Eupatoriopicrin Inhibits Pro-inflammatory Functions of Neutrophils via

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Eupatoriopicrin Inhibits Pro-inflammatory Functions of Neutrophils via Suppression of IL‑8 and TNF-alpha Production and p38 and ERK 1/2 MAP Kinases Barbara Michalak,† Jakub P. Piwowarski,† Sebastian Granica,† Birgit Waltenberger,‡ Atanas G. Atanasov,§,⊥ Shafaat Y. Khan,∥,¶ Johannes M. Breuss,∥ Pavel Uhrin,∥ Barbara Ż yżyńska-Granica,□ Anna Stojakowska,# Hermann Stuppner,‡ and Anna K. Kiss*,† †

Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw, Warsaw 02-097, Poland Institute of Pharmacy/Pharmacognosy and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck 6020, Austria § Institute of Genetics and Animal Breeding of the Polish Academy of Science, Jastrzębiec 05-552, Poland ⊥ Department of Pharmacognosy, University of Vienna, Vienna 1010, Austria ∥ Institute of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna 1090, Austria ¶ Department of Zoology, University of Sargodha, Sargodha 40010, Pakistan □ Department of Pharmacodynamics, Faculty of Pharmacy, Medical University of Warsaw, Warsaw 02-097, Poland # Institute of Pharmacology, Department of Phytochemistry, Polish Academy of Sciences, Kraków 30-024, Poland

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‡

S Supporting Information *

ABSTRACT: During chronic inflammation, neutrophils acting locally as effector cells not only activate antibacterial defense but also promote the inflammatory response. Interleukin 8 (IL-8), the main cytokine produced by activated neutrophils, positively correlates with the severity of respiratory tract diseases. By screening European plants traditionally used for treating respiratory tract diseases, we found that extracts of aerial parts of Eupatorium cannabinum inhibit IL-8 release from neutrophils. Using bioassay-guided fractionation, we identified five sesquiterpene lactones, eupatoriopicrin (1), 5′-deoxyeupatoriopicrin (2), hiyodorilactone A (3), 3-hydroxy-5′-O-acetyleupatoriopicrin = hiyodorilactone D (4), and hiyodorilactone B (5), that efficiently (IC50 < 1 μM) inhibited IL-8 and TNF-α release in lipopolysaccharide (LPS)-stimulated human neutrophils. Moreover, all these sesquiterpene lactones suppressed the adhesion of human neutrophils to an endothelial monolayer by downregulating the expression of the β2 integrin CD11b/CD18 on the neutrophil surface. Furthermore, eupatoriopicrin efficiently suppressed LPSinduced phosphorylation of p38 MAPK and ERK and attenuated neutrophil infiltration in the thioglycolate-induced peritonitis model in mice. Altogether, these results demonstrate the potential of the sesquiterpene lactone eupatoriopicrin as a lead substance for targeting inflammation.

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interaction of PMNs with the endothelium, followed by their transmigration.1 After their emigration and recruitment to the inflamed site, PMNs get activated, leading to generation of reactive oxygen species (ROS) and release of proteases (e.g., elastase and metalloproteinases (MMPs)), promoting phagocytic and microbicidal activities of PMNs.1,3 In addition, upon their activation, PMNs secrete inflammatory chemokines and cytokines (interleukins IL-8, IL-1β and tumor necrosis factor-α, TNF-α), mediating inflammatory and immune responses.1,3 In PMNs these effects are associated with the

eutrophils, also known as polymorphonuclear leukocytes (PMNs), are the most abundant blood cell type, constituting 60−70% of human peripheral white blood cell count. They form the first line of defense of the innate immune system in humans and mediate a rapid response to inflammatory stimuli.1,2 This response involves the release of chemoattractants and upregulation of surface expression of adhesion molecules on the PMNs’ surface. In addition, bacterial- and host-produced inflammatory signals induce in endothelial cells the expression of the adhesion molecule Eselectin and intercellular adhesion molecule 1 (ICAM-1). These molecules mediate interaction with PMNs expressing β2 integrins such as CD11b/CD18, resulting in adhesive © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 7, 2018

