The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.

In many equine inflammatory disease states, neutrophil activities, such as adhesion, migration, and reactive oxygen species (ROS) production become dysregulated. Dysregulated neutrophil activation causes tissue damage in horses with asthma, colitis, laminitis, and gastric glandular disease. Non-steroidal anti-inflammatory drugs do not adequately inhibit neutrophil inflammatory functions and can lead to dangerous adverse effects. Therefore, novel therapies that target mechanisms of neutrophil-mediated tissue damage are needed. One potential neutrophil-targeting therapeutic is the PGE1 analog, misoprostol. Misoprostol is a gastroprotectant that induces intracellular formation of the secondary messenger molecule cyclic AMP (cAMP), which has been shown to have anti-inflammatory effects on neutrophils. Misoprostol is currently used in horses to treat NSAID-induced gastrointestinal injury; however, its effects on equine neutrophils have not been determined. We hypothesized that treatment of equine neutrophils with misoprostol would inhibit equine neutrophil adhesion, migration, and ROS production, in vitro. We tested this hypothesis using isolated equine peripheral blood neutrophils collected from 12 healthy adult teaching/research horses of mixed breed and gender. The effect of misoprostol treatment on adhesion, migration, and respiratory burst of equine neutrophils was evaluated via fluorescence-based adhesion and chemotaxis assays, and luminol-enhanced chemiluminescence, respectively. Neutrophils were pretreated with varying concentrations of misoprostol, vehicle, or appropriate functional inhibitory controls prior to stimulation with LTB4, CXCL8, PAF, lipopolysaccharide (LPS) or immune complex (IC). This study revealed that misoprostol pretreatment significantly inhibited LTB4-induced adhesion, LTB4-, CXCL8-, and PAF-induced chemotaxis, and LPS-, IC-, and PMA-induced ROS production in a concentration-dependent manner. This data indicate that misoprostol-targeting of E-prostanoid (EP) receptors potently inhibits equine neutrophil effector functions in vitro. Additional studies are indicated to further elucidate the role of EP receptors in regulating neutrophil function. Overall, our results suggest misoprostol may hold promise as a novel anti-inflammatory therapeutic in the horse.

Pro-inflammatory cytokines including tumor necrosis factor α (TNFα), IL-1β, IL-6, and IL-8 are potent immune mediators that exacerbate multiple equine diseases such as sepsis and laminitis. Unfortunately, safe and effective cytokine-targeting therapies are lacking in horses; therefore, novel mechanisms of inhibiting cytokine production are critically needed. One potential mechanism for inhibiting cytokine synthesis is elevation of intracellular cyclic AMP (cAMP). In human leukocytes, intracellular cAMP production is induced by activation of E-prostanoid (EP) receptors 2 and 4. These receptors can be targeted by the EP2/4 agonist and prostaglandin E1 analog, misoprostol. Misoprostol is currently used as a gastroprotectant in horses but has not been evaluated as a cytokine-targeting therapeutic. Thus, we hypothesized that misoprostol treatment would inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-stimulated equine leukocytes in an in vitro inflammation model. To test this hypothesis, equine leukocyte-rich plasma (LRP) was collected from 12 healthy adult horses and used to model LPS-mediated inflammatory signaling. LRP was treated with varying concentrations of misoprostol either before (pretreated) or following (posttreated) LPS stimulation. LRP supernatants were assayed for 23 cytokines using an equine-specific multiplex bead immunoassay. Leukocytes were isolated from LRP, and leukocyte mRNA levels of four important cytokines were evaluated via RT-PCR. Statistical differences between treatments were determined using one-way RM ANOVA (Holm–Sidak post hoc testing) or Friedman’s RM ANOVA on Ranks (SNK post hoc testing), where appropriate (p < 0.05, n = 3–6 horses). These studies revealed that misoprostol pre- and posttreatment inhibited LPS-induced TNFα and IL-6 protein production in equine leukocytes but had no effect on IL-8 protein. Interestingly, misoprostol pretreatment enhanced IL-1β protein synthesis following 6 h of LPS stimulation, while misoprostol posttreatment inhibited IL-1β protein production after 24 h of LPS stimulation. At the mRNA level, misoprostol pre- and posttreatment inhibited LPS-induced TNFα, IL-1β, and IL-6 mRNA production but did not affect IL-8 mRNA. These results indicate that misoprostol exerts anti-inflammatory effects on equine leukocytes when applied before or after a pro-inflammatory stimulus. However, the effects we observed were cytokine-specific and sometimes differed at the mRNA and protein levels. Further studies are warranted to establish the inhibitory effects of misoprostol on equine cytokine production in vivo.

