key: cord-300912-w85t4zz6
authors: Wu, Beibei; Wang, Liyin; Jiang, Lili; Dong, Lili; Xu, Fengli; Lu, Yili; Jin, Jiahui; Wang, Zhanyue; Liang, Guang; Shan, Xiaoou
title: n-butanol extract from Folium isatidis inhibits the lipopolysaccharide-induced downregulation of CXCR1 and CXCR2 on human neutrophils
date: 2017-10-25
journal: Mol Med Rep
DOI: 10.3892/mmr.2017.7870
sha: 
doc_id: 300912
cord_uid: w85t4zz6

Neutrophils, immune cells crucial for protecting against invading pathogens, are important in sepsis. Neutrophil migration is regulated by chemokine receptors and their cognate ligands. Our previous study investigated the effect of n-butanol extract from Folium isatidis on lipopolysaccharide (LPS)-induced septic shock. The present study stimulated neutrophils with LPS to explore the influence of LPS on cell. Neutrophils were then pretreated with n-butanol extract from Folium isatidis followed by LPS to examine the effect of this extract on neutrophil chemotaxis. The results showed that LPS decreased the expression levels of CXC-chemokine receptor (CXCR)1, CXCR2 and L-selectin (CD62L), and increased the expression of interleukin-8 (IL-8) by neutrophils. The addition of n-butanol extract from Folium isatidis inhibited this LPS-induced downregulation of CXCR1, CXCR2 and CD62L, and decreased the expression of IL-8 on neutrophils. In addition, n-butanol extract promoted myeloperoxidase activity in neutrophils. Taken together, LPS downregulated the expression of chemokine receptors, leading to the failure of neutrophils to migrate to sites of infection. The addition of n-butanol extract, which promoted the ability of neutrophils to migrate, is a natural product and potential therapeutic agent with which to target neutrophil chemotaxis during LPS stimulation.

Sepsis is a systemic inflammatory response syndrome associated with a high rate of mortality (1) . The incidence of sepsis has been increasing over the last two decades. This increase has been more marked in developed countries, where 6-30% of patients in Intensive Care Units suffer from sepsis (2) . Sepsis remains the leading cause of mortality in patients in Intensive Care Units (3) and the associated burden of care incurs financial burden. However, the pathogenesis of sepsis remains to be fully delineated and effective therapies are lacking. A state of immunosuppression induced by sepsis has been demonstrated in clinical and experimental sepsis (3) , with the majority of patients who have succumbed to sepsis-associated mortality showing evidence of immunosuppression and unresolved septic foci (4) .

Polymorphonuclear neutrophils (PMNs) are an essential component of the innate immune system, involved in the clearance of extracellular pathogens (5) . As the most abundant subset of leukocytes, the involvement of PMN in sepsis is significant (3, 6, 7) . The migration of PMNs is regulated by chemoattractants and chemokine gradients (3, 7, 8) . CXC-chemokine receptor (CXCR)1 and CXCR2 are the major chemokine receptors on PMNs, with interleukin (IL)-8 acting as a ligand of these receptors (9) . Severe sepsis is associated with the failure of PMNs to migrate (10) . In a previous clinical study, patients with suppression of PMN receptors were predisposed to inflammatory response syndrome (11) . Previous in vitro investigations have demonstrated that CXCR2 is downregulated upon stimulation with tumor necrosis factor-α (TNF-α) (12) . The activation of Toll-like receptor 4 (TLR4) also suppresses the expression of CXCR2 (13) . In an experimental mouse model of sepsis, the failure of PMNs to migrate was shown to result in a high rate of mortality (1). Tancevski et al and Van Zee et al reported that promoting the recruitment of PMNs ameliorates sepsis and attenuates sepsis-related injury and infection, respectively (14, 15) . Therefore, in order to improve treatment of inflammatory disorders, including sepsis, the promotion of PMN chemotaxis is an attractive target (6).

