Plasmodium falciparum - Universiti Sains Malaysia · mRNA levels. Furthermore, statistical analysis showed a significant difference of IL-23 expression between untreated and rBCG-treated

Halimah et al. 2016

Health and the Environment Journal, 2016, Vol 7, No 2

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32

Interleukin-23 (IL-23) Expression in Murine Macrophage Cell Line

J774A.1 Stimulated with Lipopolysaccharide (LPS) and Recombinant BCG

(rBCG) Clone Expressing Merozoite Surface Protein 1C (MSP-1C) of

Plasmodium falciparum

Halimah M, Rapeah S and Nor Fazila CM*

School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan,

Malaysia

*Corresponding author: [email protected]

_________________________________________________________________________________________

ABSTRACT: Interleukin 23 (IL-23) is a heterodimeric cytokine composing of p40 and p19

subunits. IL-23 has an ability to stimulate the proliferation of memory T cells to become the

novel subset of CD4+ T cells known as Th17. IL-12 has been reported to have an anti-malaria

activity, but the role of this novel cytokine in malaria remains undefined. Since IL-23 and IL-

12 share the same IL-12p40 subunit and IL-12Rβ1 receptor, thus, IL-23 might also play an

important role in host defense against malaria infection. This study was conducted to

determine the expression level of IL-23 in macrophage cells stimulated with recombinant

Bacillus Calmette–Guérin (rBCG) vaccines expressing merozoite surface protein 1C (MSP-

1C) of Plasmodium falciparum. Cells of macrophage cell line J774A.1 were incubated in

DMEM and stimulated with lipopolysaccharide (LPS) or rBCG over a various time course.

Cell culture supernatants were collected for ELISA analysis while the cell pellets were

collected for RNA isolation. cDNA was then synthesized and added to IL-23p19 primer and

5x taq polymerase master mix. PCR products were run in a 3% agarose gel with ethidium

bromide. Results showed that macrophage cells were able to express IL-23 with LPS

stimulation. The highest expression of IL-23 was at 24 hours stimulation at both protein and

mRNA levels. Furthermore, statistical analysis showed a significant difference of IL-23

expression between untreated and rBCG-treated cells. As a conclusion, rBCG stimulation

was able to induce IL-23 expression at both protein and mRNA levels compared to

mailto:[email protected]

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unstimulated cells. This finding reflects the involvement of IL-23 in innate immunity towards

malaria infection.

Keywords: Interleukin-23 (IL-23), Malaria, rBCG, LPS

Introduction

Malaria is an infectious disease caused by an obligate intracellular parasite of the genus

Plasmodium and can be transmitted via the bite of an infected female Anopheles mosquito

(Malaguarnera and Musumeci, 2002). Human malaria parasite consists of four main species

which are P. falciparum, P. vivax, P. malariae and P. ovale. Among these four species, P.

falciparum has been reported to be the most virulent species of human malaria parasite as it

accounts for approximately 90% of death globally (McGilvray et al., 2000; Angulo and

Fresno, 2002; Yazdani et al., 2006). In sub-Saharan African, 80% of malaria-related

mortality cases were reported due to P. falciparum (Perkins et al., 2011). In 2010, 5,819

malaria cases were reported in Malaysia and 25% of these were attributable to the P.

falciparum infection (World Health Organization, 2011). More than 80% of the cases were

reported in Malaysia Borneo of which 58% of the cases occurred in the northern state of

Sabah (World Health Organization, WHO, 2011). Over the last decade, 20-40 cases of

malaria death were reported and the number of death cases had declined in 2010. After 1995,

malaria cases in Malaysia had reached a steady decline. This is possibly due to insecticide-

treated bed net use and community health volunteers, as well as increased laboratory

diagnostic capability (World Health Organization, 2011). However, in 2010, 12% of reported

cases were imported cases, primarily from Indonesian and Filipino workers. Uncontrolled

migration of foreign workers into Malaysia has raised a challenge in controlling malaria

transmission and eliminating malaria. Besides, people who live in the vicinity of the forest

areas have limited access to the health care setting, thus increase the challenge in controlling

malaria infection. The vector ecology and malaria transmission pattern also result in a unique

challenge for vector control management (Phillips, 2012).

