, Flávia de Paoli and Jacy Gameiro Accepted Article · 2018. 11. 6. · 2 Departamento de...

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eModulation of immune response to induced-arthritis by low-level laser therapy

Lúcia Mara Januário dos Anjos*,1, Pollyanna Amaral Salvador2, Álvaro Carneiro de Souza1,

Adenilson de Souza da Fonseca3,4, Flávia de Paoli1 and Jacy Gameiro2

*Corresponding author: [email protected]. Departamento de Morfologia, Instituto de Ciências

Biológicas, Universidade Federal de Juiz de Fora, Rua José Lourenço Kelmer, s/n, Campus Universitário,

São Pedro, Juiz de Fora, Minas Gerais, 36036900, Brazil. Tel: +55 (32) 2102-3212.

1 Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora,

Rua José Lourenço Kelmer, s/n - Campus Universitário, Juiz de Fora, Minas Gerais, 36036900, Brazil. 2 Departamento de Microbiologia, Parasitologia e Imunologia, Instituto de Ciências Biológicas,

Universidade Federal de Juiz de Fora, Rua José Lourenço Kelmer, s/n - Campus Universitário, Juiz de

Fora, Minas Gerais, 36036900, Brazil. 3 Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcântara Gomes, Universidade

do Estado do Rio de Janeiro, Boulevard Vinte e Oito de Setembro, 87, Vila Isabel, Rio de Janeiro,

20551030, Brazil. 4 Centro de Ciências da Saúde, Centro Universitário Serra dos Órgãos, Avenida Alberto Torres, 111,

Teresópolis, Rio de Janeiro, 25964004, Brazil.

Abstract

As low-level laser therapy immune cells responses are not always clarified, this study

aimed to evaluate cytokines and immune cells profile after LLLT on arthritis-induced model.

Arthritis was induced in C57BL/6 mice divided into five groups: euthanized 5 hours after

inflammation induction; untreated; dexamethasone treated; LLLT at 3 Jcm−2; LLLT at 30 Jcm−2.

Cytokine measurements by ELISA and mRNA cytokine relative levels by qRT-PCR were

performed with arthritic ankle (IL-1β, IL-6, TNF-α, IL-10 and TGF-β). Macrophages, dendritic

cells, natural killer cells, lymphocytes CD4+, CD8+, Treg and costimulatory proteins were

quantified in proximal lymph node by flow cytometry. Data showed decrease in all cytokine

levels after LLLT and alteration in mRNA relative levels, depending on the energy density used.

LLLT was able to increase of immune cell populations analyzed in the lymph node as well as

costimulatory proteins expression on macrophages and dendritic cells. Treg TCD4+ and TCD8+

This article is protected by copyright. All rights reserved.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jbio.201800120

https://doi.org/10.1002/jbio.201800120

https://doi.org/10.1002/jbio.201800120

http://crossmark.crossref.org/dialog/?doi=10.1002%2Fjbio.201800120&domain=pdf&date_stamp=2018-09-11

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epopulation enrichment was observed in LLLT at 3 Jcm−2 and 30 Jcm−2 groups, respectively.

Furthermore, Treg TCD8+ cells expressing higher levels of CD25 were observed at LLLT at 30

Jcm−2 group. Our results indicate that LLLT could change the inflammatory course of arthritis,

tending to accelerate its resolution through immune cells photobiostimulation.

Keywords: Low-level laser therapy; arthritis; cytokines; immune cells; Treg cells.

Introduction

Rheumatoid Arthritis (RA) is an inflammatory autoimmune disease characterized by

chronic degeneration of synovial joints. Tending to worsen over time as joint architecture is

modified by synovitis (inflammation of the synovial membrane) RA is commonly associated to

pain and functional disability, systemic complications, early death, and important economic

burden worldwide [1,2]. Genetic and environmental factors compound disease etiology and

pathologic process involve disruption of the innate and adaptive immunity mechanisms, with

production of autoantibodies, as well as migration of T and B cells into the synovial

compartment and subsequent chronic inflammation [3,4].

The RA triggering event seems to be innate immune response activation, which includes

arthritis-associated antigen presentation (exogenous material and autologous antigens) through

major histocompatibility complex (MHC) and costimulatory proteins as CD80/CD86 by

dendritic cells, macrophages and activated B cells to T cells, which promote its differentiation

mainly into T helper 1 (Th1) and Th17 cells phenotype [5,6]. T- and B-cell activation mediate

effector function in RA through release of cytokines and chemokines, activation of leukocyte,

macrophages, fibroblast and endothelial cells, as well as B-cell produced autoantibodies [7].

