Investigating the Role of COX-1 and COX-2 in Toll-Like Receptor Responses

Abstract

COX is the rate-limiting enzyme in the conversion of arachidonic acid to the prostanoids. It is present in humans as two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed and involved in homeostatic functions, while COX-2 expression is mostly limited to sites of inflammation. The precise function of COX isoforms is a subject of debate, particularly with respect to nonsterodial anti-inflammatory drug (NSAID)-induced toxicity.

Toll-like receptors (TLRs) trigger the immune response following recognition of pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS), which activates TLR4. The role of COX-1 and COX-2 in responses to different TLRs is incompletely understood. The main focus of my PhD was to investigate COX-1 and COX-2 function in innate immunity, using TLR agonists in mice lacking COX-1 or COX-2. I also studied this in relation to lung inflammation using lung fibroblasts, and isolated pulmonary arteries to examine the effect of prostanoids on pulmonary vascular responses.

Lung fibroblasts isolated from COX-2-/- mice were more proliferative than lung fibroblasts from wild-type or COX-1-/- mice. They also released greater amounts of cytokines when stimulated with various TLR agonists. Human lung fibroblasts were particularly sensitive to TLR3 agonists, and cytokine release was enhanced in the presence of NSAIDs. The effect of diclofenac may have been caused by inhibition of COX-related prostaglandin (PG) E2.

In in vivo studies, Cox2 gene expression was strongly induced by TLR4 activation in all organs and less so by TLR3 activation, where induction was restricted to the spleen and stomach. Mice lacking COX-2 released higher amounts of anti-viral proteins following TLR3 activation with poly (I:C). This suggests that COX-2-specific NSAIDs may boost the anti-viral response, thus proving beneficial over traditional NSAIDs during viral infection.

In the pulmonary vasculature, I found that most prostacyclin drugs were limited by actions on EP3 receptors, but that selective peroxisome proliferator-activated receptor (PPAR)β/δ agonists were active as dilators under all conditions. Deletion of COX-1 or COX-2 affected the ability of mouse pulmonary arteries to release endothelial-derived nitric oxide but not to respond to IP or PPARβ/δ agonists. Finally, activation of adenosine monophosphate kinase (AMPK) had no direct dilator effect on pulmonary vessels. However, preliminary data suggest that pretreatment of vessels with an AMPK activator enhanced (additive) dilation induced by PPARβ/δ, indicating a potential benefit in pulmonary arterial hypertension (PAH).

In summary, my in vitro and in vivo experiments using mice lacking COX-1 or COX-2 provide insight into the role of COX in immunity and the pulmonary vasculature.