Visfatin regulates the production of lipopolysaccharide-induced inflammatory cytokines through p38 signaling in murine macrophages

Accepted Manuscript Visfatin regulates the production of lipopolysaccharide-induced inflammatory cytokines through p38 signaling in murine macrophages Xin-Tong Wu, Zhi Yang, Abdur Rahman Ansari, Ke Xiao, Xin-Xin Pang, You Luo, Hui Song PII:

S0882-4010(17)31664-9

DOI:

10.1016/j.micpath.2018.02.002

Reference:

YMPAT 2767

To appear in:

Microbial Pathogenesis

Received Date: 5 December 2017 Revised Date:

2 January 2018

Accepted Date: 1 February 2018

Please cite this article as: Wu X-T, Yang Z, Ansari AR, Xiao K, Pang X-X, Luo Y, Song H, Visfatin regulates the production of lipopolysaccharide-induced inflammatory cytokines through p38 signaling in murine macrophages, Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.02.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Visfatin regulates the production of lipopolysaccharide-induced inflammatory cytokines

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through p38 signaling in murine macrophages

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Xin-Tong Wu1,#, Zhi Yang1,#, Abdur Rahman Ansari2, Ke Xiao1, Xin-Xin Pang1, You Luo1, Hui

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Song1,*

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1. College of Animal Science and Veterinary Medicine, Huazhong Agricultural University,

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Wuhan, 430070, China

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2. Section of Anatomy and Histology, Department of Basic Sciences, College of Veterinary and Animal

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Sciences (CVAS) Jhang; University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan.

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Running title: Regulation mechanism of visfatin on LPS-induced inflammation

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Authors contributed equally as first co-authors

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Corresponding Author : [email protected], [email protected]

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Abstract

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Visfatin plays an important role in regulation of inflammatory cytokines. However, the

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role of visfatin under bacterial stress condition is not fully explored yet. Therefore, the present

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study was conducted for the better understanding of the regulation mechanism of visfatin on the

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production of inflammatory cytokines under lipopolysaccharide (LPS) stress in RAW264.7

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murine macrophages. Enzyme Linked Immuno-sorbent Assay (ELISA) results showed that, as

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compared to the control group, visfatin significantly up-regulated the levels of interleukin (IL)-

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1β, IL-6, IL-10, tumor necrosis factor (TNF)-α (P <0.05). Compared to the LPS group, the levels

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of IL-1β, IL-10, TNF-α was down-regulated in visfatin+LPS group (P＜0.05). After adding p38

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inhibitor, SB203580 to culture, the production of IL-1β, IL-6, IL-10, TNF-α was significantly

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reduced as compared to visfatin only (P<0.01). The results showed that visfatin may regulate the

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production of IL-1β, IL-6, IL-10, TNF-α through the p38 signaling pathway. As compared to the

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PBS group, phosphorylayed p38 (P-p38) level in visfatin group was significantly decreased (P

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<0.05). Compared with LPS group, P-p38 level was significantly decreased in visfatin+LPS

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group (P<0.05). Hence, it is concluded that visfatin can significantly increase the levels of IL-1β,

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IL-10 and TNF-α in normal conditions, while their levels significantly decrease during

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inflammation. Moreover, visfatin participates in the inflammatory response through the p38

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mitogen-activated protein kinase (MAPK) signal pathway by the up-regulation of p38 and down-

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regulation of P-p38 levels.

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Keywords: Visfatin; inflammatory cytokine; p38 signaling pathway; lipopolysaccharide

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1. Introduction

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Visfatin, also knwon as pre-B cell colony-enhancing factor (PBEF) or nicotinamide

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phosphoribosyl transferase (NAMPT) is present in almost all living species ranging from

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bacteria to highly developed animals and humans [1-3]. Visceral adipose tissue synthesize the

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visfatin [4]. Visfatin can be considered as clinical biomarker for adipose tissue function, chronic

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inflammation and as a possible therapeutic tool in metabolic as well as cardiovascular diseases

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[5]. Visfatin acts as a critical mediator in innate immunity and inflammation [6, 7] and controls

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the production of key cytokines on human endothelial cells at the transcript level in a dose and

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time dependent fashion [8]. Visfatin plays important role in regulation of inflammatory cytokines

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during differentiation of monocytes into macrophages [3]. However, the role of visfatin under

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bacterial stress condition is not fully explored yet. Therefore, further investigation is necessary

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for better understanding of the regulation mechanism of visfatin on the levels of inflammatory

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cytokines released from macrophages under various pathogenic conditions.

