Anoectochilus formosanus is a therapeutic orchid appreciated as a traditional Chinese medicine in Asia. The extracts of A. formosanus have been reported to possess hepatoprotective, anti-inflammatory, and anti-tumor activates. A novel protein was isolated from A. formosanus, and its immunomodulatory effect on murine peritoneal macrophage was investigated. Macrophages obtained from ascites of thioglycollate-induced BALB/c were co-cultured with IPAF (0–20μg/ml) for 24h and then harvested for flow cytometry analysis. The cytokine/chemokine production was measured by real time PCR and ELISA. The interaction between IPA and toll like receptors (TLRs) was investigated by TLR gene knockout (KO) mice and fluorescence labeled IPAF. The activation of NF-κB was assessed by EMSA. IPAF stimulated the TNF-α and IL-1β production, upregulated the CD86 and MHC II expression, and enhanced the phagocytic activity of macrophages. IPAF induced gene expression of IL-12 and Th1-assosiated cytokines/chemokines. The stimulating effect of IPAF was impaired, and the IPAF–macrophage interaction was reduced in TLR4−/− C57BL/10ScNJ mice. In addition, IPAF stimulated expressions of TLR signal-related genes and the activation of NF-κB. IPAF could induce classical activated macrophage differentiation via TLR4-dependent NF-κB activation and had potential of IPAF to modulate the Th1 response. These findings provided valuable information regarding the immune modulatory mechanism of A. formosanus, and indicated the possibility of IPAF as a potential peptide drug

1. Investigating the function of a novel protein from Anoectochilus formosanus which
induced macrophage differentiation through TLR4-mediated NF-κB activation
Yen-Chou Kuan a
, Wan-Tzu Lee a
, Chih-Liang Hung a
, Ching Yang b
, Fuu Sheu a,c,
⁎
a
Department of Horticulture, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10673, Taiwan, ROC
b
Department of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10673, Taiwan, ROC
c
Center for Biotechnology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10673, Taiwan, ROC
a b s t r a c ta r t i c l e i n f o
Article history:
Received 29 February 2012
Received in revised form 10 June 2012
Accepted 16 June 2012
Available online 28 June 2012
Keywords:
Anoectochilus formosanus
Immunomodulatory protein
Macrophage activation
Toll like receptors
NF-κB
Anoectochilus formosanus is a therapeutic orchid appreciated as a traditional Chinese medicine in Asia. The ex-
tracts of A. formosanus have been reported to possess hepatoprotective, anti-inflammatory, and anti-tumor
activates. A novel protein was isolated from A. formosanus, and its immunomodulatory effect on murine peri-
toneal macrophage was investigated. Macrophages obtained from ascites of thioglycollate-induced BALB/c
were co-cultured with IPAF (0–20μg/ml) for 24h and then harvested for flow cytometry analysis. The
cytokine/chemokine production was measured by real time PCR and ELISA. The interaction between IPAF
and toll like receptors (TLRs) was investigated by TLR gene knockout (KO) mice and fluorescence labeled
IPAF. The activation of NF-κB was assessed by EMSA. IPAF stimulated the TNF-α and IL-1β production,
upregulated the CD86 and MHC II expression, and enhanced the phagocytic activity of macrophages. IPAF in-
duced gene expression of IL-12 and Th1-assosiated cytokines/chemokines. The stimulating effect of IPAF was
impaired, and the IPAF–macrophage interaction was reduced in TLR4−/−
C57BL/10ScNJ mice. In addition,
IPAF stimulated expressions of TLR signal-related genes and the activation of NF-κB. IPAF could induce clas-
sical activated macrophage differentiation via TLR4-dependent NF-κB activation and had potential of IPAF to
modulate the Th1 response. These findings provided valuable information regarding the immune modulatory
mechanism of A. formosanus, and indicated the possibility of IPAF as a potential peptide drug.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Orchids have been used as a traditional medicine for centuries to treat
various diseases and ailments worldwide [1]. Anoectochilus formosanus is
a medicinal orchid that has been appreciated as a folk remedy in Asia. As
reported by others, the extracts of A. formosanus exhibited diverse
pharmacological functions such as hepatoprotection [2], antitumor
[3,4], and immunomodulation [5]. A glycosidic constituent named
kinsenoside has been isolated from A. formosanus [6] and exhibited
hepatoprotective and anti-inflammatory activities [7,8]. Neverthe-
less, the pharmacological function mechanism of A. formosanus was
still unclear.
