Download Serotonin and melatonin, neurohormones for homeostasis, as novel

Journal of Antimicrobial Chemotherapy (2005) 56, 861–868
doi:10.1093/jac/dki331
Advance Access publication 19 September 2005
Serotonin and melatonin, neurohormones for homeostasis, as novel
inhibitors of infections by the intracellular parasite chlamydia
Mohd. Akhlakur Rahman1, Yoshinao Azuma1*, Hajime Fukunaga2, Tomoyuki Murakami2,
Kazurou Sugi2, Hideto Fukushi3, Koshiro Miura1, Harumi Suzuki1 and
Mutsunori Shirai1
1
Department of Microbiology, Yamaguchi University School of Medicine, 1-1-1, Minamikogushi,
Ube, Yamaguchi 755-8505, Japan; 2Department of Clinical Research, National Sanyou Hospital,
Yamaguchi 755-0241, Japan; 3Department of Veterinary Microbiology, Gifu University,
1-1 Yanagido, Gifushi, Gifu 501-1193, Japan
Received 4 July 2005; returned 29 July 2005; revised 16 August 2005; accepted 19 August 2005
Objectives: Chlamydiae are obligate intracellular bacteria, causing a variety of diseases, i.e. pneumonia,
sexually transmitted disease, conjunctivitis and zoonosis. Tryptophan depletion by interferon-gamma
(IFN-g) is the most important host defence system against chlamydial infection. Thus chlamydial tryptophan metabolism is thought to play key roles for IFN-g resistance, persistent infection and host/tissue
tropisms. We tested tryptophan derivatives for activity against chlamydia-infected cells.
Methods: Rates of chlamydial infection and sizes of the inclusions were evaluated by in vitro infection using
three Chlamydiaceae species, Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila
felis, which show significant divergence of tryptophan synthesis genes and different susceptibilities to IFN-g.
Results: Melatonin and serotonin, which are recognized as neural hormones for maintenance of organism
homeostasis, reduced chlamydial infection but not other bacterial growth tested here. Unlike IFN-g,
melatonin limited infection of all three chlamydiae and the effects were not recovered by tryptophan
supplementation. Melatonin treatment only of host cells could diminish infection and the infection reduction was neutralized by a pertussis toxin, an inhibitor of G proteins. Ligands of melatonin and serotonin
receptors also hampered infection.
Conclusions: Inhibition mechanisms of chlamydial infection by melatonin and serotonin appear to be
different from those of IFN-g and involve specific G-protein-coupled receptors. Melatonin is deemed to
inhibit early progression of the chlamydial development cycle, such as establishment of intracellular
infection and/or conversion from elementary body to reticulate body. Utilization of melatonin, serotonin
or their derivatives may be advantageous for harmless prevention of chlamydial infection.
Keywords: Chlamydophila pneumoniae, Chlamydia trachomatis, tryptophan, antimicrobial agents, G-protein
coupled receptor
Introduction
Chlamydiae are obligate intracellular prokaryotic pathogens,
exhibiting a unique developmental cycle consisting of two
major cell phases: the infectious but metabolically inert elementary
body (EB) and the metabolically active but non-infectious reticulate body (RB).1 The chlamydiae cause a variety of diseases
ranging from asymptomatic to serious diseases in humans and
animals.2 Chlamydia trachomatis is a highly prevalent cause of
ocular and urogenital diseases. Chlamydophila pneumoniae is
responsible for acute and chronic respiratory diseases, and is
widely considered to be a cause of atherosclerosis,3 asthma,4
Alzheimer’s disease5,6 and other inflammatory processes.
Chlamydophila species, which formerly belonged to Chlamydia
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*Corresponding author. Tel: +81-836-22-2227; Fax: +81-836-22-2415; E-mail: [email protected]
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861
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Rahman et al.
psittaci as subspecies, cause several zoonoses, for example,
Chlamydophila felis is a worldwide pathogen of cats responsible
for ocular and pulmonary diseases.7
One of the most important host defence systems against the
chlamydial infection is interferon-gamma (IFN-g) synthesis,
which induces indoleamine 2,3-dioxygenase (IDO) and thus
depletes tryptophan from human cells, since many human pathogenic chlamydiae rely on the tryptophan.8–11 Besides the function
of IFN-g-inducible tryptophan shortage as a host defence, insufficient IFN-g exposure may lead the chlamydial growth to a persistent state, which is characterized by atypical inclusions that are
generally smaller in diameter and contain larger aberrant RBs.12
This is why chlamydial IFN-g resistance and host/tissue tropism are
thought to depend on chlamydial tryptophan synthesis.13,14
Persistent chlamydial infection is thought to be involved in the
pathophysiology of some chronic inflammatory diseases, e.g.