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Macrophages belong to haematopoietic system with extraordinary plasticity and may be

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found in almost all body tissues [9, 10]. Macrophages act as a first line of host defense and

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phagocytosis is a fundamental process in preliminary immune response during pathogenic

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infections [11, 12]. Lipopolysaccharide (LPS), a toll like receptor 4 (TLR4) ligand, is a key

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constituent of Gram-negative bacterium cell wall [13] The production of inflammatory cytokines

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was severely impaired in response to the TLR4 ligand in murine macrophages [14].

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Macrophages are naturally activated by bacterial LPS exposure[15] and the activated

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macrophages have an enhanced potential for phagocytic activity during clearance of infectious

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agents [16, 17]. Mitogen-activated protein kinase (MAPK)-p38 signaling pathway mediates the

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phagocytosis in LPS-stimulated macrophages [18]. LPS stimulation causes considerable increase

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in the levels of TLR4 and pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6 and

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tumor necrosis factor (TNF)-α [19]. Several chemotherapeutic agents including ethanolic extract

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of pomegranate flower [20], emodin, an anthraquinone derivative [21], lactic acid [22], britanin,

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an isolate from the flowers of Inula japonica [23] and curcumin, an extract of Curcuma longa

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rhizomes [24] have shown anti-inflammatory effects on the level of LPS-induced inflammatory

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cytokine by producing mediator molecules implicated in the inflammatory response. However,

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studies regarding the regulation mechanism of visfatin on the production of inflammatory

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cytokines under LPS stress are still scarce. Therefore, in the current study, we investigated the

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effects of visfatin on the levels of LPS-induced inflammatory cytokines and p38 signaling

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pathway in RAW264.7 murine macrophages.

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2. Materials and methods

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2.1 Reagents

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We purchased RAW264.7 cells lines from the cell bank of Shanghai Chinese Academy of

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Sciences [25], visfatin from Adipo Bio-science, Escherichia coli LPS (O111:B4) from Sigma (St

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Louis, MO, USA) and BCA Protein Quantification Kit from Vazyme Biotech. The cytokines

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including IL-1β, IL-6, IL-10 and TNF-α were quantified using ELISA kits (NeoBioscience,

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Shenzhen, China).

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2.2 RAW264.7 cells culture, passage, cryopreservation and resuscitation

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The RAW264.7 murine macrophages were passaged and treated by LPS and visfatin by

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following same steps as described previously [26]. Briefly, the cell suspension was packed into

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T25 cell culture flask and high glucose, dulbecco's modified Eagle medium (H-DMEM) medium

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containing 10% fetal bovine serum (FBS) was added in it and observed the growth phase of the

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cells daily. After the growth of the cell culture, cell passage was performed by washing the cells

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twice with 0.1 mol/L PBS at 37 °C. After digestion with 0.25% trypsin digestion solution, the

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appropriate cell density was adjusted to maintain the normal metabolism of cells. The

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cryopreserved solution (50%H-DMEM, 40%FBS, 10%DMSO) was added and mixed into the

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frozen tube and then the cell suspension was added into each tube and finally saved into the

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liquid nitrogen. For cell resuscitation, cryopreservation tube were immediately taken out from -

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80 ℃ low temperature refrigerator or liquid nitrogen into the 37 ℃ water bath to dissolve it

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quickly, then the cell suspension was added to the fresh medium and the medium was changed

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on the next day to remove DMSO.