Additionally, polymorphonuclear cells (PMN), mobilized by chemokines and cytokines,

infiltrate the synovial compartment and produce a wide range of pro-inflammatory cytokines,

leading to an increase in cell proliferation, vasodilation, vascular permeability, and proteolytic

enzyme secretion (e.g., matrix metalloproteinases - MMPs 1, 3, 8, 13, 14 and 16) from

synovium stromal cells and from chondrocytes [8,9]. MMPs promotes collagen type II


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edegradation (particularly MMP-2, -9 and 13) and alters the glycosaminoglycan composition and

water retention capacity of joint cartilage, which results in joint biochemical and mechanical

dysfunctions [10,11].

Macrophages present an important role in RA degenerative progress due to their antigen

presentation and osteoclastogenesis activities and as the major source of pro-inflammatory

cytokines TNF-a, IL-1 and IL-6 [12]. TNF-α promotes stimulation of other cytokine expression

such as IL-1β, induces also, monocyte cytokine and protaglandin release, PMN activation,

apoptosis and oxidative burst, besides decreasing synovial fibroblast proliferation and collagen

synthesis. IL-1β increase cytokine and chemokine release of synovial fibroblast and up-

regulates cell adhesion molecules expression in endothelial cells. IL-1β and TNF-α also

stimulate release of MMPs and IL-6 production, and the latter in turn promotes T-cell and B-

cell proliferation and antibody production, haematopoiesis and thrombopoiesis induction

[7,13,14].

On the other hand, joint inflammation can be modulate by regulatory T (Treg) cells

immunosuppressive activity [15]. Treg cell immune suppression mechanisms comprise

secretion of cytokines, such as TGF-β and IL-10, direct cytotoxicity to activated effector T cells

through secretion of perforin and granzyme A, inactivation of effector T cells via cell surface

immunosuppressive molecules, such as cytotoxic T lymphocyte antigen 4 (CTLA4) and Fas

ligand. Moreover, Treg cells can inhibit DC maturation and promote downregulation of

CD80/CD86 expression and competition with effector CD4+ cells for interaction with antigen-

captured antigen-presenting cells [16].

IL-10, also produced by B cells, can inhibit T-cell cytokine release and promotes its

anergy, can induce Treg cell maturation, decrease dendritic cells activation and cytokine release

as well as decrease synovial fibroblast MMP and collagen release. More, IL-10 inhibits

proteases, upregulate Interleukin 1 receptor antagonist (IL-1Ra) and the metallopeptidase

inhibitor (TIMP) production [12]. TGF-β, also produced by synovial-fibroblast, offers

ambivalent inflammatory effects in synovitis depending on IL-6 presence or absence: in the


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epresence can it promote cell differentiation into Th17 cells; IL-6 absence can favor T cells

regulatory phenotype (Treg) [17,18].

In spite of fibroblast-like synoviocytes (FLS), macrophages and T lymphocytes are the

most abundant cell types in RA synovium and PMN are often the most abundant cellular

component in synovial fluid, neutrophils, mast cells, dendritic cells, natural killer (NK) cells,

NKT cells, B cells, osteoclasts and plasma cells have been identified in synovial compartments

and present important role in RA pathogenesis through cytokine, chemokine, and protease

production, antigen presentation and antibody production, among others [19,20].

As synovitis comprises the main RA clinical feature, its therapy has been focused on

inflammation modulation and consequently disease degenerative progression deceleration by

administration of anti-inflammatory drugs (NSAIDs), corticosteroids, drugs modifying the

course of the disease (DMARDs) synthetic and biological and immunosuppressive drugs, used

alone or in combination [21]. However, this treatment displays several important side effects,

especially in administration of NSAIDs and corticosteroids which sometimes does not show

clinical improvement and disease remission [22]. Therefore, a great deal of effort has been

invested to identify other treatment strategies which present an anti-inflammatory effect, but not

suppress the entire immune system [5]. In this context, the low-level laser therapy (LLLT) could

be considered a promising non-pharmacological alternative to RA treatment due to its local

bone healing stimulation, inflammatory process modulation and pain relief effects [23-27].

The LLLT use is suggested as a short-term alternative to reduce RA pain and morning

stiffness [25]. LLLT positive effects in RA treatment could be related to decrease of

prostaglandin E2 (PGE2), inflammatory mononuclear cells number, of protein exudate, spinal

hemorrhage, hyperemia and necrosis [28,29].

