Inhibitory effects of 405 nm irradiation on Chlamydia trachomatis growth and characterization of the ensuing inflammatory response in HeLa cells.
ABSTRACT Chlamydia trachomatis is an intracellular bacterium that resides in the conjunctival and reproductive tract mucosae and is responsible for an array of acute and chronic diseases. A percentage of these infections persist even after use of antibiotics, suggesting the need for alternative treatments. Previous studies have demonstrated anti-bacterial effects using different wavelengths of visible light at varying energy densities, though only against extracellular bacteria. We investigated the effects of visible light (405 and 670 nm) irradiation via light emitting diode (LEDs) on chlamydial growth in endocervical epithelial cells, HeLa, during active and penicillin-induced persistent infections. Furthermore, we analyzed the effect of this photo treatment on the ensuing secretion of IL-6 and CCL2, two pro-inflammatory cytokines that have previously been identified as immunopathologic components associated with trichiasis in vivo.
C. trachomatis-infected HeLa cells were treated with 405 or 670 nm irradiation at varying energy densities (0 - 20 J/cm2). Bacterial growth was assessed by quantitative real-time PCR analyzing the 16S: GAPDH ratio, while cell-free supernatants were examined for IL-6 and monocyte chemoattractant protein-1 (CCL2) production. Our results demonstrated a significant dose-dependent inhibitory effect on chlamydial growth during both active and persistent infections following 405 nm irradiation. Diminished bacterial load corresponded to lower IL-6 concentrations, but was not related to CCL2 levels. In vitro modeling of a persistent C. trachomatis infection induced by penicillin demonstrated significantly elevated IL-6 levels compared to C. trachomatis infection alone, though 405 nm irradiation had a minimal effect on this production.
Together these results identify novel inhibitory effects of 405 nm violet light on the bacterial growth of intracellular bacterium C. trachomatis in vitro, which also coincides with diminished levels of the pro-inflammatory cytokine IL-6.
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ABSTRACT: The antimicrobial effects of 405nm light have generated interest in its use as an emerging disinfection technology with potential food-related applications. The aim of this study was to assess the bactericidal efficacy of 405nm light for inactivation of Escherichia coli and Listeria monocytogenes under sub-lethally stressed environmental conditions. Bacteria were exposed to 405nm light from a light emitting diode (LED) array under various temperature, salt (NaCl) and acid conditions to determine if bacterial susceptibility to 405nm light inactivation is affected when exposed under these conditions. Non-stressed bacterial populations (10(5)CFU/mL) were exposed to increasing doses of 405nm light (~70mW/cm(2)) and the inactivation results were compared with those generated under stress conditions. Bacteria were held at various temperatures (4°C, 22°C and 45°C), acid concentrations (pH3, 3.5 and 7) and salt concentrations (0%, 0.8%, 10% and 15% NaCl), and simultaneously exposed to 405nm light. Enhanced inactivation of both E. coli and L. monocytogenes was achieved when light exposure was combined with each of the sub-lethal stresses, with significantly increased inactivation rates compared to non-stressed populations (P≤0.05). One exception was with L. monocytogenes when light-exposed in the presence of 15% salt, as this combination reduced bacterial inactivation. The greatest enhancement of 405nm light inactivation for both bacterial species was achieved when light exposure was combined with sub-lethal acid stress conditions at pH3. This was demonstrated by a 5-log10 reduction of E. coli following a 405nm light dose of 84J/cm(2) compared to 378J/cm(2) for non-stressed populations (77% reduction in dose) and by a 5-log10 reduction of L. monocytogenes achieved with a dose of 42J/cm(2) which corresponded to 50% of the dose required for the equivalent reduction of non-stressed populations. This acid-enhanced 405nm light inactivation effect was demonstrated with E. coli and L. monocytogenes when dispersed in liquid suspension and when deposited on a test surface. Overall, results from this study have shown that sub-lethally stressed bacteria have increased susceptibility to 405nm light inactivation, thereby providing a synergistic inactivation effect, findings which increase the potential of this new light-based decontamination technology for food related applications.International journal of food microbiology 11/2013; 170C:91-98. · 3.01 Impact Factor
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ABSTRACT: Background Although the germicidal properties of ultraviolet (UV) light have long been known, it is only comparatively recently that the antimicrobial properties of visible violet–blue 405 nm light have been discovered and used for environmental disinfection and infection control applications. Aim To review the antimicrobial properties of 405 nm light and to describe its application as an environmental decontamination technology with particular reference to disinfection of the hospital environment. Methods Extensive literature searches for relevant scientific papers and reports. Findings A large body of scientific evidence is now available that provides underpinning knowledge of the 405 nm light-induced photodynamic inactivation process involved in the destruction of a wide range of prokaryotic and eukaryotic microbial species, including resistant forms such as bacterial and fungal spores. For practical application, a high-intensity narrow-spectrum light environmental disinfection system (HINS-light EDS) has been developed and tested in hospital isolation rooms. The trial results have demonstrated that this 405 nm light system can provide continuous disinfection of air and exposed surfaces in occupied areas of the hospital, thereby substantially enhancing standard cleaning and infection control procedures. Conclusion Violet–blue light, particularly 405 nm light, has significant antimicrobial properties against a wide range of bacterial and fungal pathogens and, although germicidal efficacy is lower than UV light, this limitation is offset by its facility for safe, continuous use in occupied environments. Promising results on disinfection efficacy have been obtained in hospital trials but the full impact of this technology on reduction of healthcare-associated infection has yet to be determined.Journal of Hospital Infection 09/2014; · 2.78 Impact Factor