asthma and atherosclerosis for C. pneumoniae, and blindness
and infertility for C. trachomatis.15
Human tryptophan catabolism mainly consists of two pathways,
the kynurenine pathway and the serotonin pathway. In the kynurenine pathway, tryptophan is processed to N-formylkynurenine by
two types of rate-limiting enzymes, tryptophan 2,3-dioxygenase
(TDO) induced by tryptophan in the liver and the IDO induced
by IFN-g during the immune response in many tissues. Tryptophan
is finally metabolized to an essential cellular cofactor, nicotinamide adenine dinucleotide (NAD+). The other pathway is the
serotonin pathway that produces a variety of neural activators
such as a neurotransmitter, serotonin and, a pineal hormone,
melatonin.10
Serotonin is a well-known ubiquitous monoamine, which
is derived from tryptophan and functions as a neurotransmitter,
hormone and mitogenic factor that mediates a wide range of
physiological activities in different cells through multiple
receptor subtypes. Serotonin is produced and stored within several
tissues throughout the human body, such as the central neural
cells,16 blood platelets,17 gastrointestinal enterochromaffin cells18
and prostatic neuroendocrine cells.19 Furthermore, serotonin plays
a fundamental role in regulation of growth, differentiation and gene
expression.20–22 Melatonin, originally identified as an effector for
circadian rhythms,23 is now known to be a hormone involved in a
vast range of homeostasis maintenance activities, for example
seasonal timing, sexual development, the antioxidant defence system and immune response.24–26 Melatonin is synthesized from
tryptophan within the serotonin pathway mainly in the pineal
gland, and in a number of extrapineal organs such as retina, lens,
bone marrow, intestine, skin and so on.27 To date, three mammalian
melatonin receptors, G-protein coupled receptors MT1 and MT2,
and a quinone reductase family receptor, MT3, have been
identified.19,28,29
Recently, several chlamydial genome sequencing projects
have been completed and have revealed the divergence of the
gene repertoire for tryptophan synthesis.13,14,30 It is not
known why chlamydial species contain different tryptophan
gene repertoires, but it is possible that some host factors
related to tryptophan naturally restrict gene compositions of
the chlamydial tryptophan operon in chlamydial evolution.
Here we report the finding that the tryptophan derivatives
serotonin and melatonin reduce chlamydial infection in HEp2 cells in a manner different from the IFN-g-inducible host
defence.
Materials and methods
Chemicals
L-Tryptophan, and its derivative compounds, such as 5-hydroxy tryptophan, 5-hydroxy tryptophol, 5-methoxyindole acetic acid, melatonin
and serotonin, and ligands of melatonin and serotonin receptors, IIK7,
luzindole, buspirone and NAN190, were purchased from Sigma–
Aldrich (Saint Louis, MO). Gentamicin, cycloheximide, DAPI and
Dulbecco’s modified Eagle medium (DMEM) were purchased from
Sigma–Aldrich. Pertussis toxin was purchased from List Biological
Laboratories, Inc. (Campbell, CA), human recombinant IFN-g was
from Peprotech Ec Ltd (London, UK), and fetal calf serum (FCS)
was from Cansera International Inc. (Etobicoke, Canada). The family
Chlamydiaceae-specific fluorescein isothiocyanate (FITC)-labelled
antibody was from Denkaseiken (Tokyo, Japan).
Cell line and bacterial strains
HEp-2 cells (ATCC CCL-23) were grown in HEp-2 medium (DMEM
supplemented with 10% heat-inactivated FCS and 50 mg/L gentamicin) at 37 C with 5% CO2. C. pneumoniae J138 isolated in Japan in
1994,30 C. felis Japanese strain Fe/C-5631 and C. trachomatis serovar D
were used in this work. These strains were purified by sucrose-gradient
centrifugation and stored at –80 C in the SPG buffer (pH 7.2), which
consists of 250 mM sucrose, 10 mM sodium phosphate and 5 mM
glutamate. Chlamydial titres were adjusted for C. pneumoniae J138 to
2.0 · 108 inclusion formation units (IFU)/mL, and C. felis and C.
trachomatis to 1.0 · 108 IFU/mL. Other bacterial strains used here
are Escherichia coli, Staphylococcus aureus, Bacillus subtilis E192,
Streptococcus pneumoniae, Pseudomonas aeruginosa PAO1,
Helicobacter pylori CPY3401, Mycobacterium tuberculosis H37Rv,
and Legionella pneumophila ATCC33153.