Despite well-known LLLT anti-inflammatory effect and its extensive clinical use in the

treatment of chronic inflammatory conditions, the impact of this treatment on the immune

response, especially on immune cells behavior, is still poorly exploited. Therewith, our research

investigated immune response alterations after LLLT using an experimental arthritis-induced


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emodel [30], evaluating cytokine levels at inflammation site and immune cell population profile

in proximal lymph node to inflammation.

Material and Methods

Experimental groups

Forty male C57BL/6 mice, 8-10 weeks old, weighing 24-28 g each were used (Animal Ethical

Committee guidelines at Federal University of Juiz de Fora - protocol 039/2014). They were

allowed to move freely in the cages and have free access to laboratory diet and water.

Temperature (25° ± 2°C) and 12:12h light/dark cycles were controlled. The experimental

animals were randomly distributed into five groups (n=8): Arthritis induced and euthanized 5

hours after zymosan administration (5h); Arthritis induced and untreated (ZY); Arthritis induced

and treated with dexamethasone (ZY + DEXA); Arthritis induced and treated with LLLT at 3

Jcm−2 energy density (ZY + 3 Jcm−2); Arthritis induced and treated with LLLT at 30 Jcm−2

energy density (ZY + 30 Jcm−2). In order to confirm inflammatory process outset, a group was

created and euthanized 5 hours after arthritis induction (morphological analysis methods are

available on supplementary material).

Inflammatory process induction

Arthritis induction was performed as previously described by Dimitrova et al. [31].

Briefly, a solution containing 180μg of zymosan A from Saccharomyces cerevisiae (Sigma

Chemical Company, USA) dissolved in 10μL of sterile phosphate buffer solution (PBS) was

injected into the region near talocrural and subtalar joints (right and left) of mouse hind limbs.

All procedures were performed using anesthesia: a mix of 80mg kg−1 ketamine (Syntec, Brazil)

and 20mg kg−1 xylazine (Syntec, Brazil) by intraperitoneal via.

LLLT and dexamethasone protocols


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eA gallium-aluminum-arsenide (GaAlAs) low-level infrared laser (HTM Indústria de

Equipamentos Eletroeletrônicos Ltda, Brazil) was used in the following parameters: continuous

wave emission mode, 830nm wavelength, 10mW power output, 0.05cm2 spot area, irradiance at

0.2Wcm-2, energy densities at 3 and 30 Jcm−2 (total energy of 150 and 1500mJ were delivered

after 15 and 150s, respectively). Laser irradiation was applied at one point on ankle joint

(medial and external side of the ankle) and the optical pen was positioned perpendicularly to the

skin. Effective penetration of light at 830nm is around 4 cm [32]. Dexamethasone (Aché

Pharmaceutical Laboratory, Brazil) was administrated by intraperitoneal via (4 mg kg−1).

LLLT and dexamethasone administration was performed 4 times: 5, 29, 53, and 77h

after zymosan administration. The groups were euthanized twenty-four hours after the last

treatment section (101h / 4 days after zymosan administration). Their ankles, together with a

tiny portion of structures above and below ankle, such as skin, muscles and bones were removed

(right ankles were used for cytokine measurement by ELISA and the left ankle for real-time

PCR) as well as the both proximal lymph nodes (popliteal) were used for flow cytometry

analysis.

Quantification of cytokine mRNA relative levels by real-time quantitative polymerase chain

reaction assay (qRT-PCR)

Total RNA was isolated from left hide ankle after skin removal and maceration with

liquid nitrogen, using cold Trizol Reagent (Invitrogen, USA) according to the manufacturer's

instructions. Then, 2μg of total RNA were transcribed to complementary DNA (cDNA) using

the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Primers for

qRT-PCR were designed using the Primer 3 program [33], on different exons in order to avoid

genomic DNA contamination possibility. Gene primers used here (encoding the cytokines IL-

1β, IL-6, IL-10, TNF-α and TGF-β) are described in supplementary material (Table S1) as well

as that of β-actin, used as internal control. qRT-PCR assay was performed in StepOnePlus™

Real-Time PCR System instrument (Applied Biosystems, USA).


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eThe Delta-Delta Ct method (ΔΔCt) was used for gene relative levels analysis by qRT-

PCR [34]. Internal normalization was performed by β-actin and untreated samples (ZY) were

used to calculate the ΔΔCt.

Quantification of cytokines extracted from arthritic ankle

After skin removal, total proteins were extracted from hide right ankles using 100mg of

tissue/ml in PBS buffer supplemented with 0.4MNaCl, 0.05% Tween 20 and protease inhibitors

(0.1mM PMSF, 0.1mM benzethonium chloride, 10mM EDTA and 20KI aprotinin A / 100ml).