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2.3 Treatment of cell culture and detection of inflammatory cytokines by ELISA

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For the determination of inflammatory cytokines levels, the cell culture treatments were divided

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into four groups i.e., PBS group, Visfatin group, LPS group, LPS + visfatin group, (n=3 in each

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group). All the treatment chemicals are pre-dissolved with DMEM. In PBS group, complete

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medium, in visfain group medium having 200 ng/mL visfatin, in LPS group medium containing

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10 µg/mL LPS and in visfatin+LPS group medium containing 200 ng/mL visfatin and 10 µg/mL

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LPS were added. Sampling from these treated cell culture was done at 6, 12, 18, 24 hours and the

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samples were preserved at -20 ℃ refrigerator. Determination of cytokine levels in RAW264.7

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cells of different groups were performed by ELISA using paired antibodies according to the

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manufacturer’s instructions. All the assays were performed in duplicate and ELISA plates (IL-

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1β,IL-6, IL-10 and TNF-α) were read using an ELISA reader at 450 nm. The concentrations of

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cytokine samples were calculated from the standard curves.

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2.4 Detection of inflammatory factors with the inhibitor by ELISA

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For the detection of inflammatory cytokine with the inhibitor, the cell culture treatments were

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divided into three groups i.e., PBS group,visfatin group, visfatin+Inhibitor group, (n=3 in each

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group). All the treatment chemicals are pre-dissolved with DMEM. In visfain group medium

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having 200 ng/mL visfatin and in visfatin+inhibitor group medium containing 200 ng/mL

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visfatin and pre-incubated 20µM (µmol/L) p38 were added. Sampling from these treated cell

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culture was done at 6, 12, 18, 24 hours and the samples were preserved at -20 ℃ refrigerator.

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Similarly, determination of cytokine levels in RAW264.7 cells of different groups was

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accomplished by ELISA using paired antibodies according to the manufacturer’s instructions.

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All the assays were performed in duplicate and ELISA plates (IL-1β,IL-6,IL-10 and TNF-α)

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were read using an ELISA reader at 450 nm. Cytokine concentrations of the samples were

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calculated from standard curves.

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2.5 Determination of P-p38 and p38 contents by Western Blotting

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Frozen tissue preparations were homogenized with sample buffer, centrifuged and boiled. Total

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protein concentration of the tissue was quantified using the Bradford method. Protein

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concentrations were determined using the BioRad protein assay (Bio-Rad, Hercules, CA, USA).

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Equal amounts of total protein were loaded onto 1 % SDS-PAGE and then electro-phoretically

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transferred onto polyvinylidene difluoride mem-branes (IPVH00010; Millipore). Transferred

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membranes were blocked using 5% skim milk and incubated overnight with antibodies against

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p38, P-p38. The same membrane was probed with anti-GAPDH (Xianzhi, Hangzhou, China) as

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house-keeping protein. After washing with TBST three times, the blots were hybridized with

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secondary antibodies (1:5000 dilution; Boster) conjugated with horseradish peroxidase for 2h at

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room temperature. The antibody-specific protein was visualized by ECL detection system.

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Statistical analysis Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS,

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v.17.0; SPSS, Chicago, IL, USA) and Graphpad Prism 5 software (Graphpad, San Diego, CA,

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USA). The ANOVA test was used for comparisons between groups. Differences were considered

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statistically significant at P values of ≤0.05. All data were expressed as means±SD.

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3. Results

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3.1 The changes of inflammatory cytokine levels in different treatment groups of

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RAW264.7 Cells

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The results of ELISA showed that the production of IL-1β was gradually increased from 6h, 12h,

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18h to 24h in each group. The level of IL-6 in each group was increased first and then decreased,

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and peaked at 18 h. The level of IL-10 and TNF-α were also increased first and then decreased,

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and reached the peak at 18 h. Compared with PBS group, IL-1β, IL-6, IL-10 and TNF-α (P <0.05)

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(Fig.1A-D) in visfatin group were significantly up-regulated. Compared with LPS group, the

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level of IL-1β, IL-10 and TNF-α (P <0.05) in the visfatin + LPS group was significantly

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decreased, while the level of IL-6 was significantly up-regulated (P <0.05). These results

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revealed that levels of inflammatory cytokines change over time in different treatment groups.

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3.2 The effect of p38 inhibitor on inflammatory cytokine

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After the addition of p38 inhibitor SB203580, the supernatant was collected at 6h, 12h, 18h and

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24h and the levels of IL-1β, IL-6, IL-10 and TNF-α in the cells were measured with ELISA. The

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most obvious results were obtained at 18h post inhibitor treatment. IL-1β, IL-6, IL-10 and TNF-α

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were significantly (P<0.01) down-regulated in p38 inhibitor SB203580 as compared to non-

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inhibitor or visfatin group (Fig. 2).

