium (CRAC) channels Orai1 and Orai2, or treatment with a PUFA γ-linolenic acid (GLA). Conclusions: Epidermal cells isolated from AD mice can spontaneously produce TSLP through STIM/Orai-mediated calcium entry, and GLA may negatively regulate this TSLP production.

Hypoxia-inducible factor prolyl hydroxylase inhibitors suppressed thymic stromal lymphopoietin production and allergic responses in a mouse air-pouch-type ovalbumin sensitization model.

Atopic dermatitis (AD) is an allergic skin disease, triggered by excessive type 2 immune reactions. Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine that induces type 2 immune response through dendritic cell activation. Therefore, TSLP inhibitors may serve as novel antiallergic drugs. Hypoxia-inducible factor (HIF) activation in the epithelia contributes to several homeostatic phenomena, such as re-epithelialization. However, the effects of HIF activation on TSLP production and immune activation in the skin remain unclear. In this study, we found that selective HIF prolyl hydroxylase inhibitors (PHD inhibitors), which induce HIF activation, suppressed TSLP production in a mouse ovalbumin (OVA) sensitization model. PHD inhibitors also suppressed the production of tumor necrosis factor-alpha (TNF-α), which is a major inducer of TSLP production, in this mouse model and in a macrophage cell line. Consistent with these findings, PHD inhibitors suppressed OVA-specific IgE levels in the serum and OVA-induced allergic responses. Furthermore, we found a direct suppressive effect on TSLP expression in a human keratinocyte cell line mediated by HIF activation. Taken together, our findings suggest that PHD inhibitors exert antiallergic effects by suppressing TSLP production. Controlling the HIF activation system has therapeutic potential in AD.

The roles of epidermal Blimp-1 in atopic dermatitis

Abstract Keratinocytes are the main cell type constructing the epidermis of animals, which can further differentiate into corneocytes and express multiple innate immune receptors. B lymphocyte-induced maturation protein-1 (Blimp-1) plays a crucial rule in directing cornification of keratinocytes. We previously reported that keratinocyte-specific deletion of Prdm1, the gene encoding Blimp-1, induced spontaneous inflammation in the skin of adult mice. We further found that deletion of Prdm1in keratinocytes in Balb/c genetic background caused hyper-production of type 2 cytokines and thymic stromal lymphopoietin (TSLP) in the skin, and staphylococci colonization, which are characteristics of atopic dermatitis (AD). Since K5-CreER+Prdm1f/f(CKO) mice in Balb/c background exhibited AD-like symptoms, we wondered whether they are more susceptible to the disease progression in a murine AD model induced by MC903, a vitamin D3 analog enhancing TSLP production by keratinocytes. We found that CKO mice showed more immune cell infiltration in ear skin, developed more severe epidermal hyperplasia, and induced more TSLP secretion in the skin following topical application of MC903. Recent studies showed that staphylococci colonization activated the transcription of endogenous retroviruses (ERVs) in murine skin, while excess ERV expression led to skin inflammation. As staphylococci colonized on our CKO mouse skin, we hypothesized that enhanced ERV expression may also link to skin inflammation of CKO mice. Indeed, both in vivoand in vitrodeletion of Prdm1in keratinocytes resulted in elevated levels of ERV transcripts. We are studying the contribution of elevated ERV transcripts in Blimp-1-reduction-mediated AD-like skin inflammation.

A CpG-Oligodeoxynucleotide Suppresses Th2/Th17 Inflammation by Inhibiting IL-33/ST2 Signaling in Mice from a Model of Adoptive Dendritic Cell Transfer of Smoke-Induced Asthma.

