Bone Marrow Mesenchymal Stem Cells Inhibit ......Bone Marrow Mesenchymal Stem Cells Inhibit ... and...

9
Research Article Bone Marrow Mesenchymal Stem Cells Inhibit Lipopolysaccharide-Induced Inflammatory Reactions in Macrophages and Endothelial Cells Dequan Li, 1 Cong Wang, 2 Chuang Chi, 3 Yuanyuan Wang, 4 Jing Zhao, 4 Jun Fang, 1 and Jingye Pan 4 1 Department of Traumatology Medicine, e First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China 2 School of Pharmacy, Wenzhou Medical University, Wenzhou, Zhejiang 325000, China 3 Department of Cardiothoracic Surgery, e First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China 4 Department of Intensive Care Unit, e First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China Correspondence should be addressed to Jingye Pan; [email protected] Received 31 August 2015; Revised 24 December 2015; Accepted 6 January 2016 Academic Editor: Teresa Zelante Copyright © 2016 Dequan Li et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Systemic inflammatory response syndrome (SIRS) accompanied by trauma can lead to multiple organ dysfunction syndrome (MODS) and even death. Early inhibition of the inflammation is necessary for damage control. Bone marrow mesenchymal stem cells (BMSCs), as a novel therapy modality, have been shown to reduce inflammatory responses in human and animal models. Methods. In this study, we used Western blot, quantitative PCR, and enzyme-linked immunosorbent assay (ELISA) to assess the activity of BMSCs to suppress the inflammation induced by lipopolysaccharide (LPS) in human umbilical cord endothelial cells (HUVECs) and alveolar macrophages. Results. Our results demonstrated that LPS caused an inflammatory response in alveolar macrophages and HUVECs, increased permeability of HUVEC, upregulated expression of toll-like receptor (TLR) 2, TLR4, phosphorylated p65, downregulated release of IL10, and promoted release of TNF- in both cells. Coculture with BMSCs attenuated all of these activities induced by LPS in the two tested cell types. Conclusions. Together, our results demonstrate that BMSCs dosage dependently attenuates the inflammation damage of alveolar macrophages and HUVECs induced by LPS. 1. Introduction Traumatic injury is the leading cause of morbidity and mortality, which affects many parts of the body, including the brain, extremities, and internal organs. e incidence of life- threatening complications, such as systemic inflammatory response syndrome (SIRS) and acute lung injury (ALI), in severely injured trauma patients remains between 30% and 50% [1, 2]. SIRS induced by severe trauma or other injuries is a clinical syndrome initiated by dysregulation of inflammation, which could lead to various tissue injuries culminating in multiple organ dysfunction/failure syndrome (MODS/MOF). Bone marrow-derived mesenchymal stem cells (BMSCs), also referred to as marrow stromal cells, are a multipo- tential lineage characterized by the capacity for extracor- poreal expansion and the ability to differentiate into bone, cartilage, and adipose tissues. BMSCs appear to function as potent immunomodulators. Numerous studies indicated that BMSCs can be an effective modality for cell-based immunomodulatory therapies in various diseases, such as sepsis, endotoxemia, diabetes, and lung injury [3–9], espe- cially for reducing of inflammation [4, 5, 10]. Several clinical trials assessing the efficacy of BMSCs in immune-mediated diseases are currently underway. However, how BMSCs attenuate inflammatory reaction is poorly understood. Hindawi Publishing Corporation Mediators of Inflammation Volume 2016, Article ID 2631439, 9 pages http://dx.doi.org/10.1155/2016/2631439

Transcript of Bone Marrow Mesenchymal Stem Cells Inhibit ......Bone Marrow Mesenchymal Stem Cells Inhibit ... and...

Page 1: Bone Marrow Mesenchymal Stem Cells Inhibit ......Bone Marrow Mesenchymal Stem Cells Inhibit ... and TLR4 response to acute otitis through activation of NF-𝜅B [15], we hypothesized

Research ArticleBone Marrow Mesenchymal Stem Cells InhibitLipopolysaccharide-Induced Inflammatory Reactions inMacrophages and Endothelial Cells

Dequan Li1 Cong Wang2 Chuang Chi3 Yuanyuan Wang4

Jing Zhao4 Jun Fang1 and Jingye Pan4

1Department of Traumatology Medicine The First Affiliated Hospital of Wenzhou Medical University WenzhouZhejiang 325000 China2School of Pharmacy Wenzhou Medical University Wenzhou Zhejiang 325000 China3Department of Cardiothoracic Surgery The First Affiliated Hospital of Wenzhou Medical University WenzhouZhejiang 325000 China4Department of Intensive Care Unit The First Affiliated Hospital of Wenzhou Medical University WenzhouZhejiang 325000 China

Correspondence should be addressed to Jingye Pan panjingyewzhospitalcn

Received 31 August 2015 Revised 24 December 2015 Accepted 6 January 2016

Academic Editor Teresa Zelante

Copyright copy 2016 Dequan Li et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Systemic inflammatory response syndrome (SIRS) accompanied by trauma can lead to multiple organ dysfunctionsyndrome (MODS) and even death Early inhibition of the inflammation is necessary for damage control Bone marrowmesenchymal stem cells (BMSCs) as a novel therapy modality have been shown to reduce inflammatory responses in humanand animal models Methods In this study we used Western blot quantitative PCR and enzyme-linked immunosorbent assay(ELISA) to assess the activity of BMSCs to suppress the inflammation induced by lipopolysaccharide (LPS) in human umbilicalcord endothelial cells (HUVECs) and alveolar macrophages Results Our results demonstrated that LPS caused an inflammatoryresponse in alveolar macrophages and HUVECs increased permeability of HUVEC upregulated expression of toll-like receptor(TLR) 2 TLR4 phosphorylated p65 downregulated release of IL10 and promoted release of TNF-120572 in both cells Coculture withBMSCs attenuated all of these activities induced by LPS in the two tested cell types Conclusions Together our results demonstratethat BMSCs dosage dependently attenuates the inflammation damage of alveolar macrophages and HUVECs induced by LPS

1 Introduction

Traumatic injury is the leading cause of morbidity andmortality which affects many parts of the body including thebrain extremities and internal organs The incidence of life-threatening complications such as systemic inflammatoryresponse syndrome (SIRS) and acute lung injury (ALI)in severely injured trauma patients remains between 30and 50 [1 2] SIRS induced by severe trauma or otherinjuries is a clinical syndrome initiated by dysregulation ofinflammation which could lead to various tissue injuriesculminating in multiple organ dysfunctionfailure syndrome(MODSMOF)

Bone marrow-derived mesenchymal stem cells (BMSCs)also referred to as marrow stromal cells are a multipo-tential lineage characterized by the capacity for extracor-poreal expansion and the ability to differentiate into bonecartilage and adipose tissues BMSCs appear to functionas potent immunomodulators Numerous studies indicatedthat BMSCs can be an effective modality for cell-basedimmunomodulatory therapies in various diseases such assepsis endotoxemia diabetes and lung injury [3ndash9] espe-cially for reducing of inflammation [4 5 10] Several clinicaltrials assessing the efficacy of BMSCs in immune-mediateddiseases are currently underway However how BMSCsattenuate inflammatory reaction is poorly understood

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 2631439 9 pageshttpdxdoiorg10115520162631439

2 Mediators of Inflammation

Quick and efficient response to microbial infections isdriven by recognition of molecules broadly shared by avariety of pathogens which are distinguishable from hostmolecules named pathogen associated molecular patterns(PAMPs) Pattern-recognition receptors (PRRs) that containmembrane-bound PRRs including toll-like receptors (TLRs)and other cytoplasmic proteins recognize the PAMPs [10]Upon binding with the PAMPs TLR2 and TLR4 then activateNF-120581B and subsequently promote cytokine synthesis whichinclude IL-1 IL-6 and TNF-120572 [11 12] The network of TLRsand PRRs mediates the response of BMSCs to inflammatorystimuli such as LPS [13 14] However how TLRs modulatethe BMSC activity is not clearly elucidated Since TLR2and TLR4 response to acute otitis through activation ofNF-120581B [15] we hypothesized that BMSCs ameliorate theinflammation damage of alveolarmacrophages andHUVECsthrough the inhibition of TLR2 and TLR4 mediated NF-120581Bpathways

2 Materials and Methods

21 Cell Culture Alveolar macrophages and HUVECs werepurchased from Chi Scientific Inc (China) The cells werecultured in RPMI 1640 medium (Sigma Chemical Co StLouis MO) supplemented with 10 fetal bovine serum (FBS)(Gibco Carlsbad CA USA) and 1 penicillinstreptomycin(Solarbio Beijing China) at 37∘C in a humidified atmosphereof 5 CO

2 BMSCs obtained from Cyagen Biosciences

Inc (Suzhou China) were cultured in 10 FBS-DMEM-LG(Gibco Carlsbad CA USA)The surface markers for BMSCsdifferentiation of adipocytic osteogenic and chondrogeniclineages were characterized as previously reported [16] Onlypassages 3ndash7 of the cells were used for experiments

22 Endothelial Permeability Assay Monolayer permeabilitywas quantitated by spectrophotometric measurement of theflux of Evans blue-albumin across HUVECs as describedpreviously [17] HUVECs (1 times 105) were seeded in 24-wellTranswell inserts (5 120583m pore size Corning IncorporatedNY USA) and cultured with 10 FBS DMEM overnightand then changed with serum-free medium for another 24hours The various numbers of BMSCs were seeded at thebottom of the same 24-well plate with serum-free media LPSwas added to the Transwell inserts at a final concentrationof 100 ngmL for different time points (1 3 6 12 and24 h) After that the BMSCs were digested by trypsin-EDTAsolution to terminate the effects of BMSCs and 100 120583L Hankrsquossolution containing Evans blue- (EB-) conjugated bovineserum albumin (final concentration 067mgmL) was addedto the Transwell inserts while 600 120583L 4 BSA was addedto the lower plate chamber After incubation at 37∘C for1 hour 100 120583L BSA solution from the lower chamber washarvested and measured for the absorbance at 620 nm TheHUVECs permeability capacity was calculated as followsEvans blue dye-labeled albumin (EB-albumin) leak rate =OD620leaked EB-albuminOD620total EB-albumin times 100

23 Alveolar MacrophagesHUVECs-BMSC Coculture Exper-iments Alveolar macrophages or HUVECs (1 times 106well)

were seeded in a 6-well plate after 24-hour incubationBMSCs (2 times 105well) were seeded in the 6-well Transwellinserts (5 120583m pore Corning Incorporated NY USA) at theindicated time points (1 3 6 12 and 24 h) LPS (100 ngmL)was then added to the lower chamber for 1 h Alveolarmacrophages or HUVECs lysate was collected for RNA andprotein analyses and the cell-free supernatants were collectedfor ELISA detection

24 Western Blot Analysis Alveolar macrophagesHUVECswere lysed in the RIPA buffer (Thermo Scientific RockfordUSA) supplemented with 1 PMSF and 1 protein phos-phatase inhibitor mixture (P1260 Applygen Beijing China)The concentration of the proteins was measured with theBicinchoninic Acid Kit (Thermo Scientific Rockford USA)Rabbit anti-TLR2 TLR4 and phosphorylated p65 unit of NF-120581B antibodies were obtained from Cell Signaling Technol-ogy Horseradish peroxidase- (HRP-) conjugated goat anti-rabbit antibodies were purchased from Bio-Rad ECL-PlusChemiluminescent Reagent (Thermo Scientific RockfordUSA) was used to visualize the specific proteins Relativeconcentration of proteins was quantitated using the Image JSoftware (National Institutes of Health Bethesda USA)

25 Gene Expression Analysis Total RNA was isolated fromcells using the TRIzol RNA Isolation Reagents (Life Tech-nologies) The first-strand cDNAs were converted using theSuperScript III reverse transcriptase (Invitrogen CarlsbadCA) according to the manufacturerrsquos protocols Real-timeRT-PCR analysis was performed on ABI 7500 SequenceDetection System using the UltraSYBR Mixture (CW BioCo Ltd Beijing China) Transcript expression was nor-malizedwith the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) housekeeping gene The PCR primer sequencesare GAPDH forward 51015840-TGGAGTCTACTGGCGTCTT-31015840 reverse 51015840-TGTCATATTTCTCGTG GTTCA-31015840 TLR4forward 51015840-CTGCATAGAGGTAGTTCCT-31015840 reverse 51015840-TCCAGCC ACTGAAGTTCTGA-31015840 TLR2 forward 51015840-GGAGACTCTGGAAGC ATG-31015840 reverse 51015840-GCATCCTGAAGCCTGTG-31015840

26 ELISA Analysis The supernatants collected from cocul-ture system were centrifuged for 10 minutes at 1000 g(4∘C) to remove cell debris The levels of TNF-120572 and IL10were measured using commercially available enzyme-linkedimmunosorbent assay (ELISA) kits (BD Biosciences SanDiego CA USA) according to the instructions provided bythe manufacturer

27 Statistical Analysis SPSS 170 software was used to per-form the statistical analysis and values are presented as themeans plusmn standard deviations (SD) all experiments wererepeated at least three times The one-way ANOVA was usedto determine the significance of differences between variousgroups119875 lt 005was considered to be statistically significant

3 Results

31 BMSCs Inhibit LPS to Induce Hyperpermeability inHUVECs To assess how BMSCs affected HUVECs perme-ability induced by LPS HUVECs with or without coculture

Mediators of Inflammation 3

LPSLow concentrations of BMSCsMedian concentrations of BMSCsHigh concentrations of BMSCs

10

8

6

4

2

0

EB-a

lbum

in le

ak ra

te (

)

1 3 6 12 24

Time (h)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

Figure 1 Effect of BMSCs on the barrier permeability of HUVECsHUVECs were stimulated by LPS at a dosage of 100 ngmL (blackbars) In addition HUVECswere coculturedwith indicated concen-trations of BMSCs low (1 times 103 cellsmL dark grey bars) median (1times 104 cellsmL light grey bars) and high (1 times 105 cellsmL whitebars) for 1 3 6 12 and 24 h followed by measuring permeability asdescribed in Section 2 All results are means plusmn SD of at least threedifferent experiments lowast119875 lt 005 lowastlowast119875 lt 001

with BMSCs were stimulated by LPS for the indicated timesWithout coculture with BMSCs the EB-albumin leaking rateranged from 535 (at 1 h) to 772 (at 24 h) in the LPS group(Figure 1) Compared with the LPS group the EB-albuminleaking rates in the BMSC coculture group were reducedin a BMSC cell number- and time-dependent manner TheEB-albumin leaking rate in the 1 times 104mL BMSCs groupwas decreased 3 hours after coculture The effects were moresignificant after coculture for 6 hours to 24 hours The effectswere less significant in the group with 1 times 103mL BMSCsand more significant in the group with 1 times 105mL BMSCs(Figure 1)

32 BMSCsAttenuate LPS-Induced Expression of TLR2 TLR4and p65 in Alveolar Macrophages and HUVECs To deter-mine whether LPS stimulated expression of TLR2 TLR4 andp65 in macrophages and HUVECs was affected by BMSCsalveolar macrophages and HUVECs cells were coculturedwith BMSCs for the indicated time followed by LPS treat-ment Western blot (Figure 2) and real-time RT-PCR anal-yses (Figure 3) showed that LPS administration increasedexpression of TLR2 TLR4 and p65 within 1 hour after thetreatment The effect persisted even until 24 hours after thetreatment However the activity of LPS was attenuated bycoculture with BMSCs in both alveolar macrophages andHUVECs (Figures 2 and 3)

33 BMSCs Suppress Alveolar Macrophages and HUVECs toRelease IL10 and TNF-120572 in Response to LPS Stimulation To

determine whether BMSCs regulated the release of TNF-120572 and IL10 from alveolar macrophages and HUVECs inresponse to LPS stimulation the conditioned medium wascollected from the coculture system and the concentrationsof IL10 and TNF-120572 were assessed by ELISA It was clearthat concentration of TNF-120572 in the medium was increasedafter LPS stimulation within 1 hour and the effects lasted for24 hours However the increases were blunted by coculturewith BMSCs In contrast IL10 was reduced by LPS stimu-lation while coculture with BMSCs increased medium IL10production The results indicate that BMSCs suppress LPS-induced inflammation by decreasing TNF-120572 and increasingIL10 releases (Figure 4)