In humans, P. falciparum life cycle involves two main stages, which are the extra-

erythrocytic and intra-erythrocytic stages. Extra-erythrocytic stage involves the invasion of

sporozoites into the hepatocytes. Within the hepatocytes, the sporozoites replicate asexually

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and produce thousands of merozoites. Production of merozoites will initiate the intra-

erythrocytic cycles of malaria infection. Merozoites released from the liver invade the red

blood cells after entering the blood stream. Repeated invasion and replication of merozoites

within red blood cell are responsible for the clinical symptoms of malarial infection (Angulo

and Fresno, 2002). Thus, the development of asexual blood stage vaccine may arrest the

growth of the parasite and lower the complication of the disease (Wipasa et al., 2002). C-

terminus of merozoite surface protein (MSP-1C) has been found as one of the suitable

candidates for malaria vaccine development (Yazdani et al., 2006; Girard et al., 2007; Nurul

and Norazmi, 2010). It is a 19-kDa polypeptide which is a major complex protein that can be

found on the surface of merozoites (Stevenson and Riley, 2004). MSP-1C is the only part of

the MSP-1 after undergoes proteolytic activity inside red blood cells during merozoites

invasion while the remaining protein fragments are shed as soluble complex (Mazumdar et

al., 2010; Waisberg et al., 2012). In vivo study conducted on mice infected with P.

falciparum MSP-1 showed protective immune responses directly against the C-terminal 19-

kDa domain (Mazumdar et al., 2010). Development of recombinant BCG (rBCG) expressing

MSP-1C surface protein of P. falciparum is believed to be able to induce phagocytosis and

pro-inflammatory cytokine production (Rapeah et al., 2010).

Immunity towards malaria infection involves cellular and innate immunity, and also humoral

immunity. Cellular and innate immunity plays a role in controlling the parasite growth while

humoral immunity is responsible for dissolution of infection (Yazdani et al., 2006). Parasitic

invasion stimulates the release of cytokines from human peripheral mononuclear cells

(PBMC), which play an important roles in activation of monocytes, neutrophils, T cells and

natural killer (NK) cells to react against the liver and blood stage parasites (Malaguarnera and

Musumeci, 2002). Phagocytosis of macrophages plays an important role in innate immunity

towards malaria infection due to their ability to ingest infected red blood cells in the absence

of opsonizing malaria-specific antibodies (Serghides et al., 2003; Yazdani et al., 2006).

Besides that, activated macrophages have the ability to synthesize pro-inflammatory

cytokines such as interleukin-12 (IL-12), IL-23, tumor necrosis factor-alpha (TNF-α) and IL-

1β (McKenzie et al., 2006; Tan et al., 2009).

IL-23 is a key cytokine in connecting the innate immune response with the adaptive immune

response (Langrish et al., 2004). IL-23 is produced as an early response towards any

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pathogenic or antigenic materials, thus it is important in the initiation stage of local immune

response (McKenzie et al., 2006). Besides that, IL-23 also involves in the regulation of other

pro-inflammatory cytokines such as interferon gamma (IFN-γ), which is important in the

immunity towards intracellular pathogens such as malaria parasites (Langrish et al., 2004).

IL-23 possesses a unique immunological function, as this novel cytokine is able to

differentiate and expand memory T cells and stimulate the production of IL-17 (Breadling

and Slifka, 2006). IL-12 has been found to have an anti-malaria activity (Yazdani et al.,

2006). A study showed that the low IL-12 concentration is related to the increase in

parasitemia and severity of malaria pathogenesis (Yazdani et al., 2006). IL-12 and IL-23

share the same p40 subunits and IL-12Rβ1 receptor, thus, it leads to the notion that IL-23 also

may have a function in immunity towards malaria infection. In this study, the expression of

IL-23 at protein and mRNA levels in macrophage cells was determined after stimulation with

rBCG clone expressing the MSP-1C of P. falciparum.