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3.3 Detection of p38 inflammatory signaling pathway in RAW264.7 cells

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The production of p38 and P-p38 in different groups was detected by Western Blot at 15 min, 30

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min, 45 min and 60 min, in different treatment groups. It was found that the production of p38

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and P-p38 was obvious at 30 min. Other time periods also present the laws. The results showed

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that the level of p38 in visfatin group was significantly higher than that in PBS group (P <0.05)

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(Fig. 3A). Compared with the LPS group, the p38 level in the visfatin+LPS group was

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significantly higher (P <0.05) (Fig. 3A). The level of P-p38 in the visfatin group was

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significantly lower than that in the PBS group (P <0.05) (Fig. 3B) and the P-p38 level in the

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visfatin + LPS group was significantly lower than that in the LPS group (P <0.05) (Fig. 3B). In

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the study of p38 and P-p38 production changes over time, it was found that the level of p38 in

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the LPS group increased gradually over time and reached the maximum at 60 min (Fig. 4A).

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After the addition of visfatin, the level of p38 has reached its maximum in the visfatin + LPS

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group at 45 min (Fig. 4B). Similarly, the production of P-p38 attained a peak at 60 min, and this

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peak appeared at 45 min after addition of visfatin. These results showed that visfatin regulated

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the production of LPS induced inflammatory cytokines in macrophages through p38 signaling

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pathway.

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4. Discussion

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The inflammation is a strongly regulated process and macrophages are involved in its

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commencement, continuation and resolution that synthesize a lot of biologically mediators for

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both harmful and useful effects during inflammatory process [27, 28]. Adipocytokines act like

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two-edged sword, they usually regulate the body's energy homeostasis and also control the

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body's inflammatory response [29-31]. This study investigated the regulation mechanism of

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visfatin by adopting LPS-induced RAW 264.7 murine model. The results showed that visfatin

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significantly up-regulated the level of IL-1β, IL-6, IL-10 and TNF-α (P <0.05). Prior studies

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have also found that visfatin function by inducing the production of cytokines including IL-6, IL-

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1β and TNF-α [32-34]. The present study determined that LPS stimulation to macrophages

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induced the noteworthy up-regulation in the levels of IL-1β, IL-10 and TNF-α and slight

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decrease in IL-6 level. Consistent with our results, previous reports have also found that LPS

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exposure to mouse RAW264.7 cells induced the synthesis of IL-6 and TNF-α in a dose

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dependent manner [35-37]. This is contrary to the production of IL-6 in the current trial. It is

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presumed that the expression level of IL-6 might be different with time and peaked before 6 h, so

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its level was found slightly lower than the control group after 6 h. However, other studies have

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shown that IL-6 levels in serum have peaked after 6 h following LPS stimulation and existed for

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up to 36 h in the serum [38, 39]. This is consistent with the conclusion of current experimental

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research work. Several chemical agents such as britanin, ethanolic extract of pomegranate flower

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and curcumin have also shown the anti-inflammatory effects on the inflammatory mediators in

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RAW 264.7 cells under bacterial LPS stress [20, 23, 24]. As compared to the control group, the

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levels in IL-1β, IL-10 and TNF-α in visfatin+LPS stimulated group were significantly decreased,

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while IL-6 production was significantly up-regulated in the present study. Moreover, the addition

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of visfatin could decrease the levels of IL-1β, IL-10 and TNF-α in LPS-induced RAW264.7 cells.

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Hence, it is concluded that visfatin increased the production of some inflammatory factors in the

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normal conditions while it inhibited the production of same inflammatory factors after induction

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of inflammation by LPS stimulation, indicating that visfatin has a dual effect on the regulation of

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inflammatory factors. Signaling pathways play vital role in both normal tissue regeneration and repair and

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during infection in inflammatory conditions [40]. MAPK-p38 signaling pathway can regulate the

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body's inflammatory response [41] and visfatin helps in regulation of inflammatory factors [42].

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MAPK-p38 signaling also mediates the phagocytic activity in LPS-stimulated macrophages [18].

