IL-6 and IL-8 Suppression by Bacteria-adhered Mesenchymal Stem

Cells Co-cultured with PBMCs under TNF-α Exposure

Agung Putra

1,2,3

, Iffan Alif

, Mohammad Ariq Nazar

, Ardi Prasetio

Risky Chandra

Satria Irawan

, Dina Amalina

1,4

, Endah Permata Sari

, Azizah Retno Kustiyah

and Iqbal Pahlevi Adeputra Nasution

Stem Cell and Cancer Research, Medical Faculty, Universitas Islam Sultan Agung, Semarang, Indonesia

Department of Pathological Anatomy, Medical Faculty, Universitas Islam Sultan Agung, Semarang, Indonesia

Department of Postgraduate Biomedical Science, Medical Faculty, Universitas Islam Sultan Agung, Semarang, Indonesia

Pharmacy Study Program, Chemistry Department, Faculty of Mathematics and Natural Sciences, Semarang State

University, Semarang, Indonesia

Student of Medical Faculty, Universitas Islam Sultan Agung, Semarang, Indonesia

Department of Pediatrics, Medical Faculty, Universitas Islam Sultan Agung, Semarang, Indonesia

Department of Surgery, Medical Faculty, Universitas Sumatera Utara, Medan, Indonesia

Keywords: IL-6, IL-8, MSCs type-1, MSCs type-2 PBMCs, S. aureus.

Abstract: The potential of mesenchymal stem cells (MSCs) in controlling bacterial infections is an evolving field to

investigate. In terms of response to inflammatory cytokines, MSCs can polarize into MSCs type-1 and MSCs

type-2 to reach the homeostasis process, including regulating IL-8 and IL-6. MSCs are also exhibit

antimicrobial properties and regulate immune responses. This study was designed to explore the ability of

MSCs to control the inflammation produced by Staphylococcus aureus-contaminated PBMC with TNF-α

stimulation by analyzing IL-6 and IL-8 levels. We used a post-test group design with 2 study groups, consist

of vehicle control (Veh) and a treatment (1:20 comparison of MSCs: PBMCs) in triplicate supplemented with

S. aureus under 10 ng/mL TNF-α recombinant. The medium supernatant was collected after 0, 4, 8, and 12,

the IL-6 and IL-8 were measured using ELISA assay. This study showed a significant increase in IL-6 and

IL-8 at the first hour’s incubation. Interestingly, the IL-6 and IL-8 levels were significantly decreased after

12 and 8 to 12 hours of incubation, respectively. Based on our study, we conclude that MSCs may regulate

the IL-6 and IL-8 production on bacteria-contaminated PBMC with inflammation in early incubation to late

incubation.

1 INTRODUCTION

Recently, mesenchymal stem cell (MSC) therapy has

gained more attention in controlling the massive

inflammation than other stem cells. These cells have

immunomodulatory, anti-inflammatory, and

https://orcid.org/0000- 0002-9822-3119

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https://orcid.org/0000- 0003-1609-9829

https://orcid.org/0000- 0001-7919-2735

antibacterial properties in addition to differentiation

capabilities (1,2). Sepsis is a systemic inflammatory

response to infection, characterized by the excessive

release of pro-inflammatory cytokines and disordered

fibrinolysis produced by immune cells and damaged

tissue (3). Although several approaches aim to reduce

Putra, A., Alif, I., Nazar, M., Prasetio, A., Satria Irawan, R., Amalina, D., Sari, E., Kustiyah, A. and Adeputra Nasution, I.

IL-6 and IL-8 Suppression by Bacteria-adhered Mesenchymal Stem Cells Co-cultured with PBMCs under TNF- Exposure.

DOI: 10.5220/0010491903110317

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 311-317

ISBN: 978-989-758-499-2

311

mortality rates in severe sepsis, such as goal-oriented

treatments, appropriate antibiotic treatment, and

corticosteroid treatment, there is still no effective

treatment for the sepsis (4). The uncontrolled

activation of the immune response in sepsis leads the

macrophages and endothelial and epithelial cells to

produce the release of cytokine cascades such as

tumor necrosis factor-alpha (TNF-α), interleukin

(IL)-1, IL-6, IL-8, IL-12, IL-18, and interferon (IFN)-

γ (5). However, the pro-inflammatory cytokines IL-8,

the chemoattractant of neutrophil and IL-6, the

induction of the acute-phase response have been

studied extensively concerning its possible role in the

pathogenesis of sepsis (6). Therefore, investigating

the role of MSCs to control IL-8 and IL-6 under the

inflammation microenvironment is currently one of

the most promising options.