Background: Misoprostol is an E prostanoid (EP) 2, 3 and 4 receptor agonist that is anecdotally used to treat and prevent NSAID-induced GI injury in horses. Misoprostol elicits anti-inflammatory effects in vivo in men and rodents, and inhibits TNFa production in equine leucocytes in vitro. Objective: Define the pharmacokinetic parameters of oral misoprostol in horses, and determine the inhibitory effect of oral misoprostol administration on equine leucocyte TNFa production in an ex vivo inflammation model. Study design: Pharmacokinetic study, ex vivo experimental study. Methods: Six healthy adult horses of mixed breeds were used. In phase one, horses were given 5 lg/kg misoprostol orally, and blood was collected at predetermined times for determination of misoprostol free acid (MFA) by UHPLC-MS/MS. Pharmacokinetic parameters were calculated. In phase two, horses were dosed as in phase one, and blood was collected at T0, 0.5, 1 and 4 h following misoprostol administration for leucocyte isolation. Leucocytes were stimulated with 100 ng/mL LPS, and TNFa mRNA concentrations were determined via quantitative real-time PCR. Results: About 5 lg/kg oral misoprostol produced a rapid time to maximum concentration (T max) of 23.4 AE 2.4 min, with a maximum concentration (C max) of 0.29 AE 0.07 ng/mL and area under the curve (AUC 0˰) of 0.4 AE 0.12 h ng/mL. LPS stimulation of equine leucocytes ex vivo significantly increased TNFa mRNA concentrations, and there was no significant effect of misoprostol even at the T max. Main limitations: Only a single dose was used, and sample size was small. Conclusions: Misoprostol is rapidly absorbed following oral administration in horses, and a single 5 lg/kg dose had no significant inhibitory effect on ex vivo LPS-stimulated TNFa mRNA production in leucocytes. Further studies analysing different dosing strategies, including repeat administration or combination with other anti-inflammatory drugs, are warranted.

Inhibition of prostaglandin E2 (PGE2) production effectively limits inflammation in horses, however nonspecific prostaglandin blockade via cyclooxygenase (COX) inhibition elicits deleterious gastrointestinal side effects in equine patients. Thus, more selective PGE2 targeting therapeutics are needed to treat inflammatory disease in horses. One potential target is microsomal prostaglandin E-synthase-1 (mPGES-1), which is the terminal enzyme downstream of COX-2 in the inducible PGE2 synthesis cascade. This enzyme has yet to be studied in equine leukocytes, which play a pivotal role in equine inflammatory disease. The objective of this study was to determine if mPGES-1 is a PGE2-selective anti-inflammatory target in equine leukocytes. To evaluate this objective, leukocyte-rich plasma (LRP) was isolated from equine whole blood collected via jugular venipuncture of six healthy adult horses of mixed breeds and genders. LRP was primed with granulocyte-monocyte colony-stimulating factor (GM-CSF) and stimulated with lipopolysaccharide (LPS) in the presence or absence of an mPGES-1 inhibitor (MF63), a COX-2 inhibitor (NS-398), or a nonselective COX inhibitor (indomethacin). Following treatment, mPGES-1 and COX-2 mRNA and protein levels were measured via qPCR and western blot, respectively, and PGE2, thromboxane (TXA2) and prostacyclin (PGI2) levels were measured in cellular supernatants via ELISA. This study revealed that LPS significantly increased mPGES-1 mRNA, but not protein levels in equine LRP as measured by qPCR and western blot, respectively. In contrast, COX-2 mRNA and protein were coordinately induced by LPS. Importantly, treatment of LPS-stimulated leukocytes with indomethacin and NS-398 significantly reduced extracellular concentrations of multiple prostanoids (PGE2, TXA2 and PGI2), while the mPGES-1 inhibitor MF63 selectively inhibited PGE2 production only. mPGES-1 inhibition also preserved higher basal levels of PGE2 production when compared to either COX inhibitor, which might be beneficial in a clinical setting. In conclusion, this work identifies mPGES-1 as a key regulator of PGE2 production and a PGE2-selective target in equine leukocytes. This study demonstrates that mPGES-1 is a potentially safer and effective therapeutic target for treatment of equine inflammatory disease when compared to traditional non-steroidal anti-inflammatory drugs.