4 Discussion

BMSCs treatments have been shown to reduce inflammatoryresponse in human and animal models in response to injuryHowever the detailed mechanism underlying this effect isnot clear Here we reported that BMSCs alleviated HUVECdamage induced by LPSThe results also showed that BMSCsrestricted the inflammatory responses of HUVECs and alve-olar macrophage stimulated by LPS and therefore protectedHUVECs from damage induced by the endotoxin The dataindicate that attenuating LPS-induced TNF-120572 pathway acti-vation inHUVECs andmacrophage by BMSCs contributes tothe protection of HUVECs from endotoxin-induced damageby BMSCs

Our data demonstrated that LPS increased the perme-ability of HUVECs monolayers in a time-dependent mannerAlthough the extent of the activity was lower than thatafter coculture for a longer time the protection effects weredetectable within one hour The underlying mechanism ofthe protection is not clear Since BMSCs and HUVECs werecultured separating by the Transwell membrane the effectsof BMSCs must be mediated by soluble factors releasedfrom BMSCs However we did not rule out the possibilitythat BMSCs sequestrated LPS and therefore protected theendothelial cells Future efforts are needed to identify thesebeneficial secretory factors from BMSCs Although LPS stillinduced HUVECs damage in the coculture the EB-albuminleaks were reduced compared with the group without cocul-ture with BMSCs This suggests that BMSCs attenuate thedamage We observed that the protective effect of BMSCs onHUVEC was cell number-dependent High concentrationsof BMSCs were more effective than low concentrationsof BMSCs It appeared that the BMSCs released sufficientprotective molecules to protect endothelial cells within onehour Therefore immediately administration of BMSCs toSIRS patients shall have beneficial effects Furthermore theresults were in line with previous reports that LPS leadsto persistent damage of HUVECs [18 19] However it wasdifferent from the report that the damage of HUVEC by LPSis not time-dependent [17] The discrepancy is likely due todifferent culture time We cultured the cells for 24 hours toreach the confluence followed by serum starvation for 24hours and LPS treatment This method is different from thepublished one where the cells were cultured for 4 days prior

4 Mediators of Inflammation

TLR2

TLR4

(1) PBS control (2) BMSCs control(3) LPS (4) BMSCs treatment

Alv

eola

r mac

roph

ages

1 2 3 4

p-NF-120581B p65

120573-actin

PBS controlBMSCs control

LPSBMSCs treatment

TLR2

Nor

mal

ized

to120573

-act

in (T

LR2

)

00

06

12

18

24

30

63 121 24Time (h)

lowastlowastlowastlowast

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Nor

mal

ized

to120573

-act

in (T

LR4

)

00

06

12

18

24

30

6 12 2431Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowastlowast lowast

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lowastlowast lowast

(A)

(B) (C)

Nor

mal

ized

to120573

-act

in (p

-NF-120581

B p65

)

p-NF-120581B p65

00

06

12

18

24

1 3 12 246Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowast

lowastlowast lowast

lowastlowast lowastlowastlowast lowast

lowastlowast lowast

(D)

(a)

Figure 2 Continued

Mediators of Inflammation 5

TLR2

TLR4

(1) PBS control (2) BMSCs control(3) LPS (4) BMSCs treatment

HU

VEC

s

1 2 3 4

p-NF-120581B p65

120573-actin

00

06

12

18

24

30

241263Time (h)

1

TLR2

PBS controlBMSCs control

LPSBMSCs treatment

Nor

mal

ized

to120573

-act

in (T

LR2

)

lowastlowastlowastlowast

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Nor

mal

ized

to120573

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in (T

LR4

)

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06

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30

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LPSBMSCs treatment

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lowast

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mal

ized

to120573

-act

in (p

-NF-120581

B p65

)

p-NF-120581B p65

00

06

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PBS controlBMSCs control

LPSBMSCs treatment

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(E)

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(H)

(b)

Figure 2 Coculture with BMSCs attenuates the activity of LPS to induce expressions of TLR2 TLR4 and p65 in alveolar macrophages (a)and HUVECs (b) at the protein levelThe total proteins were extracted from the cultured alveolar macrophages and HUVECs treated (1) PBScontrol (2) BMSCs (3) LPS and (4) BMSCs and LPS The expressions of TLR2 TLR4 and p-NF-120581B p65 were determined by Western blotThe density of specific bands was quantitated Data are expressed as the mean plusmn SD from three experiments lowast119875 lt 005 lowastlowast119875 lt 001

6 Mediators of Inflammation

0

1

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eola

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ages

2412631

Relat

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A le

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LPSBMSCs treatment

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LPSBMSCs treatment

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A le

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LPSBMSCs treatment

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(C) (D)

(b)

Figure 3 Coculture with BMSCs attenuates the activity of LPS to induce expressions of TLR2 and TLR4 in alveolar macrophages (a) andHUVECs (b) at themRNA levelThe total RNAswere extracted from the cultured alveolarmacrophages andHUVECs treated (1) PBS control(2) BMSCs (3) LPS and (4) BMSCs and LPSThe expressions of TLR2 and TLR4 were determined by real-time RT-PCR Data are expressedas the mean plusmn SD from three experiments lowast119875 lt 005 lowastlowast119875 lt 001

to the experiments Further efforts are needed to clarify thisdiscrepancy

TLRs play a pivotal role in defense against invadingpathogens through recognizing PAMPs It is well docu-mented that cells use TLR2 and TLR4 to recognize PAMPsexpressed by bacteria Activation of TLR2 and TLR4 leadsto activation of MAPK and NF-120581B pathways and productionof inflammatory cytokines including IL-1 IL-6 and TNF-120572 [20ndash22] In this study we showed that LPS promoted

expression of TLR2 and TLR4 in alveolar macrophages andHUVECs However how expression of TLR2 expressionwas increased by LPS is unknown and deserves futureinvestigations TNF-120572 and IL10 expressions were also affectedby LPS Furthermore the LPS effects were diminishedwhen the HUVECs and macrophages were cultured withBMSCs TNF-120572 is a potent inflammatory factor and IL10 isa key anti-inflammatory cytokine Downregulation of TNF-120572 and upregulation of IL10 in HUVECs and macrophage

Mediators of Inflammation 7A

lveo

lar m

acro

phag

es

3 6 24121Time (h)

Supe

rnat

ant l

evels

of T

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(pg

mL)

TNF-120572

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Figure 4 Coculture with BMSCs attenuates the activity of LPS to induce release of cytokines in alveolar macrophages (a) and HUVECs (b)Alveolar macrophages and HUVECs with or without coculture with BMSCs were treated with LPS or PBS for 1 3 6 12 and 24 hours Thecell culture supernatant was then collected for cytokine analysis by ELISA Data are expressed as the mean plusmn SD from three experimentslowast

119875 lt 005 lowastlowast119875 lt 001

indicate that the inflammatory reactions were suppressedThe results suggest that BMSCs have the ability to alleviatepathogen-induced inflammation damage in HUVECs andmacrophages

In conclusion the results showed that BMSCs attenuatedthe activity of LPS on the inflammatory reaction of HUVECsand alveolar macrophages through inhibiting upregulation ofTLR2 TLR4 and p65 expression as well as downregulatingTNF-120572 release and upregulating IL10 release

Abbreviations

BMSC Bone marrow mesenchymal stem cellsLPS LipopolysaccharideTLR Toll-like receptorSIRS Systemic inflammatory response syndromeMODS Multiple organ dysfunction syndromePAMP Pathogen associated molecular patterns

8 Mediators of Inflammation

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Dequan Li and Jun Fang participated in study designand coordination analysis and interpretation of data andsupervised study Yuanyuan Wang Chuang Chi and JingZhao performed most of the experiments and statisticalanalyses and drafted the paper Other authors carried outthe experiments and sample collections All authors read andapproved the final paper Dequan Li and Cong Wang equallycontributed to this paper

Acknowledgments

The authors thank the Medical Scientific Research Platform(Wenzhou Medical University Wenzhou China) and theSurgery Laboratory (the First Affiliated Hospital of WenzhouMedical University Wenzhou China) for their technicalassistance This work was supported by Zhejiang ProvinceNatural Science Foundation of China (LY12H15001) Healthand Family Planning Commission of Zhejiang Province(2013KYA130) Science Technology Department of ZhejiangProvince (2011R50018-20) Wenzhou Municipal Science andTechnology Bureau (Y20130091) and the National Natu-ral Science Foundation of China (81171787 31371470 and81270761)

References

[1] J-Z Zhang Z Liu J Liu J-X Ren and T-S Sun ldquoMitochon-drial DNA induces inflammation and increases TLR9NF-120581Bexpression in lung tissuerdquo International Journal of MolecularMedicine vol 33 no 4 pp 817ndash824 2014

[2] T Tsukamoto R S Chanthaphavong andH-C Pape ldquoCurrenttheories on the pathophysiology of multiple organ failure aftertraumardquo Injury vol 41 no 1 pp 21ndash26 2010

[3] Y Zhao C Yang H Wang et al ldquoTherapeutic effects of bonemarrow-derivedmesenchymal stem cells on pulmonary impactinjury complicated with endotoxemia in ratsrdquo InternationalImmunopharmacology vol 15 no 2 pp 246ndash253 2013

[4] K Tang Y Xiao D Liu et al ldquoAutografting of bone marrowmesenchymal stem cells alleviates streptozotocin-induced dia-betes in miniature pigs real-time tracing with MRI in vivordquoInternational Journal of Molecular Medicine vol 33 no 6 pp1469ndash1476 2014

[5] S Shin Y Kim S Jeong et al ldquoThe therapeutic effect of humanadult stem cells derived from adipose tissue in endotoxemic ratmodelrdquo International Journal of Medical Sciences vol 10 no 1pp 8ndash18 2012

[6] K Nemeth A Leelahavanichkul P S T Yuen et al ldquoBonemarrow stromal cells attenuate sepsis via prostaglandin E

2

-dependent reprogramming of host macrophages to increasetheir interleukin-10 productionrdquo Nature Medicine vol 15 no1 pp 42ndash49 2009

[7] N Kusadasi and A B J Groeneveld ldquoA perspective onmesenchymal stromal cell transplantation in the treatment ofsepsisrdquo Shock vol 40 no 5 pp 352ndash357 2013

[8] S R R Hall K Tsoyi B Ith et al ldquoMesenchymal stromalcells improve survival during sepsis in the absence of hemeoxygenase-1 the importance of neutrophilsrdquo STEMCELLS vol31 no 2 pp 397ndash407 2013

[9] J S Elman M Li F Wang J M Gimble and B Parekkadan ldquoAcomparison of adipose andbonemarrow-derivedmesenchymalstromal cell secreted factors in the treatment of systemicinflammationrdquo Journal of Inflammation vol 11 no 1 p 1 2014

[10] H Yagi A Soto-Gutierrez Y Kitagawa A W Tilles R GTompkins and M L Yarmush ldquoBone marrow mesenchymalstromal cells attenuate organ injury induced by LPS and burnrdquoCell Transplantation vol 19 no 6-7 pp 823ndash830 2010

[11] D Togbe S Schnyder-Candrian B Schnyder et al ldquoToll-likereceptor and tumour necrosis factor dependent endotoxin-induced acute lung injuryrdquo International Journal of Experimen-tal Pathology vol 88 no 6 pp 387ndash391 2007

[12] W Ren Z Wang F Hua and L Zhu ldquoPlasminogen activatorinhibitor-1 regulates LPS-induced TLR4MD-2 pathway activa-tion and inflammation in alveolar macrophagesrdquo Inflammationvol 38 no 1 pp 384ndash393 2015

[13] K Nemeth B Mayer and E Mezey ldquoModulation of bonemarrow stromal cell functions in infectious diseases by toll-likereceptor ligandsrdquo Journal of Molecular Medicine vol 88 no 1pp 5ndash10 2010

[14] N V Gorbunov B R Garrison D P McDaniel et al ldquoAdaptiveredox response of mesenchymal stromal cells to stimulationwith lipopolysaccharide inflammagenmechanisms of remodel-ing of tissue barriers in sepsisrdquoOxidative Medicine and CellularLongevity vol 2013 Article ID 186795 16 pages 2013

[15] S Jesic A Jotic N Tomanovic M Zivkovic A Kolakovic andA Stankovic ldquoExpression of toll-like receptors 2 4 and nuclearfactor kappa B in mucosal lesions of human otitis pattern andrelationship in a clinical immunohistochemical studyrdquo Annalsof Otology Rhinology and Laryngology vol 123 no 6 pp 434ndash441 2014

[16] Y-H Zheng W Xiong K Su S-J Kuang and Z-G ZhangldquoMultilineage differentiation of human bone marrow mes-enchymal stem cells in vitro and in vivordquo Experimental andTherapeutic Medicine vol 5 no 6 pp 1576ndash1580 2013

[17] J L Shelton L Wang G Cepinskas et al ldquoAlbumin leak acrosshuman pulmonarymicrovascular vs umbilical vein endothelialcells under septic conditionsrdquo Microvascular Research vol 71no 1 pp 40ndash47 2006

[18] Z Zhou F Guo Y Dou J Tang and J Huan ldquoGua-nine nucleotide exchange factor-H1 signaling is involved inlipopolysaccharide-induced endothelial barrier dysfunctionrdquoSurgery vol 154 no 3 pp 621ndash631 2013

[19] J-S Bae W Lee and A R Rezaie ldquoPolyphosphate elicitspro-inflammatory responses that are counteracted by activatedprotein C in both cellular and animal modelsrdquo Journal ofThrombosis and Haemostasis vol 10 no 6 pp 1145ndash1151 2012

[20] Z Tianzhu and W Shumin ldquoEsculin inhibits the inflammationof LPS-Induced acute lung injury in mice via regulation ofTLRNF-120581B pathwaysrdquo Inflammation vol 38 no 4 pp 1529ndash1536 2015

Mediators of Inflammation 9

[21] Q S Liu Z W Cheng J G Xiong S Cheng X F He and XC Li ldquoErythropoietin pretreatment exerts anti-inflammatoryeffects in hepatic ischemiareperfusion-injured rats via suppres-sion of the TLR2NF-120581B pathwayrdquo Transplantation Proceedingsvol 47 no 2 pp 283ndash289 2015

[22] Y Chen T Liu P Langford et al ldquoHaemophilus parasuisinduces activation of NF-120581B and MAP kinase signaling path-ways mediated by toll-like receptorsrdquo Molecular Immunologyvol 65 no 2 pp 360ndash366 2015

Page 2: Bone Marrow Mesenchymal Stem Cells Inhibit ......Bone Marrow Mesenchymal Stem Cells Inhibit ... and TLR4 response to acute otitis through activation of NF-𝜅B [15], we hypothesized

2 Mediators of Inflammation

Quick and efficient response to microbial infections isdriven by recognition of molecules broadly shared by avariety of pathogens which are distinguishable from hostmolecules named pathogen associated molecular patterns(PAMPs) Pattern-recognition receptors (PRRs) that containmembrane-bound PRRs including toll-like receptors (TLRs)and other cytoplasmic proteins recognize the PAMPs [10]Upon binding with the PAMPs TLR2 and TLR4 then activateNF-120581B and subsequently promote cytokine synthesis whichinclude IL-1 IL-6 and TNF-120572 [11 12] The network of TLRsand PRRs mediates the response of BMSCs to inflammatorystimuli such as LPS [13 14] However how TLRs modulatethe BMSC activity is not clearly elucidated Since TLR2and TLR4 response to acute otitis through activation ofNF-120581B [15] we hypothesized that BMSCs ameliorate theinflammation damage of alveolarmacrophages andHUVECsthrough the inhibition of TLR2 and TLR4 mediated NF-120581Bpathways

2 Materials and Methods

21 Cell Culture Alveolar macrophages and HUVECs werepurchased from Chi Scientific Inc (China) The cells werecultured in RPMI 1640 medium (Sigma Chemical Co StLouis MO) supplemented with 10 fetal bovine serum (FBS)(Gibco Carlsbad CA USA) and 1 penicillinstreptomycin(Solarbio Beijing China) at 37∘C in a humidified atmosphereof 5 CO