The samples were macerated, homogenized and centrifuged for 15min at 10,000rpm (4ºC). IL-

1β, IL-6 and TNF-α and IL-10 levels were estimated using a commercially available ELISA

(enzyme-linked immunosorbent assay) kit (BD Biosciences, USA) according to the

manufacturer's guidelines. The plates were read using a 450nm wavelength laser. Optical

density was analyzed and a standard curve was constructed using SoftMax® Pro software

(Molecular devices, USA).

Flow Cytometry of immune cells in lymph node proximal to inflammation (popliteal)

Multicolour flow cytometry was used to identify popliteal lymph node cells and cell

surface marker expression. All monoclonal antibodies (mAbs) were obtained from BD

Biosciences (USA): anti-CD11b, anti-CD11c, anti-CD80, anti-CD86, anti-CD3, anti-NK1.1,

anti-CD4, anti-CD8, anti-CD25 and anti-FoxP3. In brief, after both rear popliteal lymph node

removal from each animal, maceration was performed joining lymph nodes of 2 animals. Cells

were counted in Neubauer's Chamber and plated at 106 per well. For extracellular labeling, cells

were washed twice in staining buffer and then stained for 30 minutes at 4°C with fluorochrome-

labeled antibodies. For intracellular staining, cells were stained for cell surface markers for 30

minutes at 4°C with fluorochrome-labeled antibodies and then fixed and permeabilized with the

FoxP3 buffer set (BD Biosciences) according to manufacturer’s instructions and stained with

antibody anti-FoxP3. Flow cytometry data were acquired at FACsCanto ™ II (BD Biosciences)


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eand analyzed with FlowJo software (version 10). Mean fluorescence intensity (MFI) were

determined to CD80, CD86 and CD25.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 7.04 (GraphPad Software

Inc., USA). Data are presented as means ± SD. Multiple comparisons were performed using the

one-way ANOVA tests followed by Bonferroni multiple contrast hypothesis test. P values lower

than 0.05 were considered significant.

Results

Morphological analysis

Arthritis was successfully induced in mouse subtalar and talocrural joints.

Iinflammation was characterized by influx of polymorphonuclear cells (PMN), particularly

neutrophils in synovial tissues and their adjacent connective tissues 5 hours after zymosan

injection (Figure S1). This inflammation was also characterized by an intense pro-inflammatory

cytokine gene expression if compared to untreated group (Figure S2). At this point, we started

treatment protocols: ZY + 3 Jcm-2, ZY + 30 Jcm-2 and ZY + DEXA groups.

The inflammation process persisted until the fourth day for the untreated group.

Macrophages, lymphocytes and intense fibrous tissue deposition could also be observed (Figure

S1).

Cytokine levels

Cytokine mRNA relative levels are shown in the Figure 1. ZY + 3 Jcm-2 and ZY +

DEXA groups demonstrated IL-1β mRNA relative levels reduced when comparing to ZY (ZY +

3 Jcm-2 p<0.0001; ZY + DEXA p<0.0001) and ZY + 30 Jcm-2 groups (ZY + 3 Jcm-2 p<0.0001;

ZY + DEXA p<0.0001). Meanwhile, ZY + 30 Jcm-2 group showed IL-1β mRNA relative levels


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ereduced comparing to ZY group (p=0.0124) (Figure 1A). Figure 1B shows IL-6 mRNA relative

levels reduced for ZY + 3 Jcm-2 and ZY + DEXA groups when compared to ZY (ZY + 3 Jcm-2

p=0.0031; ZY + DEXA p=0.0018) and ZY + 30 Jcm-2 groups (ZY + 3 Jcm-2 p<0.0001; ZY +

DEXA p<0.0001); differently, ZY + 30 Jcm-2 group showed a increase levels when compared to

ZY group (p<0.0001). Figures 1C, 1D and 1E show mRNA relative levels of TNF-α, IL-10 and

TGF-β, respectively. For these analysis, ZY + 3 Jcm-2 group presented higher levels than all

others groups (Compared to ZY: TNF-α p=0.0008, IL-10 p<0.0001 and TGF-β p=0.0042;

Compared to ZY + 30 Jcm-2: TNF-α p<0.0001, IL-10 p<0.0001 and TGF-β p=0.0120;

Compared to ZY + DEXA: TNF-α p<0.0001, IL-10 p<0.0001 and TGF-β p=0.0055).


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eFigure 1 - Cytokine mRNA relative levels in mouse ankle joints after LLLT and dexamethasone

treatments. A: IL-1 β; B: IL-6; C: TNF-α; D: IL-10; E: TGF:β. β-actin was used as an internal control.