Chlamydial infection
Chlamydial infection was performed by methods described elsewhere.32 Briefly, 2.0 · 104 HEp-2 cells in 0.10 mL of the medium
were seeded into each well of flat-bottomed 96-well tissue culture
plates, and allowed to adhere for 24 h prior to use. Infection was
performed by addition of chlamydial EBs to achieve a multiplicity
of infection (MOI) of 0.20. In this condition, inclusion formation
units, number of host cells and infection rate may turn to 1 · 105
IFU/mL, 5 · 104 cells and 20%, respectively. Relative infection
rate was determined as an average of at least three replicate tests
and confirmed with two other independent experiments performed
under the same conditions. After centrifugation at 700g for 60 min
at 22 C and then incubation for 30 min at 36 C with 5% CO2, the
inoculum was replaced with the post-infection medium (DMEM
with 5% heat-inactivated FCS, 50 mg/L gentamicin and 1 mg/mL
cycloheximide). The infected cells were incubated for 48 h at 36 C
with 5% CO2, following fixation of the infected cells for 30 min in
methanol.
For measurement of IFUs and rates of infected host cells, the
infected cells were stained with the family Chlamydiaceae-specific
FITC-labelled antibody for 60 min at 37 C, and then stained with
0.1 mg/mL of DAPI for 10 min. Numbers of host cell nuclei and
chlamydial inclusions were counted under fluorescence microscopy,
BX50 (Olympus, Japan) at a magnification of ·200. Under treatments
with chemicals and IFN-g, typical and atypical (or small) inclusions
were observed. Here, inclusions larger than approximately 1 mm in
diameter were counted as successful infections.33,34
862
Serotonin and melatonin inhibit chlamydial infection
Chemical treatment
(a)
In most experiments, treatments with 50–200 mM test compound were
started at 24 h prior to infection and ended at 48 h post-infection,
otherwise treatment schedules and concentrations of chemicals are
described in the figure legends for each experiment.
For other bacteria, E. coli, S. aureus, B. subtilis, P. aeruginosa were
cultured in LB broth, whereas S. pneumoniae was cultured in Brain
Heart Infusion and H. pylori in Brucella broth supplemented with 5%
horse serum and 0.1% b-cyclodextrin. Inocula of each strain were
incubated in their respective media treated with melatonin, gentamicin
or mock at 37 C, and then the OD at 600 nm was measured as
the indication for bacterial growth up to 8 h except H. pylori to
24 h. The BACTEC MGIT 960 Automated System (Becton Dickinson,
Cockeysville, MD) was adapted for susceptibility tests with
M. tuberculosis and L. pneumophila. For L. pneumophila growth,
Legionella BCYE growth supplement (Oxoid Ltd, Hampshire, UK)
was added to the MGIT medium for M. tuberculosis.
5-hydroxyB
tryptophan
KynurenineC
SerotoninD
MelatoninE
5-hydroxyF
tryptophol
Relative infection rate (%)
100
80
*
60
*
40
20
0
–
A
B
C
D
E
F
G
120
Relative infection rate (%)
(c)
Screening of agents against chlamydial infection in vitro
5-methoxyindoleG
acetic acid
120
(b)
Results
Elevation of IFN-g levels by melatonin has been reported during
viral infection in a mouse model.35 To investigate the relationship
between melatonin and IFN-g, excess tryptophan was added
independently to melatonin and IFN-g in chlamydial infection.
The inhibition of chlamydial infection by IFN-g was observed
as previously reported33 and was reversed by adding tryptophan,
consistent with the well-known schema of tryptophan depletion
by IFN-g.9,11 In contrast, tryptophan addition did not reverse
melatonin-mediated inhibition (Figure 2a). This suggests that
the inhibitory mechanism of melatonin is different from that of
IFN-g in this case.
L-formyl
kynurenine
NAD
To analyse the production of infectious progeny under the treatment of
melatonin, serotonin and IFN-g, chlamydial progeny were collected
from the culture medium at 72 h post-infection by centrifugation at
10 000g for 15 min at 4 C. To measure the amount of infectious
progeny, the collected chlamydiae were suspended in the DMEM
buffer and adapted to the secondary infection using HEp-2 cells growing in 96-well plates. The procedures described above were used to
determine the infection rates.