Materials and Methods

Murine macrophage cell line J774A.1 culture

The J774A.1 mouse macrophage cell line (ATCC, USA) was cultured in Dulbecco’s

modified eagle’s medium, DMEM (Sigma, USA) containing 4500 mg glucose, 110 mg

sodium pyruvate and L-glutamine. DMEM was also supplemented with penicillin and

streptomycin (Gibco®, Life Technologies, USA) and 10% fetal bovine serum (Gibco®, Life

Technologies, USA). The cell culture was grown in a humidified incubator at 37oC with 5%

CO2 (Rapeah et al., 2010).

rBCG culture

The rBCG vaccine expressing the MSP-1C of P. falciparum was kindly provided by Dr.

Nurul Asma Abdullah and Prof. Dr. Norazmi Mohd Nor (Universiti Sains Malaysia). rBCG

(Japan) was grown in Middlebrook 7H9 broth (Sigma, USA) supplemented with 10% oleic

acid/albumin/dextrose (OADC) (Middlebrook, USA) and 15 μg/mL kanamycin. The culture

was incubated at 37oC for 3 weeks (Rapeah et al., 2010).

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LPS optimization

For macrophage activation, 24-well micro titer plates were seeded with macrophage cells at a

concentration of 1 x 106

cells/well in 1 mL DMEM. Two different concentrations of LPS

(Sigma E. coli 0111:B4, USA) at 100 ng/mL and 500 ng/mL were added to each well and

incubated at 37oC with 5% CO2. The cells were incubated at a different time course at 0, 4, 8,

16 and 48 hours. The supernatant and cell pellet were collected for ELISA and PCR analyses,

respectively.

Infection of macrophages with rBCG

Macrophages (1 x 106 cells/well) were infected with 100 ng/mL of LPS in complete DMEM.

Infected cells were incubated at 37oC with 5% CO2. The supernatant and cell pellet were

collected for ELISA and PCR analysis, respectively. For rBCG stimulation, macrophages (1

x 106 cells/mL) were incubated with 2 x 10

7 cfu/mL of rBCG in complete DMEM medium at

37°C with 5% CO2 (MOI 1:20). The cells were incubated for 24 and 48 hours.

Cytokine assay by Enzyme Link immunosorbent assay (ELISA)

The IL-23 production was determined using ELISA MAX™ Deluxe set Mouse IL23

(BioLegend, San Diego, California, USA). The procedure was conducted according to

manufacturer’s instructions.

RNA extraction and cDNA synthesis

RNA was extracted from cell pellet by using RNAeasy® Plus Mini Kit (Qiagen, Texas,

USA). The procedure was conducted according to manufacturer’s instruction. The cDNA was

then synthesized from RNA template by using Omniscript® Reverse Transcription kit

(Qiagen, USA). The master mix for Reverse-Transcription reaction components consists of 2

μL of 10x buffer RT, 2 μL of dNTP mix (5 mM each dNTP), 2 μL of 10μM Oligo-dT primer,

1 μL of RNase inhibitor, 1 μL of Omniscript Reverse Transcriptase, RNase-free water and

template RNA. The mixture was then mixed thoroughly and PCR was conducted for cDNA

synthesis at 37°C for 60 minutes.

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Polymerase chain reaction (PCR) of IL-23p19

The following primers from IDT, USA were used to detect the presence of IL-23 p19: IL-

23p19 sense, 5’-TGCTGGATTGCAGCGCAGTAA-3’, anti-sense 5’-AGTCCTTGTGGG

TCACAACC-3’. The amplification was performed at 94oC for 5 minutes for denaturation,

followed by 40 cycles of amplification at 94oC, 55-58

oC, and 72

oC for 30 seconds for each

temperature and a final incubation cycle at 72oC for 5 minutes. The amplified PCR product

was analyzed using 3% agarose gel with ethidium bromide. β-actin was amplified under the

same condition with its specific primer.

Statistical analysis

All experiments were carried out in triplicates and were repeated three times. Data were

presented as the arithmetic mean ± standard deviation (SD). Where appropriate, the results

were analyzed using unpaired T-test and one-way ANOVA followed by Dunnett’s C post-hoc

analysis using SPSS software version 20. All tests were two tailed and the significance level

was set at p <0.05.

