Targeting viral induced TSLP - an airway treatment opportunity
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Abstract:
Respiratory viral infections cause exacerbations of asthma and COPD that cannot be effectively treated today. Thymic Stromal Lymphopoietin (TSLP) is an upstream epithelial cytokine linking the innate and adaptive immune system. Viral stimuli induce epithelial overexpression of TSLP in asthma and COPD. We hypothesise that TSLP switches on Th2-type inflammation in severe asthma/COPD. A deficient antiviral interferon-response has also been showed in asthmatic epithelium.
The aim of this thesis was to study effects of selected compounds on viral induced epithelial TSLP and antiviral proteins.
We show that capsazepine, a small airway relaxant, inhibits TSLP induced by the viral infection surrogate dsRNA in human bronchial epithelial cells (HBECs) from asthmatic and healthy donors. Surprisingly, simvastatin, a cholesterol-lowering compound with pleiotrophic effects, inhibits dsRNA-induced IRF3 phosphorylation and TSLP but not NFkB in HBECs from COPD-patients, healthy smokers and asthmatics. TSLP-inhibitory effects of capsazepine and simvastatin are superior to effects produced by the glucocorticoid dexamethasone. However, simvastatin, but not dexamethasone, inhibits antiviral interferon-beta (IFNβ) and IL-32. We developed a method by which repeated topical nasal dsRNA for the first time induces effects in vivo on human respiratory mucosa. We found that IFNβ, IFNλ and IL-32 were upregulated by dsRNA during but not outside birch pollen season. dsRNA challenges were below threshold for TSLP induction.
In conclusion, using HBEC we discovered that different classes of compounds were effective inhibitors of viral induced TSLP. With pharmacological tools we discovered that IRF3 phosphorylation is involved in TSLP production meaning also that difficulties arise regarding the aim of inhibiting TSLP without inhibiting interferons.
For future studies we devise novel human in vivo methods for study of pharmacology of airway antiviral protein production.Keywords:
Thymic stromal lymphopoietin
IRF3
Rhinovirus
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House dust mite impairs antiviral response in asthma exacerbation models through its effects on TLR 3
Impaired antiviral interferon expression may be involved in asthma exacerbations commonly caused by rhinovirus infections. Allergy is a known risk factor for viral-induced asthma exacerbation, but little is known whether allergens may affect interferon responses.Our hypothesis is that house dust mite (HDM) impairs viral stimulus-induced antiviral signalling.Experimental asthma exacerbations were produced in vitro in human bronchial epithelial cells (HBECs) and in mice using sequential challenges with HDM and a viral infection mimic, Poly(I:C). We examined rhinovirus pattern recognition receptors (PRRs) signalling pathways and potential mechanisms of impaired interferon response.HBECs and mice exposed to HDM prior to Poly(I:C) exhibited a reduced antiviral response compared to Poly(I:C) alone, including reduced IFN-β, IFN-λ, TLR3, RIG-I, MDA5, IRF-3 and IRF-7. Heat inactivation of HDM partially restored the TLR3-induced interferon response in vitro and in vivo. Our HBEC-data further showed that HDM directly affects TLR3 signalling by targeting the receptor glycosylation level.Direct effects of allergens such as HDM on PRRs can present as potential mechanism for defective antiviral airway responses. Accordingly, therapeutic measures targeting inhibitory effects of allergens on antiviral PRRs may find use as a strategy to boost antiviral response and ameliorate exacerbations in asthmatic patients.
Rhinovirus
TLR3
TLR7
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Epidemiological studies based on the "hygiene hypothesis" declare that the level of childhood exposure to environmental microbial products is inversely related to the incidence of allergic diseases in later life. Multiple types of immune cell-mediated immune regulation networks support the hygiene hypothesis. Epithelial cells are the first line of response to microbial products in the environment and bridge the innate and adaptive immune systems; however, their role in the hygiene hypothesis is unknown. To demonstrate the hygiene hypothesis in airway epithelial cells, we examined the effect of lipopolysaccharide (LPS; toll-like receptor 4 ligand) on the expression of the proallergic cytokines thymic stromal lymphopoietin (TSLP) and interleukin 33 (IL33) in H292 cells (pulmonary mucoepidermoid carcinoma cells). Stimulation with the TLR ligand polyI:C and human parechovirus type 1 (HPeV1) but not LPS-induced TSLP and IL33 through interferon regulatory factor 3 (IRF3) and NF-κB activity, which was further validated by using inhibitors (dexamethasone and Bay 11-7082) and short hairpin RNA-mediated gene knockdown. Importantly, polyI:C and HPeV1-stimulated TSLP and IL33 induction was reduced by LPS treatment by attenuating TANK-binding kinase 1, IRF3, and NF-κB activation. Interestingly, the basal mRNA levels of TLR signaling proteins were downregulated with long-term LPS treatment of H292 cells, which suggests that such long-term exposure modulates the expression of innate immunity signaling molecules in airway epithelial cells to mitigate the allergic response. In contrast to the effects of LPS treatment, the alarmin high-mobility group protein B1 acts in synergy with polyI:C to promote TSLP and IL33 expression. Our data support part of the hygiene hypothesis in airway epithelia cells in vitro. In addition to therapeutic targeting of TSLP and IL33, local application of non-pathogenic LPS may be a rational strategy to prevent allergies.
