Journal of Photochemistry and Photobiology B: Biology 105 (2011) 175–182

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

Enhancement of cutaneous immune response to bacterial infection after

low-level light therapy with 1072 nm infrared light: A preliminary study
Seung Yoon Celine Lee a,b,⇑, In-Wha Seong a, Ji-Seon Kim a, Kyeong-A. Cheon a, Se Hun Gu a,
Hee Hwan Kim c, Ki Ho Park d
a
Department of Microbiology, Korea University Medical School, 126-1, 5-Ga, Anam-Dong, Seongbuk-Gu, Seoul 136-705, South Korea
b
Hayan-nara Dermatology Group, 142-3, Sankok-dong, Bupyeong-ku, Incheon, South Korea
c
Department of Pathology, Korea University Medical School, 126-1, 5-Ga, Anam-Dong, Seongbuk-Gu, Seoul 136-705, South Korea
d
Quantitative Real-Time PCR Lab, Clinical Research Institute, Seoul National University Hospital, 28, Yeongun-dong, Jongno-ku, Seoul, South Korea

a r t i c l e i n f o a b s t r a c t

Article history: We investigated the photobiomodulation effects of 1072 nm infrared light on the natural immune
Received 8 April 2011 response involved in anti-bacterial and wound healing processes. Thirty mice infected with MRSA on
Received in revised form 30 August 2011 the skin were divided into two groups. The experimental group was treated with 1072 nm infrared light
Accepted 30 August 2011
(irradiance: 20 mW/cm2, fluence: 12 J/cm2 for 10 min) at 2, 4, 8, 12, 24 h, 3 and 5 days after inoculation
Available online 6 September 2011
and the control group with sham light. Serial changes of the mRNA levels of TLR2, IL-1b, TNF-a, IL-6, iNOS,
MCP-1, TGF-b, bFGF and VEGF were studied by real time RT-PCR and those of the expression level of
Keywords:
VEGF, bFGF, TGF-b and NF-jB by immunohistochemistry. The mRNA levels of the cytokines involved
Photobiomodulation
1072 nm
in the early phase of anti-bacterial immune response (IL-1b, TNF-a, IL-6, MCP-1) increased significantly
Low level light therapy in the 1072 nm group, peaking between 12 and 24 h post-inoculation. These levels normalized after 3–
LED 5 days. Immunohistochemistry revealed a notably stronger expression of VEGF in the 1072 nm group
Antibacterial from 8-h post-inoculation to 5-day post-inoculation. We concluded that 1072 nm infrared light had a
VEGF photobiomodulation effect which resulted in an enhanced biological immune response to the bacterial
infection by MRSA and also increased the expression of VEGF to a significant level.
Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction tive for the treatment of Herpes Simplex Labialis (HSL), the com-
mon vesicular eruptive disease around the lips caused by Herpes
Photobiomodulation is the process where incident photons of Simplex Virus (HSV) [9,10]. In an in vitro study, this particular wave-
light are absorbed by chromophores of living tissue, for example length has been proven to have a cytoprotective effect for lympho-
cytochrome C oxidase in the respiratory electron transport chain cytes that were exposed to insult of ultraviolet irradiation [11].
of mitochondria, which results in modulation of various cell func- Recently, a pilot study conducted in our laboratory showed that,
tions [1–3]. It has been demonstrated by many in vitro studies that in nude mice infected with Methicillin-resistant Staphylococcus aur-
photobiomodulation through low level light/laser therapy (LLLT) eus (MRSA), irradiation with 1072 nm infrared light shortened the
in the waveband of 600–1000 nm can significantly stimulate cellu- time duration for the infected wound to heal completely (unpub-
lar proliferation and cellular activities in a variety of cell lines, such lished data). We also found that direct irradiation of 1072 nm over
as keratinocytes, epithelial cells, skeletal muscle cells, and various S. aureus colonies did not show a reduction of the colony count,
dermal cells that are involved in the wound healing process includ- which suggested that this shortened healing time of the infection
ing fibroblasts, macrophages, and mast cells [3–8]. Furthermore, might involve participation of immune cell functions rather than
LLLT has been proven to facilitate the chemotactic and phagocytic a direct anti-bacterial effect of the wavelength. Herein, to investi-
activities of neutrophils and macrophages, with the latter shown gate further about this LLLT action of 1072 nm of indirect antibac-
to be enhanced in its synthetic activity of fibroblast growth factor terial and wound healing effect, we designed a placebo-controlled
(FGF) by LLLT, which resulted in increased collagen synthesis and animal study with a mouse model infected with MRSA.
faster wound healing [5–8].
Among various wavelengths that have been studied for their 2. Material and methods
photobiomodulation effects, 1072 nm has been found to be effec-
2.1. Study design
⇑ Corresponding author at: Hayan-nara Dermatology Group, 142-3, Sankok-dong,
Bupyeong-ku, Incheon, South Korea. Tel.: +82 10 9991 8406; fax: +81 31 711 8406. Thirty female BALB/c nude miceaged 6–8 week-old were ob-
E-mail address: sycelinelee@gmail.com (S.Y. Celine Lee). tained for this study (Japan SLC, Inc., Hamamatsu, Japan). Animals

