The natural dual cyclooxygenase and 5-lipoxygenase inhibitor flavocoxid is protective in EAE through effects on Th1/Th17 differentiation and macrophage/microglia activation

https://doi.org/10.1016/j.bbi.2015.11.002Get rights and content

Highlights

  • The pharmaconutrient flavocoxid (FVC), has therapeutic effects in EAE.

  • FVC reduced COX2, 5-LO, IL12, IL23, IL17, IFNγ and increased IL10 in spinal cord of EAE mice.

  • FVC reduced MHCII, CD40, CD80, CD86, COX2, 5-LO, IL12 and IL6, and increased IL-10 in primary macrophages.

  • FVC promoted Arg1, Ym1, CD206, TGFβ1, IL10 and reduced iNOS, IL-12 in macrophages and microglia.

  • FVC directly inhibited Th1/Th17 differentiation in polarizing and non-polarizing conditions.

Abstract

Prostaglandins and leukotrienes, bioactive mediators generated by cyclooxygenases (COX) and 5-lipoxygenase (5-LO) from arachidonic acid, play an essential role in neuroinflammation. High levels of LTB4 and PGE2 and increased expression of COX and 5-LO, as well as high expression of PGE2 receptors were reported in multiple sclerosis (MS) patients and in experimental autoimmune encephalomyelitis (EAE). Prostaglandins and leukotrienes have an interdependent and compensatory role in EAE, which led to the concept of therapy using dual COX/5-LO inhibitors. The plant derived flavocoxid, a dual COX/5-LO inhibitor with anti-inflammatory and antioxidant properties, manufactured as a prescription pharmaconutrient, was reported to be neuroprotective in models of transient ischemic stroke and brain injury. The present study is the first report on prophylactic and therapeutic effects of flavocoxid in EAE. The beneficial effects correlate with reduced expression of proinflammatory cytokines and of COX2 and 5-LO in spinal cords and spleens of EAE mice. The protective mechanisms include: 1. reduction in expression of MHCII/costimulatory molecules and production of proinflammatory cytokines; 2. promotion of the M2 phenotype including IL-10 expression and release by macrophages and microglia; 3. inhibition of Th1 and Th17 differentiation through direct effects on T cells. The direct inhibitory effect on Th1/Th17 differentiation, and promoting the development of M2 macrophages and microglia, represent novel mechanisms for the flavocoxid anti-inflammatory activity. As a dual COX/5-LO inhibitor with antioxidant properties, flavocoxid might be useful as a potential therapeutic medical food agent in MS patients.

Introduction

Arachidonic acid (AA), released from plasma membrane primarily by the inducible cytosolic phospholipase A2 (cPLA-2α), is enzymatically processed into eicosanoids, including prostaglandins (PG), leukotrienes (LT), thromboxanes (TX) and lipoxins (LX). PG and LT generated respectively by cyclooxygenases (COX1/2) and 5-lipoxygenase (5-LO), are major players in inflammatory processes, including neuroinflammation (Bazinet and Laye, 2014, Minghetti, 2004, Ong et al., 2015). Increased CNS expression of COX2 and 5-LO was reported in cerebral ischemia, Alzheimer’s and Parkinson’s disease, traumatic brain injury, spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS) (Phillis et al., 2006).

Multiple sclerosis (MS) is a major CNS autoimmune disease characterized by T cell infiltration in the CNS, demyelinating lesions and axonal damage/loss. Increased PGE2, LTB4/C4 levels and 5-LO expression were observed in the CSF and lesions of MS patients (Kihara et al., 2009, Neu et al., 2002, Whitney et al., 2001). Similar to MS, increased expression of COX2 and 5-LO, and of the inducible PGE2 synthase mPGES1, were observed in the CNS of EAE mice (Kihara et al., 2009, Whitney et al., 2001). The role of AA-derived bioactive mediators in EAE was confirmed in studies showing significant protection in genetically or pharmacologically cPLA2α ablated mice. However, pharmacological inhibition of either COX1/2 or 5-LO showed only partial beneficial effects (Marusic et al., 2008), leading to the conclusion that both COX and 5-LO pathways contribute to EAE, and that shunting between the two pathways may occur in the event of silencing one of them. The fact that the pathways leading to the generation of proinflammatory PG and LT are interdependent and compensatory led to the concept of dual COX/5-LO inhibitors as a preferable therapeutic approach.

