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Rhinoviral stimuli, epithelial factors and ATP signalling contribute to bronchial smooth muscle production of IL-33.

Calvén J, Akbarshahi H, Menzel M, Ayata CK, Idzko M, Bjermer L, Uller L - J Transl Med (2015)

Bottom Line: Here, we investigated the effects of epithelial-derived media and viral stimuli on IL-33 expression in human BSMCs.These effects were inhibited by dexamethasone.We suggest that epithelial-derived factors contribute to baseline BSMC IL-33 production, which is further augmented by RV infection of BSMCs and stimulation of their pathogen-recognising receptors.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Immunopharmacology, Department of Experimental Medical Science, BMC D12, Lund University, 221 84, Lund, Sweden. jenny.calven@med.lu.se.

ABSTRACT

Background: Bronchial smooth muscle cells (BSMCs) from severe asthmatics have been shown to overexpress the Th2-driving and asthma-associated cytokine IL-33. However, little is known regarding factors involved in BSMC production of IL-33. Rhinovirus (RV) infections cause asthma exacerbations, which exhibit features of Th2-type inflammation. Here, we investigated the effects of epithelial-derived media and viral stimuli on IL-33 expression in human BSMCs.

Methods: Primary human BSMCs from healthy (n = 3) and asthmatic (n = 3) subjects were stimulated with conditioned media from primary human bronchial epithelial cells (BECs), double-stranded (ds)RNA, dsRNA/LyoVec, or infected with RV. BSMCs were also pretreated with the purinergic receptor antagonist suramin. IL-33 expression was analysed by RT-qPCR and western blot and ATP levels were determined in cell supernatants.

Results: RV infection and activation of TLR3 by dsRNA increased IL-33 mRNA and protein in healthy and asthmatic BSMCs. These effects were inhibited by dexamethasone. BSMC expression of IL-33 was also increased by stimulation of RIG-I-like receptors using dsRNA/LyoVec. Conditioned media from BECs induced BSMC expression of IL-33, which was further enhanced by dsRNA. BEC-derived medium and viral-stimulated BSMC supernatants exhibited elevated ATP levels. Blocking of purinergic signalling with suramin inhibited BSMC expression of IL-33 induced by dsRNA and BEC-derived medium.

Conclusions: RV infection of BSMCs and activation of TLR3 and RIG-I-like receptors cause expression and production of IL-33. Epithelial-released factor(s) increase BSMC expression of IL-33 and exhibit positive interaction with dsRNA. Increased BSMC IL-33 associates with ATP release and is antagonised by suramin. We suggest that epithelial-derived factors contribute to baseline BSMC IL-33 production, which is further augmented by RV infection of BSMCs and stimulation of their pathogen-recognising receptors.

No MeSH data available.


Related in: MedlinePlus

Bronchial smooth muscle cell (BSMC) expression of IL-33 is induced by epithelial conditioned media and further enhanced by dsRNA. To establish an optimal concentration of epithelial conditioned media, BSMCs from healthy subjects were stimulated with different dilutions of conditioned media from either non-treated (cond med) or dsRNA-treated (dsRNA-cond med) bronchial epithelial cells and mRNA expression of IL-33 was analysed by RT-qPCR after 3 and 24 h (a, b). IL-33 expression after stimulation with 10 μg/ml dsRNA alone or in combination with epithelial conditioned media was analysed in both healthy (c, d) and asthmatic (e, f) BSMCs. Epithelial basal medium diluted 1:10 in BSMC medium was used as vehicle control. Data are presented as mean with SEM and n = 3–6 independent experiments from three BSMC donors. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 compared to vehicle if not otherwise indicated
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Fig1: Bronchial smooth muscle cell (BSMC) expression of IL-33 is induced by epithelial conditioned media and further enhanced by dsRNA. To establish an optimal concentration of epithelial conditioned media, BSMCs from healthy subjects were stimulated with different dilutions of conditioned media from either non-treated (cond med) or dsRNA-treated (dsRNA-cond med) bronchial epithelial cells and mRNA expression of IL-33 was analysed by RT-qPCR after 3 and 24 h (a, b). IL-33 expression after stimulation with 10 μg/ml dsRNA alone or in combination with epithelial conditioned media was analysed in both healthy (c, d) and asthmatic (e, f) BSMCs. Epithelial basal medium diluted 1:10 in BSMC medium was used as vehicle control. Data are presented as mean with SEM and n = 3–6 independent experiments from three BSMC donors. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 compared to vehicle if not otherwise indicated