Mesenchymal stem cells (MSCs) are non-

hematopoietic, multipotent, and plastic adherent

fibroblast-like cells capable of differentiation into

mesenchymal and nonmesenchymal lineages (7).

They are characterized by the expressions of surface

markers CD73, CD90, CD105, and the absence of

CD45, CD34, CD14 or CD11b, CD79a or CD19, and

Human Leucocyte Antigen (HLA) class II (8,9)

MSCs can differentiate into osteocytes,

chondrocytes, and adipocytes under standard in-vitro

differentiating conditions (7). They also can be

isolated from bone marrow, mobilized peripheral

blood, cord blood, umbilical cord (UC), placenta,

adipose tissue, dental pulp, and even fetal livers and

lungs (10). MSCs can regulate immune responses in

various disease models by polarizing into MSCs type-

1 as proinflammation phenotype and MSCs type-2,

anti-inflammatory cells depend on inflammation

exposure. MSCs can also regulate immune responses

in a variety of disease models through polarizing into

type-1 (proinflammation) and type-2 (anti-

inflammation), depending on inflammation exposure

(11). MSCs originating from either bone marrow or

adipose tissue are beneficial in sepsis, indicating that

MSC may upregulate antimicrobial activity in the

presence of infection by releasing Antimicrobial

Peptides (AMPs) (12-14).

MSCs have immunoregulator properties that can

control inflammatory cells by releasing anti-

inflammatory cytokine IL-10 leading to the decrease

of pro-inflammatory cytokines including IL-8 and IL-

6 (1,15,16). Theoretically, IL-8 belongs to the class

of pro-inflammatory chemokines produced by the

active macrophages post an infectious process in

which its level follows a course of time similar to that

of IL-6 (17). In line with IL-8, increased IL-6 also

shows in response to severe infection (15). Thus, the

IL-8 and IL-6 indicated as an early marker of sepsis.

However, several studies reported that MSCs could

suppress severe inflammation, leading to improved

sepsis and decreased sepsis animal models (2,18).

Thus far, the treatment of MSCs in patients with

sepsis has not been used. Furthermore, the role of

MSCs to control the level of IL-6 and IL-8 released

by inflammatory cells following bacteria

contamination and under TNF-α stimulation in

human PBMCs culture to mimic sepsis conditions

remains unclear. Therefore, in the present study, we

explored the ability of Staphylococcus aureus-

adhered MSCs co-cultured with PBMCs at a

comparison of 1:20 (MSCs and PBMCs) under 10

ng/mL TNF-α exposure in regulating the IL-6 and IL-

8 level.

2 MATERIALS AND METHODS

2.1 Research Design and Ethical

Approval

This study conducted at the Stem Cell and Cancer

Research (SCCR) Laboratory from October to

December 2019. This study used two groups: vehicle

control (Veh) and a treatment (T) group. The

institutional review board of the Committee ethic of

Medical Faculty, Sultan Agung Islamic University of

Semarang, Indonesia, approved this study.

2.2 Isolation and Culture of Human

Umbilical Cord-MSCs

MSCs were isolated from umbilical cords and cord

blood obtained from donors with written informed

consent. The isolation and expansion of MSCs were

performed, as described previously (1). Briefly, cords

were cut into smaller pieces and transferred into a T25

culture flask (Corning, Tewksbury, MA, USA)

containing DMEM (Sigma-Aldrich, Louis St, MO)

supplemented with 10% Fetal Bovine Serum (FBS)

(Gibco™ Invitrogen, NY, USA), 1% penicillin (100

U/mL)/streptomycin (100 µg/mL) (Gibco™

Invitrogen, NY, USA). Cultures were incubated at

37°C in a humidified atmosphere containing 5% CO

The medium was renewed every 3 days and passaged

after reaching 80% confluences (14 days). UC-

MSCs-like at passages 4–6 was employed.