The development of dual COX/5-LO inhibitors is still at an early stage. Licofelone, a synthetic derivative of pyrrolizine, and flavocoxid (marketed as Limbrel™), a plant-derived dual COX/5-LO inhibitor, showed efficacy in clinical trials for osteoarthritis (Levy et al., 2009, Raynauld et al., 2009). In the present study we focused on flavocoxid (FVC), a dual COX/5-LO inhibitor with anti-inflammatory and antioxidant properties, manufactured as a >90% purified mixture of baicalin flavonoids and catechin flavans extracted from Scutellaria baicalensis and Acacia catechu, respectively (Burnett et al., 2011, Burnett et al., 2007). FVC has been reported to be neuroprotective in models of transient ischemic stroke and kainic acid-induced brain injury, primarily through its anti-inflammatory and antioxidant properties (Minutoli et al., 2015, Singh and Chopra, 2014), and to exert anti-inflammatory effects in models of Duchenne muscular dystrophy, experimental pancreatitis, and sepsis [reviewed in (Bitto et al., 2014)].

Presently, there is little information on the effects of dual COX/5-LO inhibitors in EAE/MS. Two preliminary studies, using BW755c, a prototypic dual inhibitor, and more recently phenidone (1-phenyl-3-pyrazolidinone), showed EAE amelioration without addressing the cellular/molecular mechanisms (Moon et al., 2005, Simmons et al., 1992). In the present study we report on the effects of FVC in EAE and show for the first time that FVC affects Th1/Th17 differentiation, reduces expression of MHCII and costimulatory molecules on macrophages and promotes a distinct M2 phenotype in both primary macrophages and microglia. These results suggest that the dual inhibitor FVC affects both innate and adaptive immunity, primarily through effects on macrophages, microglia and CD4 T cells.

Section snippets

Mice

C57BL/6 mice (6–10 weeks old; females) were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in the Temple University School of Medicine animal facility (Philadelphia, PA) under pathogen-free conditions. Mice were handled and housed in accordance with the guidelines of the Temple University Animal Care and Use Committee.

Reagents

Lipopolysaccharide (LPS) (Escherichia coli O55:B5), pertussis toxin (PT), phorbol myristate acetate (PMA) and ionomycin, complete Freund’s adjuvant were

Flavocoxid treatment reduces EAE severity

To assess the effect of flavocoxid (FVC) in EAE, we administered FVC in a prophylactic and therapeutic treatment regimen and monitored disease severity. FVC was tested initially at two concentrations, 20 and 100 mg/kg. We observed a reduction in EAE scores at both concentrations, with more efficacy at 100 mg/kg (results not shown for 20 mg/kg). This is in agreement with the results reported for FVC in a rat model of ischemic stroke, where 100 mg/kg FVC showed the best recovery in terms of

Discussion

The plant-derived dual COX/5-LO inhibitor flavocoxid (FVC), which has both anti-inflammatory and antioxidant properties, was reported to be beneficial in models of Duchenne muscular dystrophy, experimental pancreatitis, and sepsis [reviewed in (Bitto et al., 2014)], and neuroprotective in models of transient ischemic stroke and kainic acid-induced brain injury (Minutoli et al., 2015, Singh and Chopra, 2014). The present study is the first report on prophylactic and therapeutic effects of FVC in

Conclusions

We report for the first time that prophylactic or therapeutic administration of FVC, a botanical dual COX and 5-LO inhibitor, reduces disease incidence and clinical scores in EAE. The beneficial effects correlate with reduced expression of the proinflammatory cytokines IL-12, IFNγ and IL-17, and reduced expression of COX2 and 5-LO, in spinal cords and spleens of EAE mice. In vitro experiments support the anti-inflammatory effect of FVC on macrophages and microglia, by reducing

Acknowledgments

This work was supported by U.S. Public Health Service Grants RO1AI084065 and RO1AI47325 (to D.G.). We thank Dr. Robert Levy (Primus Pharmaceutical, AZ) for providing us with the flavocoxid (Limbrel) samples used in this study. All authors declare that there are no conflicts of interests.

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