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ABSTRACT: New therapeutic strategies are needed to overcome drawbacks in treatment of infections with intracellular bacteria. Chlamydiaceae are Gram-negative bacteria implicated in acute and chronic diseases such as abortion in animals and trachoma in humans. Water-filtered infrared A (wIRA) is short wavelength infrared radiation with a spectrum ranging from 780 to 1400 nm. In clinical settings, wIRA alone and in combination with visible light (VIS) has proven its efficacy in acute and chronic wound healing processes. This is the first study to demonstrate that wIRA irradiation combined with VIS (wIRA/VIS) diminishes recovery of infectious elementary bodies (EBs) of both intra- and extracellular Chlamydia (C.) in two different cell lines (Vero, HeLa) regardless of the chlamydial strain (C. pecorum, C. trachomatis serovar E) as shown by indirect immunofluorescence and titration by subpassage. Moreover, a single exposure to wIRA/VIS at 40 hours post infection (hpi) led to a significant reduction of C. pecorum inclusion frequency in Vero cells and C. trachomatis in HeLa cells, respectively. A triple dose of irradiation (24, 36, 40 hpi) during the course of C. trachomatis infection further reduced chlamydial inclusion frequency in HeLa cells without inducing the chlamydial persistence/stress response, as ascertained by electron microscopy. Irradiation of host cells (HeLa, Vero) neither affected cell viability nor induced any molecular markers of cytotoxicity as investigated by Alamar blue assay and Western blot analysis. Chlamydial infection, irradiation, and the combination of both showed a similar release pattern of a subset of pro-inflammatory cytokines (MIF/GIF, Serpin E1, RANTES, IL-6, IL-8) and chemokines (IL-16, IP-10, ENA-78, MIG, MIP-1α/β) from host cells. Initial investigation into the mechanism indicated possible thermal effects on Chlamydia due to irradiation. In summary, we demonstrate a non-chemical reduction of chlamydial infection using the combination of water-filtered infrared A and visible light.PLoS ONE 07/2014; 9(7):e102239. · 3.53 Impact Factor
RESEARCH ARTICLEOpen Access
Inhibitory effects of 405 nm irradiation on
Chlamydia trachomatis growth and
characterization of the ensuing inflammatory
response in HeLa cells
Cassandra J Wasson1,5, Jessica L Zourelias1,6, Nathan A Aardsma2,7, Janis T Eells3, Mike T Ganger1,
Justine M Schober4and Troy A Skwor1,8*
Background: Chlamydia trachomatis is an intracellular bacterium that resides in the conjunctival and reproductive
tract mucosae and is responsible for an array of acute and chronic diseases. A percentage of these infections persist
even after use of antibiotics, suggesting the need for alternative treatments. Previous studies have demonstrated
anti-bacterial effects using different wavelengths of visible light at varying energy densities, though only against
extracellular bacteria. We investigated the effects of visible light (405 and 670 nm) irradiation via light emitting
diode (LEDs) on chlamydial growth in endocervical epithelial cells, HeLa, during active and penicillin-induced
persistent infections. Furthermore, we analyzed the effect of this photo treatment on the ensuing secretion of IL-6
and CCL2, two pro-inflammatory cytokines that have previously been identified as immunopathologic components
associated with trichiasis in vivo.
Results: C. trachomatis-infected HeLa cells were treated with 405 or 670 nm irradiation at varying energy densities
(0 – 20 J/cm2). Bacterial growth was assessed by quantitative real-time PCR analyzing the 16S: GAPDH ratio, while
cell-free supernatants were examined for IL-6 and monocyte chemoattractant protein-1 (CCL2) production. Our
results demonstrated a significant dose-dependent inhibitory effect on chlamydial growth during both active and
persistent infections following 405 nm irradiation. Diminished bacterial load corresponded to lower IL-6
concentrations, but was not related to CCL2 levels. In vitro modeling of a persistent C. trachomatis infection
induced by penicillin demonstrated significantly elevated IL-6 levels compared to C. trachomatis infection alone,
though 405 nm irradiation had a minimal effect on this production.
Conclusion: Together these results identify novel inhibitory effects of 405 nm violet light on the bacterial growth
of intracellular bacterium C. trachomatis in vitro, which also coincides with diminished levels of the
pro-inflammatory cytokine IL-6.
Keywords: 405 nm, Chlamydia trachomatis, IL-6, CCL2, Phototherapy
* Correspondence: firstname.lastname@example.org
1Biology Department, Gannon University, Erie, PA 16541, USA
8Present address: Department of Chemical and Biological Sciences, Rockford
College, 5050 E. State St, Rockford, IL 61108, USA
Full list of author information is available at the end of the article
© 2012 Wasson et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Wasson et al. BMC Microbiology 2012, 12:176
Trachoma continues to be the most common cause of
preventable blindness worldwide. It has been estimated
to visually impair between two and nine million people
globally, although this may be an underestimate due to
the lack of screening programs in endemic areas .