As an ancient Chinese herb, Folium isatidis is considered to have detoxification properties in traditional Chinese medicine. In addition to being used to cure febrile diseases, including dipsosis, fever, jaundice and hematemesis, F. isatidis is also deployed against diseases, including mumps, viral hepatitis, influenza and epidemic encephalitis B (16) (17) (18) . It was reported to be effective during the 2003 severe acute respiratory syndrome flu outbreak (19) . These antimicrobial and anti-endotoxic properties indicate its potential to be developed into a natural antibiotic (20, 21) . In our previous study (22) , the main chemical components of F. isatidis were identified using high-performance liquid chromatography. It was also demonstrated that F. isatidis increased the survival of septic mice, and ameliorated lung injury by inhibiting the production of inflammatory cytokines TNF-α and IL-6 through the myeloid differentiation primary response gene 88 and nuclear factor-κB pathways (22) . Although the beneficial immunomodulatory effects of F. isatidis have been the focus of previous investigations, whether this herb has an effect on chemokine receptors remains to be elucidated. The present study aimed to determine whether F. isatidis affects the migration of PMNs.

Isolation of neutrophils. The present study was approved by the Ethics Committee of The Second Hospital of Wenzhou Medical University (Wenzhou, China), and informed consent was obtained. Peripheral blood was collected from four 20-30-year-old female healthy volunteers between August and December 2015, who had been referred to The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University and who provided informed, written consent. The blood was transferred into heparin lithium-containing tubes (BD Bioscience, San Jose, CA, USA). The samples were processed within 1 h of collection. Whole blood was incubated with 3% dextran T-500 in the dark at room temperature for 20 min for sedimentation of red blood cells (RBCs). Following sedimentation, the white blood cell-enriched upper phase was layered over a Ficoll-Paque solution (GE Healthcare Life Science, Uppsala, Sweden). Centrifugation was performed at 427 x g at room temperature for 20 min without breaks, resulting in four distinct layers. The first three layers were discarded, and the remaining layer containing granulocytes and remnant RBCs was diluted in ACK lysis buffer (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were then washed with PBS, and the freshly isolated neutrophils were resuspended in RPMI 1640 (Gibco; Thermo Fisher Scientific, Inc.). The purity of the isolated neutrophils was >90% based on Wright's staining and differential counts.

Flow cytometry. The isolated neutrophils were diluted to 1x10 6 cells/ml with RPMI 1640 (Gibco; Thermo Fisher Scientific, Inc.). The cells were then incubated with either vehicle (0.01% DMSO) or increasing concentrations of n-butanol extract from Folium isatidis (100, 250 and 500 µg/ml) at 37˚C and 5% CO2 for 2 h. The specific method to obtain n-butanol extract from F. isatidis was described in previous study (22) . The cells were then stimulated with 0.5 µg/ml lipopolysaccharide (LPS; Sigma; Merck Millipore, Darmstadt, Germany) for 4 h at 37˚C. The cells were washed with ice-cold PBS and then resuspended in 50 µl FACS buffer. The cells were then incubated with a 1:50 dilution of the following antibodies (the concentrations suggested by the manufacturer) for 30 min at 4˚C in the dark: FITC-conjugated anti-human CD181 (CXCR1; cat. no 11-1819-42), PE-conjugated anti-human CD182 (CXCR2; cat. no 12-1829-42) and APC-conjugated anti-human CD11b (cat no. 17-0118-42) or FITC-conjugated anti-human L-selectin (CD62L; cat. no 11-0629-42), PE-conjugated anti-human TLR4 (cat no. 12-9917-41) and APC conjugated anti-human TLR2 (cat no. 17-9922-42) (all from eBioscience, San Diego, CA, USA). Antibodies of the same isotype were used as negative controls. The cells were analyzed using a FACS calibur system (BD Biosciences). The mean fluorescence intensity (MFI) for 10,000 cells in each sample was determined using CellQuest software, version 5.2 (BD Biosciences).

