The immunomodulation of dynorphin 1-17 and its biotransformation fragments on inflammatory signalling pathways

Abstract

Inflammation is a complex physiological process that involves host defense mechanisms in response to the intrusion of harmful external stimuli. Uncoordinated inflammatory responses, however, are the underlying pathophysiological cause of many chronic diseases. Inflammation is characterised by the burgeoning migration of leukocytes to the point of injury and subsequent release of pro-nociceptive mediators, generating inflammatory pain. Dynorphin 1-17 (DYN 1-17) is endogenously produced and released from leukocytes upon stimulation by local inflammatory factors in the inflamed area. This opioid peptide primarily binds to kappa-opioid receptor (KOR) to produce analgesia. Under inflammatory milieu, DYN 1-17 undergoes spontaneous degradation, yielding a variety of opioid and non-opioid fragments that may have significant implications in inflammation. The underlying cellular effects of these fragments remain unclear. This thesis seeks to explore potential mechanistic insights into selected major DYN 1-17 fragments, identified from a previous biotransformation study of DYN 1-17 in rodent inflamed tissue. This thesis examines the DYN 1-17 fragments modulation of intracellular signals associated with inflammation and thereby, gain an insight into novel therapeutic targets through exploring these endogenous mechanisms.Utilising THP-1 macrophages as an in vitro model of inflammation, this thesis begins with a semiquantitative assessment of nuclear factor-kappa B/p65 (NF-kB/p65) translocation, a major transcription factor that regulates genes responsible for immune and inflammatory responses. The findings presented in Chapter Three of this thesis demonstrated that DYN 1-17 and selected major fragments (DYN 1-6, 1-7, 1-9, 1-10, 1-11, 3-14, 6-12, 2-17 and 7-17) significantly attenuated NF- kB/p65 nuclear translocation induced by lipopolysaccharide (LPS), with the greatest reduction observed with DYN 1-7 at 10 nM. A selective KOR antagonist, ML-190, was used to examine KOR involvement in this inhibitory action. ML-190 significantly reversed the inhibition of NF-kB/p65 translocation produced by DYN 1-17, DYN 1-6, DYN 1-7 and DYN 1-9, but not DYN 1-10 and DYN 1-11.Cytokine production is linked as a downstream process to NF-kB activation; hence it is necessary to evaluate the ability of DYN 1-17 and selected biotransformation fragments in the modulation of IL- 1b and TNF-a release in differentiated THP-1 cells, as presented in Chapter Four, to gain an insight into the effects on major pro-inflammatory cytokines. DYN 1-17 and the fragments, demonstrated differential modulatory effects on LPS-induced release of IL-1b and TNF-a. DYN 1-7 and DYN 1-6, inhibited and elevated the secretion of both cytokines, respectively, in a concentration-dependent manner (10-11 to 10-7 M). DYN 1-17, however, only inhibited IL-1b release from differentiated nTHP-1 cells and had no effect on TNF-a secretion. Intriguingly, DYN 3-14 at 10-17 to 10-11 M demonstrated significant inhibition on IL-1b release and paradoxically increased TNF-a levels at 10-11 to 10-7 M. Subsequent antagonism with ML-190 only reversed the effects produced by DYN 1-17, DYN 1-6 and DYN 1-7 on IL-1b release at 10-7 M, 10-9 M and 10-11 M, respectively. Likewise, the effects of DYN 1-6 (10-7 M) and DYN 1-7 (10-11 M and 10-9 M) on TNF-a release were also blocked by ML-190. Taken together, the findings from Chapter Three and Four, collectively demonstrated that DYN 1-17 and a select of biotransformation fragments involve in the regulation of inflammatory response through inhibition of NF-kB/p65 translocation and modulation of cytokine release via KOR-dependent and -independent pathways.Chapter Five of this thesis investigates the effects of DYN 1-17 and selected biotransformation fragments at the Toll-like receptor 4 (TLR4), implicated in LPS-induced activation of NF-kB signaling pathway. Of these fragments, only DYN 3-14 showed significant concentration-dependent attenuation on TLR4 activation in HEK-Bluet-hTLR4 cells, albeit 300- old lower than LPS-RS, a potent TLR4 antagonist. This result thus describes a potential role for DYN 3-14 as an antagonist at TLR4, and as a consequence, highlights its involvement in regulating inflammatory signals through a non-opioid mechanism.In conjunction with the immunomodulatory effects seen in select fragments of DYN 1-17, Chapter Six of this thesis aims to extend previous findings of DYN 1-17 biotransformation study in rodent inflamed tissues, by further characterising the degradation of DYN 1-17 in the context of human inflammatory disease, chronic rhinosinusitis (CRS). Data obtained by LC-MS analysis revealed a similar range of fragments in inflamed human nasal tissue homogenates from those found in incubations with rodent inflamed paw tissues. Intriguingly, DYN 3-14 and DYN 1-5 were the major hydrolysis fragments produced until the end of the incubation period, suggesting that these fragments may possess important roles in the immunoregulation and therefore, may be useful as potential therapeutic agents for the treatment CRS.In conclusion, the findings presented within this thesis collectively highlight the pharmacological differences that exist between DYN 1-17 and its biotransformation fragments on inflammatory signalling pathways, through opioid- and non-opioid-dependent pathways. Furthermore, this thesis postulates that the effects of DYN 1-17 in inflammation may not be solely dependent on the parent molecule, but may also be attributed to the modulatory effects of other major biotransformation fragments.