Results

Optimization was carried out based on two main parameters, which were the concentration of

lipopolysaccharide (LPS) and the duration of exposure. According to Maitra et al. (2012),

different dosage of endotoxin such as LPS will stimulate different signaling pathway and

molecular mechanism which result in induction of either pro-inflammatory cytokines or anti-

inflammatory cytokines. This experiment was conducted to optimize the suitable dose of LPS

that needs to be used to induce the IL-23 secretion in macrophage cells. To determine the

time-and dose-dependent effect of LPS on macrophages, J774A.1 cells were incubated with

100 ng/mL or 500 ng/mL LPS at 4, 8, 16 and 24 hours.

For 100 ng/mL LPS stimulation, the expression of IL-23 rapidly increased from 4 hours to 8

hours stimulation and the highest expression level of IL-23 was shown at 24 hours

stimulation (Figure 1). By comparison, the expression of IL-23 at 24 hours was significantly

Halimah et al. 2016


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38

higher (147.11 ± 31.59 pg/mL) compared to unstimulated cell (8.336±5.44 pg/mL). With

higher dose of LPS (500ng/mL), the expression of IL-23 rapidly increased after 16 hours

stimulation. However the level of IL-23 expression decreased after 24 hours post stimulation.

Figure 1: IL-23 expression by macrophage cell line J774A.1 at protein level at different time

courses. IL-23 expression by macrophage cell line J774A.1 stimulated at different doses of

LPS at 4, 8, 16 and 24 hours. J774A.1 cells were stimulated with LPS at concentrations of

100 ng/mL and 500 ng/mL at different time course. IL-23 expression was measured by

ELISA. Data were analyzed by using one way ANOVA and represented as mean (pg/mL) ±

SD. *p<0.05 is considered as significantly different.

To determine the expression of IL-23 at mRNA level, reverse transcriptase was performed on

RNA isolated from the macrophages activated by LPS at different doses and time courses.

The mRNA isolated from the samples was reverse transcribed into cDNA and amplified with

IL-23p19 specific primer. Equivalent amount of β-actin specific cDNA had also been

0

20

40

60

80

100

120

140

160

180

200

Unstim 4 8 16 24

IL-2

3 C

on

cen

tra

tio

n (

pg/

mL)

Duration of induction (Hours)

100 ng/mL 500 ng/mL

* *

*

*

*

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39

amplified under the same condition as PCR for IL-23p19. β-actin was used as an internal

control. Incubation with 100 ng/mL of LPS for 4 hours showed no expression of IL-23p19

(Figure 2). Further incubation of J774A.1 with LPS for 16 hours resulted in an increased

expression of IL-23p19 mRNA compared to 8 hours incubation. The IL-23p19 mRNA was

highly expressed after 24 hours stimulated with LPS compared to unstimulated cells.

The expression of IL-23p19 was also tested by using a higher concentration of LPS.

Macrophage cells were treated with 500 ng/mL of LPS at different time courses. However,

no expression of IL-23p19 was detected in each time courses (Data not shown). This

indicated that the inhibition of IL-23p19 expression at mRNA level was induced by high

concentration of LPS. This result was validated by the amplification of cDNA specific primer

for β-actin which resulted in the same equivalent amount of β-actin product between the

samples. The expression of IL-23 at both mRNA and protein levels increased as the duration

of induction increased. This indicated that the expression of IL-23 at mRNA level is

correlated with the expression of IL-23 at the protein level. Therefore, a lower concentration

of LPS concentration (100 ng/mL) with 24 hours incubation time was chosen for the next

activation phase. The cytokine expression at these conditions was stable with no inhibition

observed.

The expression of IL-23 was further investigated over a period of 24 and 48 hours

stimulation. Figure 3 shows the IL-23 production in response to LPS or rBCG stimulation.

LPS was used as a positive control. The IL-23 expression by macrophages stimulated with

100 ng/mL of LPS showed an increase pattern from 24 hours stimulation to 48 hours

stimulation (Figure 3). However, in rBCG-stimulated cells, the level of IL-23 production

decreased from 24 hours to 48 hours posts stimulation. In comparison, the expression of IL-

23 stimulated with rBCG at 24 hours was significantly higher compared to untreated cells at

48 hours stimulation (Figure 3).