Untreated group - ZY, LLLT at 3 Jcm-2 (ZY + 3 Jcm-2), LLLT at 30 Jcm-2 (ZY + 30 Jcm-2) and treated

with dexamethasone (ZY + DEXA) mice. (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001 if higher than

untreated group (ZY). (ɸ) p < 0.05, (ɸɸ) p < 0.01 and (ɸɸɸ) p < 0.001 if lower than untreated group (ZY).

(#) p < 0.05, (##) p < 0.01 and (###) p < 0.001.

Levels of all cytokines were lower than those for untreated group after LLLT: ZY + 3

Jcm-2: IL1-β (p=0.0039), IL-6 (p<0.0001), IL-10 (p<0.0001) and TNF-α p<0.0001; ZY + 30

Jcm-2: IL1-β (p<0.0001), IL-6 (p<0.0001), IL-10 (p<0.0001) and TNF-α (p=0.0002).

Dexamethasone-treated groups also demonstrated an important decrease cytokine levels, when

compared to untreated group: IL1-β (p<0.0001), IL-6 (p=0.0009), IL-10 (p<0.0001) and TNF-α

(p<0.0001). On the other hand, this group showed a higher level of IL-10 than ZY + 3 Jcm-2

(p=0.0011) (Figure 2).

Figure 2 - Cytokine levels in mouse ankle joints after LLLT and dexamethasone treatments (pg/mL). A:

IL-1β; B: IL-6; C: TNF-α; D: IL-10. Untreated group - ZY, LLLT at 3 Jcm-2 (ZY + 3 Jcm-2), LLLT at 30

Jcm-2 (ZY + 30 Jcm-2) and treated with dexamethasone (ZY + DEXA) mice. (ɸɸ) p < 0.01 and (ɸɸɸ) p <

0.001 if lower than untreated group (ZY). (##) p < 0.01.


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eAntigen-presenting cells quantity and profile

ZY + 3 Jcm-2 group demonstrated higher number of cells in lymph node (p=0.025)

(Figure 3A) as well as macrophages (CD11b+ cells) (p=0.0007) and DCs (CD11c+ cells)

(p=0.0024) than ZY + DEXA group (Figure 3B and 3C). Among macrophage population, there

was higher number of cells expressing costimulatory surface protein CD86 (p=0.0049) (Figure

3D) while for DC population, more cells expressing CD80 (p=0.0314) (Figure 3E). ZY + 3

Jcm-2 group also showed higher number of DCs expressing concomitantly CD80 and CD86 than

ZY + DEXA (p=0.0185) and ZY (p=0.0240) groups (Figure 3F).

Likewise, ZY + 30 Jcm-2 group demonstrated higher number of cells in lymph node

(p=0.0037) (Figure 4A) as well as macrophages (p=0.0228) than the ZY + DEXA group (Figure

3B and 3C). Although their DC population rate was not statistically different from ZY + DEXA

group (p=0.1745) (Figure 3B and 3C), there were more DCs expressing CD86 (p=0.0467)

(Figure 3E). ZY + 30 Jcm-2 group also demonstrated higher quantity of CD80 expression on

DCs surface than ZY group (p=0.0160) (Figure 3E). Furthermore, macrophage rates expressing

concomitantly CD80 and CD86 were higher in the ZY + 30 Jcm-2 than ZY + DEXA group

(p=0.0298) (Figure 3F).

Effector cells of adaptive immune response profile

ZY + 3 Jcm-2 group showed higher NK cells rate than all other groups (Compared to ZY

- p=0.0093; ZY + 30 Jcm-2 - p=0,0366; ZY + DEXA - p=0,0019) (Figures 4A and 4B).

Similarly, T helper (CD4+) cells rates to the ZY + 3 Jcm-2 group were higher than all other

groups (Compared to ZY - p=0.0061; ZY + 30 Jcm-2 - p=0,0156; ZY + DEXA - p<0.0001)

(Figures 4C and 4D), while T cytotoxic (CD8+) (Figures 4C and 4D) and Treg CD4+

(FoxP3+CD25+) cells (Figures 4E and 4G) rates were higher than ZY (TCD8+ - p=0,0403; Treg

CD4+- p=0,0415) and ZY + DEXA (TCD8+ - p=0,0003; Treg CD4+- p=0,0086) groups.