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DOI: 10.1021/acs.jnatprod.8b00939 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

activation of mitogen-activated protein kinases (MAPKs), i.e., p38 kinase, p42/44 extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinases (JNKs),4−6 and are regulated by three major transcription factors families, i.e., NF-κB, AP-1, and C/EBP.7,8 Under physiological conditions neutrophils are cleared by apoptosis, and their recruitment to the affected site gradually subsides; the failure of PMNs to undergo apoptosis may result in chronic inflammation.1,3 These conditions represent an important hallmark of many diseases of the respiratory tract, such as asthma, cystic fibrosis, chronic obstructive pulmonary disease, acute lung injury, and acute respiratory distress syndrome.9 Moreover, excessive recruitment of neutrophils to the site of inflammation has been shown to occur in acute bronchiolitis, one of the most common respiratory diseases in children under 2 years of age. The levels of IL-8 were shown to positively correlate with the severity of several respiratory tract diseases.10−12 For example, protracted bacterial bronchitis, a common cause of chronic cough in children, is characterized by an elevated presence of neutrophils in broncho-alveolar lavage fluid as well as increased IL-8 levels.13,14 Nonallergic chronic rhinosinusitis is also characterized by neutrophil infiltration into the sinus mucosa. In these patients, sinus lavage fluid contains an increased number of neutrophils and markedly increased IL-8- and TNFα levels.15−17 A typical treatment of nonallergic bronchitis and rhinosinusitis is based on the use of antibiotics (e.g., macrolides) and corticosteroids.18,19 Furthermore, use of phytotherapy (e.g., Echinacea/Pelargonium/Sinupret) has been recommended in some cases for treating this condition.19−21 However, nowadays there is still an urgent need to discover new bioactive compounds that would be able to silence the excessive and prolonged inflammatory response without causing serious adverse effects. Expanding on our preliminary data on screening of plants traditionally used in Europe for the treatment of respiratory tract diseases, such as cough, catarrh, bronchitis, throat infection accompanied by fever, and influenza,22 in our current study we demonstrate the potency of extracts of the aerial parts of Eupatorium cannabinum L. (Asteraceae) to inhibit IL-8 release from neutrophils. Furthermore, bioassay-guided fractionation allowed us to identify sesquiterpene lactones, and especially eupatoriopicrin, as compounds responsible for these effects. In addition, these sesquiterpene lactones potently suppress the expression of integrin CD11b/CD18 on neutrophils’ surface and also attenuate release of IL-8, TNF-α, and IL-1β, as well as ROS production from PMNs. Furthermore, we show that eupatoriopicrin suppresses lipopolysaccharide (LPS)-induced phosphorylation of p38 and ERK MAPKs and inhibits, in vivo, neutrophil infiltration in the thioglycolate-induced peritonitis model.

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Figure 1. Extracts of the aerial parts of E. cannabinum suppress IL-8 production in LPS-stimulated neutrophils. IL-8 production in nonstimulated neutrophils (NST), in LPS-stimulated (ST) neutrophils, and in LPS-stimulated neutrophils pretreated with 10−50 μg/ mL of aerial parts of E. cannabinum extracts. Data are expressed as mean ± SEM (three separate experiments performed on neutrophils isolated from independent donors assayed in duplicate). Statistical significance: ***p < 0.001 versus stimulated control, #statistically significant (p < 0.001) versus nonstimulated control (Dunnettʼs post hoc test).

Bioassay-Guided Fractionation of Sesquiterpene Lactones. To isolate single components of aerial parts of E. cannabinum responsible for its biological effects, we used ethanol extraction, followed by HPLC fractionation and subsequent NMR and mass spectrometry analysis. These procedures led to the identification of five sesquiterpene lactones previously described in the literature: eupatoriopicrin (1),23 5′-deoxyeupatoriopicrin (2),24 hiyodorilactone A = 20hydroxychromolaenide (3),25 3-hydroxy-5′-O-acetyleupatoriopicrin = hiyodorilactone D (4),26 and hiyodorilactone B (5).27 Structures of these compounds are depicted in Figure 2.

Figure 2. Structures of isolated sesquiterpene lactones.

Sesquiterpene Lactones 1−5 at a 2.5 μM Concentration Are Not Cytotoxic. We tested the cytotoxicity of all five sesquiterpene lactones in human PMNs and human umbilical vein endothelial cells (HUVECs) using propidium iodide (PI) staining and FACS analysis. While all these compounds were found essentially nontoxic in PMNs and HUVECs at 2.5 μM or lower concentration, they were cytotoxic at 5 μM concentration (Tables S1, S2, Supporting Information). In accordance, when measuring lactate dehydrogenase (LDH) release from PMNs into the medium, we obtained comparable results (Table S3, Supporting Information). Solvent DMSO did not induce any cytotoxicity to the cells (data not shown). On the basis of these data, we performed all further experiments using sesquiterpene lactones at concentrations of up to 2.5 μM.