Effect of melatonin and IFN-g on chlamydial infection
Serotonin
pathway
5-hydroxyindole
acetaldehyde
Assay of infectious progeny
Tryptophan and six derivatives (Figure 1a) were administered at
50 mM onto HEp-2 cells infected with C. pneumoniae J138 at 24 h
prior to infection and maintained until the infection was terminated
by fixation. The rates of infected host cells were calculated and are
shown in Figure 1(b). Among the compounds tested here, serotonin
and melatonin reduced the chlamydial infection by 50% compared with controls. 5-Hydroxytryptophan at 200 mM also showed
an inhibitory effect on chlamydial infection (data not shown).
Optimum concentrations of melatonin and serotonin
were analysed in a range of 25–400 mM, at which no host
toxicity was observed under microscopic studies. Treatments
with melatonin and serotonin caused a dose-dependent inhibition
of C. pneumoniae infection of HEp-2 cells and at 100 mM both
reached the optimum effect (Figure 1c). In most experiments
below, melatonin and serotonin were used at 100 mM.
L-TryptophanA
Kynurenine
pathway
Melatonin
Serotonin
100
80
60
40
20
0
0
25
50
100
200
Concentration (µM)
400
Figure 1. Effects of tryptophan and its metabolites on chlamydial infection
in vitro. (a) Schematic presentation of tryptophan and its metabolites in
kynurenine and serotonin mammal pathway. (b) Effects of tryptophan and its
metabolites, indicated as A–G in panel (a), were tested at 50 mM each on infection
of C. pneumoniae J138 to human HEp-2 cells. (c) Dose tests were carried out with
25–400 mM melatonin and serotonin. In both cases of panels (b) and (c), HEp-2
cells were infected with C. pneumoniae J138 at 0.2 multiplicity of infection
(MOI). The rates of infected host cells were determined at 48 h post-infection
by staining of chlamydial inclusions with the chlamydia-specific FITC-labelled
antibody and host nuclei with DAPI. Each infection rate was normalized with
controls to the relative infection rate, while the actual infection rates of the
controls were 11.9% and 19.4% in panels (b) and (c), respectively. None of
the chemicals showed substantial toxicity to the host cells under these conditions.
The bars shown are expressed as mean – SD from triplicate and more independent
experiments. *Significant differences between each experiment and control
calculated by Student’s t-test (P < 0.01).
Inclusion sizes under melatonin treatment varied from 2.0 to
5.5 mm in diameter. In contrast, the majority of inclusions in the
control show typical sizes, 5.0 mm in diameter, and in IFN-g treatment are atypical or small sizes, 1.5 mm in diameter (Figure 2b
and c). Here, successful infections showing typical and atypical
863
Rahman et al.
inclusions larger than appropriate 1 mm in diameter were
counted,33,34 because chlamydial infection is thought to be able
to switch from the persistent to the productive replicative cycle
when the stress to sustain persistent infection is removed.11
120
Relative infection rate (%)
(a)
100
80
*
*
*
+
–
–
+
–
+
–
+
–
60
40
20
0
Melatonin
IFN-γ
Tryptophan
(b)
–
–
–
Control
–
–
+
Melatonin
–
+
+
IFN-γ
Because of homogeneity of the bacteria, the melatonin function
as an anti-chlamydial agent is suggested to be associated with host
cell status rather than that of chlamydia. In the case of melatonin
treatment up to 0.5 mM, no growth arrest of HEp-2 cells nor
obvious morphological changes (such as cell shrinkage and chromatin condensation) were observed (Figure 2b).
IFN-g exposure and melatonin treatment cause atypical chlamydial inclusions, supposedly showing the typical persistent state
of chlamydial infection and producing few progeny.12,33,34 The
re-infection ability of progeny produced under melatonin and serotonin treatment was analysed. At 72 h post-infection, infected host
cells which were treated with melatonin and serotonin had released
approximately half the infectious progeny compared with
untreated control (Figure 2d), while IFN-g-treated cells had
released one-fourth of the control. Chlamydial progeny in inclusions under melatonin and serotonin treatment are infectious, not in
the persistent state, and are released at approximately the same time
as in controls. This suggests that melatonin and serotonin may
affect the early establishment stage in the chlamydial development
cycle, but not intracellular growth or later development.