Mentions: To address our hypothesis that epithelial mediators may induce expression of IL-33 in BSMCs, we stimulated BSMCs with conditioned media obtained from non-treated or dsRNA-treated bronchial epithelial cells. Concentration-dependent effects on IL-33 mRNA expression were induced in healthy BSMCs at 3 h (Fig. 1a) and further augmented at 24 h (Fig. 1b). Healthy BSMCs were also stimulated directly with dsRNA, either alone or in combination with epithelial conditioned media in an optimal concentration of 1:10. dsRNA alone induced modest expression of IL-33 mRNA at 3 h (Fig. 1c), which was more pronounced at 24 h (Fig. 1d). At 24 h, direct stimulation of BSMCs with dsRNA in combination with conditioned media from either non-treated or dsRNA-treated epithelial cells caused an additive induction of IL-33 expression (Fig. 1d). Since BSMCs are known to be in juxtaposition with the bronchial epithelium in asthma, we investigated the effect of epithelial conditioned media also on BSMCs from asthmatic subjects. Similar to in healthy BSMCs, IL-33 expression was induced by dsRNA and/or conditioned media at 3 h (Fig. 1e) and further increased at 24 h (Fig. 1f). Although no statistical significance could be achieved, the observed IL-33 responses at 24 h tended to be more pronounced in BSMCs from asthmatic subjects (Fig. 1f) compared with healthy BSMCs (Fig. 1d). Interestingly however, in contrast to the observed effect in healthy BSMCs (Fig. 1a–d), IL-33 expression in asthmatic BSMCs at 24 h was more increased after stimulation with conditioned medium from dsRNA-treated epithelial cells compared to conditioned medium from non-treated epithelial cells (Fig. 1f).Fig. 1


Rhinoviral stimuli, epithelial factors and ATP signalling contribute to bronchial smooth muscle production of IL-33.

Calvén J, Akbarshahi H, Menzel M, Ayata CK, Idzko M, Bjermer L, Uller L - J Transl Med (2015)

Bronchial smooth muscle cell (BSMC) expression of IL-33 is induced by epithelial conditioned media and further enhanced by dsRNA. To establish an optimal concentration of epithelial conditioned media, BSMCs from healthy subjects were stimulated with different dilutions of conditioned media from either non-treated (cond med) or dsRNA-treated (dsRNA-cond med) bronchial epithelial cells and mRNA expression of IL-33 was analysed by RT-qPCR after 3 and 24 h (a, b). IL-33 expression after stimulation with 10 μg/ml dsRNA alone or in combination with epithelial conditioned media was analysed in both healthy (c, d) and asthmatic (e, f) BSMCs. Epithelial basal medium diluted 1:10 in BSMC medium was used as vehicle control. Data are presented as mean with SEM and n = 3–6 independent experiments from three BSMC donors. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 compared to vehicle if not otherwise indicated
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Related In: Results  -  Collection

