Article pubs.acs.org/jnp
Pentalinonsterol, a Constituent of Pentalinon andrieuxii, Possesses Potent Immunomodulatory Activity and Primes T Cell Immune Responses Steve Oghumu,†,# Sanjay Varikuti,‡,# Noushin Saljoughian,‡ Cesar Terrazas,‡ Andrew C. Huntsman,§ Narasimham L. Parinandi,⊥ James R. Fuchs,§ A. Douglas Kinghorn,§ and Abhay R. Satoskar*,‡,∥ †
College of Public Health, §College of Pharmacy, and ∥Department of Microbiology, The Ohio State University, Columbus, Ohio 43210, United States ‡ Department of Pathology and ⊥Department of Internal Medicine, The Ohio State University Medical Center, Columbus, Ohio 43210, United States ABSTRACT: The use of natural products as adjuvants has emerged as a promising approach for the development of effective vaccine formulations. Pentalinonsterol (PEN) is a recently isolated compound from the roots of Pentalinon andrieuxii and has been shown to possess antileishmanial activity against Leishmania spp. The objective of this study was to examine the immunomodulatory properties of PEN and evaluate its potential as an adjuvant. Macrophages and bone-marrow-derived dendritic cells (BMDCs) were stimulated with PEN and tested for gene expression, cytokine production, and their ability to activate T cells in vitro. PEN was also evaluated for its ability to generate antigen-specific Th1 and Th2 responses in vivo, following ovalbumin (OVA) immunization using PEN as an adjuvant. The results obtained demonstrate that PEN enhances the expression of NF-κB and AP1 transcription factors, promotes gene expression of Tnfα, Il6, Nos2, and Arg1, and upregulates MHCII, CD80, and CD86 in macrophages. PEN also enhanced IL-12 production in BMDCs and promoted BMDC-mediated production of IFN-γ by T cells. Further, mice immunized with OVA and PEN showed enhanced antigen-specific Th1 and Th2 cytokines in their splenocytes and lymph node cells, as well as increased levels of IgG1 and IgG2 in their sera. Taken together, this study demonstrates that PEN is a potent immunomodulatory compound and potentially can be used as an adjuvant for vaccine development against infectious diseases.
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and in vivo studies have demonstrated that P. andrieuxii root extracts not only possess direct antileishmanial activity but also display potent immunomodulatory properties, which markedly increase the antiparasitic activity of macrophages and dendritic cells. Recently, activity-guided fractionation using high-throughput screening led to the purification of the constituents of P. andreuxii roots that possess major antiparasitic activity.6 In particular, we isolated and characterized structurally a new compound, pentalinonsterol (PEN, cholest-4,20,24-trien-3one), from a hexane-soluble extract of P. andrieuxii roots. PEN displayed potent antileishmanial activity against L. mexicana promastigotes and amastigotes in vitro.6 Further, we have developed a novel synthesis protocol for PEN, which enables the efficient production of this bioactive compound in gram quantities in a cost-effective manner. The synthetic PEN demonstrated the same spectroscopic data as the naturally occurring compound.7 Synthetic PEN was found to exhibit potent inhibitory activity against Leishmania parasites both in
atural products are widely recognized for their antimicrobial and immunomodulatory properties. Bioactive compounds that are derived from these natural products are being developed for the treatment of infectious and immunological disorders. Pentalinon andrieuxii (Muell. Arg.) B.F. Hansen & Wunderlin (Apocynaceae) is a plant that grows in the subtropical rainforest region of the Yucatan peninsula, Mexico, and traditionally has been used for the treatment of Chiclero’s ulcer (cutaneous leishmaniasis), snakebites, headaches, and nervous disorders.1,2 Studies using extracts from the roots and leaves of this plant have shown remarkable antiparasitic activity. For example, in vitro studies conducted by our group have demonstrated that the hexane- and methanol-soluble extracts of P. andrieuxii roots displayed growth inhibitory activity against various Leishmania promastigotes, including L. donovani, L. braziliensis, and L. mexicana.3,4 Moreover, in macrophage host cells that were infected with L. mexicana amastigotes, a hexane-soluble extract of P. andrieuxii promoted parasite killing through mechanisms involved with macrophage activation.5 Further, it was shown that topical treatment using this P. andrieuxii root hexane extract significantly reduced parasitic loads in a cutaneous leishmaniasis mouse infection model caused by L. mexicana.5 These in vitro © XXXX American Chemical Society and American Society of Pharmacognosy
Received: May 23, 2017
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DOI: 10.1021/acs.jnatprod.7b00445 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
