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Figure f4.5: Endogenously generated PGE2, induced by LPS, acts via the EP4 receptor to suppress TNFα secretion. Top Panel: LPS-stimulated WB was assayed for the presence of PGE2 at time-points ranging from 1h-24h via ELISA (n=1 HV, 2 technical replicates, assayed (ELISA) in duplicate). Columns represent mean values. Left Panel: Both NSAID (indomethacin 10μM) and EP4 antagonist (MF498 1μM) addition to LPS-stimulated WB leads to enhanced TNFα accumulation (columns represent mean values of n=1 2 technical replicates, assayed (ELISA) in duplicate). Right Panel: Indomethacin, but not MF498, significantly suppresses PGE2 generation ex-vivo (columns as per Left Panel).

4.3.3 Specificity and restoration of PGE2 immunosuppressive effect

At physiologically relevant concentrations PGI2, in addition to PGE2 suppresses ex vivo TNFα secretion in the LPS-stimulated WB model. Carbaprostacyclin, a stable analog of PGI2, was found to inhibit TNFα release with an IC50 of 590pM (95% CI 444-785pM) in contrast to 427pM (95% CI 307-592pM) when directly compared to PGE2. Carbocyclic thromboxane A2, a stable analog of TXA2, was found to have the nearest properties possessing an IC50 of 184nM (95% CI 5.7nM to 5.8μM) (Figure

Figure f4.6: Comparative ability of alternate COX-derived prostanoids to elicit immunosuppression in the ex-vivo WB assay. Dose-dependent inhibition of LPS-stimulated (1ng/ml) TNFα release by PGD2, PGF2α, PGI2 (carbaprostacyclin, a stable analog of PGI2) and TXA2 (carbocyclic thromboxane A2, a stable analog of TXA2). WB was pre-treated with indomethacin 10μM to prevent exogenous prostanoid production. Data points represent the average of n=3 HV (2 technical repeats/volunteer, each assayed in duplicate [ELISA]), whiskers display SD. IC50’s were determined via four-parameter dose-response curves.

IFN-γ and GM-CSF represent recognised immunoadjuvant therapies, capable of restoring ex vivo cytokine secretion in immunocompromised CI patients. As such their ability to reverse PGE2-mediated cytokine secretion was assessed. Both agents were found to increase mean TNFα concentration (LPS 5482pg/mL, IFN-γ 8826pg/mL, GM-CSF 8666pg/mL) in assayed supernatants after LPS-stimulation but did not induce TNFα release when administered independently (Figure f4.7, Left Panel). They were found to ‘reverse’ PGE2 1ng/ml-mediated suppression of TNFα release to baseline (LPS 1ng/mL alone), however the co-administration of an EP4-recpetor antagonist (MF498 1μM) was found to further increase observed TNFα concentrations (Figure f4.7, Right Panel). This indicates that IFN-γ and GM-CSF act via pathways independent of the PGE2-EP4-cAMP axis and that they may represent complementary immunorestorative therapeutic strategies.

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0 1000 2000 3000 4000

log[Eicosanoid], M

TNFa pg/ml

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PGD2 PGF2α PGI2 TXA2

Figure f4.7: Comparison of established immunostimulatory agents to EP4 receptor antagonism in restoring LPS-stimulated WB TNFα secretion. Left Panel: WB was co-incubated IFNγ (100ng/ml, chess-board pattern) or GM-CSF (50ng/ml, cross-hatch pattern) and either stimulated with LPS-of not. Both agents led to significantly greater TNFα release (p<0.001). Right Panel: WB was co-incubated with PGE2 (1ng/ml) in the presence or absence of IFNγ (100ng/ml, chess-board pattern) or GM-CSF (50ng/ml, cross-hatch pattern) with the addition an EP4 receptor antagonist (MF498 1μM) where indicated. LPS 1ng/ml was used to stimulate the blood and supernatants were removed after 6hrs. WB was pre-treated with indomethacin 10μM to prevent any exogenous prostanoid production in both experiments. Bars represent the mean of n=1 HV, with 2 technical repeats/condition, each assayed (ELISA) 4 times.

4.4: DISCUSSION

PGE2 is capable of inducing monocyte deactivation at physiological-relevant concentrations with an IC50 ~300pg/mL, as determined by ex vivo WB LPS-stimulated TNFα release. The EP4 receptor appears central to this process, selective EP4 agonists and antagonists either mimicking the effect of PGE2 or abrogating it.

Like the EP2 receptor, EP4 is GS-coupled, cognate-binding serving to increase the intracellular concentration of cAMP⁴⁰⁹: a mechanism that has been repeatedly associated with the immunosuppressive action of PGE2 in vivo and in vitro199,250,410. Supportive evidence that this pathway is responsible for the reduction in observed TNFα release was provided through the replication of PGE2’s action by forskolin, a cell permeable diterpenoid that directly activates adenylyl cyclase⁴¹¹, and it’s augmentation by rolipram, a selective phosphodiesterase 4 inhibitor that reduces the catabolism of biosynthesised cAMP^412,413. LPS-stimulated but not un-stimulated WB was additionally observed to release PGE2, acting as an autocrine/paracrine immunomodulator. To prevent endogenously generated eicosanoids impairing interpretation of the effect of exogenously added PGE or alternate

immunomodulatory agents pre-treatment with COX-inhibitors (indomethacin 10μM) was instituted.

Preliminary evidence that reversal of PGE2-mediated monocyte deactivation could represent a complimentary immunorestorative strategy to those already under investigation was obtained. Both GM-CSF and IFN-γ, and NSAIDs and EP4 antagonists were observed to independently increase TNFα release in the WB model, the latter seemingly due to ablation or blockade of the aforementioned endogenously released PGE2. Whilst GM-CSF and IFN-γ, appeared to ‘antagonise’

the effects of PGE2 - restoring TNFα concentration to baseline (LPS alone) when exogenous PGE2 was added - the addition of an EP4 receptor antagonist led to a further increase in TNFα release. This indicates that the mechanism through which GM-CSF and IFN-γ work is separate to the PGE2-EP4-cAMP axis described above.

As such, ‘anti-PGE2’ therapy may act synergistically with these established immunostimulatory agents, affording greater clinical gain.

Of note, PGE2 was not the only COX-derived prostanoid to suppress WB TNFα release at physiological concentrations, PGI2 possessing a similar IC50. This is not unexpected: the IP receptor also being Gs-coupled and therefore potentially exerting this action via a common cAMP-protein kinase A axis. PGI2 is now thought to play a regulatory or anti-inflammatory role in several disease states (which may be beneficial or detrimental), primarily through modulation of dendritic cell (DC) function (see⁴¹⁴ for review). Equally, previous authors have described how synthetic analogs of PGI2 inhibit macrophage and monocyte-derived DC pro-inflammatory cytokine release, and thus the data described here may represent an extension of these findings^415,416. Care must be taken however in translating such results to the in vivo setting, artificially long-lasting analogs (in this case carbaprostacyclin) potentially being able to generate alterations in cellular function their more fleeting biological cousins cannot.

Additionally, PGE2-induced monocyte deactivation may not however be absolute.

10ng/mL PGE2 was observed to significantly reduce TNFα and IL-6 release but non-significantly increase IL-8 and IL-1β. Whilst we and others have focused on TNFα due to its central place in the inflammatory cascade and the proven link between alteration in LPS-stimulated release and clinically meaningful outcomes, it is clear that PGE2 may not universally suppress monocyte function, but phenotypically bias it.

This may necessitate a more nuanced view than one of PGE2 being entirely