In our recent report [9], we have purified a new protein molecule,
the immunomodulatory protein from A. formosanus (IPAF), through
anion exchange chromatography approach, and cloned the complete
nucleotide sequence of IPAF (ADK74829.1) by means of the rapid
amplification of cDNA ends PCR methods. The amino acid (aa) se-
quence of IPAF comprised a 25-aa signal peptide and a 138-aa mature
protein, and shared high sequence homology to the lectins from
Orchidaceae plants. We have also demonstrated that IPAF stimulation
induced the cell proliferation, activation, and immunoglobulin pro-
duction in murine CD19+
B lymphocytes in a T cell independent
manner. In the researches regarding the pharmacological activity of
A. formosanus, most studies were conducted on the polysaccharide
extracts. To our knowledge, this protein IPAF is the first and the
only one that has been reported.
Macrophages are mononuclear phagocytic cells resident in most
tissues. They are derived from peripheral blood monocytes and func-
tion as professional antigen presenting cells (APCs) and as effector
cells in humoral and cellular immunity [10]. Macrophages can be ac-
tivated by different stimuli, or combination of stimuli, and differenti-
ate into cells exerting diverse functions. As proposed by Mosser and
Edwards [11], there are at least three types of activated macrophages,
the classical activated macrophages, the wound healing macrophages,
and the regulatory macrophages. In the immune system, the classical
activated macrophages produce IL-12 to promote Th1 cell differenti-
ation, whereas the IL-10 produced by regulatory macrophages can in-
duce Th2 cell differentiation by down-regulating IL-12 secretion [12].
The classical activated macrophages are induced by the combined sig-
nals of interferon-γ (IFN-γ) and tumor necrosis factor (TNF), or cer-
tain TLR agonists that induce TNF and IFN. This population of
International Immunopharmacology 14 (2012) 114–120
⁎ Corresponding author at: Center for Biotechnology, National Taiwan University,
No. 1, Sec. 4, Roosevelt Rd., Taipei 10673, Taiwan, ROC. Tel.: +886 2 33664846; fax:
+886 2 23673103.
E-mail address: fsheu@ntu.edu.tw (F. Sheu).
1567-5769/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2012.06.014
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journal homepage: www.elsevier.com/locate/intimp

2. macrophage has enhanced microbicidal or tumoricidal capacity and
secretes high level of pro-inflammatory cytokines and mediators
[13,14].
In the present study, we focused on investigating the modulatory
effects of IPAF on murine peritoneal macrophages. We evaluated the
pro-inflammatory cytokine production, the expression level of
co-stimulatory molecules and major histocompatibility complex
class II (MHC II), and the phagocytic activity of IPAF stimulated
macrophages. To determine the effector type of IPAF-activated mac-
rophages, the cytokine and chemokine gene expressions were
analyzed by quantitative real time (qRT) PCR. In addition, we eluci-
dated the molecular receptor of IPAF by utilizing toll like receptor
gene knockout (KO) mice and fluorescence protein labeling tech-
nique. The possible mechanism responsible for the IPAF-induced
NF-κB activation in macrophages was also studied.
2. Materials and methods
2.1. Preparation of IPAF
The IPAF used in this study was prepared as described in our pre-
vious work [9]. Briefly, the whole plant of A. formosanus was homog-
enized by sonication, and the crude protein was precipitated by
applying 90% of ammonium sulfate. The IPAF was purified from
crude protein by the AKTA fast protein liquid chromatography system
using the HitrapQ anion exchange column (GE Healthcare, Bucking-
hamshire, UK). The IPAF content in different parts of A. formosanus
was determined. In brief, the roots, stems, and leaves of A. formosanus
were separated for IPAF purification. The IPAF samples were verified
by SDS-PAGE analysis, and the protein contents were measured by
the BCA Protein Assay kit (Pierce, Rockford, IL) following man-
ufacturer's instructions. Endotoxin levels in IPAF were determined
by Limulus amoebocyte lysate (LAL) assay (b0.012 EU/μg) to exclude
the possibility of LPS contamination.
2.2. Mice and cell culture
C57BL/6J and BALB/c between 6 and 8weeks of age were pur-
chased from the National Laboratory Animal Center, Taipei, Taiwan.