2 BMSCs obtained from Cyagen Biosciences

Inc (Suzhou China) were cultured in 10 FBS-DMEM-LG(Gibco Carlsbad CA USA)The surface markers for BMSCsdifferentiation of adipocytic osteogenic and chondrogeniclineages were characterized as previously reported [16] Onlypassages 3ndash7 of the cells were used for experiments

22 Endothelial Permeability Assay Monolayer permeabilitywas quantitated by spectrophotometric measurement of theflux of Evans blue-albumin across HUVECs as describedpreviously [17] HUVECs (1 times 105) were seeded in 24-wellTranswell inserts (5 120583m pore size Corning IncorporatedNY USA) and cultured with 10 FBS DMEM overnightand then changed with serum-free medium for another 24hours The various numbers of BMSCs were seeded at thebottom of the same 24-well plate with serum-free media LPSwas added to the Transwell inserts at a final concentrationof 100 ngmL for different time points (1 3 6 12 and24 h) After that the BMSCs were digested by trypsin-EDTAsolution to terminate the effects of BMSCs and 100 120583L Hankrsquossolution containing Evans blue- (EB-) conjugated bovineserum albumin (final concentration 067mgmL) was addedto the Transwell inserts while 600 120583L 4 BSA was addedto the lower plate chamber After incubation at 37∘C for1 hour 100 120583L BSA solution from the lower chamber washarvested and measured for the absorbance at 620 nm TheHUVECs permeability capacity was calculated as followsEvans blue dye-labeled albumin (EB-albumin) leak rate =OD620leaked EB-albuminOD620total EB-albumin times 100

23 Alveolar MacrophagesHUVECs-BMSC Coculture Exper-iments Alveolar macrophages or HUVECs (1 times 106well)

were seeded in a 6-well plate after 24-hour incubationBMSCs (2 times 105well) were seeded in the 6-well Transwellinserts (5 120583m pore Corning Incorporated NY USA) at theindicated time points (1 3 6 12 and 24 h) LPS (100 ngmL)was then added to the lower chamber for 1 h Alveolarmacrophages or HUVECs lysate was collected for RNA andprotein analyses and the cell-free supernatants were collectedfor ELISA detection

24 Western Blot Analysis Alveolar macrophagesHUVECswere lysed in the RIPA buffer (Thermo Scientific RockfordUSA) supplemented with 1 PMSF and 1 protein phos-phatase inhibitor mixture (P1260 Applygen Beijing China)The concentration of the proteins was measured with theBicinchoninic Acid Kit (Thermo Scientific Rockford USA)Rabbit anti-TLR2 TLR4 and phosphorylated p65 unit of NF-120581B antibodies were obtained from Cell Signaling Technol-ogy Horseradish peroxidase- (HRP-) conjugated goat anti-rabbit antibodies were purchased from Bio-Rad ECL-PlusChemiluminescent Reagent (Thermo Scientific RockfordUSA) was used to visualize the specific proteins Relativeconcentration of proteins was quantitated using the Image JSoftware (National Institutes of Health Bethesda USA)

25 Gene Expression Analysis Total RNA was isolated fromcells using the TRIzol RNA Isolation Reagents (Life Tech-nologies) The first-strand cDNAs were converted using theSuperScript III reverse transcriptase (Invitrogen CarlsbadCA) according to the manufacturerrsquos protocols Real-timeRT-PCR analysis was performed on ABI 7500 SequenceDetection System using the UltraSYBR Mixture (CW BioCo Ltd Beijing China) Transcript expression was nor-malizedwith the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) housekeeping gene The PCR primer sequencesare GAPDH forward 51015840-TGGAGTCTACTGGCGTCTT-31015840 reverse 51015840-TGTCATATTTCTCGTG GTTCA-31015840 TLR4forward 51015840-CTGCATAGAGGTAGTTCCT-31015840 reverse 51015840-TCCAGCC ACTGAAGTTCTGA-31015840 TLR2 forward 51015840-GGAGACTCTGGAAGC ATG-31015840 reverse 51015840-GCATCCTGAAGCCTGTG-31015840

26 ELISA Analysis The supernatants collected from cocul-ture system were centrifuged for 10 minutes at 1000 g(4∘C) to remove cell debris The levels of TNF-120572 and IL10were measured using commercially available enzyme-linkedimmunosorbent assay (ELISA) kits (BD Biosciences SanDiego CA USA) according to the instructions provided bythe manufacturer

27 Statistical Analysis SPSS 170 software was used to per-form the statistical analysis and values are presented as themeans plusmn standard deviations (SD) all experiments wererepeated at least three times The one-way ANOVA was usedto determine the significance of differences between variousgroups119875 lt 005was considered to be statistically significant

3 Results

31 BMSCs Inhibit LPS to Induce Hyperpermeability inHUVECs To assess how BMSCs affected HUVECs perme-ability induced by LPS HUVECs with or without coculture

Mediators of Inflammation 3

LPSLow concentrations of BMSCsMedian concentrations of BMSCsHigh concentrations of BMSCs

10

8

6

4

2

0

EB-a

lbum

in le

ak ra

te (

)

1 3 6 12 24

Time (h)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

Figure 1 Effect of BMSCs on the barrier permeability of HUVECsHUVECs were stimulated by LPS at a dosage of 100 ngmL (blackbars) In addition HUVECswere coculturedwith indicated concen-trations of BMSCs low (1 times 103 cellsmL dark grey bars) median (1times 104 cellsmL light grey bars) and high (1 times 105 cellsmL whitebars) for 1 3 6 12 and 24 h followed by measuring permeability asdescribed in Section 2 All results are means plusmn SD of at least threedifferent experiments lowast119875 lt 005 lowastlowast119875 lt 001

with BMSCs were stimulated by LPS for the indicated timesWithout coculture with BMSCs the EB-albumin leaking rateranged from 535 (at 1 h) to 772 (at 24 h) in the LPS group(Figure 1) Compared with the LPS group the EB-albuminleaking rates in the BMSC coculture group were reducedin a BMSC cell number- and time-dependent manner TheEB-albumin leaking rate in the 1 times 104mL BMSCs groupwas decreased 3 hours after coculture The effects were moresignificant after coculture for 6 hours to 24 hours The effectswere less significant in the group with 1 times 103mL BMSCsand more significant in the group with 1 times 105mL BMSCs(Figure 1)

32 BMSCsAttenuate LPS-Induced Expression of TLR2 TLR4and p65 in Alveolar Macrophages and HUVECs To deter-mine whether LPS stimulated expression of TLR2 TLR4 andp65 in macrophages and HUVECs was affected by BMSCsalveolar macrophages and HUVECs cells were coculturedwith BMSCs for the indicated time followed by LPS treat-ment Western blot (Figure 2) and real-time RT-PCR anal-yses (Figure 3) showed that LPS administration increasedexpression of TLR2 TLR4 and p65 within 1 hour after thetreatment The effect persisted even until 24 hours after thetreatment However the activity of LPS was attenuated bycoculture with BMSCs in both alveolar macrophages andHUVECs (Figures 2 and 3)

33 BMSCs Suppress Alveolar Macrophages and HUVECs toRelease IL10 and TNF-120572 in Response to LPS Stimulation To

determine whether BMSCs regulated the release of TNF-120572 and IL10 from alveolar macrophages and HUVECs inresponse to LPS stimulation the conditioned medium wascollected from the coculture system and the concentrationsof IL10 and TNF-120572 were assessed by ELISA It was clearthat concentration of TNF-120572 in the medium was increasedafter LPS stimulation within 1 hour and the effects lasted for24 hours However the increases were blunted by coculturewith BMSCs In contrast IL10 was reduced by LPS stimu-lation while coculture with BMSCs increased medium IL10production The results indicate that BMSCs suppress LPS-induced inflammation by decreasing TNF-120572 and increasingIL10 releases (Figure 4)

4 Discussion

BMSCs treatments have been shown to reduce inflammatoryresponse in human and animal models in response to injuryHowever the detailed mechanism underlying this effect isnot clear Here we reported that BMSCs alleviated HUVECdamage induced by LPSThe results also showed that BMSCsrestricted the inflammatory responses of HUVECs and alve-olar macrophage stimulated by LPS and therefore protectedHUVECs from damage induced by the endotoxin The dataindicate that attenuating LPS-induced TNF-120572 pathway acti-vation inHUVECs andmacrophage by BMSCs contributes tothe protection of HUVECs from endotoxin-induced damageby BMSCs

Our data demonstrated that LPS increased the perme-ability of HUVECs monolayers in a time-dependent mannerAlthough the extent of the activity was lower than thatafter coculture for a longer time the protection effects weredetectable within one hour The underlying mechanism ofthe protection is not clear Since BMSCs and HUVECs werecultured separating by the Transwell membrane the effectsof BMSCs must be mediated by soluble factors releasedfrom BMSCs However we did not rule out the possibilitythat BMSCs sequestrated LPS and therefore protected theendothelial cells Future efforts are needed to identify thesebeneficial secretory factors from BMSCs Although LPS stillinduced HUVECs damage in the coculture the EB-albuminleaks were reduced compared with the group without cocul-ture with BMSCs This suggests that BMSCs attenuate thedamage We observed that the protective effect of BMSCs onHUVEC was cell number-dependent High concentrationsof BMSCs were more effective than low concentrationsof BMSCs It appeared that the BMSCs released sufficientprotective molecules to protect endothelial cells within onehour Therefore immediately administration of BMSCs toSIRS patients shall have beneficial effects Furthermore theresults were in line with previous reports that LPS leadsto persistent damage of HUVECs [18 19] However it wasdifferent from the report that the damage of HUVEC by LPSis not time-dependent [17] The discrepancy is likely due todifferent culture time We cultured the cells for 24 hours toreach the confluence followed by serum starvation for 24hours and LPS treatment This method is different from thepublished one where the cells were cultured for 4 days prior

4 Mediators of Inflammation

TLR2

TLR4

(1) PBS control (2) BMSCs control(3) LPS (4) BMSCs treatment

Alv

eola

r mac

roph

ages

1 2 3 4

p-NF-120581B p65

120573-actin

PBS controlBMSCs control

LPSBMSCs treatment

TLR2

Nor

mal

ized

to120573

-act

in (T

LR2

)

00

06

12

18

24

30

63 121 24Time (h)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowast

TLR4

Nor

mal

ized

to120573

-act

in (T

LR4

)

00

06

12

18

24

30

6 12 2431Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowastlowast lowast

lowastlowast lowastlowastlowast lowast

lowastlowast lowast

(A)

(B) (C)

Nor

mal

ized

to120573

-act

in (p

-NF-120581

B p65

)

p-NF-120581B p65

00

06

12

18

24

1 3 12 246Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowast

lowastlowast lowast

lowastlowast lowastlowastlowast lowast

lowastlowast lowast

(D)

(a)

Figure 2 Continued

Mediators of Inflammation 5

TLR2

TLR4

(1) PBS control (2) BMSCs control(3) LPS (4) BMSCs treatment

HU

VEC

s

1 2 3 4

p-NF-120581B p65

120573-actin

00

06

12

18

24

30

241263Time (h)

1

TLR2

PBS controlBMSCs control

LPSBMSCs treatment

Nor

mal

ized

to120573

-act

in (T

LR2

)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

TLR4

Nor

mal

ized

to120573

-act

in (T

LR4

)

00

06

12

18

24

30

PBS controlBMSCs control

LPSBMSCs treatment

3 24121 6Time (h)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

63 241 12Time (h)

Nor

mal

ized

to120573

-act

in (p

-NF-120581

B p65

)

p-NF-120581B p65

00

06

12

18

24

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

(E)

(F) (G)

(H)

(b)

Figure 2 Coculture with BMSCs attenuates the activity of LPS to induce expressions of TLR2 TLR4 and p65 in alveolar macrophages (a)and HUVECs (b) at the protein levelThe total proteins were extracted from the cultured alveolar macrophages and HUVECs treated (1) PBScontrol (2) BMSCs (3) LPS and (4) BMSCs and LPS The expressions of TLR2 TLR4 and p-NF-120581B p65 were determined by Western blotThe density of specific bands was quantitated Data are expressed as the mean plusmn SD from three experiments lowast119875 lt 005 lowastlowast119875 lt 001

6 Mediators of Inflammation

0

1

2

3

4

5

Alv

eola

r mac

roph

ages

2412631

Relat

ive T

LR2

mRN

A le

vel

Time (h)

TLR2

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

TLR4

0

1

2

3

4

5

Relat

ive T

LR4

mRN

A le

vel

1 3 126 24Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

(A) (B)

(a)

HU

VEC

s

TLR2

0

1

2

3

4

5

Relat

ive T

LR2

mRN

A le

vel

3 121 246Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

TLR4

0

1

2

3

4

5

Relat

ive T

LR4

mRN

A le

vel

3 6 12 241Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

(C) (D)

(b)

Figure 3 Coculture with BMSCs attenuates the activity of LPS to induce expressions of TLR2 and TLR4 in alveolar macrophages (a) andHUVECs (b) at themRNA levelThe total RNAswere extracted from the cultured alveolarmacrophages andHUVECs treated (1) PBS control(2) BMSCs (3) LPS and (4) BMSCs and LPSThe expressions of TLR2 and TLR4 were determined by real-time RT-PCR Data are expressedas the mean plusmn SD from three experiments lowast119875 lt 005 lowastlowast119875 lt 001

to the experiments Further efforts are needed to clarify thisdiscrepancy

TLRs play a pivotal role in defense against invadingpathogens through recognizing PAMPs It is well docu-mented that cells use TLR2 and TLR4 to recognize PAMPsexpressed by bacteria Activation of TLR2 and TLR4 leadsto activation of MAPK and NF-120581B pathways and productionof inflammatory cytokines including IL-1 IL-6 and TNF-120572 [20ndash22] In this study we showed that LPS promoted

expression of TLR2 and TLR4 in alveolar macrophages andHUVECs However how expression of TLR2 expressionwas increased by LPS is unknown and deserves futureinvestigations TNF-120572 and IL10 expressions were also affectedby LPS Furthermore the LPS effects were diminishedwhen the HUVECs and macrophages were cultured withBMSCs TNF-120572 is a potent inflammatory factor and IL10 isa key anti-inflammatory cytokine Downregulation of TNF-120572 and upregulation of IL10 in HUVECs and macrophage

Mediators of Inflammation 7A

lveo

lar m

acro

phag

es

3 6 24121Time (h)

Supe

rnat

ant l

evels

of T

NF-120572

(pg

mL)

TNF-120572

0

500

1000

1500

2000

2500

3000

3500

4000

4500

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

0

10

20

30

40

50

60

70

80

90

2412631Time (h)

Supe

rnat

ant l

evels

of I

L10

(pg

mL)

IL10

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(A) (B)

(a)

HU

VEC

sSu

pern

atan

t lev

els o

f TN

F-120572

(pg

mL)

0

50

100

150

200

250

300

350

63 12 241Time (h)

TNF-120572

lowastlowast lowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

IL10

0

10

20

30

40

50

60

70

80Su

pern

atan

t lev

els o

f IL1

0 (p

gm

L)

3 6 241 12Time (h)

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(C) (D)

(b)

Figure 4 Coculture with BMSCs attenuates the activity of LPS to induce release of cytokines in alveolar macrophages (a) and HUVECs (b)Alveolar macrophages and HUVECs with or without coculture with BMSCs were treated with LPS or PBS for 1 3 6 12 and 24 hours Thecell culture supernatant was then collected for cytokine analysis by ELISA Data are expressed as the mean plusmn SD from three experimentslowast

119875 lt 005 lowastlowast119875 lt 001

indicate that the inflammatory reactions were suppressedThe results suggest that BMSCs have the ability to alleviatepathogen-induced inflammation damage in HUVECs andmacrophages

In conclusion the results showed that BMSCs attenuatedthe activity of LPS on the inflammatory reaction of HUVECsand alveolar macrophages through inhibiting upregulation ofTLR2 TLR4 and p65 expression as well as downregulatingTNF-120572 release and upregulating IL10 release

Abbreviations

BMSC Bone marrow mesenchymal stem cellsLPS LipopolysaccharideTLR Toll-like receptorSIRS Systemic inflammatory response syndromeMODS Multiple organ dysfunction syndromePAMP Pathogen associated molecular patterns