Chemotaxis assays. The isolated neutrophils were pretreated as described above for 2 h and then stimulated with LPS (0.5 µg/ml) for 4 h. The cells were then washed twice with PBS, and resuspended in RPMI 1640 at 1x10 6 cells/ml. The chemotaxis assays were performed using Transwell inserts (24 wells, 5 µm pore size; Corning, Inc., Corning, NY, USA), where 10,000 cells in 100 µl were loaded in the upper inserts and 600 µl of IL-8 (100 ng/ml; eBioscience; Thermo Fisher Scientific, Inc.) containing RPMI 1640 was added to the lower wells. Following co-incubation for 2 h at 37˚C and a humidified 5% CO 2 atmosphere, the inserts were removed, and the migrated neutrophils in the lower wells were collected and quantified according to myeloperoxidase (MPO) activity using a myeloperoxidase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Total RNA was isolated from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RT-qPCR analysis was performed using an M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 1 µg RNA was used for the determination. qPCR was performed in a 10 µl reaction volume containing 2 µl cDNA target, 2.25 µl SYBR Green (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 0.25 µl sense primers, 0.25 µl anti-sense primers and 5.25 µl ddH 2 O. An Eppendorf Mastercycler realplex detection system (Eppendorf, Hamburg, Germany) was used for RT-qPCR analysis: The reaction consisted of the following conditions: 95˚C for 3 min, followed by 95˚C for 15 sec, 60˚C for 30 sec, and 70˚C for 30 sec (40 cycles). The primers of the genes were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) and the sequences were as follows: Human CXCR1, The result of real-time PCR was expressed as the threshold cycle (CT). The CT represents the PCR cycle at which the reported fluorescence rises above a set baseline threshold when the DNA amplicon is replicating exponentially (12) . Results were normalized to β-actin as plotted as relative expression to the average of CON or DMSO, which were set as 100.

Quantification of IL-8. The neutrophils were pretreated for 2 h with the extract as described above, and then stimulated with LPS (0.5 µg/ml) for 1 or 4 h. The supernatant and cells were collected, and the levels of IL-8 in the supernatant were determined using enzyme-linked immunosorbent assay (ELISA) kits (cat no. 88-8086-88; eBioscience; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The total quantity of IL-8 in the supernatant was normalized to that of total protein in the viable cell pellets.

Statistical analysis. All the experiments were performed in triplicate. Data are expressed as the mean ± standard error of the mean. All the data were statistically analyzed using Student's t test. P<0.05 was considered to indicate a statistically significant difference. GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis.

and CD62L. TLR2 and TLR4/myeloid differentiation factor 2 are vital in the recognition of LPS in the host, and the triggering of these receptors can lead to neutrophil recruitment and migration via CXCR1 and CXCR2. CD11b and CD62L, which are expressed on the surface of neutrophils, are critical for the occurrence of sepsis. LPS, also known as endotoxin, is a component of the outer membrane of Gram-negative bacteria, and is known to induce septic shock when present in high quantities. Therefore, the present study measured the expression of neutrophil chemokine receptors under LPS stimulation. The isolated neutrophils were treated with either vehicle or increasing concentrations (10, 100 and 1,000 ng/ml) of LPS for 4 h, and the expression levels of CXCR1, CXCR2, TLR2, TLR4, CD11b and CD62L were measured using flow cytometry. The results showed that LPS treatment resulted in decreased expression levels of CXCR1, CXCR2 and CD62L in a dose-dependent manner (Fig. 1A-C) , which was in accordance with the results of a previous study (23) . However, no significant changes were observed in the expression levels of TLR2, TLR4 or CD11b in the LPS-treated neutrophils, compared with the vehicle-treated neutrophils (Fig. 1D-F) . Therefore, the expression levels of TLR2, TLR4 and CD11b were not examined in the remainder of the experiments.