1011-1344/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2011.08.009
176 S.Y. Celine Lee et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 175–182

were housed separately and were maintained on a 12-h light/dark Biopsy samples were evaluated for difference in the expression
cycle with food and water. On day 1, animals were anesthetized of Vascular endothelial growth factor (VEGF), basic fibroblast
with an intraperitoneal injection of Rompun (Bayer) and Zoletil growth factor (bFGF), Transforming growth factor beta (TGF-b)
50 (Laboratories Virbac). Then, a 1.5  2.5 cm wound area was cre- and Nuclear Factor kappa-light-chain-enhancer of activated B cells
ated on the back of the mice by repeated (7–10 times) tape-strip- (NF-jB) compared to the control group using immunohistochemis-
ping and subsequent gentle abrasion with sand paper to remove try. Changes in mRNA levels were studied in Toll like receptor
the epidermal barrier [12]. A 20 lL suspension of MRSA containing 2(TLR2), Interleukins 1b and 6 (IL-1b, IL-6), Tumor necrosis factor
2.4  1010 bacteria was then applied evenly to the wound area. alpha (TNF-a), inducible nitric oxide synthetase (iNOS), Monocyte
After applying the bacteria, all mice were randomly assigned to Chemotactic Protein-1 (MCP-1), TGF-b, bFGF and VEGF.
either the control group (n = 15) or the experimental group
(n = 15) and placed into individual cages separated by plastic bar- 2.2. Immunohistochemistry
riers. Free access to food and water and enough space to exercise
were allowed for all subjects. However, physical contact between Immunohistochemical staining was performed using a peroxide
the mice was strictly inhibited by the use of plastic walls. Before technique. Formalin-fixed paraffin-embedded tissue blocks were
starting the actual experiment, 2 h were allotted for the bacterial sectioned to a thickness of 4 lm. Sections were deparaffinized for
intrusion into the skin and this time point was set as ‘baseline’. 5 min three times in xylene and rehydrated for 5 min per session
Irradiation with 1072 nm LED array over the dorsum of the subject in serial-graded alcohol (100%, 95%, 80%, 70% alcohol). Antigen re-
animals commenced 2 h after the inoculation (baseline) and re- trieval was performed for VEGF, bFGF, TGF-b and NF-jB. For anti-
peated at 4, 8, 12, 24 h, 3 days and 5 days after the inoculation. gen retrieval, 10 mM citrate buffer (pH 6.0) was heated in a
The time-points for evaluation were set based on a previous publi- pressure cooker for 10 min. The container was then cooled for
cation that proved the establishment of a superficial skin infection 20 min at room temperature. Endogenous peroxide activity was
model using S. aureus in a 6–8 week-old BALB/c female mice [12] blocked by 3% hydrogen peroxide in methanol for 10 min. The
and also on the results of our preliminary study which was con- slides were washed three times in Tris-buffered saline (TBS, pH
ducted to see the natural course of skin infection in the same 7.6) for 5 min and incubated with a blocking solution (normal goat
mouse model without any intervention. The early time-points serum) at room temperature for 20 min. The antibodies then used
within the first 24 h (4, 8, 12, 24 h) were chosen to investigate were as follows: an anti-VEGF antibody (1:200, Lab Vision, Califor-
the effect of 1072 nm LLLT on the early or acute inflammatory nia, USA); an anti-bFGF antibody (1:50, Lab Vision); an anti-TGF-b
phase, the later time-points (3- and 5-day) on the late inflamma- antibody (1:100, Lab Vision); and an anti-NF-jB antibody (1:50,
tory phase and wound healing process. Lab Vision). The antibodies were incubated for 30 min at room
Prior to each evaluation point, the experimental group received temperature and washed three times in TBS for 5 min. Subse-
10 min of 1072 nm infrared light (bandwidth ±50 nm) from an LED quently, secondary antibody reaction was achieved with a Chem-
planar array positioned approximately 3 cm above the mice. The Mate DAKO EnVision Detection Kit (Dako, Denmark) for about