RESULTS AND DISCUSSION

Eupatorium cannabinum Extract Inhibits IL-8 Secretion. For preliminary assessment, we prepared extracts from the aerial parts E. cannabinum using 60% ethanol and tested them for their ability to inhibit IL-8 release from LPSstimulated human neutrophils. In agreement with our preliminary findings, these extracts efficiently suppressed the IL-8 release from LPS-stimulated human neutrophils at concentrations ranging from 10 to 50 μg/mL (Figure 1), without cytotoxic effects (not shown). B



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Figure 3. Sesquiterpene lactones inhibit cytochalasin A/f-MLP-stimulated upregulation of β2 integrin (CD11b/CD18) expression on the surface of neutrophils. (A) Effect of sesquiterpene lactones 1−5 at concentrations of 0.25, 1, and 2.5 μM on the surface expression of β2 integrin (CD11b/ CD18) in cytochalasin A/f-MLP-stimulated neutrophils. Quercetin (Q) and clarithromycin (C) at 2.5 μM were used as positive controls. Data are expressed as means ± SEM (three separate experiments performed on neutrophils isolated from independent donors). Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001 versus stimulated control (ST); #(p < 0.001) versus nonstimulated control (NST) using Dunnettʼs post hoc test. (B) Effect of eupatoriopicrin at 0.25, 1, and 2.5 μM on integrin (CD11b) expression on the surface of cytochalasin A/f-MLP-stimulated neutrophils. The overlaid unfilled histogram is of stimulated cells (ST). The shaded histograms (from left to right) correspond to nonstimulated cells (NST) and cells stimulated with 0.25, 1, and 2.5 μM eupatoriopicrin.

quercetin at 2.5 μM showed no inhibitory activity, and clarithromycin at 2.5 μM led to an only slightly reduced CD11b induction (67.7 ± 1.9%, p < 0.01) (Figure 3A). Clarithromycin only at a higher concentration of 50 μM reduced CD11b/CD18 expression to 31.5 ± 2.4% (p < 0.001). As an example, the potent inhibitory effect of eupatoriopicrin (1) on the expression of CD11b/CD18 is presented in more detail (Figure 3B). In endothelial cells, sesquiterpene lactone preincubation did not affect the expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin), as revealed by FACS analysis of nonstimulated HUVECs and HUVECs stimulated with TNF-α for 4 h at 10 ng/mL (data not shown). To investigate whether the downregulation of CD11b/ CD18 in neutrophils expression upon their pretreatment with sesquiterpene lactones (at 0.25−2.5 μM) can affect their attachment to an endothelial-cell monolayer, we followed their attachment to a monolayer of HUVECs that had been exposed to TNF-α for 4 h. Indeed, we observed a statistically significant (p < 0.01) decrease in adhesion of such pretreated neutrophils for compounds 1−3 applied at 2.5 μM (p < 0.01) and for compound 5 at 1 and 2.5 μM (Figure 4A and B). Sesquiterpene Lactones Suppress ROS Production and Elastase Release of Activated Neutrophils. Neutrophils that proceeded through the endothelium to the inflamed tissue become further activated at the site of

Sesquiterpene Lactones Prevent Upregulation of Adhesion Molecule Expression in Neutrophils as Well as the Attachment of Neutrophils to Endothelial Cells. Upon tissue inflammation, injury, or stress, neutrophils migrate from blood vessels into the inflamed tissues. This process is initiated by increased expression of β2-integrins (e.g., CD11b/ CD18) on the surface of PMNs promoting the attachment of PMNs to the blood vessel endothelium exposing intercellular adhesion molecule I (ICAM-1).3 In order to assess how sesquiterpene lactones might influence this process, we preincubated neutrophils with sesquiterpene lactones prior to their stimulation with cytochalasin A, an inhibitor of actin polymerization, which potentiates PMN degranulation,28 combined with f-MLP, a chemotactic factor of PMNs.29 For comparison of the effects of the investigated sesquiterpene lactones, we pretreated neutrophils with quercetin, a natural compound with antioxidant and anti-inflammatory activity,30,31 or clarithromycin, a macrolide antibiotic previously shown to inhibit neutrophil recruitment and IL-8 release.32,33 Preincubation of PMNs with sesquiterpene lactones 1, 2, and 5 resulted in a statistically significant reduction of CD11b expression on the cell surface. This reduction was significant at concentrations of 1 and 2.5 μM (p < 0.001) (Figure 3A), and these effects were much stronger than the effects of quercetin or clarithromycin used as positive controls. Specifically, C