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40

Unstim 4hr 8hr 16hr 24hr

IL-23p19

β-actin

Figure 2: IL-23p19 mRNA expression was measured in J774A.1 cells stimulated with 100

ng/mL of LPS at 4, 8, 16 and 24 hours. β-actin was used as an internal control. β-actin

specific cDNA was amplified using the same PCR condition.

Figure 3: IL-23 expression by macrophage cell line J774A.1 stimulated by LPS (100 ng/mL)

and rBCG at different time courses. IL-23 expression was measured by ELISA. Data were

analyzed by using one way ANOVA and represented as mean (pg/mL) ± SD. *p<0.05 is

considered as significantly different.

0

20

40

60

80

100

120

140

160

180

Unstim 24 48

IL-2

3 C

on

cen

tra

tio

n (

pg

/mL

)

Duration of induction (Hours) LPS rBCG

*

* *

*

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The expression level of IL-23 stimulated with rBCG was significantly higher compared to

LPS stimulation at 24 hours post stimulation. Even though IL-23 expression upon rBCG

stimulation at 48 hours was lower compared to 24 hours stimulation, the expression level of

IL-23 was still higher compared to the LPS stimulation. This demonstrates that the rBCG

clone expressing MSP-1C of P. falciparum enhanced the IL-23 expression in murine

macrophage cell line J774A.1.

The expression of IL-23p19 at mRNA level upon stimulation with LPS and rBCG were

determined by reverse transcription PCR method. Upon stimulation with LPS for 24 and 48

hours, IL-23p19 PCR product showed an increased intensity of the band from 24 to 48 hour

post stimulation (Figure 4a). This shows that the expression of IL-23p19 at mRNA level

increased as the duration of exposure increased. On the other hand, IL-23p19 PCR product

from J774A.1 cells stimulated with rBCG showed the highest expression at 24 hours

stimulation and the cytokine expression level decreased after 48 hours of stimulation (Figure

4b). The IL-23p19 expression at mRNA level stimulated with LPS or rBCG was correlated

with the IL-23 expression at protein level

These results demonstrated that the rBCG clone expressing MSP-1C of P. falciparum has an

ability to induce IL-23 expression in murine macrophage cell line J774A.1. This reflects the

involvement of IL-23 novel cytokine in providing early immune response towards malaria

infection.

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Unstim 24hr 48hr Unstim 24hr 48 hr

(a)

(b)

Figure 4: (a) IL-23p19 mRNA expression in J774A.1 cells stimulated with LPS (100 ng/mL)

at different time course. (b) IL-23p19 mRNA expression in J774A.1 cells stimulated with

rBCG expressing MSP-1C of P. falciparum at different time courses. β-actin was used as an

internal control in LPS or rBCG stimulation. β-actin specific cDNA was amplified using the

same PCR condition.

DISCUSSION

The expression level of interleukine-23 (IL-23) in murine macrophage cell line J774A.1

stimulated with LPS and rBCG clone expressing the MSP-1C of P. falciparum was

determined in this study. Macrophages play a significant role in immunity and immune

response during blood stage of malaria parasite infection (Bogdan et al., 1992; Serghides et

al., 2003). They exhibit a defensive role by performing phagocytosis of infected red blood

cells (Serghides et al., 2003; Yazdani et al., 2006). Activated macrophages are able to present

antigens and secrete pro-inflammatory cytokines such as TNF-α, IL-6 and IL-12 and other

immune mediators as a response to the pathogen invasion (McKenzie et al., 2006; Tan et al.,

2009; Nomura et al., 2000; Langrish et al., 2004). Macrophages act as a center regulation of

immune system that links the innate and adaptive immune responses (McKenzie et al., 2006).