One of the etiologic agents is the obligate intracellular
bacterium Chlamydia trachomatis , which is also the
leading bacterial cause of sexually transmitted infections
(STI) worldwide. These reproductive infections can lead
to clinical symptoms such as urethritis, cervicitis, and
pelvic inflammatory disease [3,4]. The ability of C. tra-
chomatis to evade the immune system (reviewed in )
results in 70-90% of infected women and 30-50% of
infected men being asymptomatic . Due to repeated
or persistent infections, or an absence of antibiotic treat-
ment, ocular and reproductive tract sequelae can de-
velop, resulting in corneal pacification and salpingitis
C. trachomatis has a unique biphasic life cycle involv-
ing both elementary and reticulate bodies. Elementary
bodies (EBs) represent a metabolically inactive infectious
phenotype capable of attaching to epithelial cells with
subsequent internalization resulting in the formation of
an inclusion body. Once inside the inclusion, the EB dif-
ferentiates into a metabolically active reticulate body
(RB) that multiplies via binary fission. As the inclusion
grows, the RBs reorganize into EBs that are released
from the host cell and can infect adjacent cells. These
varying bioforms make treatment of chlamydial infec-
tions difficult. Furthermore, antibiotic therapies, expos-
ure to IFNγ, or nutrient deprivation can lead to an
atypical, persistent, non-cultivable, and morphologically
aberrant intracellular state (reviewed in ).
Chlamydial infections in the conjunctiva and genitalia
can incite an intense inflammatory response that, if
chronic, can lead to scarring and fibrosis. Numerous
pro-inflammatory cytokines, including TNFα, IL-1α, IL-
6 and IL-18 [8,9], as well as a group of chemokines
[8,10,11] responsible for the recruitment of leukocytes
have been shown to be secreted from C. trachomatis-
infected epithelial cells. This arsenal of cytokines and
chemokines with incoming leukocytes results in the
stimulation of both cellular- and humoral-mediated im-
mune defenses. The type of host inflammatory response
that is initiated with the infection determines the out-
come of the infection. The current hypothesis is that
resolution is mediated primarily by a dominant cell-
mediated Th1 response, whereas chronic inflammation
with subsequent scarring ensues if either the humoral
(reviewed in ).
Development of chronic chlamydial infections is
believed to arise as a result of immunopathology. This
has been demonstrated by persistent elevation of pro-
inflammatory cytokines like IL-6 among infertile women
 and in tear fluid from post-scarring trachoma popu-
lations . One study identified IL-6 secretion via the
TLR2 signaling pathway after C. trachomatis infections
. This TLR2 pathway has been shown to be asso-
ciated with fallopian-tube pathology, potentially contrib-
uting to the immunopathogenesis associated with C.
trachomatis infection . The chemokine monocyte
chemoattractant protein-1 (CCL2) has also been identi-
fied in chronic chlamydial infections demonstrating ele-
vated levels in post-scarring trachoma populations .
Due to the high prevalence of worldwide trachoma,
the World Health Organization (WHO) established and
supports the use of the SAFE (surgery, antibiotics, facial
cleanliness, and environmental improvements) strategy
to reduce disease transmission in endemic areas. Mass
antibiotic therapy has been a mainstay in this program
resulting in diminished prevalence of active chlamydial
infections [15-17]. However, heightened recurrence rates
of infection 6-24 months after termination of antibiotic
therapy were evident in multiple studies [18-21]. Add-
itionally, Burnham et al. saw an increase in chlamydia-
associated STI Reinfection after a control program with
antibiotic treatment was established . The mass ad-
ministration of antibiotics may lead to the development
of antibiotic resistance in chlamydial species as well as
other pathogenic bacteria. It is apparent that research
into alternative treatments is warranted, and the use of
phototherapy may be an attractive option.
Phototherapy utilizing low power lasers or light emit-
ting diodes (LEDs) has been shown to reduce pain and
chronic inflammation, and to promote tissue regener-
ation via a photochemical mechanisms (reviewed in ).
Additionally, anti-bacterial effects due to the increased
production of reactive oxygen species resulting in mem-
brane instability and DNA damage have been evident
with phototherapy [23-27]. Its use with several discrete
wavelengths exhibits anti-bacterial activity requiring
short treatment times without inducing anti-bacterial
resistance subsequent to multiple treatment sessions .
In this study, we analyzed the effect of low-level
405 nm and 670 nm LED irradiation on the growth of
C. trachomatis and the ensuing secretion of pro-
inflammatory cytokines IL-6 and CCL2 from C. tracho-
matis-infected epithelial cells.
Inhibition of chlamydial growth post - 405 nm irradiation
This study assessed the use of 405 nm and 670 nm LEDs
as an alternative treatment against chlamydial infections.