C57BL/10ScNJ (TLR4−/−
) and B6.129-TLR2tmlkir/J
(TLR2−/−
) were
purchased from The Jackson Laboratory. The mice were maintained
in our animal facility under pathogen-free condition. All animal stud-
ies were permitted by the Institutional Animal Care and Use Commit-
tee of National Taiwan University (Approval ID: NTU-IACUC-98-112),
and performed according to the regulations of the NTU-IACUC. Mice
peritoneal macrophages were obtained as described in our previous
report [15]. In brief, thioglycollate-elicited peritoneal macrophages
were harvested from C57BL/6J, BALB/c, C57BL/10ScN, and
B6.129-TLR2tmlkir/J
mice 4days after an intraperitoneal injection of
1.5mL of 4% thioglycollate medium (Sigma-Aldrich St. Louis, MO) by
lavage of the peritoneal cavity with PBS. The cells were washed and
suspended in DMEM medium (Hyclone, Logan, UT) supplemented
with 10% fetal bovine serum (GIBCO-BRL Life Technologies, New
York, NY). The cell suspension was seeded in 24-well flat bottom plates
(Costar, Cambridge, MA) and incubated for 4h in Hera Cell (Heraeus
group, Hanau, Germany) at 37°C with 5% CO2 humidified air. The
non-adherent cells were subsequently washed off by PBS, and the
remaining adherent monolayer cells were further cultured and stimu-
lated with IPAF (0–20μg/mL), ultrapure LPS (InvivoGen, San Diego,
CA), or Pam3csk4 (InvivoGen) for 24h, in some experiments, the cells
were pre-incubated with Anti-Human/Mouse CD282 (TLR2) Functional
Grade Purified T2.5 (30μg/mL; eBioscience, San Diego, CA), Anti-Mouse
TLR4/MD-2 Complex Functional Grade Purified MTS510 (30μg/mL;
eBioscience), or isotype-matched control IgG (30μg/mL; eBioscience)
for 1h before stimulation.
2.3. Measurement of cytokine production
The levels of murine tumor necrosis factor alpha (TNF-α) and inter-
leukin 1 beta (IL-1β) production in cell culture supernatant were ana-
lyzed using OptEIA murine TNF-α and IL-1β ELISA kits (eBioscience),
respectively according to the manufacturer's instructions. The results
were acquired by measuring the absorbance at 450nm on microplate
reader 3550-UV (Bio-Rad, Hercules, CA), and the concentrations of
TNF-α and IL-1β were calculated by comparing with recombinant mu-
rine TNF-α and IL-1β as standards, respectively.
2.4. Flow cytometry analysis
For cell surface marker detection, cells were suspended in PBS
containing 2% (v/v) FBS and 0.1% sodium azide and stained with fluores-
cein isothiocyanate (FITC)-labeled anti-mouse CD80 or FITC-labeled
anti-mouse MHC class II antibodies (eBioscience, San Diego, CA) at 4°C
for 30min in dark. Data acquisition and analysis were performed with
FACScan using CellQuest software (BD Biosciences, San Jose, CA). The re-
sults are expressed as the total mean fluorescence intensity (MFI) or per-
centage of positive fluorescent cells.
2.5. Phagocytic activity assay
The peritoneal macrophages from C57BL/6J treated with or
without IPAF (20μg/mL) for 24h were harvested for phagocytosis ac-
tivity analysis using Vybrant Phagocytosis Assay Kit (Invitrogen,
Carlsbad, CA) following manufacturer's instructions. The cells were
suspended in Hanks balanced salt solution (HBSS) and incubated
with Fluorescein-labeled Escherichia coli K-12 BioParticles at room
temperature for 2h in dark. After the phagocytotic reaction, the
cells were washed twice with PBS to remove excessive E. coli parti-
cles. The cell viability was assessed by trypan blue staining, and the
phagocytic activity was assessed via flow cytometry where positive
florescence indicated the phagocytosing cells.
2.6. Quantitative real-time polymerase chain reaction (qRT PCR)
Quantitative real-time PCR was performed according to the methods
used by Chang [15]. In brief, total RNA was extracted using the TRIzol re-
agent (Invitrogen) following manufacturer's instructions. The first-strand
cDNA was synthesized from total RNA using ThermoScript RT PCR system
(Invitrogen), and was utilized as a template for evaluating gene expres-
sions in IPAF treated macrophages. Differential expression for individual
genes was assessed by qPCR on an Applied Biosystems 7300 Real-Time
PCR System (Applied Biosystems, Foster City, CA). Beta-Actin was used
to confirm the quality of reverse-transcripted cDNA and served as a
housekeeping gene. The expression level of each target gene was quanti-
fied according to cycling threshold (Ct) analysis.
2.7. Toll like receptors–IPAF interaction study
The IPAF samples were labeled with FITC using an FITC protein la-
beling kit (Thermo Scientific, Waltham, MA) following manufacturer's
instructions. Primary murine peritoneal macrophages from C57BL/6J
(WT), C57BL/10ScN (TLR4−/−
) and B6.129-TLR2tmlkir/J (TLR2−/−
)
were incubated with 20μg/mL of FITC-labeled IPAF for 40min, or
with FACS buffer alone as a control. The interactions between IPAF
and WT or TLR−/−
macrophages were analyzed by flow cytometry,
and the results were expressed as the MFI and relative fluorescent in-
tensity in total cells.