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Figure 5. Sesquiterpene lactones efficiently inhibit ROS production in f-MLP- or PMA-stimulated human neutrophils and elastase release from cytochalasin A/f-MLP-stimulated human neutrophils. Effect of sesquiterpene lactones 1−5 at concentrations of 0.25, 1, and 2.5 μM on (A) ROS release from neutrophils upon f-MLP or PMA stimulation detected by luminol or lucygenin, respectively; (B) on elastase release from cytochalasin A/f-MLP-stimulated neutrophils. Data of three separate experiments performed on neutrophils isolated from independent donors assayed in duplicate are expressed as mean ± SEM. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001 versus stimulated control, #p < 0.001 versus nonstimulated control (NST) determined using Dunnettʼs post hoc test. ST, stimulated control; Q, quercetin; C, clarithromycin.

Figure 4. Pretreatment of leukocytes with sesquiterpene lactones reduces their attachment to endothelial-cell monolayers of HUVECs. (A) Effect of sesquiterpene lactones 1−5 at 0.25, 1, and 2.5 μM on attachment of LPS-stimulated neutrophils to TNF-α-primed HUVECs. Data are expressed as mean ± SEM (three separate experiments performed on HUVECs). Statistical significance: *p < 0.05, **p < 0.01, versus stimulated control (ST) using Dunnettʼs post hoc test, NST-nonstimulated control; Q, quercetin. (B) Representative photos showing the attachment of calcein-AM-labeled neutrophils to HUVECs; NTS, nonstimulated neutrophils and TNF-α-stimulated HUVECs; ST, LPS-stimulated neutrophils and TNF-α-stimulated HUVECs; (1) LPS-stimulated neutrophils preincubated with 2.5 μM eupatoriopicrin and TNF-α-stimulated HUVECs. Left: light microscopy; right: fluorescence microscopy.

iopicrin, did not influence ROS production in concentrations tested (Figure 5A). On the other hand, all five sesquiterpene lactones significantly inhibited elastase release at 1−2.5 μM concentration in cytochalasin A/f-MLP-stimulated neutrophils (Figure 5B). However, only compounds 1 and 5 were able to suppress the enzyme release at a concentration of 0.25 μM (p < 0.01 and 0.05, respectively). Both control substances, quercetin and clarithromycin, were able to significantly decrease elastase release only at a very high concentration of 50 μM (to 26.6 ± 1.6%, p < 0.001 and 39.0 ± 1.8%, p < 0.001, respectively, in comparison to stimulated control referred to as 100% of release, Figure 5B). Sesquiterpene Lactones Induce Apoptosis in Neutrophils Exposed to LPS. As neutrophil apoptosis substantially contributes to the resolution of inflammation,35 we investigated how sesquiterpene lactones influence this process. To this end, we made use of the fact that the proinflammatory factor LPS delays the development of apoptosis in cultured neutrophils. We then tested the five different sesquiterpene lactones for their capability to prevent this apoptosis-delaying effect of LPS. We preincubated neutrophils with sesquiterpene lactones 1−5 for 1 h, exposed them to LPS at a concentration of 1 μg/mL, and 24 h later determined neutrophil apoptosis using staining with PI and annexin VFITC. We found that preincubation of neutrophils with

inflammation. Such activation induces in neutrophils oxidative burst characterized by intense ROS production and releases proteolytic enzymes such as elastase, responsible for extracellular matrix (ECM) degradation from azurophilic granules.1 We examined how sesquiterpene lactones affect ROS generation utilizing two models of ROS induction: in response to cytochalasin A/f-MLP stimulation and in response to phorbol myristic acid (PMA), a specific protein kinase C (PKC) activator.34 While in both these assays compounds 2−5 efficiently inhibited ROS release at 1−2.5 μM, their inhibitory effect on f-MLP stimulation seemed to be higher in comparison to PMA stimulation (Figure 5A), possibly indicating less significant interference of sesquiterpene lactones with the PKC pathway. Strikingly, compound 1, eupatorD



