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S18 Activation of nociceptin orphanin FQ (N/OFQ) – N/OFQ peptide (NOP) receptor system plays a key immunomodulatory role in asthma
  1. R Singh1,
  2. N Sullo2,
  3. M Matteis2,
  4. G Spaziano2,
  5. J McDonald1,
  6. R Saunders1,
  7. L Woodman1,
  8. K Urbanek2,
  9. A DeAngelis2,
  10. R DePalma2,
  11. R Berair1,
  12. M Pancholi1,
  13. V Mistry1,
  14. P Bradding1,
  15. F Rossi2,
  16. R Guerrini3,
  17. G Calo3,
  18. B D'Agostino2,
  19. C E Brightling1,
  20. D G Lambert1
  1. 1University Of Leichester, Leichester, UK
  2. 2SecondUniversity of Naples, Naples, Italy
  3. 3University of Ferrara, Ferrara, Italy

Abstract

Asthma is a complex heterogeneous disease characterised by variable airflow obstruction, bronchial hyper-responsiveness, airway inflammation and remodelling. The heptadecapeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide (NOP) receptor, a non-opioid member of the opioid receptor family. The role of N/OFQ-NOP system in asthma is uncertain. We sought to evaluate N/OFQ-NOP expression in healthy and asthmatic human airway tissues and relate this to an established animal model of asthma.

NOP expression in human airway cells was investigated predominantly by qRT-PCR. The functional role of N/OFQ on human airway structural and immune cells was then interrogated using a range of functional assays including proliferation,migration,collagen gel contraction and wound healing. We further investigated the functional role of N/OFQ in vivo using ovalbumin-sensitised mice.

NOP expression was detected in human airway smooth muscle cells (HASM; mean?C? = 11 ± 0.7,n = 13), bronchial epithelial cells (HBEC;mean?C? = 10 ± 0.49,n = 12), lung mast cells (mean?C? = 7 ± 0.64,n = 5) and peripheral blood eosinophils (mean?C? = 10.4 ± 1.2,n = 16). N/OFQ inhibited chemoattractant-induced migration of mast cells and eosinophils (see Figure). N/OFQ stimulated significant HBEC wound closure with 49.62 ± 3.58% (p < 0.001, n = 8) of the wound area healed relative to the control (30.88 ± 4.13%, n = 8)18 h post-wound. Similarly N/OFQ significantly promoted HASM wound closure (p < 0.01). Our findings showed that SCF-stimulated IL-8 release (4.43 ± 0.69 fold over control, p < 0.01,n = 7) was significantly inhibited by N/OFQ (3.32 ± 0.56 fold over control, p < 0.01, n = 7). Ex vivo human studies demonstrate significantly (p < 0.01) increased endogenous N/OFQ in asthmatic airways relative (sputum N/OFQ:59.02 ± 2.57 pg/ml,n = 26) to healthy airways (sputum N/OFQ:44.69 ± 0.43,n = 10) and identifies eosinophils as a potential source for these. Pre-treatment with N/OFQ was shown to significantly reduce agonist-induced bronchial hyper-responsiveness using in vitro (p < 0.01) and in vivo models (p < 0.001). Ex vivo animal studies show that N/OFQ significantly inhibits release of inflammatory mediators including IL4, IL5, IL12, IL13 and inflammatory cell recruitment including mast cells and eosinophils within the airways. Further mucus hyper-secretion was also reduced following N/OFQ pre-treatment in these models.

Abstract S18 Figure 1.

N/OFQ inhibits chemoattractant-induced migration of lung mast cells and eosinophils. Migration of HMC-l cells (through an 8- µm pore-size transwell membrane) was enhanced following stimulation With SCF (p<0.01, n = 5), CXCL10 (p<0.001, n = 5) and TNF-α stimulated ASM s/n (p<0.001, n = 8) and this was significantly inhibited by N/OFQ (p<0.01). HLMC migration induced by SCF (p<0.001, n = 7) was significantly attenuated by N/OFQ (p<0.001, n = 7). Migration of peripheral blood eosinophils (n = 6) stimulated by epithelial conditioned media (p<0.01) was significantly inhibited by N/OFQ (p<0.01). Cell counts were performed by a blinded observer and data was represented as fold change over control in log (2) scale (mean ± SEM). Statistical analysis was performed by one-Way ANOVA With Bonferroni’s multiple comparison test. (*: p value vs control; #: p value vs different relevant stimulation).

This is the first study to perform a comprehensive and complementary in vivo and in vitro study of the expression and actions of the N/OFQ-NOP system in the airways and provide evidence for a role of NOP activation in the management of asthma.

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