Toll-like receptor (TLR) family is an important sensory receptor on macrophage surface

functions in binding of unique microbial product such as LPS and delivers the signal for

generation of appropriate response (Huang et al., 2012). Previous study conducted by

Nomura et al. (2000) showed that stimulation of macrophages with LPS displayed a

LPS rBCG

β-actin

IL-23p19

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significant production of IL-12. Based on this finding, IL-23 cytokines are predicted to be

also secreted by macrophages since IL-12 and IL-23 share the same p40 subunit. From the

results, it showed that the macrophage cell line J774A.1 has the ability to produce IL-23 upon

stimulation with LPS or rBCG.

The expression of IL-23 in J774A.1 cells was studied in both protein and mRNA levels. The

results showed that the expression of IL-23 at protein level was correlated with the mRNA

level. This was shown by 100 ng/mL LPS stimulation on J774A.1 cell. IL-23 production at

protein level showed an increase pattern in concentration from 8 hours to 24 hours of

stimulation, while the intensity of the PCR band product also showed an increased from 8

hours to 24 hours of LPS stimulation.

A study conducted by Huang et al. (2012) showed that the cytokine production in both

protein and mRNA levels was highly dependent on the concentration of LPS, duration of

stimulation and cell specificity. Thus, for optimization, macrophage cell line J774A.1 was

incubated with two different concentrations of LPS at various time courses. The

concentrations of LPS were chosen according to the previous study conducted by Huang et

al., (2012). This study suggested that high concentration of LPS stimulation over

physiological concentration of macrophage will disturb the inflammatory response regulation

(Huang et al., 2012). Another study showed that IL-23 could be produced by dendritic cells

and macrophages within a few hours after exposure to microbial product such as LPS and

peptidoglycan (Oppmann et al., 2000; Smits et al., 2004). Therefore, a different time course

of LPS incubation was done to determine whether IL-23 expression was dependent on the

temporal effect. It was done to determine which duration of exposure would exhibit the

highest and stable expression of IL-23 at both protein and mRNA levels. Based on 100

ng/mL LPS stimulation result (Figure 1), the concentration of cytokine production increased

as the duration of exposure was extended. The highest concentration of IL-23 cytokines

expression was collected after 24 hours post-stimulation while the minimum IL-23 secretion

was collected after 4 hours of stimulation. This result showed that IL-23 production upon

LPS stimulation on macrophages was not only dependent on LPS concentration but also

affected by duration of LPS exposure.

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At 500 ng/mL LPS stimulation, the level of IL-23 expression decreased from 16 hours to 24

hours stimulation (Figure 1). This indicates that the inhibition of IL-23 expression occurred

after 16 hours of stimulation. Inhibition of cytokine production can be occurred due to the IL-

10 secretion (de Waal Malefyt et al., 1991) and decreased expression of Toll-like receptor 4

(TLR4) after in contact with LPS stimulation (Nomura et al., 2000). A study conducted by

Ong’echa et al., (2008) on a children population in sub-saharan African with P. falciparum

parasitemia, showed that high expression of IL-10 over IL-23 exhibited an increased anemia

severity among children, suggested the suppressive effect of IL-10 on protective

characteristic of IL-23 expression. IL-10 is an anti-inflammatory cytokine that can be

secreted by macrophages upon LPS stimulation, which functions in down regulating pro-

inflammatory cytokine production (de Waal Malefyt et al., 1991). This anti-inflammatory

cytokine was produced after LPS stimulation but at delayed kinetics compared to pro-

inflammatory cytokine secretions (de Waal Malefyt et al., 1991; Randow et al., 1995). High

concentrations of LPS stimulation may produce high level of IL-10 and suppress IL-23

secretion (de Waal Malefyt et al., 1991; Maitra et al., 2012).

The IL-23p19 mRNA expression in J774A.1 was detected at 100 ng/mL and the intensity of

the band was correlated with the IL-23 expression at protein level. However, upon

stimulation with a higher concentration of LPS (500 ng/mL), IL-23p19 PCR product was not

detected at any time course. It is possible that high concentration of LPS led to the induction

of both pro-inflammatory and anti-inflammatory cytokines (Maitra et al., 2012). The

secretion of anti-inflammatory cytokine such as IL-10 did give some suppression effect on

IL-23p19 expression (de Waal Malefyt et al., 1991). Inhibition of IL-23 expression in

macrophage cells by IL-10 also occurred at the transcriptional level (de Waal Malefyt et al.,

1991). Besides that, LPS stimulation was also found to have the ability in reducing TLR4

mRNA expression in the macrophage cells (Nomura et al., 2000). TLR4 is an essential

receptor for LPS in regulation of signaling pathway upon in contact with endotoxin. The

suppression of TLR4 mRNA expression was associated with the reduced activation of LPS-

signaling cascade after LPS stimulation and also corresponded to the decreased in pro-

inflammatory cytokines production (Nomura et al., 2000).