On the other hand, ZY + 30 Jcm-2 showed TCD4+ (p=0,0005) and TCD8+ (p=0,0010)

cells rates higher than the ZY + DEXA group (Figure 4D), besides Treg CD8+ (FoxP3+CD25+)

increased if compared to ZY (p=0,0380) and ZY + DEXA (p=0,0261) (Figures 4F and 4H), and


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eTreg CD8+ expressing CD25 (CD25high) elevated rates if compared to all other groups

(Compared to ZY - p=0,0129; ZY + 3 Jcm-2 - p=0,0068; ZY + DEXA - p=0,0212) (Figure 4I).

Figure 3 - Macrophages, dendritic cells and their costimulatory surface proteins in lymph node proximal

to inflammation. A: Total cell; B: Representative flow cytometry plots showing distribution of CD11b+

cells and CD11c+ cells for each group; C: CD11b+ cell and CD11c+ cell proportion; D and E: lymph node

cells were gated on CD11b or CD11c expression and stained for CD80 and CD86 costimulatory surface

proteins. Proportions of CD11b+CD80+cells/CD11c+CD80+cells and CD11b+CD86+cells/

CD11c+CD86+cells are shown for each group as well as CD80 and CD86 expression histograms and their


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elevels by MFI; F: Proportions of CD11b+ cells and CD11c+ cells double positive to CD80+ and CD86+.

Untreated group - ZY, LLLT at 3 Jcm-2 (ZY + 3 Jcm-2), LLLT at 30 Jcm-2 (ZY + 30 Jcm-2) and treated

with dexamethasone (ZY + DEXA) mice. (*) p < 0.05 when higher than untreated group (ZY). (ɸ) p <

0.05 when lower than untreated group (ZY). (#) p < 0.05, (##) p<0.01 and (###) p < 0.001.

Figure 4 - NK cells and lymphocytes of lymph node proximal to inflammation. A: Representative flow

cytometry plots showing distribution of CD3+ cells and NK1.1+ cells from lymph node total leukocytes,

for each group. B: Proportions of NK1.1+ cells for each group are shown. C: Representative flow

cytometry plots showing distribution of CD4+ cells and CD8+ cells from CD3+ cells to each group. D:

Proportions of CD4+ and CD8+ cells. E: Representative flow cytometry plots showing distribution of

CD25+ cells and FoxP3+ cells from CD4+ cells and double positive cells (Treg cells). F: Representative

flow cytometry plots showing distribution of CD25+ cells and FoxP3+ cells from CD8+ cells and double

positive cells (Treg cells). G: Proportions of Treg cells (CD4+CD25+FoxP3+). H: Proportions of Treg

cells (CD8+CD25+FoxP3+). I: Levels of CD25 expression on Treg cells CD4+ and CD8+ by MFI.


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eUntreated group - ZY, LLLT at 3 Jcm-2 (ZY + 3 Jcm-2), LLLT at 30 Jcm-2 (ZY + 30 Jcm-2) and treated

with dexamethasone (ZY + DEXA) mice. (*) p < 0.05 if higher than untreated group (ZY). (ZY). (#) p <

0.05 and (##) p<0.01.

Discussion

In a previous work, we showed that LLLT promotes apoptosis in PMN cells at joint,

which could comprise one of LLLT anti-inflammatory mechanisms [24]. Due this fact, the

present study investigated whether this treatment could also modify immune response in this

inflammatory model.

Anti-inflammatory effects were observed after LLLT through changes in pro- and anti-

inflammatory cytokine mRNA relative levels and in reduction of IL-1β, IL-6, and TNF-α

(Figures 1 and 2) at inflammation site. The LLLT at 3 Jcm-2 presented a tendency to maintain

this anti-inflammatory profile at joint since IL-10 and TGF-β mRNA relative levels were higher

than all other groups despite the IL-10 tissue levels being still reduced. Furthermore, the

elevation in mRNA relative levels of the pro-inflammatory cytokines TNF-α and IL-6 was

observed in the 3 Jcm-2 and 30 Jcm-2 groups, respectively and could not necessarily mean a

return to the pro-inflammatory state. Despite TNF-α is classically being a pro-inflammatory

cytokine, it could inhibit mature DCs and also lead to their apoptosis, resulting in antigen

presentation failure and reducing lymphocytes levels by apoptosis [35]. Recent researches has

shown that IL-6 presents pleiotropic functions in acute phase response, inducing neutrophil

apoptosis and switching from neutrophil to monocyte recruitment in inflammation site by

suppressing mainly neutrophil-attracting [36]. These IL-6 effects substantially contribute to

acute infiltration resolution, since neutrophils are the most abundant cells in our inflammation

model (Figure S1).

LLLT presents an activity on inflammation by modulation of pro- and anti-

inflammatory mediator expression, such as IL-1β, IL-6, TNF-α, TGF-β and IL-10 [37-42].