8 Mediators of Inflammation

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Dequan Li and Jun Fang participated in study designand coordination analysis and interpretation of data andsupervised study Yuanyuan Wang Chuang Chi and JingZhao performed most of the experiments and statisticalanalyses and drafted the paper Other authors carried outthe experiments and sample collections All authors read andapproved the final paper Dequan Li and Cong Wang equallycontributed to this paper

Acknowledgments

The authors thank the Medical Scientific Research Platform(Wenzhou Medical University Wenzhou China) and theSurgery Laboratory (the First Affiliated Hospital of WenzhouMedical University Wenzhou China) for their technicalassistance This work was supported by Zhejiang ProvinceNatural Science Foundation of China (LY12H15001) Healthand Family Planning Commission of Zhejiang Province(2013KYA130) Science Technology Department of ZhejiangProvince (2011R50018-20) Wenzhou Municipal Science andTechnology Bureau (Y20130091) and the National Natu-ral Science Foundation of China (81171787 31371470 and81270761)

References

[1] J-Z Zhang Z Liu J Liu J-X Ren and T-S Sun ldquoMitochon-drial DNA induces inflammation and increases TLR9NF-120581Bexpression in lung tissuerdquo International Journal of MolecularMedicine vol 33 no 4 pp 817ndash824 2014

[2] T Tsukamoto R S Chanthaphavong andH-C Pape ldquoCurrenttheories on the pathophysiology of multiple organ failure aftertraumardquo Injury vol 41 no 1 pp 21ndash26 2010

[3] Y Zhao C Yang H Wang et al ldquoTherapeutic effects of bonemarrow-derivedmesenchymal stem cells on pulmonary impactinjury complicated with endotoxemia in ratsrdquo InternationalImmunopharmacology vol 15 no 2 pp 246ndash253 2013

[4] K Tang Y Xiao D Liu et al ldquoAutografting of bone marrowmesenchymal stem cells alleviates streptozotocin-induced dia-betes in miniature pigs real-time tracing with MRI in vivordquoInternational Journal of Molecular Medicine vol 33 no 6 pp1469ndash1476 2014

[5] S Shin Y Kim S Jeong et al ldquoThe therapeutic effect of humanadult stem cells derived from adipose tissue in endotoxemic ratmodelrdquo International Journal of Medical Sciences vol 10 no 1pp 8ndash18 2012

[6] K Nemeth A Leelahavanichkul P S T Yuen et al ldquoBonemarrow stromal cells attenuate sepsis via prostaglandin E

2

-dependent reprogramming of host macrophages to increasetheir interleukin-10 productionrdquo Nature Medicine vol 15 no1 pp 42ndash49 2009

[7] N Kusadasi and A B J Groeneveld ldquoA perspective onmesenchymal stromal cell transplantation in the treatment ofsepsisrdquo Shock vol 40 no 5 pp 352ndash357 2013

[8] S R R Hall K Tsoyi B Ith et al ldquoMesenchymal stromalcells improve survival during sepsis in the absence of hemeoxygenase-1 the importance of neutrophilsrdquo STEMCELLS vol31 no 2 pp 397ndash407 2013

[9] J S Elman M Li F Wang J M Gimble and B Parekkadan ldquoAcomparison of adipose andbonemarrow-derivedmesenchymalstromal cell secreted factors in the treatment of systemicinflammationrdquo Journal of Inflammation vol 11 no 1 p 1 2014

[10] H Yagi A Soto-Gutierrez Y Kitagawa A W Tilles R GTompkins and M L Yarmush ldquoBone marrow mesenchymalstromal cells attenuate organ injury induced by LPS and burnrdquoCell Transplantation vol 19 no 6-7 pp 823ndash830 2010

[11] D Togbe S Schnyder-Candrian B Schnyder et al ldquoToll-likereceptor and tumour necrosis factor dependent endotoxin-induced acute lung injuryrdquo International Journal of Experimen-tal Pathology vol 88 no 6 pp 387ndash391 2007

[12] W Ren Z Wang F Hua and L Zhu ldquoPlasminogen activatorinhibitor-1 regulates LPS-induced TLR4MD-2 pathway activa-tion and inflammation in alveolar macrophagesrdquo Inflammationvol 38 no 1 pp 384ndash393 2015

[13] K Nemeth B Mayer and E Mezey ldquoModulation of bonemarrow stromal cell functions in infectious diseases by toll-likereceptor ligandsrdquo Journal of Molecular Medicine vol 88 no 1pp 5ndash10 2010

[14] N V Gorbunov B R Garrison D P McDaniel et al ldquoAdaptiveredox response of mesenchymal stromal cells to stimulationwith lipopolysaccharide inflammagenmechanisms of remodel-ing of tissue barriers in sepsisrdquoOxidative Medicine and CellularLongevity vol 2013 Article ID 186795 16 pages 2013

[15] S Jesic A Jotic N Tomanovic M Zivkovic A Kolakovic andA Stankovic ldquoExpression of toll-like receptors 2 4 and nuclearfactor kappa B in mucosal lesions of human otitis pattern andrelationship in a clinical immunohistochemical studyrdquo Annalsof Otology Rhinology and Laryngology vol 123 no 6 pp 434ndash441 2014

[16] Y-H Zheng W Xiong K Su S-J Kuang and Z-G ZhangldquoMultilineage differentiation of human bone marrow mes-enchymal stem cells in vitro and in vivordquo Experimental andTherapeutic Medicine vol 5 no 6 pp 1576ndash1580 2013

[17] J L Shelton L Wang G Cepinskas et al ldquoAlbumin leak acrosshuman pulmonarymicrovascular vs umbilical vein endothelialcells under septic conditionsrdquo Microvascular Research vol 71no 1 pp 40ndash47 2006

[18] Z Zhou F Guo Y Dou J Tang and J Huan ldquoGua-nine nucleotide exchange factor-H1 signaling is involved inlipopolysaccharide-induced endothelial barrier dysfunctionrdquoSurgery vol 154 no 3 pp 621ndash631 2013

[19] J-S Bae W Lee and A R Rezaie ldquoPolyphosphate elicitspro-inflammatory responses that are counteracted by activatedprotein C in both cellular and animal modelsrdquo Journal ofThrombosis and Haemostasis vol 10 no 6 pp 1145ndash1151 2012

[20] Z Tianzhu and W Shumin ldquoEsculin inhibits the inflammationof LPS-Induced acute lung injury in mice via regulation ofTLRNF-120581B pathwaysrdquo Inflammation vol 38 no 4 pp 1529ndash1536 2015

Mediators of Inflammation 9

[21] Q S Liu Z W Cheng J G Xiong S Cheng X F He and XC Li ldquoErythropoietin pretreatment exerts anti-inflammatoryeffects in hepatic ischemiareperfusion-injured rats via suppres-sion of the TLR2NF-120581B pathwayrdquo Transplantation Proceedingsvol 47 no 2 pp 283ndash289 2015

[22] Y Chen T Liu P Langford et al ldquoHaemophilus parasuisinduces activation of NF-120581B and MAP kinase signaling path-ways mediated by toll-like receptorsrdquo Molecular Immunologyvol 65 no 2 pp 360ndash366 2015

Page 3: Bone Marrow Mesenchymal Stem Cells Inhibit ......Bone Marrow Mesenchymal Stem Cells Inhibit ... and TLR4 response to acute otitis through activation of NF-𝜅B [15], we hypothesized

Mediators of Inflammation 3

LPSLow concentrations of BMSCsMedian concentrations of BMSCsHigh concentrations of BMSCs

10

8

6

4

2

0

EB-a

lbum

in le

ak ra

te (

)

1 3 6 12 24

Time (h)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

Figure 1 Effect of BMSCs on the barrier permeability of HUVECsHUVECs were stimulated by LPS at a dosage of 100 ngmL (blackbars) In addition HUVECswere coculturedwith indicated concen-trations of BMSCs low (1 times 103 cellsmL dark grey bars) median (1times 104 cellsmL light grey bars) and high (1 times 105 cellsmL whitebars) for 1 3 6 12 and 24 h followed by measuring permeability asdescribed in Section 2 All results are means plusmn SD of at least threedifferent experiments lowast119875 lt 005 lowastlowast119875 lt 001

with BMSCs were stimulated by LPS for the indicated timesWithout coculture with BMSCs the EB-albumin leaking rateranged from 535 (at 1 h) to 772 (at 24 h) in the LPS group(Figure 1) Compared with the LPS group the EB-albuminleaking rates in the BMSC coculture group were reducedin a BMSC cell number- and time-dependent manner TheEB-albumin leaking rate in the 1 times 104mL BMSCs groupwas decreased 3 hours after coculture The effects were moresignificant after coculture for 6 hours to 24 hours The effectswere less significant in the group with 1 times 103mL BMSCsand more significant in the group with 1 times 105mL BMSCs(Figure 1)

32 BMSCsAttenuate LPS-Induced Expression of TLR2 TLR4and p65 in Alveolar Macrophages and HUVECs To deter-mine whether LPS stimulated expression of TLR2 TLR4 andp65 in macrophages and HUVECs was affected by BMSCsalveolar macrophages and HUVECs cells were coculturedwith BMSCs for the indicated time followed by LPS treat-ment Western blot (Figure 2) and real-time RT-PCR anal-yses (Figure 3) showed that LPS administration increasedexpression of TLR2 TLR4 and p65 within 1 hour after thetreatment The effect persisted even until 24 hours after thetreatment However the activity of LPS was attenuated bycoculture with BMSCs in both alveolar macrophages andHUVECs (Figures 2 and 3)

33 BMSCs Suppress Alveolar Macrophages and HUVECs toRelease IL10 and TNF-120572 in Response to LPS Stimulation To

determine whether BMSCs regulated the release of TNF-120572 and IL10 from alveolar macrophages and HUVECs inresponse to LPS stimulation the conditioned medium wascollected from the coculture system and the concentrationsof IL10 and TNF-120572 were assessed by ELISA It was clearthat concentration of TNF-120572 in the medium was increasedafter LPS stimulation within 1 hour and the effects lasted for24 hours However the increases were blunted by coculturewith BMSCs In contrast IL10 was reduced by LPS stimu-lation while coculture with BMSCs increased medium IL10production The results indicate that BMSCs suppress LPS-induced inflammation by decreasing TNF-120572 and increasingIL10 releases (Figure 4)

4 Discussion

BMSCs treatments have been shown to reduce inflammatoryresponse in human and animal models in response to injuryHowever the detailed mechanism underlying this effect isnot clear Here we reported that BMSCs alleviated HUVECdamage induced by LPSThe results also showed that BMSCsrestricted the inflammatory responses of HUVECs and alve-olar macrophage stimulated by LPS and therefore protectedHUVECs from damage induced by the endotoxin The dataindicate that attenuating LPS-induced TNF-120572 pathway acti-vation inHUVECs andmacrophage by BMSCs contributes tothe protection of HUVECs from endotoxin-induced damageby BMSCs

Our data demonstrated that LPS increased the perme-ability of HUVECs monolayers in a time-dependent mannerAlthough the extent of the activity was lower than thatafter coculture for a longer time the protection effects weredetectable within one hour The underlying mechanism ofthe protection is not clear Since BMSCs and HUVECs werecultured separating by the Transwell membrane the effectsof BMSCs must be mediated by soluble factors releasedfrom BMSCs However we did not rule out the possibilitythat BMSCs sequestrated LPS and therefore protected theendothelial cells Future efforts are needed to identify thesebeneficial secretory factors from BMSCs Although LPS stillinduced HUVECs damage in the coculture the EB-albuminleaks were reduced compared with the group without cocul-ture with BMSCs This suggests that BMSCs attenuate thedamage We observed that the protective effect of BMSCs onHUVEC was cell number-dependent High concentrationsof BMSCs were more effective than low concentrationsof BMSCs It appeared that the BMSCs released sufficientprotective molecules to protect endothelial cells within onehour Therefore immediately administration of BMSCs toSIRS patients shall have beneficial effects Furthermore theresults were in line with previous reports that LPS leadsto persistent damage of HUVECs [18 19] However it wasdifferent from the report that the damage of HUVEC by LPSis not time-dependent [17] The discrepancy is likely due todifferent culture time We cultured the cells for 24 hours toreach the confluence followed by serum starvation for 24hours and LPS treatment This method is different from thepublished one where the cells were cultured for 4 days prior

4 Mediators of Inflammation

TLR2

TLR4

(1) PBS control (2) BMSCs control(3) LPS (4) BMSCs treatment

Alv

eola

r mac

roph

ages

1 2 3 4

p-NF-120581B p65

120573-actin

PBS controlBMSCs control

LPSBMSCs treatment

TLR2

Nor

mal

ized

to120573

-act

in (T

LR2

)

00

06

12

18

24

30

63 121 24Time (h)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowast

TLR4

Nor

mal

ized

to120573

-act

in (T

LR4

)

00

06

12

18

24

30

6 12 2431Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowastlowast lowast

lowastlowast lowastlowastlowast lowast

lowastlowast lowast

(A)

(B) (C)

Nor

mal

ized

to120573

-act

in (p

-NF-120581

B p65

)

p-NF-120581B p65

00

06

12

18

24

1 3 12 246Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowast

lowastlowast lowast

lowastlowast lowastlowastlowast lowast

lowastlowast lowast

(D)

(a)

Figure 2 Continued

Mediators of Inflammation 5

TLR2

TLR4

(1) PBS control (2) BMSCs control(3) LPS (4) BMSCs treatment

HU

VEC

s

1 2 3 4

p-NF-120581B p65

120573-actin

00

06

12

18

24

30

241263Time (h)

1

TLR2

PBS controlBMSCs control

LPSBMSCs treatment

Nor

mal

ized

to120573

-act

in (T

LR2

)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

TLR4

Nor

mal

ized

to120573

-act

in (T

LR4

)

00

06

12

18

24

30

PBS controlBMSCs control

LPSBMSCs treatment

3 24121 6Time (h)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

63 241 12Time (h)

Nor

mal

ized

to120573

-act

in (p

-NF-120581

B p65

)

p-NF-120581B p65

00

06

12

18

24

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

(E)

(F) (G)

(H)

(b)

Figure 2 Coculture with BMSCs attenuates the activity of LPS to induce expressions of TLR2 TLR4 and p65 in alveolar macrophages (a)and HUVECs (b) at the protein levelThe total proteins were extracted from the cultured alveolar macrophages and HUVECs treated (1) PBScontrol (2) BMSCs (3) LPS and (4) BMSCs and LPS The expressions of TLR2 TLR4 and p-NF-120581B p65 were determined by Western blotThe density of specific bands was quantitated Data are expressed as the mean plusmn SD from three experiments lowast119875 lt 005 lowastlowast119875 lt 001

6 Mediators of Inflammation

0

1

2

3

4

5

Alv

eola

r mac

roph

ages

2412631

Relat

ive T

LR2

mRN

A le

vel

Time (h)

TLR2

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

TLR4

0

1

2

3

4

5

Relat

ive T

LR4

mRN

A le

vel

1 3 126 24Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

(A) (B)

(a)

HU

VEC

s

TLR2

0

1

2

3

4

5

Relat

ive T

LR2

mRN

A le

vel

3 121 246Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

TLR4

0

1

2

3

4

5

Relat

ive T

LR4

mRN

A le

vel

3 6 12 241Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

(C) (D)

(b)

Figure 3 Coculture with BMSCs attenuates the activity of LPS to induce expressions of TLR2 and TLR4 in alveolar macrophages (a) andHUVECs (b) at themRNA levelThe total RNAswere extracted from the cultured alveolarmacrophages andHUVECs treated (1) PBS control(2) BMSCs (3) LPS and (4) BMSCs and LPSThe expressions of TLR2 and TLR4 were determined by real-time RT-PCR Data are expressedas the mean plusmn SD from three experiments lowast119875 lt 005 lowastlowast119875 lt 001

to the experiments Further efforts are needed to clarify thisdiscrepancy

TLRs play a pivotal role in defense against invadingpathogens through recognizing PAMPs It is well docu-mented that cells use TLR2 and TLR4 to recognize PAMPsexpressed by bacteria Activation of TLR2 and TLR4 leadsto activation of MAPK and NF-120581B pathways and productionof inflammatory cytokines including IL-1 IL-6 and TNF-120572 [20ndash22] In this study we showed that LPS promoted

expression of TLR2 and TLR4 in alveolar macrophages andHUVECs However how expression of TLR2 expressionwas increased by LPS is unknown and deserves futureinvestigations TNF-120572 and IL10 expressions were also affectedby LPS Furthermore the LPS effects were diminishedwhen the HUVECs and macrophages were cultured withBMSCs TNF-120572 is a potent inflammatory factor and IL10 isa key anti-inflammatory cytokine Downregulation of TNF-120572 and upregulation of IL10 in HUVECs and macrophage