During macrophages activation, low concentration of LPS can suppress the anti-

inflammatory phosphoinositide 3-kinase (P13K) pathway resulting in the inhibition of anti-

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inflammatory cytokine secretion (Maitra et al., 2012). Since anti-inflammatory cytokine

secretion is inhibited, the expression of pro-inflammatory cytokine such as IL-23 will be

prolonged as had been observed in the macrophage J774A.1 cell stimulated with 100 ng/mL.

Even though IL-10 can down-regulate the pro-inflammatory cytokine production, this

cytokine is needed in controlling the pathological effect of macrophages during malarial

pathogenesis (Rhee et al., 2001; Dodoo et al., 2002; Yazdani et al., 2006). Maintaining the

balance between pro-inflammatory and anti-inflammatory cytokines is a crucial step in

controlling parasite infection and reducing the immunopathology effects (Yazdani et al.,

2006).

Based on Figure 3, macrophage has an ability to secrete IL-23 cytokine upon stimulation with

rBCG clone expressing MSP-1C of P. falciparum. This demonstrates that IL-23 involves in

the immunity towards malarial infection. BCG is able to be ingested by macrophages due to

the presence of lipoarabinomannan on BCG, a potential ligand for macrophage receptor

(Prinzis et al., 1993). Ingestion of BCG by macrophage is also attributed to the various

receptors found on the surface of macrophage such as Fc receptors (FcR) and phagocytic

pattern recognition receptors (PRR) (Rapeah et al., 2010). A study showed that the cloning of

MSP-1C into the BCG vector had increased the phagocytosis activity and secretion of pro-

inflammatory cytokines in stimulated cells (Rapeah et al., 2010). The enhancement of

phagocytosis of macrophages might due to the presence of MSP-1C on the surface of BCG

which introduces more ligand recognition site on macrophage surface (Rapeah et al., 2010).

MSP-1C has been identified as one of the most suitable antigen candidates for developing

malaria vaccines (Yazdani et al., 2006). The rBCG clone expressing MSP-1C has the ability

in inducing both humoral and cellular immune response (Nurul and Norazmi, 2010). The

rBCG expressing MSP-1C also has the ability to regulate immune-modulatory activity of

macrophages (Rapeah et al., 2010).

IL-12 has been found to have a protective role towards intracellular malaria infection

(Malaguarnera and Musumeci, 2002; Yazdani et al., 2006). The IL-12 secretion was

correlated with the reduced risk of parasitemia in malaria infection (Dodoo et al., 2002). It

has also been proven that P. cynomolgi infection in the rhesus monkey can be protected in the

presence of IL-12 (Hoffman et al., 1997). IL-12 is a 70 kDa heterodimer cytokine, which

consists of p40 and p35 subunits. IL-12 can be secreted by dendritic cells, monocytes and

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macrophages (Hoffman et al., 1997; Langrish et al., 2004; Yazdani et al., 2006; Tan et al.,

2009). This cytokine has an important involvement in the regulation of T-helper 1 cell (Th1)

responses (Langrish et al., 2004). Additional heterodimer cytokines have been discovered to

have a similarity close to IL-12 during searching for IL-6 family member (Oppmann et al.,

2000; Langrish et al., 2004). This new cytokine has been named as IL-23, comprises of p19

subunit that is covalently linked to IL-12p40 subunit (Oppmann et al., 2000). In addition,

both IL-12 and IL-23 can be suppressed by IL-10 (Bogdan et al., 1992) and IL-12p40

homodimers (Ong’echa et al., 2008).