2.8. Electrophoretic mobility shift assay (EMSA)
The EMSA analysis was performed as described by Chang et al.
[15]. Briefly, nuclear protein extracts were prepared using NE-PER
115Y-C. Kuan et al. / International Immunopharmacology 14 (2012) 114–120

3. nuclear and cytoplasmic extraction reagents (Pierce). DNA–protein
interactions were detected using Light Shift Chemiluminescent
EMSA Kit (Pierce). The nuclear protein extracts were incubated with
double-stranded, 3′-end-biotinylated oligonucleotides containing
the consensus NF-κB binding sequence, and transcription factor bind-
ing analysis was performed according to Tamassia et al. [16]. The
specificity of the identified NF-κB DNA binding activity in the nuclear
extracts was confirmed by using a 200-fold molar excess of unlabeled
NF-κB.
2.9. Data analysis
All experimental results are presented as mean±SD of three inde-
pendent experiments performed in triplicates (n=3). Statistical com-
parisons were performed by one way ANOVA using PC-SAS (SAS
institute Inc., Cary, NC). Differences between experimental data
were determined significant at P valueb0.05.
3. Results
3.1. IPAF presented universally in all parts of A. formosanus
To identify the presence of IPAF in different plant parts of
A. formosanus, we separated the roots, stems, and leaves for IPAF
purification. The percent plant weight of each part was 30.1%,
23.1%, and 46.8% for roots, stems, and leaves respectively. As dem-
onstrated by the SDS-PAGE analysis (Fig. 1A), IPAF existed in all
parts of A. formosanus. The IPAF content in each plant part was
31.9, 15.9, and 59.5mg/kg in the roots, stems, and leaves, respec-
tively (Fig. 1B) as measured by the BCA assay. These results
suggested that A. formosanus leaf contained higher level of IPAF
and was a good source for immunomodulatory protein extraction.
3.2. IPAF stimulated the activation and pro-inflammatory cytokines
production of murine peritoneal macrophages
In our preliminary research, we discovered that IPAF could induce
the activation of murine macrophage cell line RAW264.7 (Supple-
mentary data S1). We then tested the stimulatory effect of IPAF on
primary cells. The murine peritoneal macrophages were stimulated
Fig. 2. IPAF stimulated the activation of murine macrophages. A, B: the TNF-α and IL-1β
production in the culture supernatant of macrophages stimulated with or without IPAF
(5–20μg/mL); C, D: the CD86 and MHC II expression on the surface of macrophages
stimulated with or without IPAF (20μg/mL); E: the phagocytic activity of macrophages
stimulated with or without IPAF (20μg/mL). The flow cytometry data (C–E) were
expressed as percent of positive fluorescence cells in total cells and the mean fluores-
cence intensity (MFI).
Fig. 1. The IPAF content in different plant parts of A. formosanus. A: SDS-PAGE analysis
of IPAF samples purified from the roots (lane 2), stems (lane 3), and leafs (lane 4) of
A. formosanus; pre-stained protein marker was loaded in lane 1 as a molecular weight
reference, and PBS was loaded in lane 5 as a negative control; B: The protein concentra-
tion of the IPAF samples purified from different plant parts of A. formosanus.
116 Y-C. Kuan et al. / International Immunopharmacology 14 (2012) 114–120

4. with IPAF (0–20μg/ml) for 24h and then harvested for flow cytome-
try analysis. The pro-inflammatory cytokine content in the culture su-
pernatant was measured by ELISA. As a result, a dose-dependent
increase in TNF-α production was observed (Fig. 2A). Similar result
was also found in IL-1β production (Fig. 2B). The up-regulation of sur-
face molecules such as CD86 and MHC II could be viewed as an
indicator of macrophage activation [17]. We discovered that the ex-
pression level of CD86 and MHC II was increased on macrophages
stimulated with IPAF.
Furthermore, the phagocytic activity of IPAF-induced macro-
phages was 2.4-fold greater than the control cells, where the popula-
tion of FITChigh
cell lifted from 24.1% to 58% in the cells treated with
IPAF (Fig. 2E). Herein, we confirmed that IPAF could activate primary
murine macrophages.