inflammation-associated genes including cytokines and growth factors. A number of previously determined plant-derived immunomodulatory agents are known to modulate NF-κB and AP1 activity.8−11 The present results demonstrate that this new lead compound, PEN, promotes macrophage activation via NFκB- and AP-1-dependent mechanisms. The activation of NF-κB results in enhanced antigen presentation and increased expression of costimulatory molecules.12 Levels of MHC-II and costimulatory molecule expression play an important role in shaping the nature and magnitude of an immune response.13 Earlier, it was shown that a P. andrieuxii root hexane extract increased the expression of MHC-II and the costimulatory molecule CD80.5 Since synthetic PEN activates NF-κB, its impact on antigen presentation and expression of costimulatory molecules was investigated. RAW macrophages were treated with 25 or 50 μM PEN and analyzed for CD40, CD80, CD86, and MHC-II expression by flow cytometry. It was observed that PEN (at 25 and 50 μM) significantly enhanced the expression of CD40 after 72 h (Figure 2A and E) and MHC-II at 48 and 72 h (Figure 2B and F). Expression of CD80 and CD86 was increased by 50 μM PEN at 48 h and by both concentrations of PEN at 72 h (Figure 2C, D, F, and G). These results demonstrated that PEN enhances macrophage activation and potentiates its antigen-presenting capabilities. The expression of MHC-II and the costimulatory molecules CD80 and CD86 on antigen-presenting cells (APCs) is critical for subsequent T cell activation.14,15 Furthermore, CD40 expressed on macrophages binds to its ligand (CD40L) on activated T cells, leading to macrophage activation, expression of pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6, and release of reactive oxygen species and nitric oxide. These pro-inflammatory activities are also linked to NF-κB activation.16 Since increased NF-κB activity was observed in PEN-stimulated macrophages, the gene expression of proinflammatory mediators after PEN stimulation of these cells was analyzed. The results obtained indicate that PEN stimulation increases the mRNA levels of both Tnfα and Il6 at 48 h (Figure 3A and C). Also observed was an increased trend of Il1b gene expression at 24 and 48 h, although this increase was not statistically significant (Figure 3B). Next, it was determined whether or not activation of macrophages by PEN resulted in a specific polarization pattern characteristic of classical or alternative activation. Depending on the signaling pathway of activation as well as the surrounding cytokine microenvironment, activation of macrophages can result in distinct phenotypes known as classically activated (M1) macrophages or alternatively activated (M2) macrophages.17−19 M1 macrophages are pro-inflammatory, and their activation is induced by IFN-γ, TNF-α, LPS, and other TLR ligands. M1 macrophages display their effector functions by expressing enzymes such as Nos2, as well as cytokines such as TNF-α, IL-1, IL-15, and IL-18. RAW macrophage stimulation by PEN resulted in significantly increased gene transcript levels of Nos2 after 48 h (Figure 3D), while IL-15 and IL-18 gene transcripts were increased by 25 μM PEN after 24 h of stimulation (Figure 3E and F). It appears from these data that activation of RAW macrophages by PEN promotes the M1 macrophage polarization. M2 macrophages are anti-inflammatory and are characterized by high Arg1 and IL-10 production. Chitinase-like 3 protein (Chil3) is also produced by M2 macrophages.20,21 While increased transcript levels of Arg1 were observed, levels of Il10 (at 48 h) and Chil3 (at 24 and 48
vivo and in vitro. A liposomal formulation of synthesized PEN inhibited parasite burdens in a mouse model of visceral leishmaniasis caused by L. donovani.7 Taken together, the compelling data obtained thus far demonstrate the considerable therapeutic potential of PEN as an antiparasitic drug. Much like the hexane-soluble extract of P. andreuxii roots, the results from our recent in vitro and in vivo studies with PEN suggest that, in addition to its antiparasitic properties, this substance may exhibit immunomodulatory activity. For example, in L. donovani-infected mice, treatment with liposomal PEN was associated with increased antigen-specific proliferation of T cells and enhanced IFN-γ cytokine production, compared to infected mice treated with empty liposomes.7 This indicates that PEN potentially can modulate host immune responses, a property that can expand the application of this compound in immune-associated diseases. The use of naturally occurring compounds or synthesized pharmacological agents as immunomodulators has significant applications in infectious and immune-associated disease treatment, as well as in vaccine design. Given the beneficial characteristics of PEN, the objective of this study was to examine extensively the immunomodulatory properties of this steroid derivative using both in vitro and in vivo models, so as to evaluate its potential utility as an adjuvant in vaccination against infectious diseases.
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RESULTS AND DISCUSSION We examined the immune-modulatory effects of PEN, a recently identified compound (Figure 1A) that was originally
Figure 1. (A) Structure of pentalinonsterol (PEN). (B) NF-κB and AP1 transcription factor activity on the RAW-Blue reporter cell line after stimulation with 25 μM PEN or DMSO (vehicle control) for 24 h. Alkaline phosphatase activity was analyzed by spectrophotometry (at 655 m) using Quanti-blue substrate (* represents p value of