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Table 1. Sesquiterpene Lactones 1−5 Counteract Decrease in Apoptosis in LPS-Treated Human Neutrophilsa cells (%) viable NST ST 1 2 3 4 5 quercetin 2.5 clarithromycin 2.5

51.0 75.4 42.4 26.8 43.7 58.7 56.9 58.8 49.8

± ± ± ± ± ± ± ± ±

1.9 1.1# 18.0** 13.5** 12.7** 7.5** 3.8** 19.3* 14.3*

early apoptotic

late apoptotic

± ± ± ± ± ± ± ± ±

19.9 ± 2.8 9.5 ± 1.8# 25.0 ± 8.4* 22.1 ± 9.3 14.3 ± 4.6 14.0 ± 4.4 13.0 ± 2.9 15.4 ± 2.2 13.3 ± 4.1

26.3 13.0 31.2 49.5 39.7 25.0 25.9 27.0 33.7

1.9 1.9# 12.5* 5.7** 8.8** 6.3* 2.8* 7.7* 4.2*

necrotic 2.9 2.1 1.5 1.7 2.3 2.3 4.2 1.0 1.1

± ± ± ± ± ± ± ± ±

0.7 0.4 0.9 0.7 0.6 1.0 2.1 0.4 0.5

a

Using FACs analysis, human neutrophils were assigned as viable, early, and late apoptotic or necrotic. While treatment of human neutrophils with LPS at 1 μg/mL for 24 h significantly decreased percentage of apoptotic cells at the end of the cultivation period (ST) compared to nontreated neutrophils (NST), preincubation of human neutrophils with sesquiterpene lactones 1−5 at a concentration of 0.25 μM increased the percentage of apoptotic neutrophils primed with LPS. Quercetin at 2.5 μM and clarithromycin at 2.5 μM were used as controls. Data are expressed as mean ± SEM (three separate experiments performed on neutrophils isolated from independent donors). Statistical significance: *p < 0.05, **p < 0.01 versus stimulated control (Dunnettʼs post hoc test); #statistically significant (p < 0.001) versus nonstimulated control.

processes including gene expression, adhesion, migration, and cell survival.6,38 We first investigated whether treatment of eupatoriopicrin influences phosphorylation of signaling molecules p38 MAPK, p42/44, and JNK. For this purpose, eupatoriopicrin-pretreated PMNs were stimulated with LPS, and phosphorylation of signaling molecules was analyzed in cell lysates using the Western blot method. Both forms of proteins, phosphorylated and total, were analyzed. Indeed, pretreatment with eupatoriopicrin (1) between 0.25 and 2.5 μM significantly decreased the LPS-induced phosphorylation of p38 MAPK and ERK1/2 but not of JNK (Figure 7A). Second, we examined the effect of eupatoriopicrin (1) on NF-κB translocation and activation in human neutrophils. Upon treatment with LPS, the level of p65 subunit increased in the nucleus. These results evidence that eupatoriopicrin (1) interferes with the translocation of p65 subunit of NF- NF-κB from the cytoplasm to the nucleus at 1 and 2.5 μM (Figure 7B). Effect of Eupatoriopicrin (1) on Polymorphonuclear Leukocyte Recruitment in Vivo. Undoubtedly, the complexity of the inflammatory response cannot be solely assessed by in vitro studies. We therefore used an in vivo model, the murine thioglycolate-induced peritonitis model, in which we tested whether eupatoriopicrin would influence recruitment of PMNs into inflamed tissue. We pretreated male C57BL/6J male mice i.p. with eupatoriopicrin at a dose of approximately 3 mg/kg; 30 min later we injected animals i.p. with 4% sterile thioglycolate with eupatoriopicrin; and 5 h after the second injection mice were sacrificed. We found that eupatoriopicrin substantially reduced the recruitment of neutrophils to the peritoneum during the inflammatory response (Figure 8). Natural products comprise great structural diversity and offer a wide range of pharmacophores and stereochemical variation; thus they play a significant role in the discovery of leads.39 Additionally, natural products may have the advantage over synthetic compounds of being natural metabolites that may be more prone to protein−protein interaction interference and might be more easily delivered to their intracellular site of action.40,41 Despite some skepticism about this source of therapeutics, during the period 1981−2014, about 50% of all new approved drugs came from natural sources or are derived from natural compounds.42