In Figure 1A, HeLa cells were infected with C. tracho-
matis at a multiplicity of infection (MOI) of 5. Irradi-
ation treatment with violet 405 nm LEDs demonstrated
Wasson et al. BMC Microbiology 2012, 12:176
Page 2 of 10
chlamydial growth inhibition at energy densities as low
as 5 J/cm2(Figure 1B, P<0.005). Anti-chlamydial prop-
erties were dose dependent with the strongest effect vis-
ible at 20 J/cm2(Figure 1B, P<0.005). Fluorescent
staining (FITC) for chlamydial antigens supported an
impairment of chlamydial growth and inclusion body ex-
pansion after 405 nm exposure (Figure 2D-F, 20 J/cm2)
(Figure 2A-C). We also analyzed the effect of irradiation
application time post-infection to determine if it was
growth phase specific. At 24 h post-infection, irradiation
with 405 nm (20 J/cm2) LEDs still demonstrated a sig-
nificant growth inhibition (Figure 1B, P<0.005). C. tra-
chomatis-infected cells treated with red 670 nm LEDs at
similar energy densities (5-20 J/cm2) showed no signifi-
cant effect on growth (data not shown).
Considering many chronic chlamydial infections are in
a persistent stage of growth, we tested the effect of
405 nm on chlamydial growth after penicillin-induced
persistence. As shown in Figure 1C, 405 nm retarded
chlamydial growth during a persistent state (P<0.05) at
20 J/cm2, though the result was not as pronounced as it
was in the active state. Once again, no effect was seen
with 670 nm treatment (data not shown).
The effect of 405 nm irradiation on IL-6 production in
C. trachomatis-infected HeLa cells
Previous studies have identified IL-6 as a pro-inflammatory
cytokine associated with immunopathologic effects in
chronic C. trachomatis infections [12,13]. In this study,
we demonstrated elevated IL-6 levels post-chlamydial
infection compared to uninfected cells (Figure 3A,
P<0.005). C. trachomatis infected HeLa cells were
treated with varying 405 nm energy densities (5-20 J/
cm2) promptly after infection. Irradiation with 405 nm
at energy densities of 5, 10, and 20 J/cm2diminished
IL-6 secretion in a dose-dependent manner 48 h
post-C. trachomatis infection when compared to C.
Figure 1 Effects of 405 nm irradiance on chlamydial growth in
HeLa cells. (A) HeLa cells were infected with C. trachomatis serovar
E at a MOI of 5. (B) Infected cells were then exposed to varying
doses of 405 nm at a range of energy densities (5-20 J/cm2) either
promptly after infection or 24 h post-infection (24 h post).
Treatments are grouped based on post-hoc comparisons for
convenience. The effect of 405 nm on chlamydial growth was
assessed during active and persistent stages induced with penicillin
(B and C). Growth was determined using quantitative real-time PCR
to determine the ratio of chlamydial and eukaryotic housekeeping
genes (16S: GAPDH respectively) 48 h post-infection on cDNA
reverse transcribed from RNA. Mean ± standard deviation are
plotted for the two replicated experiments. Statistical significance
was determined post-hoc using a Bonferonni adjustment comparing
all groups against C. trachomatis-infected HeLa cells alone (CTE);
* P<0.05, ** P<0.005.
Wasson et al. BMC Microbiology 2012, 12:176
Page 3 of 10
P<0.05, and P<0.005 respectively). Considering the
potential for clinical therapies, we tested whether the
effect of this phototherapy was dependent upon the
405 nm application time post-chlamydial infection. If
applied 24 h post-infection rather than two hours, the
significant 405 nm effect on IL-6 was lost (Figure 3B).
Due to the elevated levels of IL-6 with chlamydia-
induced chronic grades of disease, we determined
whether penicillin-induced persistence of a C. trachoma-
tis infection in vitro would mimic the above clinical in-
flammatory signs. We demonstrated that persistence
induction by penicillin significantly increased IL-6 pro-
duction compared to C. trachomatis infection alone
(Figure 3C, P<0.05). The absence of IL-6 production
above mock-infected levels from HeLa cells stimulated
with 200 U/ml of penicillin alone indicates this effect
was not cumulative (data not shown). No significant
effects were evident on IL-6 production after 405 nm
(Figure 3C) or 670 nm (data not shown) irradiation in
this penicillin-induced persistent state.
infectionalone (Figure 3B,P<0.05,
The effect of 405 nm irradiation on CCL2 production in
C. trachomatis infected HeLa cells
Due to the involvement of CCL2 with acute and chronic
grades of chlamydial infections [13,29] and its associ-
ation with a Th2-mediated response , we evaluated
the effect of 405 nm photo treatment on its production.