Tobacco smoke exposure is a major environmental risk factor that facilitates the development and progression of asthma. Our previous study showed that CpG oligodeoxynucleotide (CpG-ODN) inhibits thymic stromal lymphopoietin (TSLP)-dendritic cells (DCs) to reduce Th2/Th17-related inflammatory response in smoke-related asthma. However, the mechanism underlying CpG-ODN -downregulated TSLP remains unclear. A combined house dust mite (HDM)/cigarette smoke extract (CSE) model was used to assess the effects of CpG-ODN on airway inflammation, Th2/Th17 immune response, and amount of IL-33/ST2 and TSLP in mice with smoke-related asthma induced by adoptive transfer of bone-marrow-derived dendritic cells (BMDCs) and in the cultured human bronchial epithelium (HBE) cells administered anti-ST2, HDM, and/or CSE. In vivo, compared to the HDM alone model, the combined HDM/CSE model had aggravated inflammatory responses, while CpG-ODN attenuated airway inflammation, airway collagen deposition, and goblet cell hyperplasia and reduced the levels of IL-33/ST2, TSLP, and Th2/Th17-cytokines in the combined model. In vitro, IL-33/ST2 pathway activation promoted TSLP production in HBE cells, which could be inhibited by CpG-ODN. CpG-ODN administration alleviated Th2/Th17 inflammatory response, decreased the infiltration of inflammatory cells into the airway, and improved the remodeling of smoke-related asthma. The underlying mechanism may be that CpG-ODN inhibits the TSLP-DCs pathway by downregulating the IL-33/ST2 axis.

Montelukast Increased IL-25, IL-33, and TSLP via Epigenetic Regulation in Airway Epithelial Cells.

The epithelium-derived cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP) are important mediators that initiate innate type 2 immune responses in asthma. Leukotriene receptor antagonists (LTRAs) are commonly used to prevent asthma exacerbations. However, the effects of LTRAs on epithelium-derived cytokines expression in airway epithelial cells are unclear. This study aimed to investigate the effects of LTRAs on the expression of epithelium-derived cytokines in human airway epithelial cells and to explore possible underlying intracellular processes, including epigenetic regulation. A549 or HBE cells in air-liquid interface conditions were pretreated with different concentrations of LTRAs. The expression of epithelium-derived cytokines and intracellular signaling were investigated by real-time PCR, enzyme-linked immunosorbent assay, and Western blot. In addition, epigenetic regulation was investigated using chromatin immunoprecipitation analysis. The expression of IL-25, IL-33, and TSLP was increased under LTRAs treatment and suppressed by inhaled corticosteroid cotreatment. Montelukast-induced IL-25, IL-33, and TSLP expression were mediated by the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways and regulated by histone H3 acetylation and H3K36 and H3K79 trimethylation. LTRAs alone might increase inflammation and exacerbate asthma by inducing the production of IL-25, IL-33, and TSLP; therefore, LTRA monotherapy may not be an appropriate therapeutic option for asthma.

Evidence that oncostatin M synergizes with IL-4 signaling to induce TSLP expression in chronic rhinosinusitis with nasal polyps

Oncostatin M (OSM) may promote type 2 inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP) by inducing thymic stromal lymphopoietin (TSLP). We sought to study the impact of OSM on TSLP synthesis and release from nasal epithelial cells (NECs). OSM receptors, IL-4 receptors (IL-4R), and TSLP were evaluated in mucosal tissue and primary NECs from patients with CRSwNP by quantitative PCR and immunofluorescence. Air-liquid interface-cultured NECs were stimulated with cytokines, including OSM, and quantitative PCR, ELISA, Western blot, and flow cytometry were used to assess the expression of OSM receptors, IL-4R, and TSLP. Increased levels of OSM receptor β chain (OSMRβ), IL-4Rα, and TSLP were observed in nasal polyp tissues and primary epithelial cells from nasal polyps of patients with CRSwNP compared with control tissues or cells from control subjects. The level of expression of OSMRβ in tissue was correlated with levels of both IL-4Rα and TSLP. OSM stimulation of NECs increased the expression of OSMRβ and IL-4Rα. Stimulation with IL-4 plus OSM augmented the production of TSLP; the response was suppressed by a signal transducer and activator of transcription 6 inhibitor. Stimulation of NECs with IL-4 plus OSM increased the expression of proprotein convertase subtilisin/kexin 3, an enzyme that truncates and activates TSLP. OSM increases the expression of IL-4Rα and synergizes with IL-4 to induce the synthesis and release of TSLP in NECs. Because the combination of IL-4 and OSM also augmented the expression of proprotein convertase subtilisin/kexin 3, these results suggest that OSM can induce both synthesis and posttranslational processing/activation of TSLP, promoting type 2 inflammation.