sesquiterpene lactones already at a low concentration of 0.25 μM counteracted the LPS-induced delay of apoptosis and that their effects were more pronounced than those of the positive controls quercetin and clarithromycin (Table 1). Sesquiterpene Lactones Suppress Chemokine and Cytokine Secretion by Neutrophils. Neutrophils, in response to different stimuli comprising f-MLP, PMA, granulocyte colony-stimulating factor (GM-CSF), complement component C5a, zymosan, and LPS, secrete TNF-α, IL-1β, and IL-8.2,36 LPS stimulation of neutrophils is mediated via toll-like receptors (TLRs), and this process includes activation of both NF-κB and MAP kinase pathways.37,38 As we were interested in studying how sesquiterpenes influence the process of LPS stimulation, we pretreated human neutrophils before their priming with LPS. Using ELISA, we determined levels of IL-8, TNF-α, and IL-1β in culture medium 24 h after LPS stimulation. Such preincubation of human neutrophils with sesquiterpene lactones significantly inhibited their production of IL-8 upon LPS stimulation. Specifically compounds 1, 2, and 5 at concentrations of 0.25− 2.5 μM almost totally abolished the IL-8 production (Figure 6A, upper panel), and 50% (IC50) of the IL-8 production was determined as 0.09, 0.10, and 0.13 μM, respectively (Figure 6A, lower panel). In addition, at 2.5 μM, the sesquiterpene lactones 1, 2, and 5 prohibited almost completely TNF-α production in LPS-stimulated human neutrophils (Figure 6B, upper panel), and the calculated IC50 values were 0.09, 0.10, and 0.12 μM, respectively (Figure 6B, lower panel). Clarithromycin, as expected, was able to decrease the IL-8 production. Neither of the control substances clarithromycin or quercetin inhibited TNF-α production in LPS-stimulated human neutrophils at 2.5 μM (Figure 6A and B), and only a slight inhibitory effect was found at 50 μM (data not shown). Interestingly, sesquiterpene lactones at 1 and 2.5 μM only moderately suppressedIL-1β production in LPS-stimulated human neutrophils (Figure 6C). Effects of Eupatoriopicrin (1) Influence on Molecular Signaling Pathways. Treatment of human neutrophils with LPS is known to cause rapid phosphorylation of the signaling molecules p38 MAPK, p42/44 extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK) as well as NF-κB activation, which subsequently influence many cellular E



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Figure 6. Sesquiterpene lactones and primarily eupatoriopicrin efficiently inhibit IL-8 and TNFα production in LPS-stimulated human neutrophils. Effects of sesquiterpene lactones 1−5 at 0.25, 1, and 2.5 μM on (A) IL-8, (B) TNFα, and (C) IL-1β production in LPS-stimulated neutrophils. Data from three separate experiments performed on neutrophils isolated from independent donors assayed in duplicate are expressed as mean ± SEM. Statistical significance: ***p < 0.001 versus stimulated control, #p < 0.001 versus nonstimulated control (NST) using Dunnettʼs post hoc test. ST, stimulated control; Q, quercetin; C, clarithromycin.

In this study, we report that eupatoriopicrin and other sesquiterpene lactones derived from E. cannabinum display

potent anti-inflammatory effects on human neutrophils. Until now, only very fragmentary studies concerning the cytostatic F



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Figure 7. Eupatoriopicrin (1) potently inhibits the phosphorylation of p38 MAPK and p42/44 ERK in LPS-stimulated human neutrophils. (A) Western blots using antibodies against phosphorylated and total p38, p42/44 ERK, and JNK proteins in LPS-stimulated human neutrophils preincubated with eupatoriopicrin (0.25, 1, and 0.25 μM) quantified in three independent experiments shown as bar graphs. (B) Western blots of the p65 subunit of NF-κB in the cytosol and nucleus in LPS-stimulated human neutrophils incubated with eupatoriopicrin (0.25, 1, and 0.25 μM) quantified in three independent experiments shown as bar graphs.