3.3. IPAF induced classical activated macrophage differentiation
The activated macrophage could differentiate into classical acti-
vated, wound healing, and regulatory macrophages depending on
the stimuli and the situation presented. Since differently activated
macrophages played entirely different or even opposite roles in
hosts, it was crucial to identify which type of cell did IPAF-induced
macrophages differentiated into. Taking advantage of the different
cytokine/chemokine systems expressed by diverse form of macro-
phages [18], we address the issue by screening the mRNA expression
of cytokine/chemokine by the IPAF-induced cells. As shown in Fig. 3,
gene expressions of seven cytokines, seven chemokines, and nitric
oxide synthase (iNOS) were measured in time-course experiments.
The gene expression of iNOS increased 8.3-fold, which corresponded
to our previous finding that IPAF induced nitric oxide production by
RAW264.7 (Supplementary data S1). In accordance to the ELISA
data (Fig. 2A, B), mRNA expression of TNF-α and IL-1β was
up-regulated 28- and 5.6-fold, respectively. The Th1-assosiated cyto-
kine IL-6, IL-12, and IL-18 gene expressions were induced within 4h,
where IL-12p35 showed the most significant 31-fold up-regulation.
Although in a lesser degree, the immune suppressive IL-10 gene ex-
pression was induced at 2h. Of the chemokines examined, only the
Th1-associated chemokines CCL3, CCL4, and CCL24 showed more ob-
vious changes, where the gene expressions were enhanced 25-, 17-,
and 13-fold, respectively. Taken together, these data suggested that
IPAF induction led to classical activated macrophage differentiation.
3.4. IPAF-induced cytokine production was TLR4-dependent
After confirming that IPAF induced classical activated macrophage
differentiation, we then sought the molecular receptor responsible for
IPAF-induced cell activation. As mentioned in the Introduction, liga-
tion of TLR ligands with TLR could lead to classical activation of mac-
rophages. Of the 12 TLRs identified in murine, TLR2 and TLR4 were
responsible for protein/peptide recognition; therefore, we investigat-
ed the involvement of TLR2 and TLR4 in the IPAF-induced macro-
phage activation. As the first step, we examined whether the
cytokine inducing effect of IPAF persisted in the TLR2−/−
and
TLR4−/−
mice. We found that, in the TLR2−/−
mice, IPAF-stimulated
macrophages produced significantly higher amount of TNF-α and
IL-1β than did the control cells (Fig. 4A, B). Interestingly, IPAF in-
duced neither TNF-α nor IL-1β production by the TLR4−/−
macro-
phages under the same experimental condition (Fig. 4C, D). These
Fig. 3. IPAF stimulated classical activated macrophage differentiation. The gene expression of iNOS, cytokines, and chemokines of IPAF-stimulated macrophages was measured by
means of real time PCR.
117Y-C. Kuan et al. / International Immunopharmacology 14 (2012) 114–120

5. results indicated that the IPAF-induced cytokine production was
TLR4-dependent.
3.5. IPAF stimulated murine macrophage through TLR4-dependent
activation of NF-κB
To show the direct involvement of TLR4 in the IPAF-induced mac-
rophage activation, we used anti-TLR2 and anti-TLR4 antibodies to
block the receptors, and found that only anti-TLR4 antibodies signifi-
cantly blocked the TNF-α production of IPAF-stimulated macro-
phages (Fig. 5A).
We then investigated the binding between IPAF and macrophages
from C57BL/6 (WT), TLR2−/−
, and TLR4−/−
mice. The cells were
co-incubated with or without FITC-labeled IPAF (20μg/106
cells) on
ice for 40min, and the IPAF binding was analyzed by flow cytometry.
The binding between IPAF and macrophages was significant in WT
and TLR2−/−
mice, whereas the binding between IPAF and macro-
phages from TLR4−/−
mice was below the detection limit (Fig. 5B
and C). With this discovery, we concluded that IPAF activated macro-
phage through ligation to TLR4.
To elucidate the mechanism of IPAF-stimulated macrophage activa-
tion, we detected the expression of the molecules downstream the TLR4
signaling pathway. We analyzed the expression of toll-interleukin 1 re-
ceptor domain containing adaptor protein (TIRAP), myeloid differentia-
tion primary response gene 88 (MyD88), TNF receptor-associated factor
6 (TRAF6), and NF-κB. As shown in Fig. 6A, the mRNA expression of
TIRAP, MyD88, TRAF6, and NF-κB was up-regulated within 4h of IPAF
stimulation.
Finally, we analyzed NF-κB activation by EMSA. A clear and
dose-dependent shift of NF-κB-nucleotide complex was observed in
nuclear protein extracts from cell treated with 0–20μg/ml of IPAF
(Fig. 6B, lanes 2–5). Collectively, these data indicated that IPAF in-
duced murine macrophage through TLR4-dependent activation of
NF-κB.