light source had an irradiance of 20 mW/cm2 and fluence of 12 J/ 30 min at room temperature. After washing with TBS, the samples
cm2 for the treatment duration of 10 min, and was equipped with were stained with 3, 30-diaminobenzidine for chromogenic reac-
a cooling fan. These parameters were almost identical to those at tion and counter-stained with hematoxylin for 30 s.
the skin level of the subjects which were located at a distance of
3 cm (attenuation of the irradiance less than 3%). At this level of 2.3. Real time RT-PCR
irradiance and fluence, with the addition of a cooling fan, heat pro-
duction was too low to cause any temperature change of the sub- Total RNA was extracted from the skin samples with a Trizol kit
jects and experimental environment. The subjects of the control (Invitrogen Corp., Carlsbad, California). For synthesis of cDNA, re-
group were treated with sham LED panels (no light) equipped with verse PCR was performed with 20 lL reaction mixtures containing
the same cooling fans as the actual device. This ensured that all 1 lL of 10 mM dNTP (Finnzymes Corp., Finland), 0.5 lL of M-MLV
subjects were treated under the same environment and levels of Reverse transcriptase (Promega Corp. Madison, WI, USA), 4 lL of
stress. M-MLV 5X buffer (Promega), 2 lL of random hexamer (10 lM),
Biopsy specimens were taken 20 min after the light treatment and 10 lL of total RNA template. Amplification was achieved by
at every treatment time-point. Two subjects from each group the following cycle; 10 min at 25 °C, 60 min at 37 °C, 10 min at
(control group and experimental group) were taken for biopsy 72 °C and finally 15 min at 4 °C; in Peltier thermal cycler PTC-
at every time-point. Each subject was used for only one biopsy 200 (MJ Research Inc. USA). For real time PCR, mixtures were pre-
at a given time-point, not for multiple biopsies over multiple pared with each 1.0 lL of forward and reverse primer (9 lM), 5 lL
time-points, because multiple biopsies on the same animal over of fluorescent probe (2.5 lM), 10 lL of Taqman PCR 2X master
time could change the wound environment and also systemic re- mixture (Perkin–Elmer, Applied Biosystens, Lincoln, CA, USA) and
sponses (e.g. propagation of bacteria into deeper skin layers, pen- 4 lL of PCR products. The PCR primers and probes used are listed
etration of the bacteria from the skin into the blood vessels and in Table 1. Five microliters of reverse transcription reaction mix-
the internal organs, causing systemic introduction of the organ- ture was added as a PCR template. Relative quantitative real-time
ism and possible sepsis). First, the animals were sacrificed by in- PCR was performed using the above reagents using an ABI Prism
stant cervical dislocation to prevent any possible physiologic 7000 Sequence Detection System (Perkin–Elmer Applied Biosys-
change caused by the biopsy process and also to avoid inflicting tems, Lincoln, CA). The following procedure was used. After initial
unnecessary pain on the experimental animals. The skin of the activation of uracyl-N-glycosylase at 50 °C for 2 min, AmpliTaq
whole wound area (1.5  2.5 cm) was excised for specimen prep- Gold (Applied Biosystems) was activated at 95 °C for 10 min. PCR
aration. Immediately after the excision, about a 1 cm2 sized piece consisted of 45 amplification cycles (denaturation at 95 °C for
of skin was fixed in phosphate-buffered formalin (4%) for further 15 s, annealing at 60 °C for 1 min, and extension at 60 °C for
process for immunohistochemical staining, and the remaining 1 min). During PCR amplification, the amplified product amount
tissue was immediately frozen with liquid nitrogen for RT-PCR. was monitored by continuous measurement of fluorescence. The
This size of the skin sample provided a sufficient specimen tissue expression of the genes was normalized versus a GAPDH (VIC/
that could produce enough amount of RNA for high-quality MGB probe, primer limited) as follows; the cycle number at which
RT-2PCR. the transcripts of the genes were detectable (threshold cycle, Ct)
 S.Y. Celine Lee et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 175–182 177