Topical Skullcapflavone II attenuates atopic dermatitis in a mouse model by directly inhibiting associated cytokines in different cell types.

Skullcapflavone II (SFII), a flavonoid derived from Scutellaria baicalensis, is an anticancer agent. We aimed to validate SFII for atopic dermatitis (AD) therapy by demonstrating the anti-inflammatory effects of SFII in an AD mouse model produced by the topical application of the vitamin D3 analog MC903. We showed that topical treatment with SFII significantly suppressed MC903-induced serum IgE levels compared with topical hydrocortisone (HC) treatment. Topical SFII also prevents MC903-induced pruritus, skin hyperplasia, and inflammatory immune cell infiltration into lesional skin comparable to topical HC. In addition, MC903-induced immune cell chemoattractants and AD-associated cytokine production in skin lesions were effectively suppressed by topical SFII. The production of MC903-induced effector cytokines influencing T helper (Th)2 and Th17 polarization in lesioned skin is significantly inhibited by topical SFII. Furthermore, we showed that SFII can directly inhibit the production of AD-associated cytokines by human primary keratinocytes, mouse bone marrow-derived cells (BMDCs), and mouse CD4+ T cells in vitro. Lastly, we demonstrated that topical SFII more effectively suppressed serum IgE levels, the production of IL-4 and thymic stromal lymphopoietin (TSLP), and infiltration of CD4+ T cells and Gr-1+ cells (neutrophils) into lesion skin compared to topical baicalein (a flavonoid derived from Scutellaria baicalensis), which has anti-inflammatory effects. Taken together, our findings suggest that SFII may have promising therapeutic potential for this complex disease via the regulation of multiple AD-associated targets.

TSLP activates the production of IFN-γ via CD8-positive T cells in recurrent tonsillitis

The role of thymic stromal lymphopoietin (TSLP) in chronic infection is not well understood. We report an increased number of cluster of differentiation 8 (CD8+ T) cells and higher expression of TSLP in the tonsil tissues of patients with recurrent tonsillitis as compared to that in the tonsil tissues of patients with tonsillar hypertrophy. Stimulation of tonsil CD8+ T cells with TSLP in the recurrent tonsillitis group resulted in higher production of interferon gamma (IFN-γ) and increased cell proliferation when compared to that in the tonsillar hypertrophy group. These results suggest the possibility that recurrent infection of tonsils may enhance local CD8+ T cell proliferation and IFN-γ and TSLP production. Anti-TSLP antibodies, which are potential therapeutic agents in allergic diseases, could have beneficial effects in patients with recurrent tonsillitis.

Anti-Itching and Anti-Inflammatory Effects of Kushenol F via the Inhibition of TSLP Production.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that results from eczema, itching, disrupted barrier function and aberrant cutaneous immune responses. The aim of the present study was to assess the efficacy of kushenol F as an effective treatment for AD via the suppression of thymic stromal lymphopoietin (TSLP) production. The results of the present study demonstrated that the clinical symptoms of AD were less severe and there was reduced ear thickening and scratching behavior in kushenol F-treated Dermatophagoides farinae extract (DFE)/1-chloro-2,4-dinitrochlorobenzene (DNCB)-induced AD mice. Histopathological analysis demonstrated that kushenol F decreased the DFE/DNCB-induced infiltration of eosinophil and mast cells and TSLP protein expression levels. Furthermore, kushenol F-treated mice exhibited significantly lower concentrations of serum histamine, IgE and IgG2a compared with the DFE/DNCB-induced control mice. Kushenol F also significantly decreased phosphorylated NF-κB and IKK levels and the mRNA expression levels of IL-1β and IL-6 in cytokine combination-induced human keratinocytes. The results of the present study suggested that kushenol F may be a potential therapeutic candidate for the treatment of AD via reducing TSLP levels.

Pyrroloquinoline Quinone Administration Alleviates Allergic Airway Inflammation in Mice by Regulating the JAK-STAT Signaling Pathway.