n-butanol extract from Folium isatidis prevents the LPS-induced downregulation of CXCR1, CXCR2 and CD62L. CXCR1, CXCR2 and CD62L may be important in sepsis, however, whether n-butanol extract affects these chemokine receptors remains to be elucidated. Therefore, the present study assessed the effect of the extract on the expression of CXCR1, CXCR2 and CD62L. The isolated neutrophils were pre-incubated with either vehicle or increasing concentrations of extract (100, 250 and 500 µg/ml) for 2 h, and were subsequently treated with LPS (0.5 µg/ml) for 4 h. Subsequent analysis of the cells using flow cytometry indicated that the extract inhibited the LPS-induced downregulation of CXCR1, CXCR2 and CD62L ( Fig. 2A-C) in a dose-dependent manner.

Effect of n-butanol extract from Folium isatidis on the gene expression levels of CXCR1, CXCR2 and CD62L. As the n-butanol extract obtained from F. isatidis had a significant effect on chemokine receptor protein levels, the present study investigated the gene expression levels of these receptors. The isolated neutrophils were pretreated with extract and then treated with LPS (0.5 µg/ml) for 1 or 4 h (Fig. 3A-F) . The extract increased the gene expression levels of CXCR1, CXCR2 and CD62L in a dose-dependent manner.

IL-8, as the ligand of CXCR1 and CXCR2, is important in the regulation of neutrophil migration. Following exposure of the neutrophils to LPS for 4 h, the mRNA expression of IL-8 was significantly increased (Fig. 4A) . Therefore, the present study examined whether n-butanol extract affected the production of IL-8. The isolated neutrophils were pretreated with extract and then incubated with LPS (0.5 µg/ml) for 1 h (Fig. 4B ) or 4 h (Fig. 4C) . The expression levels of IL-8 were quantified using ELISA. A decrease in IL-8 was observed following exposure to LPS for 1 and 4 h when pretreated with the extract. Specifically, following exposure to LPS for 4 h, the cytokine expression of IL-8 was decreased >5-fold when the cells were treated with the extract at a dose of 500 µg/ml, compared with LPS stimulation (Fig. 4C) . MPO levels are a measure of neutrophil migration. Therefore, the present study examined the effect of the extract on the activity of MPO. Neutrophils were pretreated with n-butanol extract for 2 h followed by stimulation with LPS for 4 h. The activity of MPO increased in a dose-dependent manner following treatment with extract, compared with the control (Fig. 4D) , suggesting that n-butanol extract promoted the function of neutrophils.

Sepsis remains a threat to human health, primarily due to a lack of effective therapies (3). With current treatment options, the mortality rate of patients with sepsis remains high at ~30%, and mortality rates increase directly with disease severity (24) . A previous worldwide survey of relevant pathogens in intensive care units found that the majority of patients with sepsis had blood cultures positive for Gram-negative bacteria (25) . LPS, a component of the outer leaflet of the outer membrane of Gram-negative bacteria, is a key molecule in the induction of sepsis. Therefore, the present study used LPS to simulate sepsis induced by Gram-negative bacteria infection.

As previously reported, the present study observed that LPS stimulation decreased the expression of CXCR1, CXCR2 and CD62L on neutrophils. In addition to being effector cells involved in the acute phase of the inflammatory response, neutrophils are also important in the resolution of inflammation. The failure of neutrophils to migrate can lead to an inability to control infection due to weakened neutrophil phagocytic and bactericidal abilities (26) . CXCR1 and CXCR2 are the major chemokine receptors on neutrophils. Chemokine receptors regulate the migration of neutrophils to the site of infection to assist in controlling invading pathogens and protecting the body. Duerschmied et al (27) demonstrated that survival following LPS-induced endotoxic shock improved upon the promotion of neutrophil recruitment during acute inflammation. n-butanol extract was found to have significant antiseptic abilities in our previous study. However, whether the extract affected neutrophil migration remained unclear. In the present study, it was demonstrated that n-butanol extract prevented the downregulation of CXCR1, CXCR2 and CD62L.