In Figure 4, C. trachomatis infection increased produc-
tion of CCL2 in HeLa cells relative to uninfected cells
(Figure 4A, P<0.05). Though a diminishing pattern was
evident for CCL2 production with increasing 405 nm
energy densities (Figure 4B), 405 nm treatment failed to
demonstrate any significant difference in CCL2 produc-
tion compared to C. trachomatis infection alone. Unlike
IL-6, penicillin-induced C. trachomatis persistence did
not significantly influence CCL2 levels (Figure 4C)
Multiple studies have demonstrated inadequate long-
term protection of azithromycin for treating trachoma-
tous trichiasis [19-21]. Suboptimal efficacy of antibiotics
was also evident amongst a chlamydia-associated react-
ive arthritis population where persistent chlamydial bod-
ies were identified in fibroblasts and macrophages one
month after doxycycline treatment . Further support
for the poor antibiotic efficacy against chronic C. tracho-
matis infections was demonstrated in a population of
remained infected despite antibiotic treatment . Uro-
genital chlamydial re-infections have been identified as
probable treatment failure with azithromycin or doxy-
cycline using ompA genotyping in approximately 8 and
Figure 2 Anti-chlamydial properties of 405 nm irradiance. (A-C) HeLa cells were infected with C. trachomatis serovar E at a MOI of 5 without
exposure to photodiodes. (D-F) Infected cells were exposed to 405 nm LEDs at 20 J/cm2promptly after infection to evaluate anti-chlamydial
effects during an acute chlamydial infection. Cells were fixed and stained with dapi (blue) (B and E) and anti-chlamydial (green) (C and F)
antibody 48 hours post-infection. Bar=10μm.
Wasson et al. BMC Microbiology 2012, 12:176
Page 4 of 10
13.7% of cases [33,34]. Together the suboptimal efficacy
of therapeutic antibiotics in the treatment of active and
persistent chlamydial infections indicates the need for al-
One potential alternative treatment utilizing 405 nm
irradiation was evaluated in this study and demonstrated
photo inactivation of C. trachomatis during active and
persistent states. Small dosages starting at 5 J/cm2had a
significant growth inhibiting effect, with increasing en-
ergy densities positively correlating with growth inhib-
ition. Therapeutic utility and clinical safety have been
described using LED phototherapy at 405 nm against
acne vulgaris  and gastric Helicobacter pylori infec-
tions, with the latter applied to the gastrointestinal mu-
cosa via a light wand . In vitro anti-bacterial activity
of 405 nm irradiation has been demonstrated against
multiple medically relevant Gram-positive and Gram-
negative extracellular pathogens like Staphylococcus aur-
eus (including methicillin-resistant
Streptococcus pyogenes, Pseudomonas aeruginosa, Clos-
tridium perfringens, Campylobacter jejuni, Salmonella
enteritidis and Escherichia coli [24,37]. Overall Gram-
negative bacteria appear more resistant to 405 nm irradi-
ation than Gram-positive, with the exception of Enterococcus
faecalis. The majority of Gram-positive bacteria appeared
to require less than 11 J/cm2for a log10 reduction in
colony forming units, whereas Gram-negative bacterioci-
dal effects were apparent between 25 and 96 J/cm2.
The portable LEDs used in this study were battery oper-
ated with 88 second dosing times, therefore making it
difficult to obtain higher energy densities. Comparing
the log10 reduction levels of other Gram negative
bacteria with C. trachomatis is difficult due to its intra-
cellular nature and considering it may exist as two-
uncultivable life cycle forms: RBs and aberrant persistent
forms. After irradiation with an energy density of 20 J/cm2
we demonstrated a nearly 70% reduction in chlamydial
growth, reflecting levels similar to other Gram-negative
bacteria. To our knowledge, this is the first data de-
monstrating chlamydial growth inhibition caused by
405 nm irradiation.
Figure 3 Effect of 405 nm on IL-6 production in C. trachomatis-
infected epithelial cells. (A) HeLa cells were infected with C.
trachomatis serovar E at a MOI of 5 (CTE5). (B) Infected cells were
then exposed to varying doses of 405 nm at a range of energy
densities (5-20 J/cm2) either promptly after infection or 24 h post-
infection (post-24 h). The effect of 405 nm on IL-6 production was
assessed during active (A and B) and penicillin-induced persistent
stages (C). Supernatants were collected and measured for IL-6
production using an ELISA. Treatments are grouped based on post-
hoc comparisons for convenience. Mean ± SEM are plotted for the
two replicated experiments. Statistical differences were determined
post-hoc using a Bonferonni adjustment comparing all groups to C.
trachomatis infected cells (CTE); *, P<0.05; ** P<0.005.
Wasson et al. BMC Microbiology 2012, 12:176
Page 5 of 10
Photo inactivation by 405 nm irradiation is believed to
be caused by excitation of androgenic porphyrins, result-
ing in oxygen free radical production and subsequent
bacterial membrane disruption . Endogenously pro-
duced porphyrins, like coproporphyrin, uroporphyrins,
and protoporphyrin IX, have been shown to be produced
by both Gram positive and negative bacteria [25,39]
though, to our knowledge, porphyrin production by C.