Table 1 that of the control group after 12 h post-inoculation, and 11 times

Assay IDs of the probes and primers for real time higher after 24 h post-inoculation. It decreased back to the level of
RT-PCR.
that of the control group at day 3.
Assay ID IL-1b showed more sustained increasing tendency than TLR2. Its
TLR-2 Mm00442346_m1 mRNA level was twice as high as that of the control group after
IL-1b Mm00434228_m1 12 h from inoculation. It further increased as high as 23 times, 27
TNF-a Mm00443258_m1 times and 22 times compared to the control group after 24 h,
IL-6 Mm00446190_m1
MCP-1 Mm99999056_m1
3 days, and 5 days respectively after the inoculation.
iNOS Mm01309898_m1 TNF-a, another primary cytokine along with IL-1b, also showed
bFGF Mm01289197_m1 a remarkable increases starting from as early as 4 h post-inocula-
TGF-b1 Mm00441724_m1 tion compared to the control. The mRNA levels of TNF-a had con-
VEGF Mm01281449_m1
tinued to increase, peaking at 24 h after the inoculation (26 times
higher compared to the control) and normalized again to the con-
trol level at 3 days after the inoculation (1.2 times compared to the
was normalized against the Ct of GAPDH, which is referred to as
control).
delta Ct. The expression of the genes relative to a reference was ex-
IL-6, which is known to increase in the acute phase of inflam-
pressed as 2 deltadeltaCt, where deltadeltaCt refers to the difference
mation, also showed an enhanced response compared to the con-
in the values of deltaCt between the test group and the reference
trol. It was three times as high as the control at 1 h post-
(control).
inoculation and increased up to 17 times compared to the control
after 12 h post-inoculation. IL-6 mRNA level was normalized to the
3. Results control level at 24-h after the inoculation (1.9 times of the level of
the control).
3.1. Immunohistochemistry MCP-1 is known as a chemoattractant that recruits monocytes
to the site of infection and helps their traffic across endothelial bar-
The most significant difference between the control and the rier. In the 1072 nm group, the mRNA of MCP-1 was markedly in-
experimental groups was observed in the expression level of VEGF. creased at 12 h from the inoculation which was nine times higher
In the 1072 nm treatment group, the VEGF expression started to than that of the control group. The level continued to be higher
appear remarkably strong from 8 h after the inoculation, which than the control group until 3 days after the inoculation (5.8 times
was significantly stronger than the same time-point result of the at 24 h, 2 times at 3 days). The level dropped again at 5 days after
control group (Fig. 1A). The location of this strong VEGF expression the inoculation.
was mostly distributed in the papillary dermis and upper reticular iNOS, the inducible form of nitric oxide synthase, started
dermis, although it was observed intensively in the deep dermis as increasing from 24 h from inoculation. It was 1.7 times as high as
well 5-day post-inoculation. Conversely, no remarkable expression the control group at 24 h and 3 days after the inoculation and fur-
of VEGF was found in the control group after 5 days. ther increased to 2.7 times after 5 days.
The expression of bFGF was not significantly different between The mRNA levels of VEGF were lower in the treatment group
the control and experimental groups until 3 days after the inocula- than the control group for the first 4 h after the inoculation. How-
tion. However, after 5 days from inoculation, the bFGF expression ever, it started increasing from 12-h post-inoculation, and in-
in the 1072 nm group appeared notably stronger than the control creases as high as 14 times higher than the control at 24-h after
group, distributing in the papillary dermis and the upper reticular the inoculation were seen. VEGF levels decreased again to the con-
dermis, the former being visibly denser than the latter (Fig. 1B). trol level at 3 and 5 days after the inoculation, the level of which
With regards to the expression of TGF-b, we could not observe was 1.6 times and 1.3 times compared to the control.
any meaningful expression during the first 24 h. However, moder- The change of the mRNA levels of TGF-b showed a similar pat-
ate expression was observed on day 3 in the control group and on tern as that of VEGF. The levels were lower than the control during
day 5 in the experimental group, mostly in the papillary dermis in the first 4 h, and started increasing from 12-h post-inoculation (1.9
both groups (Fig. 1C). times compared to the control) until it peaked at 24-h post-inocu-
Nuclear translocation of NF-jB represents the activation and lation whose level was 14 times as high as that of the control
initiation of transcription genes that are involved in the rapid re- group. It decreased again to 5.8 times compared to the control after
sponse to bacterial infection. Nuclear translocation of NF-jB was 3 days and further decreased to 1.7 times 5-day post-inoculation.
markedly observed as early as 12 h after the inoculation in the bFGF showed a somewhat different pattern than other
1072 nm group, whereas this feature was not found at this time- cytokines or growth factors. It was not detected for the first 12 h.
point in the control group. In the control group, the nuclear tran- The level in the experimental group remained relatively similar
scription was observed at day 3 (Fig. 1D). to those of the control group (0.83 times and 1.775 times com-
pared to the control after 24 h and 3 days from the inoculation,
3.2. Real time RT-PCR (Fig. 2) respectively).