human peripheral blood mononuclear cells and pulmonary epithelial cells.44,45 The mechanism of LPS stimulation of human neutrophils is connected with a functional response comprising the activation of MAPKs: p38 and p42/44 (ERK) associated with the elevated synthesis of TNF-α and IL-8. In addition, LPS also induces PMN adhesion to the endothelium as well as delays apoptosis.4−6 JNK phosphorylation is strictly correlated with monocyte chemoattractant protein 1 (MCP-1) expression, but not with IL-8 nor TNF-α expression.5 In our study, we showed that eupatoriopicrin inhibits phosphorylation of p38 MAPK and of ERK, while the phosphorylation of JNK was not affected by this compound (Figure 7A). NF-κB activation is

and antitrypasomal activity of eupatoriopicrin were conducted.23,43 We now show that all tested sesquiterpene lactones, but mostly the compounds eupatoriopicrin (1), 5′deoxyeupatoriopicrin (2), and hiyodorilactone B (5) were able in the low nanomolar range (IC50 < 0.13 μM) to inhibit LPSdriven IL-8 and TNF-α production by human neutrophils. What might be of interest is the fact that inhibitory activities of sesquiterpene lactones were much more pronounced in comparison with clarithromycin (Figure 6A and B). This antibiotic is also known to modulate the pro-inflammatory response in vitro by inhibiting the production of proinflammatory cytokines (IL-1, IL-6, IL-8) in synovial fibroblast-like cells as well as suppress NF-κB translocation in G



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Figure 8. Pretretment of mice with eupatoriopicrin suppresses the recruitment of leukocytes into the peritoneum in a thioglycolate-induced peritonitis model in mice. Two independent experiments with each comprising 3 control-treated mice (control), 3 mice treated with thioglycolate (TG), and 3 mice treated with a combination of thioglycolate and eupatoriopicrin (TG + Eupat 3 mg/kg) were performed. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001 using Dunnettʼs post hoc test.

and in vivo suppresses neutrophil migration in a thioglycolateinduced inflammation model. Our findings suggest that eupatoriopicrin could be developed as a lead substance to targeting respiratory tract diseases.

connected with upregulation of the p38 MAPK pathway, although in neutrophils the MAPK pathway appears to affect cytokine production also independently of NF-κB.38 While, some sesquiterpene lactones are known as potent NF-κB inhibitors,46 only higher concentrations of eupatoriopicrin affected the NF-κB (p65) translocation to the nucleus in response to LPS stimulation (Figure 7B). The inhibition of ERK in our study by eupatoriopicrin also correlated with the abolition of the LPS-delayed neutrophil apoptosis (Table 1), which is under control of ERK activator kinase (MEK) and ERK pathways and is an important step to the resolution of inflammation.6,47 Although eupatoriopicrin and other sesquiterpene lactones decreased CD11b expression at 2.5 μM (Figure 3), they did not affect the ICAM-1 expression in stimulated HUVECs (not shown), and the decrease of neutrophil adhesion to endothelium was only observed in the highest concentration tested (Figure 4). Eupatoriopicrin was also able, in the concentration range 0.25−2.5 μM, to inhibit elastase release (Figure 5B), probably without affecting the enzyme activity itself, as this compound was proven to be only a weak elastase inhibitor (IC50 = 144 μM).48 Strikingly, eupatoriopicrin did not affect such a prime function of PMNs as the oxidative burst (Figure 5A). Our results furthermore show that other functions of neutrophils such as adhesion and degranulation are influenced by sesquiterpene lactones at higher concentrations (1 and 2.5 μM), while IL-8 and TNF-α expression are already inhibited at the lower concentration assessed, 0.25 μM. Moreover, this effect seems to be cell-specific, as we did not observe any effect of sesquiterpene lactones on IL-8 release from TNF-α-stimulated HUVECs at 5 μM (data not shown). In vivo experiments showed that eupatoriopicrin at a dose of 3 mg/kg efficiently suppressed PMN recruitment in a thioglycolate-induced peritonitis animal model. To further characterize the newly identified eupatoriopicrin bioactivity, it would be very interesting that the effect of the compound would be examined in future studies with intravital microscopical analysis, which can provide more detailed information on neutrophil−endothelium interaction, including the identification of the stage of interaction (rolling/firm adhesion/ transmigration) that is mostly affected by an inhibitory compound.49 Altogether, our work shows that sesquiterpene lactones derived from E. cannabinum potently suppress production of inflammatory cytokines by human neutrophils. Among these compounds, eupatoriopicrin, the most abundant sesquiterpene lactone, inhibits p38 and ERK 1/2 MAPK molecules in vitro