Mediators of Inflammation 7A

lveo

lar m

acro

phag

es

3 6 24121Time (h)

Supe

rnat

ant l

evels

of T

NF-120572

(pg

mL)

TNF-120572

0

500

1000

1500

2000

2500

3000

3500

4000

4500

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

0

10

20

30

40

50

60

70

80

90

2412631Time (h)

Supe

rnat

ant l

evels

of I

L10

(pg

mL)

IL10

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(A) (B)

(a)

HU

VEC

sSu

pern

atan

t lev

els o

f TN

F-120572

(pg

mL)

0

50

100

150

200

250

300

350

63 12 241Time (h)

TNF-120572

lowastlowast lowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

IL10

0

10

20

30

40

50

60

70

80Su

pern

atan

t lev

els o

f IL1

0 (p

gm

L)

3 6 241 12Time (h)

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(C) (D)

(b)

Figure 4 Coculture with BMSCs attenuates the activity of LPS to induce release of cytokines in alveolar macrophages (a) and HUVECs (b)Alveolar macrophages and HUVECs with or without coculture with BMSCs were treated with LPS or PBS for 1 3 6 12 and 24 hours Thecell culture supernatant was then collected for cytokine analysis by ELISA Data are expressed as the mean plusmn SD from three experimentslowast

119875 lt 005 lowastlowast119875 lt 001

indicate that the inflammatory reactions were suppressedThe results suggest that BMSCs have the ability to alleviatepathogen-induced inflammation damage in HUVECs andmacrophages

In conclusion the results showed that BMSCs attenuatedthe activity of LPS on the inflammatory reaction of HUVECsand alveolar macrophages through inhibiting upregulation ofTLR2 TLR4 and p65 expression as well as downregulatingTNF-120572 release and upregulating IL10 release

Abbreviations

BMSC Bone marrow mesenchymal stem cellsLPS LipopolysaccharideTLR Toll-like receptorSIRS Systemic inflammatory response syndromeMODS Multiple organ dysfunction syndromePAMP Pathogen associated molecular patterns

8 Mediators of Inflammation

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Dequan Li and Jun Fang participated in study designand coordination analysis and interpretation of data andsupervised study Yuanyuan Wang Chuang Chi and JingZhao performed most of the experiments and statisticalanalyses and drafted the paper Other authors carried outthe experiments and sample collections All authors read andapproved the final paper Dequan Li and Cong Wang equallycontributed to this paper

Acknowledgments

The authors thank the Medical Scientific Research Platform(Wenzhou Medical University Wenzhou China) and theSurgery Laboratory (the First Affiliated Hospital of WenzhouMedical University Wenzhou China) for their technicalassistance This work was supported by Zhejiang ProvinceNatural Science Foundation of China (LY12H15001) Healthand Family Planning Commission of Zhejiang Province(2013KYA130) Science Technology Department of ZhejiangProvince (2011R50018-20) Wenzhou Municipal Science andTechnology Bureau (Y20130091) and the National Natu-ral Science Foundation of China (81171787 31371470 and81270761)

References

[1] J-Z Zhang Z Liu J Liu J-X Ren and T-S Sun ldquoMitochon-drial DNA induces inflammation and increases TLR9NF-120581Bexpression in lung tissuerdquo International Journal of MolecularMedicine vol 33 no 4 pp 817ndash824 2014

[2] T Tsukamoto R S Chanthaphavong andH-C Pape ldquoCurrenttheories on the pathophysiology of multiple organ failure aftertraumardquo Injury vol 41 no 1 pp 21ndash26 2010

[3] Y Zhao C Yang H Wang et al ldquoTherapeutic effects of bonemarrow-derivedmesenchymal stem cells on pulmonary impactinjury complicated with endotoxemia in ratsrdquo InternationalImmunopharmacology vol 15 no 2 pp 246ndash253 2013

[4] K Tang Y Xiao D Liu et al ldquoAutografting of bone marrowmesenchymal stem cells alleviates streptozotocin-induced dia-betes in miniature pigs real-time tracing with MRI in vivordquoInternational Journal of Molecular Medicine vol 33 no 6 pp1469ndash1476 2014

[5] S Shin Y Kim S Jeong et al ldquoThe therapeutic effect of humanadult stem cells derived from adipose tissue in endotoxemic ratmodelrdquo International Journal of Medical Sciences vol 10 no 1pp 8ndash18 2012

[6] K Nemeth A Leelahavanichkul P S T Yuen et al ldquoBonemarrow stromal cells attenuate sepsis via prostaglandin E

2

-dependent reprogramming of host macrophages to increasetheir interleukin-10 productionrdquo Nature Medicine vol 15 no1 pp 42ndash49 2009

[7] N Kusadasi and A B J Groeneveld ldquoA perspective onmesenchymal stromal cell transplantation in the treatment ofsepsisrdquo Shock vol 40 no 5 pp 352ndash357 2013

[8] S R R Hall K Tsoyi B Ith et al ldquoMesenchymal stromalcells improve survival during sepsis in the absence of hemeoxygenase-1 the importance of neutrophilsrdquo STEMCELLS vol31 no 2 pp 397ndash407 2013

[9] J S Elman M Li F Wang J M Gimble and B Parekkadan ldquoAcomparison of adipose andbonemarrow-derivedmesenchymalstromal cell secreted factors in the treatment of systemicinflammationrdquo Journal of Inflammation vol 11 no 1 p 1 2014

[10] H Yagi A Soto-Gutierrez Y Kitagawa A W Tilles R GTompkins and M L Yarmush ldquoBone marrow mesenchymalstromal cells attenuate organ injury induced by LPS and burnrdquoCell Transplantation vol 19 no 6-7 pp 823ndash830 2010

[11] D Togbe S Schnyder-Candrian B Schnyder et al ldquoToll-likereceptor and tumour necrosis factor dependent endotoxin-induced acute lung injuryrdquo International Journal of Experimen-tal Pathology vol 88 no 6 pp 387ndash391 2007

[12] W Ren Z Wang F Hua and L Zhu ldquoPlasminogen activatorinhibitor-1 regulates LPS-induced TLR4MD-2 pathway activa-tion and inflammation in alveolar macrophagesrdquo Inflammationvol 38 no 1 pp 384ndash393 2015

[13] K Nemeth B Mayer and E Mezey ldquoModulation of bonemarrow stromal cell functions in infectious diseases by toll-likereceptor ligandsrdquo Journal of Molecular Medicine vol 88 no 1pp 5ndash10 2010

[14] N V Gorbunov B R Garrison D P McDaniel et al ldquoAdaptiveredox response of mesenchymal stromal cells to stimulationwith lipopolysaccharide inflammagenmechanisms of remodel-ing of tissue barriers in sepsisrdquoOxidative Medicine and CellularLongevity vol 2013 Article ID 186795 16 pages 2013

[15] S Jesic A Jotic N Tomanovic M Zivkovic A Kolakovic andA Stankovic ldquoExpression of toll-like receptors 2 4 and nuclearfactor kappa B in mucosal lesions of human otitis pattern andrelationship in a clinical immunohistochemical studyrdquo Annalsof Otology Rhinology and Laryngology vol 123 no 6 pp 434ndash441 2014

[16] Y-H Zheng W Xiong K Su S-J Kuang and Z-G ZhangldquoMultilineage differentiation of human bone marrow mes-enchymal stem cells in vitro and in vivordquo Experimental andTherapeutic Medicine vol 5 no 6 pp 1576ndash1580 2013

[17] J L Shelton L Wang G Cepinskas et al ldquoAlbumin leak acrosshuman pulmonarymicrovascular vs umbilical vein endothelialcells under septic conditionsrdquo Microvascular Research vol 71no 1 pp 40ndash47 2006

[18] Z Zhou F Guo Y Dou J Tang and J Huan ldquoGua-nine nucleotide exchange factor-H1 signaling is involved inlipopolysaccharide-induced endothelial barrier dysfunctionrdquoSurgery vol 154 no 3 pp 621ndash631 2013

[19] J-S Bae W Lee and A R Rezaie ldquoPolyphosphate elicitspro-inflammatory responses that are counteracted by activatedprotein C in both cellular and animal modelsrdquo Journal ofThrombosis and Haemostasis vol 10 no 6 pp 1145ndash1151 2012

[20] Z Tianzhu and W Shumin ldquoEsculin inhibits the inflammationof LPS-Induced acute lung injury in mice via regulation ofTLRNF-120581B pathwaysrdquo Inflammation vol 38 no 4 pp 1529ndash1536 2015

Mediators of Inflammation 9

[21] Q S Liu Z W Cheng J G Xiong S Cheng X F He and XC Li ldquoErythropoietin pretreatment exerts anti-inflammatoryeffects in hepatic ischemiareperfusion-injured rats via suppres-sion of the TLR2NF-120581B pathwayrdquo Transplantation Proceedingsvol 47 no 2 pp 283ndash289 2015

[22] Y Chen T Liu P Langford et al ldquoHaemophilus parasuisinduces activation of NF-120581B and MAP kinase signaling path-ways mediated by toll-like receptorsrdquo Molecular Immunologyvol 65 no 2 pp 360ndash366 2015

Page 4: Bone Marrow Mesenchymal Stem Cells Inhibit ......Bone Marrow Mesenchymal Stem Cells Inhibit ... and TLR4 response to acute otitis through activation of NF-𝜅B [15], we hypothesized

4 Mediators of Inflammation

TLR2

TLR4

(1) PBS control (2) BMSCs control(3) LPS (4) BMSCs treatment

Alv

eola

r mac

roph

ages

1 2 3 4

p-NF-120581B p65

120573-actin

PBS controlBMSCs control

LPSBMSCs treatment

TLR2

Nor

mal

ized

to120573

-act

in (T

LR2

)

00

06

12

18

24

30

63 121 24Time (h)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowast

TLR4

Nor

mal

ized

to120573

-act

in (T

LR4

)

00

06

12

18

24

30

6 12 2431Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowastlowast lowast

lowastlowast lowastlowastlowast lowast

lowastlowast lowast

(A)

(B) (C)

Nor

mal

ized

to120573

-act

in (p

-NF-120581

B p65

)

p-NF-120581B p65

00

06

12

18

24

1 3 12 246Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowast

lowastlowast lowast

lowastlowast lowastlowastlowast lowast

lowastlowast lowast

(D)

(a)

Figure 2 Continued

Mediators of Inflammation 5

TLR2

TLR4

(1) PBS control (2) BMSCs control(3) LPS (4) BMSCs treatment

HU

VEC

s

1 2 3 4

p-NF-120581B p65

120573-actin

00

06

12

18

24

30

241263Time (h)

1

TLR2

PBS controlBMSCs control

LPSBMSCs treatment

Nor

mal

ized

to120573

-act

in (T

LR2

)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

TLR4

Nor

mal

ized

to120573

-act

in (T

LR4

)

00

06

12

18

24

30

PBS controlBMSCs control

LPSBMSCs treatment

3 24121 6Time (h)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

63 241 12Time (h)

Nor

mal

ized

to120573

-act

in (p

-NF-120581

B p65

)

p-NF-120581B p65

00

06

12

18

24

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

(E)

(F) (G)

(H)

(b)

Figure 2 Coculture with BMSCs attenuates the activity of LPS to induce expressions of TLR2 TLR4 and p65 in alveolar macrophages (a)and HUVECs (b) at the protein levelThe total proteins were extracted from the cultured alveolar macrophages and HUVECs treated (1) PBScontrol (2) BMSCs (3) LPS and (4) BMSCs and LPS The expressions of TLR2 TLR4 and p-NF-120581B p65 were determined by Western blotThe density of specific bands was quantitated Data are expressed as the mean plusmn SD from three experiments lowast119875 lt 005 lowastlowast119875 lt 001

6 Mediators of Inflammation

0

1

2

3

4

5

Alv

eola

r mac

roph

ages

2412631

Relat

ive T

LR2

mRN

A le

vel

Time (h)

TLR2

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

TLR4

0

1

2

3

4

5

Relat

ive T

LR4

mRN

A le

vel

1 3 126 24Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

(A) (B)

(a)

HU

VEC

s

TLR2

0

1

2

3

4

5

Relat

ive T

LR2

mRN

A le

vel

3 121 246Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

TLR4

0

1

2

3

4

5

Relat

ive T

LR4

mRN

A le

vel

3 6 12 241Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

(C) (D)

(b)

Figure 3 Coculture with BMSCs attenuates the activity of LPS to induce expressions of TLR2 and TLR4 in alveolar macrophages (a) andHUVECs (b) at themRNA levelThe total RNAswere extracted from the cultured alveolarmacrophages andHUVECs treated (1) PBS control(2) BMSCs (3) LPS and (4) BMSCs and LPSThe expressions of TLR2 and TLR4 were determined by real-time RT-PCR Data are expressedas the mean plusmn SD from three experiments lowast119875 lt 005 lowastlowast119875 lt 001

to the experiments Further efforts are needed to clarify thisdiscrepancy

TLRs play a pivotal role in defense against invadingpathogens through recognizing PAMPs It is well docu-mented that cells use TLR2 and TLR4 to recognize PAMPsexpressed by bacteria Activation of TLR2 and TLR4 leadsto activation of MAPK and NF-120581B pathways and productionof inflammatory cytokines including IL-1 IL-6 and TNF-120572 [20ndash22] In this study we showed that LPS promoted

expression of TLR2 and TLR4 in alveolar macrophages andHUVECs However how expression of TLR2 expressionwas increased by LPS is unknown and deserves futureinvestigations TNF-120572 and IL10 expressions were also affectedby LPS Furthermore the LPS effects were diminishedwhen the HUVECs and macrophages were cultured withBMSCs TNF-120572 is a potent inflammatory factor and IL10 isa key anti-inflammatory cytokine Downregulation of TNF-120572 and upregulation of IL10 in HUVECs and macrophage

Mediators of Inflammation 7A

lveo

lar m

acro

phag

es

3 6 24121Time (h)

Supe

rnat

ant l

evels

of T

NF-120572

(pg

mL)

TNF-120572

0

500

1000

1500

2000

2500

3000

3500

4000

4500

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

0

10

20

30

40

50

60

70

80

90

2412631Time (h)

Supe

rnat

ant l

evels

of I

L10

(pg

mL)

IL10

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(A) (B)

(a)

HU

VEC

sSu

pern

atan

t lev

els o

f TN

F-120572

(pg

mL)

0

50

100

150

200

250

300

350

63 12 241Time (h)

TNF-120572

lowastlowast lowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

IL10

0

10

20

30

40

50

60

70

80Su

pern

atan

t lev

els o

f IL1

0 (p

gm

L)

3 6 241 12Time (h)

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(C) (D)

(b)

Figure 4 Coculture with BMSCs attenuates the activity of LPS to induce release of cytokines in alveolar macrophages (a) and HUVECs (b)Alveolar macrophages and HUVECs with or without coculture with BMSCs were treated with LPS or PBS for 1 3 6 12 and 24 hours Thecell culture supernatant was then collected for cytokine analysis by ELISA Data are expressed as the mean plusmn SD from three experimentslowast

119875 lt 005 lowastlowast119875 lt 001

indicate that the inflammatory reactions were suppressedThe results suggest that BMSCs have the ability to alleviatepathogen-induced inflammation damage in HUVECs andmacrophages

In conclusion the results showed that BMSCs attenuatedthe activity of LPS on the inflammatory reaction of HUVECsand alveolar macrophages through inhibiting upregulation ofTLR2 TLR4 and p65 expression as well as downregulatingTNF-120572 release and upregulating IL10 release

Abbreviations

BMSC Bone marrow mesenchymal stem cellsLPS LipopolysaccharideTLR Toll-like receptorSIRS Systemic inflammatory response syndromeMODS Multiple organ dysfunction syndromePAMP Pathogen associated molecular patterns