trachomatis has not yet been demonstrated. Considering
the intracellular nature of C. trachomatis, a second
photo inactivation mechanism might be associated with
altered expression of eukaryotic proteins in response to
405 nm irradiation. Boncompain et al. demonstrated a
transient upregulation of reactive oxygen species within
C. trachomatis-infected HeLa cells for approximately six
hours after infection, with subsequent basal levels ensu-
ing nine hours post-infection. The regulation of reactive
oxygen species appears to be mediated by C. trachomatis
sequestration of the NADPH oxidase subunit, Rac1, to
the inclusion membrane . Considering the significant
growth inhibition effect when 405 nm was applied
promptly two hours post-infection rather than 24 h, the
irradiation might have altered chlamydial protein expres-
sion thus influencing its ability to sequester host Rac1,
thereby increasing reactive oxygen species within the
epithelial cells. An alteration in protein expression may
have also delayed the formation and secretion of bacter-
ial type III effector proteins, such as CPAF, that have
previously been shown to be involved in binding and de-
grading eukaryotic proteins like cytokeratin 8, adhesion
protein nectin-1, host transcription factor RFX5, and
multiple host pro-apoptotic BH3 proteins [41-44]. Alter-
natively, the lack of 405 nm photo inactivation effect on
chlamydial growth at 24 h post-infection might be due
to the exponentially higher bacterial burdens within the
inclusion body 24 h post-infection relative to two hours
post-infection, potentially causing the differences after
treatment to be less pronounced. It is also possible that
multiple mechanisms co-exist and provide a cumulative
anti-proliferative effect on C. trachomatis, though fur-
ther studies are warranted.
Figure 4 Effect of 405 nm on CCL2 production in C.
trachomatis-infected epithelial cells. (A) HeLa cells were infected
with C. trachomatis serovar E at a MOI of 5 (CTE5). (B) Infected cells
were then exposed to varying doses of 405 nm at a range of energy
densities (5-20 J/cm2) either promptly after infection or 24 h post-
infection (post-24 h). The effect of 405 nm on CCL2 was assessed
during active (B) and persistent stages induced with penicillin (C).
Supernatants were collected and measured for CCL2 production
using an ELISA. Treatments are grouped based on post-hoc
comparisons for convenience. Mean ± SEM are plotted for the two
replicated experiments. Statistical differences were determined post-
hoc using a Bonferonni adjustment comparing all groups to C.
trachomatis infected cells (CTE); *, P<0.001.
Wasson et al. BMC Microbiology 2012, 12:176
Page 6 of 10
The immunopathologic sequelae from conjunctival and
genital chlamydial infections are likely mediated through
the secretion of a group of pro-inflammatory cytokines. In
trachoma, we demonstrated elevated levels of IL-6 during
both acute and chronic grades of infection, with detectable
chlamydial cases exhibiting more pronounced concen-
trations . The role of IL-6 in immunopathologenesis
was also evident in women with ectopic pregnancies
 and positively correlated with antibody titers against
patients . In an attempt to mimic chronic chlamydial
infections, Macaca nemestrina fallopian tubes received
repeated C. trachomatis infections, which resulted in
fibrosis and elevated IL-6, IL-10, IL-2, and IFNγ levels .
In TLR2 -/- KO mice infected with mouse pneumonitis
(MoPn), decreased fibrosis and inflammation with in
oviducts and mesosalpinx correlated with abated IL-6
concentrations . To determine the immunologic
correlation of persistence in vitro with clinical presentation,
we quantified IL-6 in penicillin-induced C. trachomatis
persistent infections in HeLa cells. We demonstrated simi-
lar increases in IL-6 production in persistent infections
compared to active infections in vitro. A previous study
looked at persistent infections with C. pneumoniae in the
presence of iron-depletion, IFNγ and penicillin, and
demonstrated slightly diminished production of IL-6 after
24 h and 48 h . However, multiple experimental differ-
ences between these studies, including the use of different
chlamydial species, might provide an explanation for the
differences in results. For example, Peters et al. added
penicillin 30 min after infection, followed by daily
media change. This is in contrast to our study which
added penicillin 24 h post-infection without a daily
media change. Wang et al. provided more molecular
details of this persistent state, demonstrating attenuated
production of secreted chlamydial proteins from ampicillin-
induced persistence of C. trachomatis infected HeLa cells
, suggesting that secreted type III effector proteins like
CPAF , Tarp , CT311 , and CT795  may be
involved in regulating IL-6 levels. We are unaware of any
other studies that examine inflammatory differences
associated with penicillin-induced persistence. The eleva-
tion of IL-6 after penicillin-induced persistence supports
the importance of this model in elucidating other inflam-
matory mediators that may be associated with chronic
infections in vivo. Further research on molecular charac-
terizations and their immunostimulatory properties is
needed to understand this in vitro antibiotic-induced
Considering the immunopathologic response to chro-
nic chlamydial infections, we were interested in deter-
mining the role of 405 nm irradiation on cytokines
previously associated with immunopathogenesis. In our
study, irradiation with 405 nm had a dose-dependent
effect on IL-6 production with 20 J/cm2causing a 64%
reduction. However, this effect was most likely asso-
ciated with a decreased bacterial burden since previous
studies demonstrated elevated IL-6 from UV-A (340-
450 nm) exposed fibroblasts [53,54] and minimal effects
of UV-A (1 J/cm2) treated keratinocytes on IL-6 produc-
tion . Interestingly, attenuation of IL-6 after 405 nm
treatment was only evident if 405 nm irradiation was ap-
plied promptly after infection; the effect was lost if ap-
plied 24 h post-infection. We believe that at this later
time point, multiple chlamydial proteins were already
secreted by type III secretory pathways into the host
cytoplasm and interacted with pattern recognition
receptors (PRRs) resulting in IL-6 production.