In our previous study, it was demonstrated that n-butanol extract from Folium isatidis significantly inhibited the activation of TLR4 and its signaling pathways (22) . It has been reported that the systemic activation of TLRs and high levels of TNF-α are involved in the reduction of neutrophil recruitment through the downregulation of CXCR2 in neutrophils (28) . Therefore, it was hypothesized that n-butanol extract from F. isatidis prevents the downregulation of CXCR1 and CXCR2 through the activation of TLR4 and secretion of TNF-α.

The primary function of CD62L, a vascular adhesion molecule, is directing neutrophil migration. In addition to CXCR1 and CXCR2, neutrophil migration requires the assistance of CD62L. In the present study, the expression of CD62L decreased following incubation with LPS for 4 h (Fig. 1C) , whereas n-butanol extract promoted the expression of CD62L in a dose-dependent manner (Fig. 2C ). This suggested that the extract promoted the migration of neutrophils by inhibiting the sepsis-induced downregulation of CD62L.

IL-8 is a ligand of CXCR1 and CXCR2, and is rapidly secreted upon cell stimulation. The mRNA expression of IL-8 significantly increased upon stimulation with LPS (Fig. 4A) . However, n-butanol extract decreased the expression of IL-8 in a dose-dependent manner ( Fig. 4B and C) , and prevented the decreases in the expression of CXCR1 and CXCR2. This suggested that the extract increased the activity of neutrophils and upregulated the expression of chemokine receptors, including CXCR1 and CXCR2, rendering neutrophils more sensitive to the ligand IL-8. The present study quantified MPO activity as a measure of neutrophil migration. It was found the extract promoted the activity of MPO. The extract also promoted the ability of neutrophils to migrate by increasing the expression of CXCR1 and CXCR2 on the surface of neutrophils.

To date, the treatment of sepsis consists primarily of supportive measures and experimental therapeutic approaches (29) . Therefore, novel pharmacological strategies are urgently required to promote the treatment of sepsis (30) . With the ability to promote the expression of CXCR1, CXCR2 Figure 2 . n-butanol extract from Folium isatidis inhibits the downregulated protein levels of CXCR1, CXCR2 and CD62L. Following pretreatment with a vehicle control (DMSO) or n-butanol extract at the indicated concentration for 2 h, neutrophils were incubated with LPS (0.5 µg/ml) for 4 h. Expression levels of (A) CXCR1, (B) CXCR2 and (C) CD62L were measured using flow cytometry. The corresponding mean fluorescence intensity was calculated. Each bar represents the mean ± standard error of the mean of three independent experiments. Statistical significance relative to the LPS group was determined. ** P<0.01. LPS, lipopolysaccharide; CXCR1, CXC-chemokine receptor 1; CXCR2, CXC-chemokine receptor 2; CD62L, L-selectin. and CD62L, n-butanol extract itself is a potential candidate for the treatment of LPS-induced sepsis. However, due to the presence of multiple bioactive components in the extract and limited approaches for preparing extracts from F. isatidis, it is difficult to identify the precise agent inhibiting LPS-induced chemokine receptor downregulation. Although the main active chemical components have been identified, there are more than eight major compounds in n-butanol extract (22) . Further investigation is required to identify the active compound in F. isatidis.

In conclusion, the present study demonstrated that n-butanol extract obtained from F. isatidis prevented the LPS-induced downregulation of CXCR1, CXCR2 and CD62L on neutrophils. The extract promoted neutrophil migration in response to IL-8. These results indicated that n-butanol extract from F. isatidis, which targets neutrophil chemotaxis during LPS-induced sepsis, is a potential candidate for the treatment of sepsis.

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This study was supported by the Provincial Natural Science Foundation of Zhejiang (grant no. Y2100530).