DAPI
Specificity of melatonin on microbes
To determine melatonin specificity for chlamydial species, infection experiments were performed with two more chlamydiae
with worldwide distribution, C. trachomatis and C. felis, which
cause a human sexually transmitted disease and cat conjunctivitis,
respectively. Infection rates of the two chlamydiae were reduced
depending on melatonin concentrations. C. felis is resistant to IFNg but susceptible to melatonin, and C. trachomatis is relatively
resistant to both melatonin and IFN-g, whereas C. pneumoniae
is susceptible to both IFN-g and melatonin (Figure 3). This indicates that the mechanism of melatonin to inhibit chlamydial
growth is different from that of IFN-g, and the chlamydial gene
Inclusion
(magnified)
Distribution of inclusion sizes (%)
(c)
IFN-γ
30
25
Melatonin
20
15
10
5
0
(d)
Control
1.0
2.0 3.0 4.0 5.0 6.0
Diameter of inclusion (µm)
7.0
Relative growth of
C. pneumoniae (%)
120
100
80
60
40
20
0
0 1.0
IFN-γ
50 100 200 400
50 100 200 400
Melatonin
Serotonin
Concentration (µM)
Figure 2. Comparison of melatonin and IFN-g on chlamydial infection.
(a) C. pneumoniae J138 infection of HEp-2 cells was carried out in the presence
of melatonin (100 mM) and IFN-g (1.0 ng/mL) alone, with and without tryptophan
(200 mM). HEp-2 cells were infected with C. pneumoniae J138 at 0.2 MOI. The
rates of infected host cells were determined at 48 h post-infection by staining of
chlamydial inclusions with the chlamydia specific FITC-labelled antibody and
host nuclei with DAPI. Average of the actual infection rates of the controls
was 15.0%. The bars shown are expressed as mean – SD from triplicate
and more independent experiments. *Significant differences between each
experiment and control calculated by Student’s t-test (P < 0.01). (b) Distribution
of sizes of C. pneumoniae J138 inclusions. HEp-2 cell nuclei (upper row) and
C. pneumoniae J138 inclusions were visualized by staining with chlamydiaspecific FITC-labelled antibody and DAPI, respectively. Inclusions are
shown in the second and third rows. The areas in the boxes of the second row
are magnified three times and small inclusions are marked with arrows in the third
row. White bars indicate 10 mm. This figure is available in colour in the online
version of this article. (c) Distributions of inclusion sizes are illustrated under
melatonin treatment (thick solid line), IFN-g treatment (thin solid line) and
control (dashed line). Sizes of approximately 200 inclusions were measured
in each condition using the NIH image 1.63 program. (d) Growth of
C. pneumoniae J138 in HEp-2 cells was tested in the presence of melatonin
(50–400 mM), serotonin (50–400 mM), and IFN-g (1.0 ng/mL). Cells were infected with C. pneumoniae for 72 h, and progenies were harvested from the triplicate
independent culture supernatants for each condition. The number of IFU was
individually assessed with the progeny using HEp-2 cells. The data presented are
the averages and the standard deviations. A colour version of Figure 2(b) is
available as online Supplementary data at http://jac.oxfordjournals.org/.
864
Serotonin and melatonin inhibit chlamydial infection
concentrations of 25–200 mM. Melatonin agonist and antagonist,
IIK7 and luzindole, respectively, decreased chlamydial infection as
effectively as melatonin, however at high concentrations luzindole
exhibited toxicity to host cells. Luzindole is generally an antagonist
of a melatonin receptor, but in certain cases, it has been reported to
work as a melatonin agonist as well.37 Serotonin receptor agonist
and antagonist, buspirone and NAN190, respectively, partially
decreased chlamydial infection. Other types of serotonin receptors,
which do not bind buspirone and NAN190, may be involved in the
serotonin inhibition of chlamydial infection.
Relative infection rate (%)
120
100
80
60
40
20
0
Melatonin
IFN-γ
– –
+
+ – – – –
– –
+
+ – – – –
+
– –
+ – – – –
Discussion
C. pneumoniae
C. felis
C. trachomatis
A new function of two well-known neurohormones, serotonin and
melatonin, has been found, namely inhibition by 50% of chlamydial infection of human epithelial HEp-2 cells in vitro. Both
effects of melatonin and serotonin are insensitive to addition of
tryptophan, which is a metabolic source of serotonin and melatonin
synthesis. On the other hand, inhibition of chlamydial infection
by IFN-g is ceased by addition of extra tryptophan. This suggests
that serotonin and melatonin are not antagonists of tryptophan for
protein synthesis or any other metabolic intermediate production,
and the inhibition mechanisms by melatonin and serotonin are
independent of IFN-g.