Previously, we have identified CCL2 as a risk factor for
trichiasis , and therefore analyzed the effect of
405 nm irradiation on C. trachomatis induced CCL2
production. To our knowledge, our findings are the first
to demonstrate elevated levels of CCL2 after C. tracho-
matis infection in HeLa cells. In vivo analysis has shown
elevated mRNA levels of CCL2 at two days post-
(MoPn) strain . Unlike IL-6, the use of 405 nm
phototherapy on C. trachomatis infected HeLa cells did
not have a significant effect on CCL2 production. More
studies are needed to further understand the relationship
between C. trachomatis infection and CCL2 production
resulting in these inflammatory differences.
With increasing evidence to support persistent infec-
tions amongst a percentage of chlamydial infections
post-antibiotic treatment [18-21,32-34], it is important
to look for alternative treatments. In this study, we have
provided the first in vitro evidence for anti-bacterial
effects against an intracellular bacterium, C. trachomatis,
using 405 nm irradiation administered by portable LEDs.
The reduction in bacterial numbers and IL-6 concentra-
tions, and the clinical safety of 405 nm irradiation, sup-
ports further studies evaluating its use as a phototherapy
against chlamydial infections within the conjunctival and
reproductive tract mucosae. The ability of photo treat-
ment to penetrate mucosal tissue layers was demon-
strated within the gastric mucosa against Helicobacter
pylori using 408 nm light . Together, these data
provide a plausible alternative treatment against chla-
mydial infections and expands the anti-bacterial prop-
erties of 405 nm irradiation to include intracellular
Cell line and bacterial stock
Human cervical adenocarcinoma cell line HeLa 229
(HeLa) and C. trachomatis serovar E were kindly
Wasson et al. BMC Microbiology 2012, 12:176
Page 7 of 10
provided by Dr. Deborah Dean (Children’s Hospital
Oakland Research Institute, Oakland, CA) and were
used following previous protocols [56,57]. HeLa cells
were cultured and maintained in minimal essential
medium (MEM; Sigma Aldrich Corp., St. Louis, MO)
lacking phenol red and supplemented with 2 mM L –
glutamine, 1 mM sodium pyruvate and 10% fetal bovine
Chlamydia trachomatis serovar E reference strain was
propagated in HeLa cells as previously described .
Elementary bodies (EB) were isolated after homo-
genization with subsequent gradient ultracentrifugation
and resuspended in 0.25 M sucrose, 10 mM sodium
phosphate and 5 mM L-glutamic acid (pH 7.2), and
stored at -80°C. Determination of inclusion forming
units (IFUs) was performed as previously described
using fluorescent microscopy .
Establishment of active and persistent C. trachomatis
HeLa cells were cultured at 1 x 105cells/ml in 6-well tis-
sue culture plates and incubated for 20-24 h at 37°C+5%
CO2prior to infection. Cells were then infected with C.
trachomatis serovar E at a multiplicity of infection (MOI)
of 5 (CTE5) in sucrose-phosphate-glutamate (SPG) buffer
(220 mM sucrose, 3.8 mM KH2PO4,10 mM Na2HPO4,
5 mM glutamate, 10 μg/ml gentamicin [MP Biomedical],
100 μg/ml vancomycin [Across Organics, Morris Plains,
NJ], and 25 U/ml nystatin [MP Biomedical] at pH 7.4) or
mock-infected with SPG alone for two hours while on an
orbital shaker. Media was then aspirated, washed, and
replacedwith C-MEM. Persistent
induced 24 h post-infection by the addition of 200 U/ml
of penicillin G (Sigma Aldrich Corp.).
Photo treatment of C. trachomatis-infected cells
405 nm and 670 nm were emitted from a WARP 10W
LED (Quantum Devices, Inc., Barneveld, WI) with an ir-
radiance of 60 mW/cm2delivering 5 J/cm2in an 88 sec-
ond dosing time within a 10 cm2area. Measurements
were performed by a Gigahertz-Optic Integrate Sphere
with a BTS256 - LED tester (Gigahertz-Optic, Turken-
feld, Germany) following LED standards set by the Na-
trachomatis-infected cells were exposed to 0, 5, 10, or
20 J/cm2of 405 nm or 670 nm LEDs as previously
described  at 2 h or 24 h post-infection. Infected
cells not exposed to 405 nm or 670 nm LED and unin-
fected cells mock infected with SPG alone were per-
formed on separate plates to ensure no LED exposure.
Quantification of IL-6 and CCL2
Supernatants were harvested at 48 h post-infection and
centrifuged 16,000 x g in a micro centrifuge to remove
all bacterial and cellular debris. Cell-free supernatants
were frozen at -80°C until further analyzed. Undiluted
supernatants were quantified for IL-6 and CCL2 using
ELISA Ready-SET-GoWplates following manufacturer’s
protocol (eBioscience, Inc., San Diego, CA). Standard
curves were performed with seven two-fold serial dilu-
tions (IL-6: 3.12 – 200 pg/ml; CCL2: 16.2 – 1000 pg/ml
with the respective recombinant human IL-6 or CCL2)
and used to determine sample concentrations.