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2.3 MSCs Characteristic

The of UC-MSCs-like cells was confirmed by

analyzing MSCs specific markers and the capability

to differentiate into mature cells. The 5th passage of

RUC-MSCs-like was stained with fluorescence-

labelled specific MSCs antibody including PE-CD44

(Clone G44-26, 555479; BD Biosciences), APC-

CD73 (Clone AD2, 560847; BD Biosciences), FITC-

CD90 (Clone 5E10, 561969 BD Biosciences), PerCP-

CD105 (Clone 266, 560819, BD Biosciences), and

PE-Lin negative (CD45/CD34/CD11b/CD19/HLA-

DR) antibodies, then incubated for 30 minutes at

room temperature, washed twice with stain buffer

(554657, BD Biosciences) and examined using a BD

C6 Plus flow cytometer (BD Biosciences) and BD

Accuri C6 Plus Software (BD Biosciences).

2.4 Differentiation of hUC-MSCs

To characterize the isolated cells, we further

performed the osteogenic differentiation assay in the

fourth passage. Osteogenesis was induced by

osteogenic induction medium containing 10 mmol/L β

glycerophosphate, 10−7 mol/L/ 0.1 μM

dexamethasone, 50μmol/L ascorbate-2-phosphate

(Sigma-Aldrich, Louis St, MO) and supported with

10% FBS (Gibco™ Invitrogen, NY, USA) in DMEM

(Sigma-Aldrich, Louis St, MO) at 37°C and 5% CO2.

Calcium deposition was shown by Alizarin Red

staining (Sigma-Aldrich, Louis St, MO) after 21 days

incubation.

2.5 Bacteria Preparation

We used S. aureus as the source of infection in this

study. The bacteria were obtained from the

Laboratory of Microbiology, Faculty of Medicine,

Unissula. S. aureus was propagated in LB medium

(BD Falcon) overnight at 37°C and used to coat mesh

implanted material during the log phase of growth.

2.6 Isolation of Human Peripheral

Blood Mononuclear Cells

(hPBMCs)

Human Peripheral Blood Mononuclear Cells

(PBMCs) were isolated by Ficoll-Paque (Axis-Shield)

density gradient centrifugation from health

volunteers’ venous blood after informed consent.

PBMCs were cultured in 2 ml of advanced Roswell

Park Memorial Institute medium (RPMI) 1640 culture

medium (Invitrogen, Grand Island, NY, USA)

supplemented with 10% FBS, 2 mM glutamine, 100

U/ml penicillin, and streptomycin, and allowed to

adhere at 37°C and 5% carbon dioxide incubator for

12 h.

2.7 Co-culturing MSCs-adhered

Bacteria with PBMCs under TNF-α

Stimulation

For the T group, S. aureus and hUC-MSCs (4 × 10

cells) were co-cultured in coverslip and put in a T25

culture flask in DMEM (Sigma-Aldrich, Louis St,

MO) at 37°C and 5% CO

for 12 h. The co-cultured

cells were then transferred to the culture flask, which

contains 8x105 PBMCs (1:10 comparison), combined

with DMEM-LG and RPMI 1640 culture medium

(Invitrogen, Grand Island, NY, USA), 10% FBS, 2

mM glutamine, and 100 U/ml penicillin and

streptomycin. The co-cultured cells were also

supplemented with TNF-α recombinant (10 ng/mL)

(BioLegend, San Diego, CA). The medium

supernatant was collected after 1, 4, 8, and 12 h

incubation for ELISA analysis. On the other hand, the

co-culture between PBMCs and S. aureus was

performed for the Veh group.

2.8 Quantification of Cytokines

The levels of both IL-6 and IL-8 were quantified in

the cell culture supernatants by enzyme-linked

immunosorbent assay (ELISA) from the various

treatment groups. IL-6 and IL-8 were calculated

according to a standard curve constructed for each

assay, and each assay performed in triplicate. The

colorimetric absorbance was recorded at a

wavelength of 450 nm. The measurement was done

entirely according to the manufacturer’s protocol

(QAYEE, Wuhan, China).

2.9 Data Analysis

Data are presented as the means ± standard deviation.