4. Discussion
In the present study, we found that IPAF existed universally in all
parts of A. formosanus and was most abundant in the leaves at 59.5mg
per kg leaves (Fig. 1). The universal expression of IPAF in all plant
parts was similar to the lectins in other herbs. In Dendrobium
officinale, a Chinese medicinal orchid, an anti-fungal lectin was
found to constitutively express in the roots, stems, and leaves [19].
Another anti-fungal lectin was also found to express in the root,
bulb, leaf, rachise, flower and fruit tissues of Crinum asiaticum, a
Chinese medicinal plant [20]. Most of the pharmacological studies
conducted on A. formosanus used the whole plant for extraction;
however, it was obvious that some component in plant might not
be present equally in all plant parts. Our finding provided information
regarding the diverse content of active component in different plant
parts.
Most of the studies evaluating the pharmacological activities of
medicinal orchids used crude extracts or focused on the alkaloids
and glycosidic constituents. To our knowledge, a few study investi-
gated the role of protein. In the present research, we focused on
evaluating the immunomodulatory function and mechanism of a pro-
tein from A. formosanus. We found that IPAF could stimulate
pro-inflammatory cytokine production (Fig. 2A, B), enhance the
co-stimulatory molecule and MHC II expression (Fig. 2C, D), and in-
crease the phagocytic activity of murine peritoneal macrophages
(Fig. 2E). In addition, the production of TNF-α and IL-1β as well as
the gene expression of Th1-related cytokines and chemokines were
Fig. 4. The stimulating activity of IPAF was TLR4-dependent. A, B: the IL-1β and TNF-α production of TLR2−/−
mice; C, D: the IL-1β and TNF-α production of TLR4−/−
mice. The
asterisk indicated a significant difference (Pb0.05) between the treatment and control.
118 Y-C. Kuan et al. / International Immunopharmacology 14 (2012) 114–120

6. induced by IPAF. These data demonstrated that IPAF could stimulate
classical-activated macrophage differentiation.
XThe immunomodulatory protein LZ-8 derived from Reishi
(Ganoderma lucidium) could activate murine macrophage, and the
LZ-8-stimulated macrophages could induce the activation and Th1
cytokine production by MACS purified CD3+
T cells in a mixed leuko-
cyte reaction, suggesting that LZ-8-activated macrophages could
stimulate Th1 response [21]. Based on the similar immunostimulatory
characteristics between LZ-8- and IPAF-activated macrophages, it
could be inferred that IPAF could activate Th1 response.
In this study, we elucidated the molecular mechanism of IPAF-induced
macrophage activation. We found that the macrophage-stimulating activ-
ity of IPAF was dependent on TLR4 expression and was blocked by
anti-TLR4 antibodies (Figs. 4 and 5A). Moreover the interaction between
IPAF and macrophage was impaired in TLR4−/−
mice (Fig. 5B and C).
The gene expression of molecules downstream the TLR4 signaling path-
way was induced, and the NF-κB activity was enhanced by IPAF
stimulation (Fig. 6A and B). These data indicated that IPAF activated mu-
rine macrophages through TLR4-dependent NF-κB activation. Intriguing-
ly, the IPAF-induced B cell activation was not qualitatively affected by
TLR4 deficiency [9]. This implied that TLR4 might participate partially in
the interaction between IPAF and B cells, and that other receptors such
as the B cell receptor might be involved in the IPAF-induced B cell
activation.
The polysaccharides purified from dried safflower petals (Carthamus
tinctorius L.) could induce cytokine production by murine peritoneal
macrophages by activating NF-κB via TLR4 [22]. The acidic polysaccha-
ride Angelan isolated from Angelica gigas Nakai could induce dendritic
cell maturation through TLR4 [23]. In addition to the polysaccharides,
protein from medicinal mushrooms/plants such as LZ-8, PCP from
Poria cocos, ACA from Antrodia camphorate, and Korean mistletoe lectin
could induce TLR4-mediated activation of murine APCs [15,24–26]. We
demonstrated here that protein from medicinal orchid could exert
immunostimulatory function through TLR4-dependent NF-κB activation.