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EXPERIMENTAL SECTION

Materials. Phosphate-buffered saline (PBS) was purchased from Biomed (Lublin, Poland). Hanks’ balanced salt solution (HBSS), RPMI 1640, f-MLP (formyl-Met-Leu-phenylalanine), LPS (from Escherichia coli 0111:B4), cytochalasin A, HEPES solution, Lglutamine, propidium iodide, MgCl2·6H2O, EGTA, IGEPAL CA630, luminol, and N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (SAAVNA) were bought from Sigma−Aldrich Chemie GmbH (Steinheim, Germany). Fetal bovine serum (FBS) was obtained from Gibco (Grand Island, NY, USA), and penicillin−streptomycin from PAA Laboratories (Pasching, Austria). Human Quantikine Pancoll human, density 1.077, was purchased from PAN-Biotech (Aidenbach, Germany), and Accutase Cell detachment solution from BD Biosciences (Franklin Lakes, NJ, USA). Lucigenin was obtained from Carl Roth (Karlsruhe, Germany), and calcein-AM from MoBiTec GmbH (Gö ttingen, Germany). Anti-pp38 (#9211), ppJNK (#9251), pJNK (#9252), pERK1/2 (#9102), NFκB (p65) (#8242S), and β-actin (#4967) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-ppERK1/2 (#V8031) was purchased from Promega (Fitchburg, WI, USA), and anti-p38 (#SC-535) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Inhibitors of p38MAPK-SB202190, ERK1/2-PD98059, and JNK-SP600125 were purchased from Tocris Biosciences (Bristol, UK). TNF recombinant human TNF-α and human Quantikine ELISA kits were purchased from R&D System (Minneapolis, MN, USA). Anti-human CD11b (conjugated with phycoerythrin, PE) was purchased from eBioscience (San Diego, CA, USA). Anti-human CD54 (conjugated with allophycocyanin, APC), anti-human CD106 (conjugated with phycoerythrin, PE), and anti-human CD62E (conjugated with phycoerythrin, PE) were purchased from BD Pharmingen (San Diego, CA, USA). Plant Material and Isolation of Sesquiterpene Lactones. Aerial parts of Eupatorium cannabinum L. were collected in July 2015 from a natural habitat near Piduń, Poland (53°30′ N, 20°47′ E). The plant material was authenticated according to Flora Europaea50 by Anna K. Kiss. A voucher specimen (no. EC072015) is deposited in the Plant Collection, Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw, Poland. The air-dried aerial parts (∼500 g) were extracted three times with 60% ethanol (1:30) at 70 °C for 2 h. The methanol from combined extracts was evaporated under reduced pressure, and the aqueous residue was lyophilized and yielded ∼112 g of extract. The crude extract was dissolved in water (500 mL) and partitioned with petroleum ether (5 × 500 mL) and with ethyl acetate (6 × 500 mL). The ethyl acetate fraction (15 g) was subjected to silica gel (Merck) H



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column chromatography (10 × 15 cm) and eluted with an CH2Cl2− MeOH gradient (100:0 → 0:100) of eight steps, 1 L each, to obtain 45 fractions of 200 mL, which were pooled into 12 main fractions (F1−F12) based on their thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) profiles. Fractions F2 (420 mg), F3 (400 mg), and F5 (3 400 mg) were rechromatographed on a Sephadex LH-20 (Pharmacia, Uppsala, Sweden) column (2.5 × 150 cm) with MeOH. From fractions F2_2 and F3_2 (600 mg) compounds 1 (47 mg; retention time, tR = 26.5−27.1 min) and 2 (87 mg; tR = 29.8−30.8 min) and from fraction F5_2 (3 050 mg) compounds 3 (56 mg; tR = 22.1−22.6 min), 4 (390 mg; tR = 25.1− 26.1 min), and (5) (625 mg; tR = 27.9−28.9 min) were isolated using preparative HPLC with a 0.1% HCOOH in H2O (A)−0.1% HCOOH in MeCN (B) gradient (80:20 → 40:60) in 35 min and Zorbax SBC18 (Agilent, Santa Clara, CA, USA, particle size 5.0 μm, 150 × 21.2 mm) at a flow rate of 5.0 mL/min. All tested compounds were dissolved in DMSO (10 mM stock solution) and then diluted with (Ca2+)-free HBSS and (Ca2+)-free PBS buffers at pH 7.4 or RPMI 1640 medium. Prepared dilutions were added directly to the cell cultures. The concentration of DMSO used (

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