The results of real time RT-PCR, which showed the mRNA levels
of the measured cytokines and growth factors, and hence their le- 4. Discussion
vel of transcription, presented a relatively consistent tendency
(Fig. 2). In the 1072 nm treatment group, the cytokines and growth Even though we now understand that low-level light can affect
factors relating to the innate immune response to infection in- various biological processes after absorption of photons of light by
creased significantly compared to the control group between 4-h cells [1–3], the exact roles of which specific wavelengths can affect
post-treatment and 3-day post-treatment, and then started to nor- which biological responses has yet to be fully elucidated. The most
malize to the level of the control group’s measurements between widely accepted example of this connection is that wavelengths
3- and 5-day post-treatment. around the red and near-infrared waveband can enhance the
The level of TLR2 mRNA started to increase 12 h after inocula- wound healing process through photobiomodulation [2,6–8]. Pho-
tion. In the 1072 nm group, its level was four times higher than tobiomodulation is the process where incident photons are ab-
178 S.Y. Celine Lee et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 175–182

Fig. 1. Serial change of VEGF, TGF-b, bFGF and NF-jB in immunohistochemistry.

sorbed by chromophores, for example cytochrome C oxidase in the tons react with a photosensitizer, either exogenous or endogenous,
respiratory electron transport chain of mitochondria, to modulate that leads to the creation of singlet oxygen and free radicals which
various cell functions, and is believed to result in new collagen in turn destroy the cellular structures [13]. Blue light around
synthesis to exert the effects leading to the healing of the wound 415 nm is known to provoke the photodynamic reaction in
[1–3]. Other examples are photodynamic reaction, where the pho- Propionibacterium acnes (P. acnes), the causative bacteria of acne
 S.Y. Celine Lee et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 175–182 179

Fig. 2. Serial changes in mRNA levels of the cytokines that are expressed in bacterial infection.