The current asthma therapies are inadequate for many patients with severe asthma. Pyrroloquinoline quinone (PQQ) is a naturally-occurring redox cofactor and nutrient that can exert a multitude of physiological effects, including anti-inflammatory and antioxidative effects. We sought to explore the effects of PQQ on allergic airway inflammation and reveal the underlying mechanisms. In vitro, the effects of PQQ on the secretion of epithelial-derived cytokines by house dust mite- (HDM-) incubated 16-HBE cells and on the differentiation potential of CD4+ T cells were investigated. In vivo, PQQ was administered to mice with ovalbumin- (OVA-) induced asthma, and lung pathology and inflammatory cell infiltration were assessed. The changes in T cell subsets and signal transducers and activators of transcription (STATs) were evaluated by flow cytometry. Pretreatment with PQQ significantly decreased HDM-stimulated thymic stromal lymphopoietin (TSLP) production in a dose-dependent manner in 16-HBE cells and inhibited Th2 cell differentiation in vitro. Treatment with PQQ significantly reduced bronchoalveolar lavage fluid (BALF) inflammatory cell counts in the OVA-induced mouse model. PQQ administration also changed the secretion of IFN-γ and IL-4 as well as the percentages of Th1, Th2, Th17, and Treg cells in the peripheral blood and lung tissues, along with inhibition the phosphorylation of STAT1, STAT3, and STAT6 while promoting that of STAT4 in allergic airway inflammation model mice. PQQ can alleviate allergic airway inflammation in mice by improving the immune microenvironment and regulating the JAK-STAT signaling pathway. Our findings suggest that PQQ has great potential as a novel therapeutic agent for inflammatory diseases, including asthma.

A population of dermal Langerin+ dendritic cells promote the inflammation in mouse model of atopic dermatitis.

Cutaneous dendritic cells (DCs) have been implicated in the pathogenesis of atopic dermatitis (AD). However, the specific role of different subsets of DCs has not been well defined. This study aimed to investigate the contributions of Langerhans cells (LCs), resident dermal Langerin+ DCs (r-Langerin+ dDCs), and newly infiltrated inflammatory dermal Langerin+ DCs (i-Langerin+ dDCs) in an AD mouse model induced by the topical application of MC903. The result showed that depletion of i-Langerin+ dDCs in DTR mice after multiple diphtheria toxin (DT) injection significantly reduced thymic stromal lymphopoietin (TSLP) production in lesions and skin inflammation alleviation. However, depletion of LCs or r-Langerin+ dDCs didn’t resulted in significant changes in skin inflammation of DTA or single DT injection-treated DTR mice compared with the wild-type (WT) mice. DT-treated DTR-WT chimeric mice with the depletion of bone marrow (BM)-derived i-Langerin+ dDCs resulted in markedly decreased skin inflammation than controls, while PBS-treated chimeric mice (DTR-WT) with only the depletion of r-Langerin+ dDCs showed inflammation comparable to that in WT mice. Furthermore, TSLP contributed to the upregulation of Langerin expression in BM-derived DCs and promoted the maturation of Langerin+ DCs. In summary, the present study demonstrated that the newly infiltrated inflammatory dermal Langerin+ DCs were essential for AD development and local TSLP production, and TSLP further promoted the production of BM-derived i-Langerin+ dDCs, which might maintain AD inflammation.

Roseburia intestinalis stimulates TLR5-dependent intestinal immunity against Crohn's disease.