8 Mediators of Inflammation

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Dequan Li and Jun Fang participated in study designand coordination analysis and interpretation of data andsupervised study Yuanyuan Wang Chuang Chi and JingZhao performed most of the experiments and statisticalanalyses and drafted the paper Other authors carried outthe experiments and sample collections All authors read andapproved the final paper Dequan Li and Cong Wang equallycontributed to this paper

Acknowledgments

The authors thank the Medical Scientific Research Platform(Wenzhou Medical University Wenzhou China) and theSurgery Laboratory (the First Affiliated Hospital of WenzhouMedical University Wenzhou China) for their technicalassistance This work was supported by Zhejiang ProvinceNatural Science Foundation of China (LY12H15001) Healthand Family Planning Commission of Zhejiang Province(2013KYA130) Science Technology Department of ZhejiangProvince (2011R50018-20) Wenzhou Municipal Science andTechnology Bureau (Y20130091) and the National Natu-ral Science Foundation of China (81171787 31371470 and81270761)

References

[1] J-Z Zhang Z Liu J Liu J-X Ren and T-S Sun ldquoMitochon-drial DNA induces inflammation and increases TLR9NF-120581Bexpression in lung tissuerdquo International Journal of MolecularMedicine vol 33 no 4 pp 817ndash824 2014

[2] T Tsukamoto R S Chanthaphavong andH-C Pape ldquoCurrenttheories on the pathophysiology of multiple organ failure aftertraumardquo Injury vol 41 no 1 pp 21ndash26 2010

[3] Y Zhao C Yang H Wang et al ldquoTherapeutic effects of bonemarrow-derivedmesenchymal stem cells on pulmonary impactinjury complicated with endotoxemia in ratsrdquo InternationalImmunopharmacology vol 15 no 2 pp 246ndash253 2013

[4] K Tang Y Xiao D Liu et al ldquoAutografting of bone marrowmesenchymal stem cells alleviates streptozotocin-induced dia-betes in miniature pigs real-time tracing with MRI in vivordquoInternational Journal of Molecular Medicine vol 33 no 6 pp1469ndash1476 2014

[5] S Shin Y Kim S Jeong et al ldquoThe therapeutic effect of humanadult stem cells derived from adipose tissue in endotoxemic ratmodelrdquo International Journal of Medical Sciences vol 10 no 1pp 8ndash18 2012

[6] K Nemeth A Leelahavanichkul P S T Yuen et al ldquoBonemarrow stromal cells attenuate sepsis via prostaglandin E

2

-dependent reprogramming of host macrophages to increasetheir interleukin-10 productionrdquo Nature Medicine vol 15 no1 pp 42ndash49 2009

[7] N Kusadasi and A B J Groeneveld ldquoA perspective onmesenchymal stromal cell transplantation in the treatment ofsepsisrdquo Shock vol 40 no 5 pp 352ndash357 2013

[8] S R R Hall K Tsoyi B Ith et al ldquoMesenchymal stromalcells improve survival during sepsis in the absence of hemeoxygenase-1 the importance of neutrophilsrdquo STEMCELLS vol31 no 2 pp 397ndash407 2013

[9] J S Elman M Li F Wang J M Gimble and B Parekkadan ldquoAcomparison of adipose andbonemarrow-derivedmesenchymalstromal cell secreted factors in the treatment of systemicinflammationrdquo Journal of Inflammation vol 11 no 1 p 1 2014

[10] H Yagi A Soto-Gutierrez Y Kitagawa A W Tilles R GTompkins and M L Yarmush ldquoBone marrow mesenchymalstromal cells attenuate organ injury induced by LPS and burnrdquoCell Transplantation vol 19 no 6-7 pp 823ndash830 2010

[11] D Togbe S Schnyder-Candrian B Schnyder et al ldquoToll-likereceptor and tumour necrosis factor dependent endotoxin-induced acute lung injuryrdquo International Journal of Experimen-tal Pathology vol 88 no 6 pp 387ndash391 2007

[12] W Ren Z Wang F Hua and L Zhu ldquoPlasminogen activatorinhibitor-1 regulates LPS-induced TLR4MD-2 pathway activa-tion and inflammation in alveolar macrophagesrdquo Inflammationvol 38 no 1 pp 384ndash393 2015

[13] K Nemeth B Mayer and E Mezey ldquoModulation of bonemarrow stromal cell functions in infectious diseases by toll-likereceptor ligandsrdquo Journal of Molecular Medicine vol 88 no 1pp 5ndash10 2010

[14] N V Gorbunov B R Garrison D P McDaniel et al ldquoAdaptiveredox response of mesenchymal stromal cells to stimulationwith lipopolysaccharide inflammagenmechanisms of remodel-ing of tissue barriers in sepsisrdquoOxidative Medicine and CellularLongevity vol 2013 Article ID 186795 16 pages 2013

[15] S Jesic A Jotic N Tomanovic M Zivkovic A Kolakovic andA Stankovic ldquoExpression of toll-like receptors 2 4 and nuclearfactor kappa B in mucosal lesions of human otitis pattern andrelationship in a clinical immunohistochemical studyrdquo Annalsof Otology Rhinology and Laryngology vol 123 no 6 pp 434ndash441 2014

[16] Y-H Zheng W Xiong K Su S-J Kuang and Z-G ZhangldquoMultilineage differentiation of human bone marrow mes-enchymal stem cells in vitro and in vivordquo Experimental andTherapeutic Medicine vol 5 no 6 pp 1576ndash1580 2013

[17] J L Shelton L Wang G Cepinskas et al ldquoAlbumin leak acrosshuman pulmonarymicrovascular vs umbilical vein endothelialcells under septic conditionsrdquo Microvascular Research vol 71no 1 pp 40ndash47 2006

[18] Z Zhou F Guo Y Dou J Tang and J Huan ldquoGua-nine nucleotide exchange factor-H1 signaling is involved inlipopolysaccharide-induced endothelial barrier dysfunctionrdquoSurgery vol 154 no 3 pp 621ndash631 2013

[19] J-S Bae W Lee and A R Rezaie ldquoPolyphosphate elicitspro-inflammatory responses that are counteracted by activatedprotein C in both cellular and animal modelsrdquo Journal ofThrombosis and Haemostasis vol 10 no 6 pp 1145ndash1151 2012

[20] Z Tianzhu and W Shumin ldquoEsculin inhibits the inflammationof LPS-Induced acute lung injury in mice via regulation ofTLRNF-120581B pathwaysrdquo Inflammation vol 38 no 4 pp 1529ndash1536 2015

Mediators of Inflammation 9

[21] Q S Liu Z W Cheng J G Xiong S Cheng X F He and XC Li ldquoErythropoietin pretreatment exerts anti-inflammatoryeffects in hepatic ischemiareperfusion-injured rats via suppres-sion of the TLR2NF-120581B pathwayrdquo Transplantation Proceedingsvol 47 no 2 pp 283ndash289 2015

[22] Y Chen T Liu P Langford et al ldquoHaemophilus parasuisinduces activation of NF-120581B and MAP kinase signaling path-ways mediated by toll-like receptorsrdquo Molecular Immunologyvol 65 no 2 pp 360ndash366 2015

Page 5: Bone Marrow Mesenchymal Stem Cells Inhibit ......Bone Marrow Mesenchymal Stem Cells Inhibit ... and TLR4 response to acute otitis through activation of NF-𝜅B [15], we hypothesized

Mediators of Inflammation 5

TLR2

TLR4

(1) PBS control (2) BMSCs control(3) LPS (4) BMSCs treatment

HU

VEC

s

1 2 3 4

p-NF-120581B p65

120573-actin

00

06

12

18

24

30

241263Time (h)

1

TLR2

PBS controlBMSCs control

LPSBMSCs treatment

Nor

mal

ized

to120573

-act

in (T

LR2

)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

TLR4

Nor

mal

ized

to120573

-act

in (T

LR4

)

00

06

12

18

24

30

PBS controlBMSCs control

LPSBMSCs treatment

3 24121 6Time (h)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

63 241 12Time (h)

Nor

mal

ized

to120573

-act

in (p

-NF-120581

B p65

)

p-NF-120581B p65

00

06

12

18

24

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

(E)

(F) (G)

(H)

(b)

Figure 2 Coculture with BMSCs attenuates the activity of LPS to induce expressions of TLR2 TLR4 and p65 in alveolar macrophages (a)and HUVECs (b) at the protein levelThe total proteins were extracted from the cultured alveolar macrophages and HUVECs treated (1) PBScontrol (2) BMSCs (3) LPS and (4) BMSCs and LPS The expressions of TLR2 TLR4 and p-NF-120581B p65 were determined by Western blotThe density of specific bands was quantitated Data are expressed as the mean plusmn SD from three experiments lowast119875 lt 005 lowastlowast119875 lt 001

6 Mediators of Inflammation

0

1

2

3

4

5

Alv

eola

r mac

roph

ages

2412631

Relat

ive T

LR2

mRN

A le

vel

Time (h)

TLR2

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

TLR4

0

1

2

3

4

5

Relat

ive T

LR4

mRN

A le

vel

1 3 126 24Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

(A) (B)

(a)

HU

VEC

s

TLR2

0

1

2

3

4

5

Relat

ive T

LR2

mRN

A le

vel

3 121 246Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

TLR4

0

1

2

3

4

5

Relat

ive T

LR4

mRN

A le

vel

3 6 12 241Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

(C) (D)

(b)

Figure 3 Coculture with BMSCs attenuates the activity of LPS to induce expressions of TLR2 and TLR4 in alveolar macrophages (a) andHUVECs (b) at themRNA levelThe total RNAswere extracted from the cultured alveolarmacrophages andHUVECs treated (1) PBS control(2) BMSCs (3) LPS and (4) BMSCs and LPSThe expressions of TLR2 and TLR4 were determined by real-time RT-PCR Data are expressedas the mean plusmn SD from three experiments lowast119875 lt 005 lowastlowast119875 lt 001

to the experiments Further efforts are needed to clarify thisdiscrepancy

TLRs play a pivotal role in defense against invadingpathogens through recognizing PAMPs It is well docu-mented that cells use TLR2 and TLR4 to recognize PAMPsexpressed by bacteria Activation of TLR2 and TLR4 leadsto activation of MAPK and NF-120581B pathways and productionof inflammatory cytokines including IL-1 IL-6 and TNF-120572 [20ndash22] In this study we showed that LPS promoted

expression of TLR2 and TLR4 in alveolar macrophages andHUVECs However how expression of TLR2 expressionwas increased by LPS is unknown and deserves futureinvestigations TNF-120572 and IL10 expressions were also affectedby LPS Furthermore the LPS effects were diminishedwhen the HUVECs and macrophages were cultured withBMSCs TNF-120572 is a potent inflammatory factor and IL10 isa key anti-inflammatory cytokine Downregulation of TNF-120572 and upregulation of IL10 in HUVECs and macrophage

Mediators of Inflammation 7A

lveo

lar m

acro

phag

es

3 6 24121Time (h)

Supe

rnat

ant l

evels

of T

NF-120572

(pg

mL)

TNF-120572

0

500

1000

1500

2000

2500

3000

3500

4000

4500

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

0

10

20

30

40

50

60

70

80

90

2412631Time (h)

Supe

rnat

ant l

evels

of I

L10

(pg

mL)

IL10

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(A) (B)

(a)

HU

VEC

sSu

pern

atan

t lev

els o

f TN

F-120572

(pg

mL)

0

50

100

150

200

250

300

350

63 12 241Time (h)

TNF-120572

lowastlowast lowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

IL10

0

10

20

30

40

50

60

70

80Su

pern

atan

t lev

els o

f IL1

0 (p

gm

L)

3 6 241 12Time (h)

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(C) (D)

(b)

Figure 4 Coculture with BMSCs attenuates the activity of LPS to induce release of cytokines in alveolar macrophages (a) and HUVECs (b)Alveolar macrophages and HUVECs with or without coculture with BMSCs were treated with LPS or PBS for 1 3 6 12 and 24 hours Thecell culture supernatant was then collected for cytokine analysis by ELISA Data are expressed as the mean plusmn SD from three experimentslowast

119875 lt 005 lowastlowast119875 lt 001

indicate that the inflammatory reactions were suppressedThe results suggest that BMSCs have the ability to alleviatepathogen-induced inflammation damage in HUVECs andmacrophages

In conclusion the results showed that BMSCs attenuatedthe activity of LPS on the inflammatory reaction of HUVECsand alveolar macrophages through inhibiting upregulation ofTLR2 TLR4 and p65 expression as well as downregulatingTNF-120572 release and upregulating IL10 release

Abbreviations

BMSC Bone marrow mesenchymal stem cellsLPS LipopolysaccharideTLR Toll-like receptorSIRS Systemic inflammatory response syndromeMODS Multiple organ dysfunction syndromePAMP Pathogen associated molecular patterns

8 Mediators of Inflammation

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Dequan Li and Jun Fang participated in study designand coordination analysis and interpretation of data andsupervised study Yuanyuan Wang Chuang Chi and JingZhao performed most of the experiments and statisticalanalyses and drafted the paper Other authors carried outthe experiments and sample collections All authors read andapproved the final paper Dequan Li and Cong Wang equallycontributed to this paper

Acknowledgments

The authors thank the Medical Scientific Research Platform(Wenzhou Medical University Wenzhou China) and theSurgery Laboratory (the First Affiliated Hospital of WenzhouMedical University Wenzhou China) for their technicalassistance This work was supported by Zhejiang ProvinceNatural Science Foundation of China (LY12H15001) Healthand Family Planning Commission of Zhejiang Province(2013KYA130) Science Technology Department of ZhejiangProvince (2011R50018-20) Wenzhou Municipal Science andTechnology Bureau (Y20130091) and the National Natu-ral Science Foundation of China (81171787 31371470 and81270761)

References

[1] J-Z Zhang Z Liu J Liu J-X Ren and T-S Sun ldquoMitochon-drial DNA induces inflammation and increases TLR9NF-120581Bexpression in lung tissuerdquo International Journal of MolecularMedicine vol 33 no 4 pp 817ndash824 2014

[2] T Tsukamoto R S Chanthaphavong andH-C Pape ldquoCurrenttheories on the pathophysiology of multiple organ failure aftertraumardquo Injury vol 41 no 1 pp 21ndash26 2010

[3] Y Zhao C Yang H Wang et al ldquoTherapeutic effects of bonemarrow-derivedmesenchymal stem cells on pulmonary impactinjury complicated with endotoxemia in ratsrdquo InternationalImmunopharmacology vol 15 no 2 pp 246ndash253 2013

[4] K Tang Y Xiao D Liu et al ldquoAutografting of bone marrowmesenchymal stem cells alleviates streptozotocin-induced dia-betes in miniature pigs real-time tracing with MRI in vivordquoInternational Journal of Molecular Medicine vol 33 no 6 pp1469ndash1476 2014

[5] S Shin Y Kim S Jeong et al ldquoThe therapeutic effect of humanadult stem cells derived from adipose tissue in endotoxemic ratmodelrdquo International Journal of Medical Sciences vol 10 no 1pp 8ndash18 2012

[6] K Nemeth A Leelahavanichkul P S T Yuen et al ldquoBonemarrow stromal cells attenuate sepsis via prostaglandin E

2

-dependent reprogramming of host macrophages to increasetheir interleukin-10 productionrdquo Nature Medicine vol 15 no1 pp 42ndash49 2009

[7] N Kusadasi and A B J Groeneveld ldquoA perspective onmesenchymal stromal cell transplantation in the treatment ofsepsisrdquo Shock vol 40 no 5 pp 352ndash357 2013

[8] S R R Hall K Tsoyi B Ith et al ldquoMesenchymal stromalcells improve survival during sepsis in the absence of hemeoxygenase-1 the importance of neutrophilsrdquo STEMCELLS vol31 no 2 pp 397ndash407 2013

[9] J S Elman M Li F Wang J M Gimble and B Parekkadan ldquoAcomparison of adipose andbonemarrow-derivedmesenchymalstromal cell secreted factors in the treatment of systemicinflammationrdquo Journal of Inflammation vol 11 no 1 p 1 2014

[10] H Yagi A Soto-Gutierrez Y Kitagawa A W Tilles R GTompkins and M L Yarmush ldquoBone marrow mesenchymalstromal cells attenuate organ injury induced by LPS and burnrdquoCell Transplantation vol 19 no 6-7 pp 823ndash830 2010