vulgaris, by reacting with the bacterial coproporphyrin III and The mRNA levels of the cytokines appeared to peak around 12–
protoporphyrin IX that acts as the endogenous photosensitizer 24 h from the inoculation and then decreased to almost the same
[14–16]. Photodynamic reaction with exogenously administered level as the control group at 3-day post-inoculation. The exception
photosensitizer is also considered as a new method to treat local was IL-1b, which started decreasing from 3 days after the inocula-
infection including acne vulgaris [17–19] and some neoplastic con- tion but was not down to the control level until 5 days after the
ditions such as basal cell carcinoma [20–22]. Until now, most stud- inoculation. This homeostasis is important to note, because sus-
ies that investigated the possibilities of utilizing the light-tissue tained increases in cytokines may lead to an overreacting immune
interaction for infection were focused on destruction of the tissues response, which can be harmful to the host. The increase of TGB-b,
by photothermolysis or photodynamic reaction [23–25]. Our study, which activates regulatory T-cells, and the increase of IL-6, which
on the other hand, was designed to investigate how low-level light exerts not only a pro-inflammatory action in the acute phase but
can affect the natural anti-bacterial response of the host. We used also has an anti-inflammatory effect by inhibiting IL-1b and TNF-
the wavelength of 1072 nm, which is within the infrared range and a, may have a role in normalizing the increased mRNA levels of
is known to be effective for shortening the duration of herpes sim- these cytokines to those of the control group [29]. MCP-1, the re-
plex infection of the lips [9,10]. The results of our study revealed cruiter of monocytes which transform to macrophages after trans-
that low level light at this wavelength was able to influence the locating to the site of infection [30], showed similar pattern as
biological processes of the host against the bacterial infection. other primary cytokines.
The most prominent changes can be summarized as two points; The significant increase of VEGF was one of the most prominent
(1) enhanced response of primary cytokines activated against differences of the experimental group compared to the control
infection and (2) significant increases of VEGF compared to the group. VEGF is the key protein that stimulates the production
control. As seen in Fig. 2, the most significant increase in primary and the growth of new blood vessels when blood circulation is
cytokines was observed with IL-1b, TNF-a and IL-6. These primary not sufficient to supply enough oxygen. VEGF is known to increase
cytokines that act as the first responders to the infection are acti- in wound healing after injury or in local infections to provide more
vated immediately after the infection is detected by local immune oxygen, nutrients and immune cells that are needed for the anti-
cells such as Langerhans cells [26]. IL-6 is also an important medi- bacterial actions and wound healing processes. VEGF also acts as
ator of the acute phase response that can be secreted by macro- a chemoattractant for macrophages and granulocytes and as a
phages to specific microbial molecules that are detected by vasodilator with the involvement of nitric oxide [31–36]. These ef-
receptors of the host cells including toll-like receptor [27]. Activa- fects of VEGF help the immune system to combat the infection and
tion of these primary cytokines stimulates other immune cells to heal the wound site so that the body system can restore the
commence anti-bacterial actions such as production of antibodies homeostasis. In the 1072 nm light treatment group, the immuno-
and phagocytosis of the pathogens, and also to produce secondary histochemistry showed a remarkably stronger expression of VEGF
cytokines and growth factors [28]. Therefore, increases in these from 8-h post-inoculation compared to the control group. This ten-
primary cytokines with the treatment of 1072 nm may suggest dency continued through the last point of measurement, which
that the natural immune response against the infection was was 5 days after the inoculation. In the earlier time-points, the
boosted by photobiomodulation effect of this wavelength. One strong expression was observed mainly in the papillary dermis
more aspect that should be noted is the pattern in which these and the superficial to mid-dermis, however, in the 5-day post-inoc-
cytokines normalized to the level of the control in the later phases. ulation pictures, it was observed even in the deep dermis and the
180 S.Y. Celine Lee et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 175–182