However, the possible explanation for LLLT cellular mechanisms that have driven to these


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eresults is still poorly understood. It is known that the photobiological effects induced by LLLT

are due to specific molecule excitation, the so-called photoacceptors, by absorption of laser

photons [43], triggering the secondary messenger production (reactive oxygen species - ROS,

lymphokines, cytokines and nitric oxide - NO) able to initiate a cascade of intracellular signals

and initiate, inhibit or accelerate biological processes, such as wound healing and inflammation

resolution [44,45].

Due to PMN cell additional mechanism for free radical production and their very short

half-life during inflammation, LLLT might have greater and specific effects on these cells,

accelerating cellular functions, as cytokine production, and the consequent cell death by

apoptosis [24,44]. Since the inflammatory cells were exposed to repeated biostimulation, in the

LLLT treatment sections, it was expected that inflammatory cytokines profile to be altered. In

fact, the decrease of pro-inflammatory cytokine production, as observed after the intense

elevation of its levels, agrees with the LLLT biphasic dose-response, as previously reported

[46], and the anti-inflammatory profile found at final inflammatory process resolution stages

[47].

In our arthritis-induced model, inflammatory response triggered by zymosan is linked to

its phagocytosis, a process mediated by cell surface receptors. In immune response induction

phase (sensitization or afferent phase), the phagocytes (monocytes, macrophages and dendritic

cells) recognize zymosan by receptor binding, resulting in the activation of NF-κB and the

production of the inflammatory cytokines as well as the expression of costimulatory molecule

CD80. Additionally, zymosan is able to elicit adaptive immune responses through DC

maturation and IL-2 production stimulation, leading to mature DCs migration to regional lymph

nodes and induction of T lymphocytes activation as well as proliferation through antigen

(zymosan) presentation. In the elicitation (efferent) phase, repeated contact with zymosan

induces T lymphocytes recruitment. They produce a variety of cytokines, amplifying the

background inflammatory response into a more vigorous process [48-51]. This intense pro-

inflammatory microenvironment was observed in non-treated group due to elevated levels of

cytokines (Figure 2).


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eDifferently, dexamethasone treatment showed an important reduction in the cytokine

levels (Figure 2) as well as in the number of all immune cells analyzed from lymph node

proximal to inflammation (Figure 3A). These results were already expected, since

corticosteroids, such as dexamethasone, switch off multiple inflammatory genes (encoding

cytokines, chemokines, adhesion molecules, inflammatory enzymes, receptors and proteins),

which have been activated during inflammation, and consequently decreasing pro-inflammatory

cells and mediators in a non-specific manner [52]. As dexamethasone is commonly used for

articular inflammatory treatments, as arthritis, flow cytometry results from this group (ZY +

DEXA) were taken as a reference to compare with LLLT groups.

In view that T cell differentiation induction into CD4+ or CD8+ cells (cytotoxic T cells)

subsets occurs through antigen presentation with MHC and costimulatory proteins CD80/CD86

by DCs and macrophages [6], there was greater antigen presentation at lymph node of LLLT

groups, as macrophage population was elevated in both energy densities (ZY + 3 Jcm-2 and ZY

+ 30 Jcm-2) and DCs population at ZY + 3 Jcm-2 group, as well as the number of costimulatory

surface proteins expressed on those LLLT group cells (Figure 3). The macrophages and DCs

elevated rates also suggest an increase in cytokine milieu at lymph node. Both results are crucial

to activation and clonal expansion of lymphocytes [12], as observed in rates of lymphocytes

subsets, CD4+, CD8+ and Treg cells from two LLLT groups.

Increased T cell populations, particularly CD4+ subsets, and their cytokines and

chemokines production promote positive feedback loops on inflammation leading to synovitis

chronicity [5]. On the other hand, CD8+ T cells present two opposing roles in the overall

immune response in the joint: contributing directly to the upkeep of the chronic inflammatory

process through secretion of high levels of pro-inflammatory cytokines and lytic enzymes,

which comprise their cytotoxic function, and suppressing the inflammatory response in the

arthritic joints by secreting IL-10 [53].

This study is the first which shows Treg cells enrichment at lymph node after that LLLT

irradiation of inflammation site (increased Treg CD4+ cells rate in the LLLT + 3 Jcm-2 group

and Treg CD8+ cells in the LLLT + 30 Jcm-2 group). Moreover, it demonstrated higher rates of


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eTreg CD8+ cells expressing higher levels of CD25 (receptor of IL-2 on cell surface of Treg), an

important marker to its regulatory potency [54]. Thus, Treg cells population increase, especially

presenting CD8+FoxP3+CD25high phenotype, induced by laser at 30 Jcm-2 could favor negative

immune response modulation in inflammatory microenvironment leading to inflammation

resolution. In fact, Treg accumulation presenting activated phenotype represents positive factor

in the prognosis of rheumatic disorders, as they are commonly associated with compromised

Treg function [55,56], and in some patients the successful treatment of RA are related to Treg

effector function reversion [57].