All calculations were carried out using IBM SPSS

22.0 (IBM Corp., Armonk, NY, USA) was used for

statistical analysis. The statistical significance of the

differences between the groups was assessed using

the paired t-test. p values: *, p < 0.05.

IL-6 and IL-8 Suppression by Bacteria-adhered Mesenchymal Stem Cells Co-cultured with PBMCs under TNF- Exposure

313

3 RESULTS

3.1 Isolation and Differentiation of

hUC-MSCs

Isolation of hUC-MSCs was performed based on the

capacity to plastic attachment under standard culture

conditions. Isolated cells were cultured for 2–3 weeks

in monolayer and used for differentiation analysis

after 4 to 5 passages. The hUC-MSCs were initially

characterized by their elongated fibroblastic cellular

phenotype (figure 1(a)); moreover, osteogenesis was

confirmed at day 21 of culture by immunodetection

with Alizarin Red staining (figure 1(b)).

Figure 1. (a) UC-MSCs characterized by their peculiar fibroblast-like (spindle shape) morphology (100x magnification). (b)

and osteogenic differentiation with Alizarin Red staining appears red color (400x magnification).

3.2 Characteristics of hUC-MSCs

According to the International Society of Cellular

Therapy (ISCT), MSCs have specific marker profiles,

such as CD73, CD105, CD90 and negative of Lin-

(CD45/CD34/CD11b/CD19/HLA-DR) and the

capability to differentiate into several mesodermal

germ layers, including osteocytes. The result showed

that UC-MSCs expressed a high level of CD90

(99.4%), CD105 (96.9%), CD73 (99.8%) and lacked

the expression of Lin- (1.3%). (Figure 2).

Figure 2. MSCs phenotypes was positive for CD73, CD90 and CD105 and negative for Lin.

3.3 The Appearance of MSC-Adhered

Bacteria and PBMCs

We firstly co-cultured the MSCs with S. aureus

(figure 3(a)) in 12 hours incubation to determine the

IL-6 and IL-8 concentration at first-hour incubation,

with and without PBMCs. Next, we co-cultured the

MSCs-adhered bacteria with PBMCs and observed its

appearance at 12 hours incubation (figure 3(b)).

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314

Figure 3. (a) MSCs adhered bacteria (black arrow) and PBMCs (blue arrow) appearance at 0 and (b) 12 hours incubation.

(Magnification: Veh = 40x; T = 10x).

3.4 IL-6 and IL-8 Levels

We subsequently quantify the level of IL-6 and IL-8

(Figure 3) by ELISA in triplicate. The IL-6 and IL-8

level analysis showed a significant increase in the T

group at the first-hour incubation (p < 0.05).

Interestingly, the level of IL-6 gradually decreased,

depending on time, which became significant after

12-hour incubation (p < 0.05). In line with this, the

level of IL-8 was also gradually decreased in time,

which reached significant after 8- and 12-hours

incubation (p < 0.05).

Figure 4. ELISA assays for the T group in 0,4,8 and 12 hours post-co-culture showed the gradual decrease of IL-6 and IL-8

depend on time. (a) The level of IL-6 was significantly after 12 hours of co-culture incubation. (b) In line with this, the level

of IL-8 was also significantly decreased after 8- and 12-hours incubation. *, p < 0.05.

4 DISCUSSION

Inﬂammation serves as a systemic or localized

protective response caused by injury, infection, or

tissue destruction and attends to eliminate pathogens

and preserve host integrity, particularly in sepsis

model infection. MSCs may exhibit antimicrobial

properties and regulate both the innate and the

adaptive immune responses, resulting in both the pro-

inflammatory and anti-inflammatory effects when

those MSCs interact with an immune system or

exposed by various cytokines (19). Several studies

have shown that MSCs respond to the inflammatory

milieu by polarizing either into MSCs type-1 with the

pro-inflammatory phenotype or MSCs type-2 with

anti-inflammatory properties depending on Toll-like

Receptors (TLRs) type activation (1,20). Another

study also reported that MSCs under bacterial

exposure might increase phagocytosis of neutrophil

and monocyte cells, suggesting that MSCs may

increase pro-inflammatory molecules (21). Although

MSCs have been widely demonstrated

experimentally on their immune properties in

suppressing inflammation, the study of MSCs in

100

200

300

0 hour 4 hours 8 hours 12 hours

IL-8 level (pg/mL)