Pre-infection melatonin treatment of host cells, but not chlamydial cells, inhibits chlamydial infection. Post-infection treatment
with melatonin also inhibited chlamydial infection, illustrating that
melatonin may not block interaction between cell surfaces of chlamydiae and host cells. Moreover, addition of pertussis toxin with
melatonin (but not serotonin) treatment, cancelled the melatonin
effect, and ligands of melatonin and serotonin receptors also inhibit
chlamydial infection, suggesting that some melatonin and
serotonin receptors are involved in the inhibition of chlamydial
infection.
Melatonin is synthesized within the serotonin pathway from
tryptophan mainly in the pineal gland and also in a number of
tissues, such as retina, intestine, and so on.27 Three mammalian
melatonin receptors have been identified to date. MT1 and MT2
receptors are G-protein-coupled receptors sensitive to the pertussis
toxin28,29 and MT3 receptor is a quinone reductase family receptor.19 It is reported that the receptors are widely exhibited on
peripheral tissues, such as brain, liver and kidney, and an epithelial
cell line, HEK-293.38,39 Gene expression of MT1 and MT2 receptors was not detected by RT–PCR in our experiments using a human
epithelial cell line, HEp-2 (data not shown). In addition, distribution analysis of inclusion sizes demonstrates distinctive groups
of inclusions. One of these may be a group (50%) in which
susceptibility to melatonin is high and prevented from visible
inclusion formation. The other two groups may be resistant and
partially susceptible to melatonin, resulting in typical and small
inclusion formations, respectively. It may implicate the existence
of a melatonin-specific receptor(s), whose signal transduction system or gene expression determines the susceptibility to melatonin
relying on the host cell cycle.
In contrast to melatonin, chlamydial growth inhibition by
serotonin is insensitive to pertussis toxin. However, it is still
highly possible that the inhibitory mechanism of serotonin
involves specific receptor(s) coupled with G-proteins resistant to
pertussis toxin, firstly because ligands of serotonin receptors have
inhibited chlamydial infection, and secondly because most
Figure 3. Host susceptibilities to different chlamydial species were tested in
response to 25, 50 and 100 mM of melatonin treatment, compared with 1.0 ng/mL
of IFN-g treatment. Prior to use, C. felis Fe/C-56 and C. trachomatis serovar D
were adapted to HEp-2 cells by three passages and numbers of IFU were assessed
in HEp-2 cells. C. pneumoniae J138, C. felis Fe/C-56 and C. trachomatis serovar
D were used for infection to HEp-2 cells at 0.2 MOI. The rates of infected host
cells were determined at 48 h post-infection. Each infection rate was normalized
with controls to the relative infection rate, while the actual infection rates of
the controls were 25.3%, 25.0% and 16.6% for C. pneumoniae, C. felis and
C. trachomatis, respectively. The bars shown are expressed as means of duplicate
independent experiments.
repertoires, but not tryptophan synthesis, may affect susceptibility
to melatonin.
To examine whether or not melatonin affects only intracellular
chlamydiae, eight other bacteria (E. coli, S. aureus, B. subtilis,
S. pneumoniae, P. aeruginosa, H. pylori, L. pneumophila and
M. tuberculosis) were tested separately at different melatonin concentrations in suitable culture medium. These bacteria represent
different groups within the kingdom Eubacteria, including normal
resident microflora and pathogenic bacteria, and Gram-positive
and -negative bacteria. In contrast to gentamicin, to which all
bacteria tested were susceptible, none of them showed effects of
melatonin treatment on their growth (data not shown).
Target(s) of melatonin and serotonin
It has been reported that melatonin influences the hydrophobic
characteristics of Escherichia coli cell surfaces.36 Therefore, chlamydial cells and host cells were pre-treated with melatonin and
serotonin before infection. Chlamydial infection was reduced by
pre-treatment of HEp-2 cells with melatonin and also serotonin.
Pre-treatment of chlamydial cells showed no effect (Figure 4a). In
addition, post-infection treatment with melatonin also reduced
chlamydial infection (data not shown). This suggests that melatonin and serotonin interact with human cells but not with chlamydial
cells, and some melatonin and serotonin receptors are involved in
the resistance to chlamydial infection.