RNA isolation, reverse transcription and real-time PCR
RNA was isolated from adherent cells, as well as the cell
pellet from supernatants post-centrifugation as men-
tioned above, using the QIAshredder and RNeasy kit
(Qiagen, Valencia, CA) following manufacturer’s instruc-
tions. RNA was then treated with DNase (Promega,
Madison, WI) to digest any contaminating genomic
DNA and reverse transcribed with script cDNA synthe-
sis reagents (Bio-Rad, Hercules, CA). Negative controls
were included that were not exposed to reverse tran-
scriptase. SYBRWGreen PCR Master Mix (Applied Bios
stems, Carlsbad, CA) amplified the cDNA with the fol-
AACAGCGACACCCACTCCTC – 3’, GAPDH reverse
5’ –CATACCAGGAAATGAGCTTGACAA– 3’, chla-
mydia 16 F 5’ – TCGAGAATCTTTCGCAATGG AC –
3’, and chlamydia 16R 5’ – CGCCCTTTACGCCCAA
TAAA – 3’ as previously described [59,60]. Arbitrary
units were assigned using standard curves with five 1:3
serial dilutions for each target gene. Samples were
reported as ratios of 16S: GAPDH.
Immunocytochemistry and microscopy
C. trachomatis-infected HeLa cells with or without
405 nm were fixed with ice-cold methanol for 10 min.
After aspiration, culture wells were washed with PBS
and then stained with rabbit anti-C. trachomatis EBs
(Virostat, Portland, ME) for 1 h. Wells were washed five
times with PBS and counterstained with 4’, 6-diamidino-
2’-phenylindole, dihydrochloride (Dapi; Thermo Scien-
tific, Rockford, IL) for 10 min. Photos were obtained
using the Olympus IX51 Fluorescent Microscope with
differential interference contrast (DIC) filters.
Due to different light intensities used for the 405 nm
and 670 nm experiments, data were analyzed separately.
In addition, both the replicated 405 nm and 670 nm
experiments were repeated and therefore variation was
partitioned between the separate experiments using a
blocking factor . Separate one-factor analyses of vari-
ance (ANOVA) were used to determine if 16S: GAPDH
ratio, IL-6, and CCL2 production varied with treatment.
Wasson et al. BMC Microbiology 2012, 12:176
Page 8 of 10
For 405 nm treatments, post-hoc contrasts consisted of
comparing C. trachomatis infected cells with uninfected
cells and also examining C. trachomatis-infected cells
exposed to different 405 nm densities (5-20 J/cm2). Add-
itionally, penicillin-induced C. trachomatis infection was
compared to C. trachomatis infected HeLa cells alone
and penicillin-induced C. trachomatis infection with
405 nm treatment. The Bonferonni method (40) was
used to establish a critical P- value.
LED: Light emitting diodes; EBs: Elementary bodies; RBs: Reticulate bodies;
CCL-2: Monocyte chemoattractant protein-1; CTE5: Chlamydia trachomatis
serovar E MOI 5; SPG: Sucrose-phosphate-glutamate buffer.
The authors declare that they have no competing interests.
CJW and JLZ: performed the experiments, acquired, analyzed and interpreted
the data, and drafted the manuscript. NAA and MTG: made substantial
contributions to the conception and design of experiments, interpretation of
results, and drafted and critically revised the manuscript. JTE and JMS: made
substantial contributions to the conception and design of experiments,
interpretation of results, and critically revised the manuscript. TAS: performed
the experiments, acquired, analyzed and interpreted the data, drafted and
critically revised the manuscript. All authors read and approved the final
This work was supported by the Lake Erie College of Osteopathic Medicine
(LECOM) and the Lake Erie Consortium for Osteopathic Medical Training
Grant (TS, NA, JS). It was also funded by James J. Duratz Undergraduate
Student Research Awards (JZ, CW) and a Faculty Research Grant (TS) through
Gannon University, and a research grant from the Beta Beta Beta Research
We would like to thank Sean Beckmann and Naraporn Somboonna for their
review of the manuscript, as well as Ashley Wimer for her assistance in the
1Biology Department, Gannon University, Erie, PA 16541, USA.2Lake Erie
College of Osteopathic Medicine, Erie, PA 16509, USA.3Department of
Biomedical Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI
53201, USA.4Department of Urology, University of Pittsburgh Medical Center
Hamot, Erie, PA 16507, USA.5Present address: Midwestern University,
Downers Grove, IL 60515, USA.6Present address: University of Buffalo, Buffalo,
NY 14260, USA.7Present address: Department of Pathology, University of
Illinois at Chicago, Chicago, IL 60612, USA.8Present address: Department of
Chemical and Biological Sciences, Rockford College, 5050 E. State St,
Rockford, IL 61108, USA.
Received: 4 May 2012 Accepted: 13 August 2012
Published: 15 August 2012
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Cite this article as: Wasson et al.: Inhibitory effects of 405 nm irradiation
on Chlamydia trachomatis growth and characterization of the ensuing
inflammatory response in HeLa cells. BMC Microbiology 2012 12:176.
Wasson et al. BMC Microbiology 2012, 12:176
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