Interestingly, we noticed that while IPAF stimulated macrophage
differentiation, kinsenoside, a glycosidic fraction of A. formosanus
suppressed immune response by NF-κB inhibition [8]. Moreover, an-
other study reported that the standardized aqueous extracts of A.
formosanus (SAEAF) exhibited anti-hypersensitivity function in an
OVA-inhaled murine model by promoting Th1 cell differentiation
[5]. In that report, the authors suggested that the water-soluble poly-
saccharides in SAEAF might be responsible for the stimulation of Th1
response since polysaccharide from Ganoderma Lucidium (PS-G)
A
B
C
Fig. 5. IPAF activated macrophages through TLR4. A: TNF-α production of macrophages
pre-treated with antibodies for 1h and then stimulated with IPAF for 24h, a group was
treated with medium alone as a control (open bar). The asterisk indicates a significant
difference (Pb0.05) between groups pre-treated with anti-TLR4 mAb and IgG2a. B, C:
the mean fluorescence intensity and the relative fluorescence intensity of macrophages
from WT, TLR2−/−
, or TLR4−/−
mice co-cultured with FITC-IPAF for 40min. B: the
numbers indicate the mean fluorescence intensity; C: the asterisks indicate a signifi-
cant difference (Pb0.05) between the FITC-IPAF treated groups and the control groups.
Fig. 6. IPAF stimulated TLR-mediated NF-κB activation. A: the gene expression of TLR
signaling-related molecules of IPAF-stimulated macrophages; B: the NF-κB activity de-
termined by EMSA, the content in each lane was negative control loaded without nu-
clear protein (lane 1), nuclear protein extract from macrophages treated with
medium alone (lane 2), 5μg/mL IPAF (lane 3), 10μg/mL IPAF (lane 4), and 20μg/mL
IPAF (lane 5), respectively.
119Y-C. Kuan et al. / International Immunopharmacology 14 (2012) 114–120

7. could induce Th1 response [27]. Still, it was also possible that the
water-soluble protein and IPAF in the SAEAF might have done this con-
tribution. This scenario has been addressed in G. lucidum, where PS-G
originally thought to be Th1 promoting failed to activate T cells after
deproteinization. This observation suggested that the LZ-8 content in
the PS-G should have been the major Th1-promoting ingredient in G.
lucidum [21]. Herein, we showed that IPAF exhibited diverse immuno-
logical function from glycosidic fractions of A. formosanus, and that
IPAF might have contributed to the Th1-promoting effect of SAEAF.
In conclusion, we demonstrated that the novel Orchidaceae pro-
tein IPAF could induce classical activated macrophage differentiation,
which suggested the potential of IPAF to modulate the Th1/Th2 re-
sponse. We elucidated the molecular receptor of IPAF and confirmed
that IPAF stimulated macrophages through TLR4-dependent NF-κB
activation. Furthermore, we demonstrated that the protein in medic-
inal orchid played important and distinct pharmacological role. In
conclusion, our data provided valuable information regarding the im-
munomodulatory mechanism of A. formosanus, and indicated the po-
tential of IPAF to be developed into a peptide drug.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.intimp.2012.06.014.
Acknowledgment
This study was supported by grants from the National Sciences
Council of Taiwan, Republic of China (NSC 99-2628-B-002-003-MY3).
References
[1] Hossain MM. Therapeutic orchids: traditional uses and recent advances — an
overview. Fitoterapia 2011;82:102–40.
[2] Fang HL, Wu JB, Lin WL, Ho HY, Lin WC. Further studies on the hepatoprotective
effects of Anoectochilus formosanus. Phytother Res 2008;22:291–6.
[3] Shyur LF, Chen CH, Lo CP, Wang SY, Kang PL, Sun SJ, et al. Induction of apoptosis in
MCF-7 human breast cancer cells by phytochemicals from Anoectochilus
formosanus. J Biomed Sci 2004;11:928–39.
[4] Tseng CC, Shang HF, Wang LF, Su B, Hsu CC, Kao HY, et al. Antitumor and
immunostimulating effects of Anoectochilus formosanus Hayata. Phytomedicine
2006;13:366–70.
[5] Hsieh CC, Hsiao HB, Lin WC. A standardized aqueous extract of Anoectochilus
formosanus modulated airway hyperresponsiveness in an OVA-inhaled murine
model. Phytomedicine 2010;17:557–62.
[6] Wu JB, Lin WL, Hsieh CC, Ho HY, Tsay HS, Lin WC. The hepatoprotective activity of
kinsenoside from Anoectochilus formosanus. Phytother Res 2007;21:58–61.
[7] Hsieh WT, Tsai CT, Wu JB, Hsiao HB, Yang LC. Kinsenoside, a high yielding constit-
uent from Anoectochilus formosanus, inhibits carbon tetrachloride induced Kupffer
cells mediated liver damage. J Ethnopharmacol 2011;135:440–9.