Pertussis toxin is a well-known inhibitor of signal transduction
through G protein-coupled receptors.28,29 The melatonin-mediated
reduction in chlamydial infection in HEp-2 cells was abrogated by
treatment with 20 ng/mL of pertussis toxin. No such inhibitory
effect was observed in the case of serotonin (Figure 4b). Because
of these results implying the involvement of certain G proteincoupled melatonin receptors, agonists and antagonists of melatonin
and serotonin receptors were tested in chlamydial infection at
865
Rahman et al.
(b)
100
80
60
*
*
40
20
0
Melatonin
Serotonin
Treatment to
120
Relative infection rate (%)
120
Relative infection rate (%)
(a)
100
80
*
60
*
40
*
20
0
–
–
+
–
–
+
–
–
Host cells
+
–
–
+
Melatonin
Serotonin
Pertussis toxin
–
–
–
–
–
+
+
–
–
+
–
+
–
+
–
–
+
+
Chlamydial cells
Relative infection rate (%)
(c) 120
Melatonin
100
IIK7
80
Luzindole
60
Serotonin
40
Buspirone
20
0
NAN190
0
25
50
100
Concentration (M)
200
Figure 4. Targets of melatonin and serotonin in HEp-2 cell infection with C. pneumoniae. (a) HEp-2 cells and C. pneumoniae J138 were separately treated with
melatonin and serotonin. Treatments of chlamydial cells were performed at 200 mM for 90 min before infection in the same culture media as used for HEp-2 cells. Both
C. pneumoniae J138 cells treated and mock treated were used for infection at 0.2 MOI to untreated HEp-2 cells and the actual infection rates of the controls were 6.7%
[right half of panel (a)]. Treatments of host cells were performed at 200 mM for 24 h and the chemicals were washed with the culture medium before infection. Host cells
treated or mock treated were infected with C. pneumoniae J138 at 0.2 MOI [left half of panel (a)]. The actual infection rates of the controls were 8.8%. (b) Effects of the
pertussis toxin, an inhibitor of G-protein (20 ng/mL), were tested on the melatonin and serotonin treatments. Treatments with the pertussis toxin, melatonin and
serotonin were carried out from 24 h prior to infection to 48 h post-infection. HEp-2 cells were infected with C. pneumoniae J138 at 0.2 MOI. The actual infection rate of
control was 17.3%. No substantial toxicity was observed under these conditions. (c) Antagonists and agonists were applied to the C. pneumoniae J138 in vitro infection.
Treatments at 25–200 mM of each chemical were carried out from 24 h prior to infection to 48 h post-infection and C. pneumoniae J138 was infected at 0.2 MOI. The
actual infection rate of control was 23.0%. No substantial toxicity was observed under these conditions, except with 100 and 200 mM luzindole. In the cases of panels
(a) to (c), the rates of infected host cells were determined at 48 h post-infection. The bars shown in panels (a) and (b) and points in (c) are expressed as mean – SD from
triplicate and more independent experiments. *Significant differences between each experiment and control calculated by Student’s t-test (P < 0.01).
physiological serotonin reactions are processed through G
protein-coupled serotonin-specific receptors. In the large family
of G protein-coupled serotonin receptors, Ga12/13, has already been
reported to be a type of G protein insensitive to pertussis toxin.40
As common features, the melatonin and serotonin signal transductions through each specific receptor result in an increase in
intracellular Ca2+ concentration and regulation of adenylyl cyclase
activity, which produces adenosine 30 :50 -cyclic monophosphate
(cAMP). Both concentrations of Ca2+ and cAMP produced by
adenylyl cyclase subsequently modulate the gene expression status
of the host cell.41,42 Interestingly, chlamydial infection is closely
related to intracellular levels of cAMP and Ca2+. Chlamydial infection is inhibited by high concentrations of extracellular cAMP or
cGMP.43 Ca2+ mobility across the cell membrane is rapidly
increased by chlamydial attachment on to host cells.43 Recently,
Ca2+-dependent phagolysosomal fusion has been illustrated as a
host defence strategy, in which host cells may monitor Ca2+ influx
accompanying injuries by invasion of intracellular pathogens and
the Ca2+ influx may promote lysosomal exocytosis.44 The type III
secretion system is a candidate to injure the host cell membrane and
is thought to be an invasion system of chlamydiae as well. It is
likely that melatonin modulates intracellular Ca2+ and/or cAMP
levels and leads to increased phagolysosomal activity of the host
cells against the chlamydial infection.
To detect modification of gene expression in HEp-2 cells by the
melatonin and serotonin treatments, microarray analysis was
carried out with AceGene oligo-DNA chips (HitachiSoft, Japan)
arraying approximately ten thousand known human genes.