Veh

0 hour 4 hours 8 hours 12 hours

IL-6 level (pg/mL)

Veh

IL-6 and IL-8 Suppression by Bacteria-adhered Mesenchymal Stem Cells Co-cultured with PBMCs under TNF- Exposure

315

treating severe infections by exploring the IL8 and

IL-6 is comparatively less performed (18). Therefore,

in the present study, we demonstrate the utility of

activated MSC in treating the S. aureus-contaminated

PBMC under TNF stimulation by examining the IL-8

and IL-6 levels as the one marker of severe infections.

This study found a significant increase of IL-8

level in treatment groups starting at first to 4 hours

incubation on S. aureus-contaminated PBMC with

TNF stimulation. Our finding suggested that MSCs

exposed by bacteria could enhance neutrophils to

recognise and kill bacteria S. aureus due to expressed

IL-8 following MSCs treatment is the robust

chemoattractant for neutrophils. This study

confirmed and extended our understanding of the

direct antibacterial activity of MSC as previously

reported when neutrophils incubated with the

activated MSCs can induce significantly greater

Neutrophil Extracellular Trap (NET) area formation

(22). The increased IL-8 in the first hours after

exposure indicated MSCs could stimulate

neutrophils, the innate immune system, to

phagocytize bacteria preventing the bacteria from

spreading into tissues (23). Interestingly, we also

found that the IL-8 level gradually decreased in time

and reached significant after 8- and 12-hours

incubation. We suggest the polarization of MSCs

caused the gradual decrease of IL-8 into MSCs type-

2 following exposed bacteria as described in our

previous study (1).

In line with the increase of IL-8, there was also a

significant increase of IL-6 in treatment groups

starting at first to 4 hours of incubation on S. aureus-

contaminated PBMC with TNFα stimulation.

Likewise, with 8 to 12 hours of incubation, we also

found the decreased IL-6 decreased and reached

significant after 12 hours incubation, similar to an

episode of IL-8. We supposed that there were the

indirect mechanisms of MSC in regulating IL-8 and

IL-6, in which MSCs initially increase those

cytokines, subsequently became decreased gradually

in time. This is due to the activation of MSCs with

TLRs ligand, known as MSCs polarization. In terms

of bacteria stimulation and TNF exposure, the TLR-4

of MSCs type-1 was activated initially to induce the

activation of MyD88-dependent pathway, then to NF-

kb pathway resulting in the release of pro-

inflammatory cytokines including IL-8 and IL-6.

These released cytokines trigger the inflammation

states crucial for innate cells to eliminate the bacteria

and antigen (24). However, along with time, the

increased IL-8 and IL-6 induce the upregulation of

COX2, which increases PGE2 secretion. Binding

PGE2 to EP2 and EP4 receptor leads the shift of

MSCs from MyD88-dependent pro-inflammatory to

TRIF-TRAM mediated anti-inflammatory signal,

known as MSCs type-2 by P110δ isoform of PI3K

kinase. This MSCs type-2 can secrete anti-

inflammatory cytokines such as IL-10 (1). This

condition leads to decreased IL-8 and IL-6 levels.

Our findings indicate that the regulation of IL-6

and IL-8 by MSCs type-1 is needed for the

neutrophils to migrate and attach to the inflammatory

niche. However, the excessive release of IL-6 and IL-

8 is also needed to suppress MSCs type-2 to reach the

homeostasis process. Unfortunately, we did not

measure the concentration of NET and a direct

bacterial killing assay of MSCs and the anti-

inflammatory cytokines. Therefore, the exact

mechanism of MSCs polarization regarding pro-

inflammatory cytokines and anti-inflammatory

cytokine remains unclear.

5 CONCLUSION

Based on our study, we conclude that MSCs may

regulate the IL-6 and IL-8 production on bacteria-

contaminated PBMC with inflammation in early

incubation to late incubation.

ACKNOWLEDGMENTS

The authors gratefully acknowledge that the present

research is fully supported and carried out by Stem

Cell and Cancer Research (SCCR) Laboratory,

Medical Faculty, Sultan Agung Islamic University

(Unissula), Semarang for all Facility to finish this

research.

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