In this study, IL-23 was able to be detected at both protein and mRNA levels upon

stimulation with rBCG clone expressing MSP-1C of P. falciparum. Stimulation of rBCG on

macrophage acts as a clone resemble to the intra-erythrocytic malaria infection, and the

successful in detecting IL-23 helps to prove the involvement of IL-23 in immunity towards

malaria infection. IL-23p19 is closely resembled with IL-12p40 in sequence level. This

similarity also involves functional characteristics of IL-23p19 as this subunit also formed a

disulfide-linked heterodimer with IL-12p40 subunit (Oppmann et al., 2000). IL-12 receptor

(IL-12R) is comprised of two subunits, IL-12Rβ1 and IL-12Rβ2 (Beadling and Slifka, 2006).

IL-23 is structurally similar to IL-12 and has been found to interact with IL-12Rβ1 and IL-23

receptors (IL-23R) to begin the signal-transduction cascade (Oppmann et al., 2000; Lankford

and Frucht, 2003; Langrish et al., 2004). IL-23R complex is expressed on memory T cells, T

cell clones, natural killer cell lines and at low level on dendritic cell population, monocytes

and macrophages (Parham et al., 2002).

Although IL-12 and IL-23 share the same subunit and receptor binding sites, these cytokines

have distinct immunological functions and responses (Belladonna et al., 2002; Langrish et

al., 2004; Beadling and Slifka, 2006; McKenzie et al., 2006). IL-12 plays an important

function in the differentiation of naïve T cells characterized by IFN-γ production while IL-23

acts on memory T cells to provide long-lasting cellular immune responses (Lankford and

Frucht, 2003). The function of IL-23 in promoting the survival and maintaining memory T

cells is vital in clearing persistent intracellular infection (Lankford and Frucht, 2003). IL-23

also plays a role in resolving intracellular pathogen without the presence of IL-12 (Langrish

et al., 2004). One of the unique characteristics of this novel cytokine is the ability of IL-23 in

the development of alternative T cell subunits, which results in the production of pro-

Halimah et al. 2016


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47

inflammatory IL-17-related cytokines IL-17A and IL-17F (Langrish et al., 2004; McKenzie

et al., 2006). This unique characteristic was found when IL-23 interacts with CD4+ T cells,

which resulted in the different genes and cytokines expression from prototype Th1 and Th2

(Langrish et al., 2004; Beadling and Slifka, 2006; McKenzie et al., 2006). Thus, this new T

cell population has been referred as Th17 due to their attribution in IL-17 production.

Despite that, IL-23 plays a significant role in connecting innate and adaptive immune

responses (Tan et al., 2009). IL-17 together with IL-23 has abilities in recruitment of

neutrophils from bone marrow to the sites of infection (Langrish et al., 2004; McKenzie et

al., 2006) and induces the expression of IL-1, IL-6 and TNF-α (Langrish et al., 2004). These

cytokines are important in immediate protection during acute infection. The production of IL-

17 and other cytokines secreted by Th17 perhaps is able to provide a long term immune

response towards bacterial infection (McKenzie et al., 2006). IL-23 and IL-17 are involved in

providing protection during bacterial infection (Langrish et al., 2004; Beadling and Slifka,

2006; Tan et al., 2009), parasite infection (Ishida et al., 2013) and a number of inflammation

conditions (Barrie and Plevy, 2005; McKenzie et al., 2006; Tan et al., 2009). However, the

functions of IL-23 and IL-17 in malarial infection are still not well understood. These

preliminary results demonstrated the ability of macrophage to express IL-23 at protein and

mRNA levels as a response to the LPS stimulation. rBCG clone expressing MSP-1C of P.

falciparum stimulation is also capable to induce the significant expression of IL-23 on

macrophage J774A.1 at both protein and mRNA levels. These findings demonstrated that the

present of IL-23 in providing immunity towards malaria infection.

Acknowledgement

This work was supported by the Short Term Grant of Universiti Sains Malaysia, Grant no:

304.PPSK.61312105.

Halimah et al. 2016


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48

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Plasmodium falciparum - Universiti Sains Malaysia · mRNA levels. Furthermore, statistical analysis showed a significant difference of IL-23 expression between untreated and rBCG-treated