Positively and negatively regulated genes in the treated cells
were nominated when the signals are more than 1.5 times or
less than 0.6 times compared with untreated cells, respectively.
Melatonin and serotonin treatments resulted in modifying
mRNA accumulation moderately, except for some genes, e.g.
myosin and kinesin family members and platelet-derived growth
factors, implying that the melatonin and serotonin treatments may
remodel the intracellular matrix to a state inadequate for chlamydial intracellular growth (data not shown).
Melatonin, originally identified as an effector for circadian
rhythms,23 is now known as a hormone involved in a vast range
of homeostasis maintenance, i.e. human mood,24 seasonal timing,45
866
Serotonin and melatonin inhibit chlamydial infection
anti-ageing,46 oncostatic,47 antioxidant defence,27,48 and immunomodulation.49 Many of the melatonin effects seem to relate to
mitochondrial functions as a direct scavenger for a variety of toxic
oxygen and nitrogen-based reactants.50 Enhancements of the nonspecific and specific immune responses by exogenous melatonin
are reported as stimulation for proliferation/production of NK cells
and monocytes,51 and prevention from the reduction in B- and Tcell proliferation and Th1 cytokine secretions caused by retroviral
infection.52 Despite the many investigations on melatonin and
serotonin that have been carried out, they have not been generally
recognized as antimicrobial agents, apart from a few reports for
melatonin showing protective effects in mice against viral infections by Venezuelan equine encephalomyelitis virus,53 Semliki
Forest virus54 and West Nile virus,55 and against infection by a
helminth parasite, Schistosoma mansoni.56 Melatonin is used in
Mycobacterium tuberculosis infection to increase the chemotherapeutic activity of isoniazid (pyridine-4-carbohydrazide), which is
an inhibitor of tryptophan metabolism to NAD.57 So, the inhibition
of infection for the intracellular bacterium chlamydia is a new
function of melatonin and serotonin.
Variation of tryptophan biosynthesis genes is the most remarkable feature in the comparison of gene repertoires among chlamydial genomes. Considering that tryptophan depletion by IFN-g is a
crucial host defence mechanism against chlamydiae, it evokes all
evolutional relationships between strategies of host defence and
parasite infection, leading to chlamydial host and tissue tropisms.
There is an apparent paradox: C. trachomatis and C. pneumoniae,
worldwide common parasites to human but not to animals, contain
partial and no tryptophan biosynthesis genes in their genomes,
respectively, and they are susceptible to both IFN-g and melatonin;
on the other hand, C. felis, a worldwide common parasite of cats but
rarely humans, seems to possess an almost complete gene set for
tryptophan biosynthesis (Y. Azuma, H. Hirakawa, A. Yamashita,
Y. Cai, M. A. Rahman, H. Suzuki, S. Mitaku, H. Toh, T. Murakami,
K. Sugi, H. Hayashi, H. Fukushi, M. Hattori, S. Kuhara and
M. Shirai, unpublished data) and show marked resistance to
IFN-g, but not to melatonin. Whereas, tryptophan might play a
key role for the molecular basis of host–parasite interaction in
terms of IFN-g resistance, the gene repertoires of tryptophan
biosynthesis cannot entirely explain host tropisms of individual
chlamydial species. The different susceptibilities to melatonin or
serotonin, or any tryptophan derivatives may be involved in determination of the host tropisms. Different melatonin susceptibilities
among chlamydial species may depend on gene variations of the
chlamydial species responsible for host defence systems through
melatonin receptors. Genes discriminating C. trachomatis from
C. felis and C. pneumoniae consist of 69 genes, whose products
are annotated as 60 unknown proteins, a membrane thiol protease,
two transporters, two polymorphic outer membrane proteins and
four phospholipase D superfamily proteins. Although our studies
have provided evidence of possible anti-chlamydial functions of
melatonin and serotonin, further investigations are still needed to
determine the chlamydial factors that are involved in the various
diseases and host/tissue tropism.
Acknowledgements
This study was supported by the Japan Society for the Promotion of
Science, Research for the Future Program (JSPS-RETF 00L01411),
a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology of Japan
(KAKENHI: 14013043, 15019069, 15659172, 16012245,
16790255), the New Frontier Project of Yamaguchi University
School of Medicine, and the Venture Business Laboratory,
Yamaguchi University.
Supplementary data
Figure 2(b) is available in colour as online supplementary data at
http://jac.oxfordjournals.org/.
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