[8] Hsiao HB, Wu JB, Lin H, Lin WC. Kinsenoside isolated from Anoectochilus
formosanus suppresses LPS-stimulated inflammatory reactions in macrophages
and endotoxin shock in mice. Shock 2011;35:184–90.
[9] Kuan YC, Wu TJ, Kuo CY, Hsu JC, Chang WY, Sheu F. Molecular cloning of a new
immunomodulatory protein from Anoectochilus formosanus which induces B cell
IgM secretion through a T-independent mechanism. PLoS One 2011;6:e21004.
[10] Parham P. Elements of the immune system and their roles in defense. In: Foltin J,
Masson S, editors. The immune system. New York: Garland Science; 2009.
p. 1–30.
[11] Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation.
Nat Rev Immunol 2008;8:958–69.
[12] Gerber JS, Mosser DM. Reversing lipopolysaccharide toxicity by ligating the mac-
rophage Fcγ receptors. J Immunol 2001;66:6861–8.
[13] Mackaness GB. Cellular immunity and the parasite. Adv Exp Med Biol 1977;93:
65–73.
[14] O'Shea JJ, Murray PJ. Cytokine signaling modules in inflammatory responses. Im-
munity 2008;28:477–87.
[15] Chang HH, Yeh CH, Sheu F. A novel immunomodulatory protein from Poria cocos
induces toll-like receptor 4-dependent activation within mouse peritoneal mac-
rophages. J Agric Food Chem 2009;57:6129–39.
[16] Tamassia N, Le Moigne V, Calzetti F, Donini M, Gasperini S, Ear T, et al. The
MyD88-independent pathway is not mobilized in human neutrophils stimulated
via TLR4. J Immunol 2007;178:7344–56.
[17] Mosser DM. The many faces of macrophage activation. J Leukoc Biol 2003;3:
209–12.
[18] Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine sys-
tem in diverse forms of macrophage activation and polarization. Trends Immunol
2004;25:677–86.
[19] Chen ZH, Sun XF, Tang KX. Molecular cloning and characterization of a
mannose-binding lectin gene from Dendrobium officinale. J Plant Biochem Bio-
technol 2005;14:33–6.
[20] Chai YR, Pang YZ, Liao ZH, Zhang L, Sun XF, Lu YQ, et al. Molecular cloning and char-
acterization of a mannose-binding lectin gene from Crinum asiaticum. J Plant Physiol
2003;160:913–20.
[21] Yeh CH, Chen HC, Yang JJ, Chuang WI, Sheu F. Polysaccharides PS-G and protein
LZ-8 from Reishi (Ganoderma lucidum) exhibit diverse functions in regulating mu-
rine macrophages and T lymphocytes. J Agric Food Chem 2010;58:8535–44.
[22] Ando I, Tsukumo Y, Wakabayashi T, Akashi S, Miyake K, Kataoka T, et al. Safflower
polysaccharides activate the transcription factor NF-κB via Toll-like receptor 4
and induce cytokine production by macrophages. Int Immunopharmacol
2002;2:1155–62.
[23] Kim JY, Yoon YD, Ahn JM, Kang JS, Park SK, Lee K, et al. Angelan isolated from
Angelica gigas Nakai induces dendritic cell maturation through toll-like receptor
4. Int Immunopharmacol 2007;7:78-7.
[24] Lin YL, Liang YC, Tseng YS, Huang HY, Chou SY, Hseu RS, et al. An immunomodu-
latory protein, Ling Zhi-8, induced activation and maturation of human
monocyte-derived dendritic cells by the NF-kappaB and MAPK pathways. J Leukoc
Biol 2009;86:877–89.
[25] Sheu F, Chien PJ, Hsieh KY, Chin KL, Huang WT, Tsao CY, et al. Purification, cloning
and functional characterization of a novel immunomodulatory protein from
Antrodia camphorata (bitter mushroom) that exhibits TLR2-dependent NF-κB ac-
tivation and M1-polarization within murine macrophages. J Agric Food Chem
2009;57:4130–41.
[26] Park HJ, Hong JH, Kwon HJ, Kim Y, Lee KH, Kim JB. Song SK TLR4-mediated activa-
tion of mouse macrophages by Korean mistletoe lectin-C (KML-C). Biochem
Biophys Res Commun 2010;396:721–5.
[27] Lin YL, Lee SS, Hou SM, Chiang BL. Polysaccharide purified from Ganoderma
lucidum induces gene expression changes in human dendritic cells and promotes
T helper 1 immune response in BALB/c Mice. Mol Pharmacol 2006;70:637–44.
120 Y-C. Kuan et al. / International Immunopharmacology 14 (2012) 114–120