[11] D Togbe S Schnyder-Candrian B Schnyder et al ldquoToll-likereceptor and tumour necrosis factor dependent endotoxin-induced acute lung injuryrdquo International Journal of Experimen-tal Pathology vol 88 no 6 pp 387ndash391 2007

[12] W Ren Z Wang F Hua and L Zhu ldquoPlasminogen activatorinhibitor-1 regulates LPS-induced TLR4MD-2 pathway activa-tion and inflammation in alveolar macrophagesrdquo Inflammationvol 38 no 1 pp 384ndash393 2015

[13] K Nemeth B Mayer and E Mezey ldquoModulation of bonemarrow stromal cell functions in infectious diseases by toll-likereceptor ligandsrdquo Journal of Molecular Medicine vol 88 no 1pp 5ndash10 2010

[14] N V Gorbunov B R Garrison D P McDaniel et al ldquoAdaptiveredox response of mesenchymal stromal cells to stimulationwith lipopolysaccharide inflammagenmechanisms of remodel-ing of tissue barriers in sepsisrdquoOxidative Medicine and CellularLongevity vol 2013 Article ID 186795 16 pages 2013

[15] S Jesic A Jotic N Tomanovic M Zivkovic A Kolakovic andA Stankovic ldquoExpression of toll-like receptors 2 4 and nuclearfactor kappa B in mucosal lesions of human otitis pattern andrelationship in a clinical immunohistochemical studyrdquo Annalsof Otology Rhinology and Laryngology vol 123 no 6 pp 434ndash441 2014

[16] Y-H Zheng W Xiong K Su S-J Kuang and Z-G ZhangldquoMultilineage differentiation of human bone marrow mes-enchymal stem cells in vitro and in vivordquo Experimental andTherapeutic Medicine vol 5 no 6 pp 1576ndash1580 2013

[17] J L Shelton L Wang G Cepinskas et al ldquoAlbumin leak acrosshuman pulmonarymicrovascular vs umbilical vein endothelialcells under septic conditionsrdquo Microvascular Research vol 71no 1 pp 40ndash47 2006

[18] Z Zhou F Guo Y Dou J Tang and J Huan ldquoGua-nine nucleotide exchange factor-H1 signaling is involved inlipopolysaccharide-induced endothelial barrier dysfunctionrdquoSurgery vol 154 no 3 pp 621ndash631 2013

[19] J-S Bae W Lee and A R Rezaie ldquoPolyphosphate elicitspro-inflammatory responses that are counteracted by activatedprotein C in both cellular and animal modelsrdquo Journal ofThrombosis and Haemostasis vol 10 no 6 pp 1145ndash1151 2012

[20] Z Tianzhu and W Shumin ldquoEsculin inhibits the inflammationof LPS-Induced acute lung injury in mice via regulation ofTLRNF-120581B pathwaysrdquo Inflammation vol 38 no 4 pp 1529ndash1536 2015

Mediators of Inflammation 9

[21] Q S Liu Z W Cheng J G Xiong S Cheng X F He and XC Li ldquoErythropoietin pretreatment exerts anti-inflammatoryeffects in hepatic ischemiareperfusion-injured rats via suppres-sion of the TLR2NF-120581B pathwayrdquo Transplantation Proceedingsvol 47 no 2 pp 283ndash289 2015

[22] Y Chen T Liu P Langford et al ldquoHaemophilus parasuisinduces activation of NF-120581B and MAP kinase signaling path-ways mediated by toll-like receptorsrdquo Molecular Immunologyvol 65 no 2 pp 360ndash366 2015

Page 6: Bone Marrow Mesenchymal Stem Cells Inhibit ......Bone Marrow Mesenchymal Stem Cells Inhibit ... and TLR4 response to acute otitis through activation of NF-𝜅B [15], we hypothesized

6 Mediators of Inflammation

0

1

2

3

4

5

Alv

eola

r mac

roph

ages

2412631

Relat

ive T

LR2

mRN

A le

vel

Time (h)

TLR2

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

TLR4

0

1

2

3

4

5

Relat

ive T

LR4

mRN

A le

vel

1 3 126 24Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

(A) (B)

(a)

HU

VEC

s

TLR2

0

1

2

3

4

5

Relat

ive T

LR2

mRN

A le

vel

3 121 246Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast lowastlowast lowastlowast lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

TLR4

0

1

2

3

4

5

Relat

ive T

LR4

mRN

A le

vel

3 6 12 241Time (h)

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

(C) (D)

(b)

Figure 3 Coculture with BMSCs attenuates the activity of LPS to induce expressions of TLR2 and TLR4 in alveolar macrophages (a) andHUVECs (b) at themRNA levelThe total RNAswere extracted from the cultured alveolarmacrophages andHUVECs treated (1) PBS control(2) BMSCs (3) LPS and (4) BMSCs and LPSThe expressions of TLR2 and TLR4 were determined by real-time RT-PCR Data are expressedas the mean plusmn SD from three experiments lowast119875 lt 005 lowastlowast119875 lt 001

to the experiments Further efforts are needed to clarify thisdiscrepancy

TLRs play a pivotal role in defense against invadingpathogens through recognizing PAMPs It is well docu-mented that cells use TLR2 and TLR4 to recognize PAMPsexpressed by bacteria Activation of TLR2 and TLR4 leadsto activation of MAPK and NF-120581B pathways and productionof inflammatory cytokines including IL-1 IL-6 and TNF-120572 [20ndash22] In this study we showed that LPS promoted

expression of TLR2 and TLR4 in alveolar macrophages andHUVECs However how expression of TLR2 expressionwas increased by LPS is unknown and deserves futureinvestigations TNF-120572 and IL10 expressions were also affectedby LPS Furthermore the LPS effects were diminishedwhen the HUVECs and macrophages were cultured withBMSCs TNF-120572 is a potent inflammatory factor and IL10 isa key anti-inflammatory cytokine Downregulation of TNF-120572 and upregulation of IL10 in HUVECs and macrophage

Mediators of Inflammation 7A

lveo

lar m

acro

phag

es

3 6 24121Time (h)

Supe

rnat

ant l

evels

of T

NF-120572

(pg

mL)

TNF-120572

0

500

1000

1500

2000

2500

3000

3500

4000

4500

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

0

10

20

30

40

50

60

70

80

90

2412631Time (h)

Supe

rnat

ant l

evels

of I

L10

(pg

mL)

IL10

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(A) (B)

(a)

HU

VEC

sSu

pern

atan

t lev

els o

f TN

F-120572

(pg

mL)

0

50

100

150

200

250

300

350

63 12 241Time (h)

TNF-120572

lowastlowast lowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

IL10

0

10

20

30

40

50

60

70

80Su

pern

atan

t lev

els o

f IL1

0 (p

gm

L)

3 6 241 12Time (h)

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(C) (D)

(b)

Figure 4 Coculture with BMSCs attenuates the activity of LPS to induce release of cytokines in alveolar macrophages (a) and HUVECs (b)Alveolar macrophages and HUVECs with or without coculture with BMSCs were treated with LPS or PBS for 1 3 6 12 and 24 hours Thecell culture supernatant was then collected for cytokine analysis by ELISA Data are expressed as the mean plusmn SD from three experimentslowast

119875 lt 005 lowastlowast119875 lt 001

indicate that the inflammatory reactions were suppressedThe results suggest that BMSCs have the ability to alleviatepathogen-induced inflammation damage in HUVECs andmacrophages

In conclusion the results showed that BMSCs attenuatedthe activity of LPS on the inflammatory reaction of HUVECsand alveolar macrophages through inhibiting upregulation ofTLR2 TLR4 and p65 expression as well as downregulatingTNF-120572 release and upregulating IL10 release

Abbreviations

BMSC Bone marrow mesenchymal stem cellsLPS LipopolysaccharideTLR Toll-like receptorSIRS Systemic inflammatory response syndromeMODS Multiple organ dysfunction syndromePAMP Pathogen associated molecular patterns

8 Mediators of Inflammation

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Dequan Li and Jun Fang participated in study designand coordination analysis and interpretation of data andsupervised study Yuanyuan Wang Chuang Chi and JingZhao performed most of the experiments and statisticalanalyses and drafted the paper Other authors carried outthe experiments and sample collections All authors read andapproved the final paper Dequan Li and Cong Wang equallycontributed to this paper

Acknowledgments

The authors thank the Medical Scientific Research Platform(Wenzhou Medical University Wenzhou China) and theSurgery Laboratory (the First Affiliated Hospital of WenzhouMedical University Wenzhou China) for their technicalassistance This work was supported by Zhejiang ProvinceNatural Science Foundation of China (LY12H15001) Healthand Family Planning Commission of Zhejiang Province(2013KYA130) Science Technology Department of ZhejiangProvince (2011R50018-20) Wenzhou Municipal Science andTechnology Bureau (Y20130091) and the National Natu-ral Science Foundation of China (81171787 31371470 and81270761)

References

[1] J-Z Zhang Z Liu J Liu J-X Ren and T-S Sun ldquoMitochon-drial DNA induces inflammation and increases TLR9NF-120581Bexpression in lung tissuerdquo International Journal of MolecularMedicine vol 33 no 4 pp 817ndash824 2014

[2] T Tsukamoto R S Chanthaphavong andH-C Pape ldquoCurrenttheories on the pathophysiology of multiple organ failure aftertraumardquo Injury vol 41 no 1 pp 21ndash26 2010

[3] Y Zhao C Yang H Wang et al ldquoTherapeutic effects of bonemarrow-derivedmesenchymal stem cells on pulmonary impactinjury complicated with endotoxemia in ratsrdquo InternationalImmunopharmacology vol 15 no 2 pp 246ndash253 2013

[4] K Tang Y Xiao D Liu et al ldquoAutografting of bone marrowmesenchymal stem cells alleviates streptozotocin-induced dia-betes in miniature pigs real-time tracing with MRI in vivordquoInternational Journal of Molecular Medicine vol 33 no 6 pp1469ndash1476 2014

[5] S Shin Y Kim S Jeong et al ldquoThe therapeutic effect of humanadult stem cells derived from adipose tissue in endotoxemic ratmodelrdquo International Journal of Medical Sciences vol 10 no 1pp 8ndash18 2012

[6] K Nemeth A Leelahavanichkul P S T Yuen et al ldquoBonemarrow stromal cells attenuate sepsis via prostaglandin E

2

-dependent reprogramming of host macrophages to increasetheir interleukin-10 productionrdquo Nature Medicine vol 15 no1 pp 42ndash49 2009

[7] N Kusadasi and A B J Groeneveld ldquoA perspective onmesenchymal stromal cell transplantation in the treatment ofsepsisrdquo Shock vol 40 no 5 pp 352ndash357 2013

[8] S R R Hall K Tsoyi B Ith et al ldquoMesenchymal stromalcells improve survival during sepsis in the absence of hemeoxygenase-1 the importance of neutrophilsrdquo STEMCELLS vol31 no 2 pp 397ndash407 2013

[9] J S Elman M Li F Wang J M Gimble and B Parekkadan ldquoAcomparison of adipose andbonemarrow-derivedmesenchymalstromal cell secreted factors in the treatment of systemicinflammationrdquo Journal of Inflammation vol 11 no 1 p 1 2014

[10] H Yagi A Soto-Gutierrez Y Kitagawa A W Tilles R GTompkins and M L Yarmush ldquoBone marrow mesenchymalstromal cells attenuate organ injury induced by LPS and burnrdquoCell Transplantation vol 19 no 6-7 pp 823ndash830 2010

[11] D Togbe S Schnyder-Candrian B Schnyder et al ldquoToll-likereceptor and tumour necrosis factor dependent endotoxin-induced acute lung injuryrdquo International Journal of Experimen-tal Pathology vol 88 no 6 pp 387ndash391 2007

[12] W Ren Z Wang F Hua and L Zhu ldquoPlasminogen activatorinhibitor-1 regulates LPS-induced TLR4MD-2 pathway activa-tion and inflammation in alveolar macrophagesrdquo Inflammationvol 38 no 1 pp 384ndash393 2015

[13] K Nemeth B Mayer and E Mezey ldquoModulation of bonemarrow stromal cell functions in infectious diseases by toll-likereceptor ligandsrdquo Journal of Molecular Medicine vol 88 no 1pp 5ndash10 2010

[14] N V Gorbunov B R Garrison D P McDaniel et al ldquoAdaptiveredox response of mesenchymal stromal cells to stimulationwith lipopolysaccharide inflammagenmechanisms of remodel-ing of tissue barriers in sepsisrdquoOxidative Medicine and CellularLongevity vol 2013 Article ID 186795 16 pages 2013

[15] S Jesic A Jotic N Tomanovic M Zivkovic A Kolakovic andA Stankovic ldquoExpression of toll-like receptors 2 4 and nuclearfactor kappa B in mucosal lesions of human otitis pattern andrelationship in a clinical immunohistochemical studyrdquo Annalsof Otology Rhinology and Laryngology vol 123 no 6 pp 434ndash441 2014

[16] Y-H Zheng W Xiong K Su S-J Kuang and Z-G ZhangldquoMultilineage differentiation of human bone marrow mes-enchymal stem cells in vitro and in vivordquo Experimental andTherapeutic Medicine vol 5 no 6 pp 1576ndash1580 2013

[17] J L Shelton L Wang G Cepinskas et al ldquoAlbumin leak acrosshuman pulmonarymicrovascular vs umbilical vein endothelialcells under septic conditionsrdquo Microvascular Research vol 71no 1 pp 40ndash47 2006

[18] Z Zhou F Guo Y Dou J Tang and J Huan ldquoGua-nine nucleotide exchange factor-H1 signaling is involved inlipopolysaccharide-induced endothelial barrier dysfunctionrdquoSurgery vol 154 no 3 pp 621ndash631 2013

[19] J-S Bae W Lee and A R Rezaie ldquoPolyphosphate elicitspro-inflammatory responses that are counteracted by activatedprotein C in both cellular and animal modelsrdquo Journal ofThrombosis and Haemostasis vol 10 no 6 pp 1145ndash1151 2012

[20] Z Tianzhu and W Shumin ldquoEsculin inhibits the inflammationof LPS-Induced acute lung injury in mice via regulation ofTLRNF-120581B pathwaysrdquo Inflammation vol 38 no 4 pp 1529ndash1536 2015

Mediators of Inflammation 9

[21] Q S Liu Z W Cheng J G Xiong S Cheng X F He and XC Li ldquoErythropoietin pretreatment exerts anti-inflammatoryeffects in hepatic ischemiareperfusion-injured rats via suppres-sion of the TLR2NF-120581B pathwayrdquo Transplantation Proceedingsvol 47 no 2 pp 283ndash289 2015

[22] Y Chen T Liu P Langford et al ldquoHaemophilus parasuisinduces activation of NF-120581B and MAP kinase signaling path-ways mediated by toll-like receptorsrdquo Molecular Immunologyvol 65 no 2 pp 360ndash366 2015

Page 7: Bone Marrow Mesenchymal Stem Cells Inhibit ......Bone Marrow Mesenchymal Stem Cells Inhibit ... and TLR4 response to acute otitis through activation of NF-𝜅B [15], we hypothesized

Mediators of Inflammation 7A

lveo

lar m

acro

phag

es

3 6 24121Time (h)

Supe

rnat

ant l

evels

of T

NF-120572

(pg

mL)

TNF-120572

0

500

1000

1500

2000

2500

3000

3500

4000

4500

PBS controlBMSCs control

LPSBMSCs treatment

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast lowastlowast

0

10

20

30

40

50

60

70

80

90

2412631Time (h)

Supe

rnat

ant l

evels

of I

L10

(pg

mL)

IL10

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(A) (B)

(a)

HU

VEC

sSu

pern

atan

t lev

els o

f TN

F-120572

(pg

mL)

0

50

100

150

200

250

300

350

63 12 241Time (h)

TNF-120572

lowastlowast lowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

IL10

0

10

20

30

40

50

60

70

80Su

pern

atan

t lev

els o

f IL1

0 (p

gm

L)

3 6 241 12Time (h)

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

lowastlowast lowastlowast

PBS controlBMSCs control

LPSBMSCs treatment

(C) (D)

(b)