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Previous study has determined that p38 inhibitor SB203580 significantly inhibited the

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production of IL-1β, IL-1Ra, IL-6, IL-10 and TNF-α in human CD14 + monocytes [32]. In the

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current investigation, p38 signal pathway was blocked by using p38 inhibitor SB203580 that

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caused significant decrease in the levels of IL-1β, IL-6, IL-10 and TNF-α, however, their levels

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were significantly up-regulated by the addition of visfatin. Hence, it seems that visfatin may

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regulate the inflammatory response in murine macrophages through the p38 signaling pathway.

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Phosphorylated p38 (P-p38) is activated form of p38 [43], that usually results through the

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exposure of bacterial LPS [43, 44]. P-p38 helps in turning on the responsive genes by mediating

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the signal transduction into the cell nucleus [45]. P-p38 may provoke cells to initiate production

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of such mediators that cause activation of downstream transcription factors [46]. Visfatin in

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synovial fibroblasts may activate P-p38, while visfatin inhibitors may result in its in-activation

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[47, 48]. In the current investigation, as compared to both control (PBS) as well as LPS groups,

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visfatin caused down-regulation of P-p38 level. Hence, it is proposed that visfatin possibly

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regulated the body's inflammatory response by up-regulating the production of p38 and down-

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regulating the production of P-p38.

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In the study of p38 and P-p38 production changes over time, we found that the level of p38 and

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P-p38 increased gradually with time in LPS-stimulated RAW264.7 cells and reached at the

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maximum level in 60 min. In a prior report, the production of P-p38 gradually increased in 60

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min following LPS (100 ng/mL) stimulation to RAW264.7 cells [49]. P-p38 was activated in 30

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min and reached at the peak level in 60 min after LPS (200 mg/mL) treatment to RAW264.7

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cells [35]. In the present research work, as compared to the LPS group, the levels of p38 and P-

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p38 reached at the peak level in 45 min after visfatin treatment. Thus, it is speculated that the

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production of p38 reached at the peak level by the up-regulation of visfatin.

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Conclusion

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Visfatin can significantly increase the levels of IL-1β, IL-10 and TNF-α in normal

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conditions, while their levels significantly decrease during inflammation. Moreover, visfatin

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participates in the inflammatory response through the p38 MAPK signal pathway by the up-

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regulation of p38 and down-regulation of P-p38 levels.

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Abbreviations

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IL, interleukin; TNF, tumor necrosis factor; LPS, lipopolysaccharide; ELISA, Enzyme Linked

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Immuno-sorbent Assay; P-p38, phosphorylayed p38; MAPK, mitogen-activated protein kinase;

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DMEM, Dulbecco's Modified Eagle Medium; FBS, Fetal bovine serum; DMSO, Dimethyl

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sulfoxide; TLRs, toll like receptors; h, hour/hours; PBS, phosphate buffered saline.

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Conflict of Interest

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The authors declared no conflict of interests.

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Acknowledgments

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This study was supported by the Fundamental Research Funds for the Central Universities No.

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2662015PY063 and National Natural Science Fund Project of China No.31101776.

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Fig. 1: The levels of inflammatory cytokines in different treatment groups

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A: The levels of IL-1β in different groups, B: The levels of IL-6 in different groups, C: The

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PBS group, * indicates P＜0.05,** indicates P＜0.01, Compared with LPS group, # indicates P

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Fig 2 The effect of p38 inhibitor to inflammatory cytokine

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The production levels of different inflammatory cytokines in PBS, visfatin and visfatin+p38

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inhibitor groups. Compared with visfatin group, **indicates P＜0.01, n=3

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Fig 3 The level of p38 and P-p38 in different treatment groups

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A: The level of p38 in different treatment groups, B: The level of P-p38 in different treatment

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Fig 4 The level of p38 and P-p38 at different time points

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P-p38 at different time points in Visfatin + LPS group

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Highlights The role of visfatin under bacterial stress condition is not fully understood yet This study determined the role of visfatin on inflammatory cytokines under LPS stress in

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murine macrophages

Visfatin inhibited the expression of inflammatory factors after induction of inflammation by LPS stimulation

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Visfatin has a dual effect on the regulation of inflammatory factors

Visfatin participated in the inflammatory response through the p38 MAPK signal

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pathway