SummaryBackgroundCrohn's disease (CD) is a chronic inflammatory disorder characterized by intestinal immune dysfunction. Multiple factors, including gut dysbiosis, are involved in the pathogenesis of CD. However, the effect of commensal bacteria on controlling the inflammatory response in individuals with CD remains unclear.MethodsWe detected Toll-like receptor 2 (TLR2), TLR4, and TLR5 expression in Roseburia intestinalis (R. intestinalis)-treated mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Then, we quantified the signs of colonic inflammation, the proportion of regulatory T cells (Tregs) and the expression of thymic stromal lymphopoietin (TSLP) and transforming growth factor (TGF)-β in TLR-5-deficient (Tlr5−/−) mice, bone marrow chimera mice (generated using wild-type (WT) and Tlr5−/− mice), and anti-TSLP/anti-TGFβ-treated C57BL/6 mice with colitis induced by TNBS. In vitro, we used the lipopolysaccharide (LPS)-stimulated human intestinal epithelial cell line Caco-2 as an inflammatory colon cell model treated with or without the TLR5-siRNA intervention in the presence of R. intestinalis and incubated human monocyte-derived dendritic cells (DCs) with the supernatant of Caco-2 cells. Then, we cocultured human CD4+ T cells with the aforementioned DCs to determine the differentiation of Tregs. Additionally, samples from patients with CD were collected to analyse the correlation between TLR5/TSLP/TGFβ expression and the percentage of R. intestinalis.FindingsHere, we show that R. intestinalis inhibits the development of CD by increasing the differentiation of anti-inflammatory Tregs. Mechanistically, R. intestinalis stimulates TSLP production in intestinal epithelial cells (IECs) through TLR5 but not TLR2 or TLR4. TSLP produced by IECs specifically induces the secretion of interleukin-10 (IL-10) and TGFβ from DCs, which is necessary for subsequent Treg differentiation. Consequently, the depletion of TLR5 (using Tlr5−/− mice) or inhibition of TSLP (using anti-TSLP neutralizing antibodies) attenuates the protective effect of R. intestinalis on experimental colitis in mice. Importantly, the expression of TSLP in patients with CD is positively correlated with the level of R. intestinalis.InterpretationThese findings reveal the previously unknown mechanism of R. intestinalis-mediated intestinal immune regulation, which may provide the basis for new therapeutic strategies for CD.FundingThis work was funded by the National Natural Science Foundation of China (81670504 and 81970494), the Key Project of Research and Development Plan of Hunan Province (2019SK2041) and the Changsha Municipal Natural Science Foundation (kq2014258).

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.

Mast cells instruct keratinocytes to produce thymic stromal lymphopoietin: Relevance of the tryptase/protease-activated receptor 2 axis

Thymic stromal lymphopoietin (TSLP) promotes TH2 inflammation and is deeply intertwined with inflammatory dermatoses like atopic dermatitis. The mechanisms regulating TSLP are poorly defined. We investigated whether and by what mechanisms mast cells (MCs) foster TSLP responses in the cutaneous environment. Exvivo and invivo skin MC degranulation was induced by compound 48/80 in wild-type protease-activated receptor 2 (PAR-2)- and MC-deficient mice in the presence or absence of neutralizing antibodies, antagonists, or exogenous mouse MC protease 6 (mMCP6). Primary human keratinocytes and murine skin explants were stimulated with lysates/supernatants of human skin MCs, purified tryptase, or MC lysate diminished of tryptase. Chymase and histamine were also used. TSLP was quantified by ELISA, real-time quantitative PCR, and immunofluorescence staining. Mas-related G protein-coupled receptor X2 (Mrgprb2) activation elicited TSLP in intact skin, mainly in the epidermis. Responses were strictly MC dependent and relied on PAR-2. Complementarily, TSLP was elicited by tryptase in murine skin explants. Exogenous mMCP6 could fully restore responsiveness in MC-deficient murine skin explants. Conversely, PAR-2 knockout mice were unresponsive to mMCP6 while displaying increased responsiveness to other inflammatory pathways, such as IL-1α. Indeed, IL-1α acted in concert with tryptase. In primary human keratinocytes, MC-elicited TSLP generation was likewise abolished by tryptase inhibition or elimination. Chymase and histamine did not affect TSLP production, but histamine triggered IL-6, IL-8, and stem cell factor. MCs communicate with kerationocytes more broadly than hitherto suspected. The tryptase/PAR-2 axis is a crucial component of this cross talk, underlying MC-dependent stimulation of TSLP in neighboring kerationocytes. Interference specifically with MC tryptase may offer a treatment option for disorders initiated or perpetuated by aberrant TSLP, such as atopic dermatitis.