Figure 4 Coculture with BMSCs attenuates the activity of LPS to induce release of cytokines in alveolar macrophages (a) and HUVECs (b)Alveolar macrophages and HUVECs with or without coculture with BMSCs were treated with LPS or PBS for 1 3 6 12 and 24 hours Thecell culture supernatant was then collected for cytokine analysis by ELISA Data are expressed as the mean plusmn SD from three experimentslowast

119875 lt 005 lowastlowast119875 lt 001

indicate that the inflammatory reactions were suppressedThe results suggest that BMSCs have the ability to alleviatepathogen-induced inflammation damage in HUVECs andmacrophages

In conclusion the results showed that BMSCs attenuatedthe activity of LPS on the inflammatory reaction of HUVECsand alveolar macrophages through inhibiting upregulation ofTLR2 TLR4 and p65 expression as well as downregulatingTNF-120572 release and upregulating IL10 release

Abbreviations

BMSC Bone marrow mesenchymal stem cellsLPS LipopolysaccharideTLR Toll-like receptorSIRS Systemic inflammatory response syndromeMODS Multiple organ dysfunction syndromePAMP Pathogen associated molecular patterns

8 Mediators of Inflammation

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Dequan Li and Jun Fang participated in study designand coordination analysis and interpretation of data andsupervised study Yuanyuan Wang Chuang Chi and JingZhao performed most of the experiments and statisticalanalyses and drafted the paper Other authors carried outthe experiments and sample collections All authors read andapproved the final paper Dequan Li and Cong Wang equallycontributed to this paper

Acknowledgments

The authors thank the Medical Scientific Research Platform(Wenzhou Medical University Wenzhou China) and theSurgery Laboratory (the First Affiliated Hospital of WenzhouMedical University Wenzhou China) for their technicalassistance This work was supported by Zhejiang ProvinceNatural Science Foundation of China (LY12H15001) Healthand Family Planning Commission of Zhejiang Province(2013KYA130) Science Technology Department of ZhejiangProvince (2011R50018-20) Wenzhou Municipal Science andTechnology Bureau (Y20130091) and the National Natu-ral Science Foundation of China (81171787 31371470 and81270761)

References

[1] J-Z Zhang Z Liu J Liu J-X Ren and T-S Sun ldquoMitochon-drial DNA induces inflammation and increases TLR9NF-120581Bexpression in lung tissuerdquo International Journal of MolecularMedicine vol 33 no 4 pp 817ndash824 2014

[2] T Tsukamoto R S Chanthaphavong andH-C Pape ldquoCurrenttheories on the pathophysiology of multiple organ failure aftertraumardquo Injury vol 41 no 1 pp 21ndash26 2010

[3] Y Zhao C Yang H Wang et al ldquoTherapeutic effects of bonemarrow-derivedmesenchymal stem cells on pulmonary impactinjury complicated with endotoxemia in ratsrdquo InternationalImmunopharmacology vol 15 no 2 pp 246ndash253 2013

[4] K Tang Y Xiao D Liu et al ldquoAutografting of bone marrowmesenchymal stem cells alleviates streptozotocin-induced dia-betes in miniature pigs real-time tracing with MRI in vivordquoInternational Journal of Molecular Medicine vol 33 no 6 pp1469ndash1476 2014

[5] S Shin Y Kim S Jeong et al ldquoThe therapeutic effect of humanadult stem cells derived from adipose tissue in endotoxemic ratmodelrdquo International Journal of Medical Sciences vol 10 no 1pp 8ndash18 2012

[6] K Nemeth A Leelahavanichkul P S T Yuen et al ldquoBonemarrow stromal cells attenuate sepsis via prostaglandin E

2

-dependent reprogramming of host macrophages to increasetheir interleukin-10 productionrdquo Nature Medicine vol 15 no1 pp 42ndash49 2009

[7] N Kusadasi and A B J Groeneveld ldquoA perspective onmesenchymal stromal cell transplantation in the treatment ofsepsisrdquo Shock vol 40 no 5 pp 352ndash357 2013

[8] S R R Hall K Tsoyi B Ith et al ldquoMesenchymal stromalcells improve survival during sepsis in the absence of hemeoxygenase-1 the importance of neutrophilsrdquo STEMCELLS vol31 no 2 pp 397ndash407 2013

[9] J S Elman M Li F Wang J M Gimble and B Parekkadan ldquoAcomparison of adipose andbonemarrow-derivedmesenchymalstromal cell secreted factors in the treatment of systemicinflammationrdquo Journal of Inflammation vol 11 no 1 p 1 2014

[10] H Yagi A Soto-Gutierrez Y Kitagawa A W Tilles R GTompkins and M L Yarmush ldquoBone marrow mesenchymalstromal cells attenuate organ injury induced by LPS and burnrdquoCell Transplantation vol 19 no 6-7 pp 823ndash830 2010

[11] D Togbe S Schnyder-Candrian B Schnyder et al ldquoToll-likereceptor and tumour necrosis factor dependent endotoxin-induced acute lung injuryrdquo International Journal of Experimen-tal Pathology vol 88 no 6 pp 387ndash391 2007

[12] W Ren Z Wang F Hua and L Zhu ldquoPlasminogen activatorinhibitor-1 regulates LPS-induced TLR4MD-2 pathway activa-tion and inflammation in alveolar macrophagesrdquo Inflammationvol 38 no 1 pp 384ndash393 2015

[13] K Nemeth B Mayer and E Mezey ldquoModulation of bonemarrow stromal cell functions in infectious diseases by toll-likereceptor ligandsrdquo Journal of Molecular Medicine vol 88 no 1pp 5ndash10 2010

[14] N V Gorbunov B R Garrison D P McDaniel et al ldquoAdaptiveredox response of mesenchymal stromal cells to stimulationwith lipopolysaccharide inflammagenmechanisms of remodel-ing of tissue barriers in sepsisrdquoOxidative Medicine and CellularLongevity vol 2013 Article ID 186795 16 pages 2013

[15] S Jesic A Jotic N Tomanovic M Zivkovic A Kolakovic andA Stankovic ldquoExpression of toll-like receptors 2 4 and nuclearfactor kappa B in mucosal lesions of human otitis pattern andrelationship in a clinical immunohistochemical studyrdquo Annalsof Otology Rhinology and Laryngology vol 123 no 6 pp 434ndash441 2014

[16] Y-H Zheng W Xiong K Su S-J Kuang and Z-G ZhangldquoMultilineage differentiation of human bone marrow mes-enchymal stem cells in vitro and in vivordquo Experimental andTherapeutic Medicine vol 5 no 6 pp 1576ndash1580 2013

[17] J L Shelton L Wang G Cepinskas et al ldquoAlbumin leak acrosshuman pulmonarymicrovascular vs umbilical vein endothelialcells under septic conditionsrdquo Microvascular Research vol 71no 1 pp 40ndash47 2006

[18] Z Zhou F Guo Y Dou J Tang and J Huan ldquoGua-nine nucleotide exchange factor-H1 signaling is involved inlipopolysaccharide-induced endothelial barrier dysfunctionrdquoSurgery vol 154 no 3 pp 621ndash631 2013

[19] J-S Bae W Lee and A R Rezaie ldquoPolyphosphate elicitspro-inflammatory responses that are counteracted by activatedprotein C in both cellular and animal modelsrdquo Journal ofThrombosis and Haemostasis vol 10 no 6 pp 1145ndash1151 2012

[20] Z Tianzhu and W Shumin ldquoEsculin inhibits the inflammationof LPS-Induced acute lung injury in mice via regulation ofTLRNF-120581B pathwaysrdquo Inflammation vol 38 no 4 pp 1529ndash1536 2015

Mediators of Inflammation 9

[21] Q S Liu Z W Cheng J G Xiong S Cheng X F He and XC Li ldquoErythropoietin pretreatment exerts anti-inflammatoryeffects in hepatic ischemiareperfusion-injured rats via suppres-sion of the TLR2NF-120581B pathwayrdquo Transplantation Proceedingsvol 47 no 2 pp 283ndash289 2015

[22] Y Chen T Liu P Langford et al ldquoHaemophilus parasuisinduces activation of NF-120581B and MAP kinase signaling path-ways mediated by toll-like receptorsrdquo Molecular Immunologyvol 65 no 2 pp 360ndash366 2015

Page 8: Bone Marrow Mesenchymal Stem Cells Inhibit ......Bone Marrow Mesenchymal Stem Cells Inhibit ... and TLR4 response to acute otitis through activation of NF-𝜅B [15], we hypothesized

8 Mediators of Inflammation

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Dequan Li and Jun Fang participated in study designand coordination analysis and interpretation of data andsupervised study Yuanyuan Wang Chuang Chi and JingZhao performed most of the experiments and statisticalanalyses and drafted the paper Other authors carried outthe experiments and sample collections All authors read andapproved the final paper Dequan Li and Cong Wang equallycontributed to this paper

Acknowledgments

The authors thank the Medical Scientific Research Platform(Wenzhou Medical University Wenzhou China) and theSurgery Laboratory (the First Affiliated Hospital of WenzhouMedical University Wenzhou China) for their technicalassistance This work was supported by Zhejiang ProvinceNatural Science Foundation of China (LY12H15001) Healthand Family Planning Commission of Zhejiang Province(2013KYA130) Science Technology Department of ZhejiangProvince (2011R50018-20) Wenzhou Municipal Science andTechnology Bureau (Y20130091) and the National Natu-ral Science Foundation of China (81171787 31371470 and81270761)

References

[1] J-Z Zhang Z Liu J Liu J-X Ren and T-S Sun ldquoMitochon-drial DNA induces inflammation and increases TLR9NF-120581Bexpression in lung tissuerdquo International Journal of MolecularMedicine vol 33 no 4 pp 817ndash824 2014

[2] T Tsukamoto R S Chanthaphavong andH-C Pape ldquoCurrenttheories on the pathophysiology of multiple organ failure aftertraumardquo Injury vol 41 no 1 pp 21ndash26 2010

[3] Y Zhao C Yang H Wang et al ldquoTherapeutic effects of bonemarrow-derivedmesenchymal stem cells on pulmonary impactinjury complicated with endotoxemia in ratsrdquo InternationalImmunopharmacology vol 15 no 2 pp 246ndash253 2013

[4] K Tang Y Xiao D Liu et al ldquoAutografting of bone marrowmesenchymal stem cells alleviates streptozotocin-induced dia-betes in miniature pigs real-time tracing with MRI in vivordquoInternational Journal of Molecular Medicine vol 33 no 6 pp1469ndash1476 2014

[5] S Shin Y Kim S Jeong et al ldquoThe therapeutic effect of humanadult stem cells derived from adipose tissue in endotoxemic ratmodelrdquo International Journal of Medical Sciences vol 10 no 1pp 8ndash18 2012

[6] K Nemeth A Leelahavanichkul P S T Yuen et al ldquoBonemarrow stromal cells attenuate sepsis via prostaglandin E

2

-dependent reprogramming of host macrophages to increasetheir interleukin-10 productionrdquo Nature Medicine vol 15 no1 pp 42ndash49 2009

[7] N Kusadasi and A B J Groeneveld ldquoA perspective onmesenchymal stromal cell transplantation in the treatment ofsepsisrdquo Shock vol 40 no 5 pp 352ndash357 2013

[8] S R R Hall K Tsoyi B Ith et al ldquoMesenchymal stromalcells improve survival during sepsis in the absence of hemeoxygenase-1 the importance of neutrophilsrdquo STEMCELLS vol31 no 2 pp 397ndash407 2013

[9] J S Elman M Li F Wang J M Gimble and B Parekkadan ldquoAcomparison of adipose andbonemarrow-derivedmesenchymalstromal cell secreted factors in the treatment of systemicinflammationrdquo Journal of Inflammation vol 11 no 1 p 1 2014

[10] H Yagi A Soto-Gutierrez Y Kitagawa A W Tilles R GTompkins and M L Yarmush ldquoBone marrow mesenchymalstromal cells attenuate organ injury induced by LPS and burnrdquoCell Transplantation vol 19 no 6-7 pp 823ndash830 2010

[11] D Togbe S Schnyder-Candrian B Schnyder et al ldquoToll-likereceptor and tumour necrosis factor dependent endotoxin-induced acute lung injuryrdquo International Journal of Experimen-tal Pathology vol 88 no 6 pp 387ndash391 2007

[12] W Ren Z Wang F Hua and L Zhu ldquoPlasminogen activatorinhibitor-1 regulates LPS-induced TLR4MD-2 pathway activa-tion and inflammation in alveolar macrophagesrdquo Inflammationvol 38 no 1 pp 384ndash393 2015

[13] K Nemeth B Mayer and E Mezey ldquoModulation of bonemarrow stromal cell functions in infectious diseases by toll-likereceptor ligandsrdquo Journal of Molecular Medicine vol 88 no 1pp 5ndash10 2010

[14] N V Gorbunov B R Garrison D P McDaniel et al ldquoAdaptiveredox response of mesenchymal stromal cells to stimulationwith lipopolysaccharide inflammagenmechanisms of remodel-ing of tissue barriers in sepsisrdquoOxidative Medicine and CellularLongevity vol 2013 Article ID 186795 16 pages 2013

[15] S Jesic A Jotic N Tomanovic M Zivkovic A Kolakovic andA Stankovic ldquoExpression of toll-like receptors 2 4 and nuclearfactor kappa B in mucosal lesions of human otitis pattern andrelationship in a clinical immunohistochemical studyrdquo Annalsof Otology Rhinology and Laryngology vol 123 no 6 pp 434ndash441 2014

[16] Y-H Zheng W Xiong K Su S-J Kuang and Z-G ZhangldquoMultilineage differentiation of human bone marrow mes-enchymal stem cells in vitro and in vivordquo Experimental andTherapeutic Medicine vol 5 no 6 pp 1576ndash1580 2013

[17] J L Shelton L Wang G Cepinskas et al ldquoAlbumin leak acrosshuman pulmonarymicrovascular vs umbilical vein endothelialcells under septic conditionsrdquo Microvascular Research vol 71no 1 pp 40ndash47 2006

[18] Z Zhou F Guo Y Dou J Tang and J Huan ldquoGua-nine nucleotide exchange factor-H1 signaling is involved inlipopolysaccharide-induced endothelial barrier dysfunctionrdquoSurgery vol 154 no 3 pp 621ndash631 2013

[19] J-S Bae W Lee and A R Rezaie ldquoPolyphosphate elicitspro-inflammatory responses that are counteracted by activatedprotein C in both cellular and animal modelsrdquo Journal ofThrombosis and Haemostasis vol 10 no 6 pp 1145ndash1151 2012

[20] Z Tianzhu and W Shumin ldquoEsculin inhibits the inflammationof LPS-Induced acute lung injury in mice via regulation ofTLRNF-120581B pathwaysrdquo Inflammation vol 38 no 4 pp 1529ndash1536 2015

Mediators of Inflammation 9

[21] Q S Liu Z W Cheng J G Xiong S Cheng X F He and XC Li ldquoErythropoietin pretreatment exerts anti-inflammatoryeffects in hepatic ischemiareperfusion-injured rats via suppres-sion of the TLR2NF-120581B pathwayrdquo Transplantation Proceedingsvol 47 no 2 pp 283ndash289 2015

[22] Y Chen T Liu P Langford et al ldquoHaemophilus parasuisinduces activation of NF-120581B and MAP kinase signaling path-ways mediated by toll-like receptorsrdquo Molecular Immunologyvol 65 no 2 pp 360ndash366 2015

Page 9: Bone Marrow Mesenchymal Stem Cells Inhibit ......Bone Marrow Mesenchymal Stem Cells Inhibit ... and TLR4 response to acute otitis through activation of NF-𝜅B [15], we hypothesized

Mediators of Inflammation 9

[21] Q S Liu Z W Cheng J G Xiong S Cheng X F He and XC Li ldquoErythropoietin pretreatment exerts anti-inflammatoryeffects in hepatic ischemiareperfusion-injured rats via suppres-sion of the TLR2NF-120581B pathwayrdquo Transplantation Proceedingsvol 47 no 2 pp 283ndash289 2015

[22] Y Chen T Liu P Langford et al ldquoHaemophilus parasuisinduces activation of NF-120581B and MAP kinase signaling path-ways mediated by toll-like receptorsrdquo Molecular Immunologyvol 65 no 2 pp 360ndash366 2015