subcutis layer. Although this study was not designed for comparing rate after exposure to ultraviolet irradiation. The authors hypothe-
the wound sizes between the experimental and the control groups, sized that increased iNOS might lead to greater production of nitric
the wound status of these two groups were visibly different. The oxide, which is known as a potent inhibitor of apoptosis in various
wounds in the experimental group showed a markedly more ad- cell types [41–43], and this increased nitric oxide may have ex-
vanced reepithelialization and smaller area of erythema and active erted a cytoprotective effect for the PBMCs. Although the exact
inflammation than the control (Fig. 3). In the 1072 nm treatment mechanism of how 1072 nm increases the amount of iNOS is not
group, at 5-day post-inoculation, the sanguineous crusts had al- yet known, our study results can be considered as supporting evi-
most fully detached leaving reepithelialized skin beneath. The re- dence of their findings, showing that the mRNA of iNOS is in-
sults of immunohistochemistry corresponded with those of real creased by 1072 nm.
time RT-PCR, where the mRNA levels of VEGF started exceeding Low-level light therapy with 1072 nm infrared light has been
those of the control group from 12 h from the infection and in- proven beneficial for the treatment of herpes simplex labialis,
creased up to 14 times higher than the control group at 24-h shortening the duration of the disease [9,10]. The mechanism of ac-
post-inoculation. Since mRNA is synthesized when there is a great- tion by which 1072 nm infrared light effects the healing time of
er need for the coded proteins than pre-existing ones, the increase herpes simplex labialis is not fully elucidated. Hypothetically, the
of mRNA of VEGF in the 1072 nm group may mean that the fact that 1072 nm light treatment can elicit a cytoprotective effect
1072 nm infrared treatment might have enhanced the production for lymphocytes, which play the major role in combating viral dis-
of the protein VEGF, which was confirmed by the increased immu- eases, can be considered as one of the possible underlying mecha-
noreactivity of the VEGF protein in the immunohistochemistry fea- nisms. The response to a bacterial infection, in contrast, is
tures. This is an important finding because it suggests that somewhat different than that to a viral infection, in the fact that
1072 nm infrared light may be beneficial for wound healing, espe- it relies on both innate and adaptive immune components [26].
cially for those that lacks sufficient blood supply such as chronic In our study results, 1072 nm infrared light treatment demon-
ulcers seen in diabetes mellitus or venous stasis. strated the established classic effects of low-level light therapy
iNOS, the inducible form of nitric oxide synthase, is believed to on the innate immunity. These classic effects are exerted through
be activated in the presence of inflammation or infection and to photobiomodultion where the photons of light absorbed by the
play an important role in the host immune defense against patho- mitochondria or other subcellular organelles increase the produc-
gens. The induction of iNOS usually happens in an oxidative envi- tion of adenosine triphosphate (ATP), reactive oxygen species
ronment, and hence, the resultant synthesis of nitric oxide leads to (ROS) and nitric oxide (NO), which facilitates the gene transcrip-
the creation of superoxide products and cell toxicity, which con- tion of the given cells (e.g. leukocytes, macrophages, endothelial
tributes to the anti-bacterial response of the host [37–40]. In the cells, fibroblasts etc.) through NK-jB and activator protein-1 (AP-
treatment group, the mRNA level was increased by up to 17 times 1) signaling pathways [1–3]. As a result, these cells become acti-
higher than the control group. This result corresponds with the vated and enhanced in producing their responsible proteins or per-
finding of a previous study that investigated the photobiological forming their given roles; for example, the lymphocytes secrete
action of 1072 nm infrared light in in vitro environment [11]. In antibodies, fibroblasts produce collagen and extracellular matrix,
this study, the authors found that, when pre-treated with mast cells degranulate, and macrophages increase phagocytic
1072 nm infrared light, human peripheral blood mononuclear cells activities. Our study results showed that 1072 nm infrared light
(PBMCs) produced a 4.9-fold increase in iNOS compared to the con- treatment increased the mRNA levels of primary cytokines in-
trol, which was measured by quantitative immunoblotting. They volved in the innate immune response and also increased the
also found that the pre-treated PBMCs showed a higher survival expression of the growth factors that participate in the wound

Fig. 3. Clinical photos of the infected wound at 5-day post-inoculation of MRSA. The wound treated with 1072 nm LLLT showed a significantly advanced reepithelialization
(B) compared to that of the control group (A). The crust on the wound of 1072 nm group was almost fully detached at the time of biopsy, while that of control group was still
adherent to the skin (pictures in the small boxes). (The crusts were removed for clinical photography. In figure (A), the weak skin of the upper margin of the wound was torn
during the process of cervical dislocation.)
 S.Y. Celine Lee et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 175–182 181

healing process. These finding suggest that low-level light therapy [11] A. Bradford, A. Barlow, P.L. Chazot, Probing the differential effects of infrared
light sources IR1072 and IR880 on human lymphocytes: evidence of selective
at the wavelength of 1072 nm may have photobiomodulation ef-
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also for the healing of the wound. One limitation of this study is Establishment of a superficial skin infection model in mice by using
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