Thymic stromal lymphopoietin
Innate lymphoid cell
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Thymic stromal lymphopoietin
Rhinovirus
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Thymic stromal lymphopoietin
Allergic Inflammation
Interleukin 13
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Viral infection is the main cause of asthma and COPD (chronic obstructive pulmonary disease) exacerbation and accumulate inflammatory cells to airway tissue. We have reported poly I:C, a mimic product of the virus and ligand of toll-like receptor 3 (TLR3), induced inflammatory chemokines from airway epithelial cells and found prior incubation with corticosteroids diminishes the effect of TLR3 activation. In clinical practice, mild asthma is recommended as-needed budesonide (BUD) when symptoms occur following a viral infection, etc. However, many questions still surround BUD's usefulness if taken after a virus has already infected airway tissue.The aim of this study was to investigate the inhibitory effects of BUD on inflammatory cytokines induced by viral infection. Methods: Normal human bronchial epithelial (NHBE) cells were stimulated with poly I:C or infected with human rhinovirus-16 (HRV16) and BUD was added after the initial stimulation. Expression of both thymic stromal lymphopoietin (TSLP) and CCL26/eotaxin-3 was quantified by real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Knockdown study was performed. Results: Pre-or post-incubation with BUD inhibited both poly I:C- and HRV16-induced mRNAs and proteins of both thymic stromal lymphopoietin (TSLP) and CCL26 with significance. Knockdown of the glucocorticoid receptor diminished these effects of BUD. Under the same conditions of BUD's experiment, post-incubation with neither fluticasone propionate nor dexamethasone suppressed expression of both TSLP and CCL26, which induced by poly I:C.Post-addition of BUD inhibited the virus-induced TSLP and CCL26 from the airway epithelial cells. These results suggest that inhalation of BUD after viral infection has beneficial effects on asthma.Late addition of BUD may benefit among patient with viral infection and type 2 allergic airway disease such as asthma.
Thymic stromal lymphopoietin
Fluticasone propionate
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The epithelial cell–derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) initiate type 2 inflammation in allergic diseases, including asthma. However, the signaling pathway regulating these cytokines expression remains elusive. Since microRNAs are pivotal regulators of gene expression, we profiled microRNA expression in bronchial epithelial brushings from type 2–low and type 2–high asthma patients. miR-206 was the most highly expressed epithelial microRNA in type 2–high asthma relative to type 2–low asthma but was downregulated in both subsets compared with healthy controls. CD39, an ectonucleotidase degrading ATP, was a target of miR-206 and upregulated in asthma. Allergen-induced acute extracellular ATP accumulation led to miR-206 downregulation and CD39 upregulation in human bronchial epithelial cells, forming a feedback loop to eliminate excessive ATP. Airway ATP levels were markedly elevated and strongly correlated with IL-25 and TSLP expression in asthma patients. Intriguingly, airway miR-206 antagonism increased Cd39 expression; reduced ATP accumulation; suppressed IL-25, IL-33, and Tslp expression and group 2 innate lymphoid cell expansion; and alleviated type 2 inflammation in a mouse model of allergic airway inflammation. In contrast, airway miR-206 overexpression had opposite effects. Overall, epithelial miR-206 upregulates airway IL-25 and TSLP expression by targeting the CD39–extracellular ATP axis, which represents a potentially novel therapeutic target in type 2–high asthma.
Thymic stromal lymphopoietin
Innate lymphoid cell
Allergic Inflammation
Proinflammatory cytokine
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Thymic stromal lymphopoietin
Rhinovirus
Humanized mouse
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Citations (43)
Background: Possibly reflecting anti-inflammatory properties, statin treatment may ameliorate COPD exacerbations. Viral infections apparently cause TH2 type COPD exacerbations. We have shown previously that thymic stromal lymphopoietin (TSLP), a cytokine linking innate and adaptive immunity and switching on TH2 type inflammation, is overproduced in viral stimulated epithelial cells from GOLD stage IV COPD donors. Objective: Explore effects of simvastatin on viral-induced TSLP in epithelial cells from patients with GOLD stage II COPD. Methods: Primary bronchial epithelial cells, obtained by fibre optic bronchoscopy from COPD (n=4) and healthy individual (n=3) donors, were grown in 12-well plates and stimulated with a synthetic viral surrogate, double-stranded RNA (dsRNA, 10μg/ml) to induce cytokine expression (3h, RT-qPCR) and production (24 h, ELISA). Simvastatin (0,2-5μg) with or without mevalonate (13μg/ml) was added 18 h prior to dsRNA. Alternatively, dexamethasone (1μM) was added 1 h prior to dsRNA. Results: dsRNA induced TSLP, TNF-alpha and IL-8 mRNA and protein expression (p 0.05). Conclusion: Independent of the mevalonic pathway, simvastatin selectively inhibited dsRNA-induced TSLP-production in COPD cells. These data support exploration of statin treatment in viral-induced COPD exacerbations. The pharmacology of simvastatin may unravel paths of selective inhibition of TSLP-production in COPD epithelium.
Thymic stromal lymphopoietin
Salmeterol
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Background Thymic stromal lymphoproetin (TSLP) is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral) and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state. Methods Primary human bronchial epithelial cells (HBEC) from control (n = 3) and asthmatic (n = 3) donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI) conditions and treated apically with dsRNA (viral surrogate) or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC) from normal (n = 3) and asthmatic (n = 3) donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20) vs. non-asthmatic uninfected controls (n = 20). Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay. Results Our data demonstrate that: 1) Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2) TSLP exposure induces unidirectional (apical) secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3) Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1. Conclusions There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations.
Thymic stromal lymphopoietin
CCL11
Respiratory tract
Allergic Inflammation
Interleukin 13
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Capsazepine
Thymic stromal lymphopoietin
TLR3
Interleukin 8
Proinflammatory cytokine
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