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Fig1: Bronchial smooth muscle cell (BSMC) expression of IL-33 is induced by epithelial conditioned media and further enhanced by dsRNA. To establish an optimal concentration of epithelial conditioned media, BSMCs from healthy subjects were stimulated with different dilutions of conditioned media from either non-treated (cond med) or dsRNA-treated (dsRNA-cond med) bronchial epithelial cells and mRNA expression of IL-33 was analysed by RT-qPCR after 3 and 24 h (a, b). IL-33 expression after stimulation with 10 μg/ml dsRNA alone or in combination with epithelial conditioned media was analysed in both healthy (c, d) and asthmatic (e, f) BSMCs. Epithelial basal medium diluted 1:10 in BSMC medium was used as vehicle control. Data are presented as mean with SEM and n = 3–6 independent experiments from three BSMC donors. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 compared to vehicle if not otherwise indicated
Mentions: To address our hypothesis that epithelial mediators may induce expression of IL-33 in BSMCs, we stimulated BSMCs with conditioned media obtained from non-treated or dsRNA-treated bronchial epithelial cells. Concentration-dependent effects on IL-33 mRNA expression were induced in healthy BSMCs at 3 h (Fig. 1a) and further augmented at 24 h (Fig. 1b). Healthy BSMCs were also stimulated directly with dsRNA, either alone or in combination with epithelial conditioned media in an optimal concentration of 1:10. dsRNA alone induced modest expression of IL-33 mRNA at 3 h (Fig. 1c), which was more pronounced at 24 h (Fig. 1d). At 24 h, direct stimulation of BSMCs with dsRNA in combination with conditioned media from either non-treated or dsRNA-treated epithelial cells caused an additive induction of IL-33 expression (Fig. 1d). Since BSMCs are known to be in juxtaposition with the bronchial epithelium in asthma, we investigated the effect of epithelial conditioned media also on BSMCs from asthmatic subjects. Similar to in healthy BSMCs, IL-33 expression was induced by dsRNA and/or conditioned media at 3 h (Fig. 1e) and further increased at 24 h (Fig. 1f). Although no statistical significance could be achieved, the observed IL-33 responses at 24 h tended to be more pronounced in BSMCs from asthmatic subjects (Fig. 1f) compared with healthy BSMCs (Fig. 1d). Interestingly however, in contrast to the observed effect in healthy BSMCs (Fig. 1a–d), IL-33 expression in asthmatic BSMCs at 24 h was more increased after stimulation with conditioned medium from dsRNA-treated epithelial cells compared to conditioned medium from non-treated epithelial cells (Fig. 1f).Fig. 1

Bottom Line: Here, we investigated the effects of epithelial-derived media and viral stimuli on IL-33 expression in human BSMCs.These effects were inhibited by dexamethasone.We suggest that epithelial-derived factors contribute to baseline BSMC IL-33 production, which is further augmented by RV infection of BSMCs and stimulation of their pathogen-recognising receptors.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Immunopharmacology, Department of Experimental Medical Science, BMC D12, Lund University, 221 84, Lund, Sweden. jenny.calven@med.lu.se.

ABSTRACT

Background: Bronchial smooth muscle cells (BSMCs) from severe asthmatics have been shown to overexpress the Th2-driving and asthma-associated cytokine IL-33. However, little is known regarding factors involved in BSMC production of IL-33. Rhinovirus (RV) infections cause asthma exacerbations, which exhibit features of Th2-type inflammation. Here, we investigated the effects of epithelial-derived media and viral stimuli on IL-33 expression in human BSMCs.

Methods: Primary human BSMCs from healthy (n = 3) and asthmatic (n = 3) subjects were stimulated with conditioned media from primary human bronchial epithelial cells (BECs), double-stranded (ds)RNA, dsRNA/LyoVec, or infected with RV. BSMCs were also pretreated with the purinergic receptor antagonist suramin. IL-33 expression was analysed by RT-qPCR and western blot and ATP levels were determined in cell supernatants.

Results: RV infection and activation of TLR3 by dsRNA increased IL-33 mRNA and protein in healthy and asthmatic BSMCs. These effects were inhibited by dexamethasone. BSMC expression of IL-33 was also increased by stimulation of RIG-I-like receptors using dsRNA/LyoVec. Conditioned media from BECs induced BSMC expression of IL-33, which was further enhanced by dsRNA. BEC-derived medium and viral-stimulated BSMC supernatants exhibited elevated ATP levels. Blocking of purinergic signalling with suramin inhibited BSMC expression of IL-33 induced by dsRNA and BEC-derived medium.

Conclusions: RV infection of BSMCs and activation of TLR3 and RIG-I-like receptors cause expression and production of IL-33. Epithelial-released factor(s) increase BSMC expression of IL-33 and exhibit positive interaction with dsRNA. Increased BSMC IL-33 associates with ATP release and is antagonised by suramin. We suggest that epithelial-derived factors contribute to baseline BSMC IL-33 production, which is further augmented by RV infection of BSMCs and stimulation of their pathogen-recognising receptors.

No MeSH data available.


Related in: MedlinePlus