The elevated Treg cells rates after LLLT could be related to higher expression of

surface costimulatory proteins CD80 and CD86 at DCs and macrophages surface observed in

both groups treated with lasers, since it has been reported in the literature that generation and

maintenance of different Treg subsets requires not only cytokines signaling but also strong

costimulation by CD80 and CD86 during T cell antigen presentation [58].

LLLT at the lower fluence was also able to induce higher rate of NK cells. These cells

are commonly expanded in inflamed joints and are responsible for pro-inflammatory cytokine

production amplification, interacting with the macrophage/monocyte population infiltrating the

joint [59].

The different results of the two energy densities seem to be related to biostimulation

promoted by them. Since irradiation is more intense at 30 Jcm-2, it is plausible to conclude that

cell effects from this treatment would be more pronounced than those observed at 3 Jcm-2.

However, biochemical mechanisms underlying the LLLT positive effects on inflammation have

commonly shown biphasic dose-response: cell activation is limited and, after it achieves its

threshold, it is followed by consequent cellular activity reduction and possible the cell death

[46]. Keeping this in mind, the quantitatively smaller changes in lymph node immune cell

profile observed in the group treated with LLLT at 30 Jcm-2 could be a repercussion of intense

photobiostimulation promoted by this therapy, which accelerates cellular activities leading to

faster inflammation resolution. Thus, the immune cells populations observed in the group

treated with LLLT at 30 Jcm-2 could indicate the cell profile at decline stage of inflammation


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ewhen polymorphonuclear (PMN) apoptotic cells phagocytosis by macrophages and the

contraction of lymphocytes population are also observed [47,60]. This hypothesis is

corroborated by our previous study reporting an important anti-inflammatory effect of LLLT at

30 Jcm-2 through photobiostimulation and consequent PMN cells apoptosis induction at arthritic

site. This anti-inflammatory effect was not observed in the group treated with LLLT at 3 Jcm-2,

a trend towards PMN cell apoptosis induction being observed though [24].

The changes to the immune cell profile after LLLT on lymph node proximal to

inflammation, with an increase of antigen presentation to T cells and their clonal expansion

could be attributed to lymphangiogenesis and lymphatic motility stimulation induced by LLLT

[41] and both can contribute to immune cell migration from joint to lymph node (or in the

opposite direction).

As the development of new treatments for inflammation which achieve an effective

anti-inflammatory behavior without a deleterious effect on innate immunity, the host defense

mechanism against infections comprises an important challenge [61], LLLT presents a

promising potential, reducing cytokine levels in the inflammation site and modifing T cell

lymph node populations (including Treg cell increase) (Figure 5), differently from non-specific

immune system depression induced by dexamethasone treatment.


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e

Figure 5 - LLLT effects on immune response to induced-arthritis. LLLT was able to reduce cytokine

levels on inflamed ankle tissues and change cytokine mRNA expression on inflammation site cells.

Lymph node cell population was also modified after LLLT, demonstrating macrophage, dendritic cell,

NK cell, TCD4, TCD8 and Treg cells populations increased. Together, LLLT effects promoted an anti-

inflammatory microenvironment, in which could accelerate inflammation resolution through immune cell

photobiostimulation.

Conclusion

LLLT anti-inflammatory effect, reported in experimental and clinical studies, have

mainly been attributed to inflammation mediator modulation. Furthermore, we showed that

there are alterations in populations of antigen-presenting cells, lymphocytes and Treg cells

expressing CD25 at high levels in proximal lymph node to inflammation. Taken together, our

results indicate that LLLT is an alternative for treatment of rheumatic disorders since it is able

to change the inflammatory course of arthritis, tending to accelerate its resolution through

immune cell photobiostimulation.

Acknowledgements


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eWe thank to Brazilian funding agencies FAPEMIG (#APQ 02123/15) and CNPq

(#474405/2013-3) for the financial support of this research. The authors would like to thank Dr.

Gilson Costa Macedo for his help in flow cytometry data acquisition and interpretation.

Authors' contributions

LMJA and FP contributed equally to the concept and design, data acquisition, analysis and

interpretation of data and drafting the manuscript. PAS and ACS were involved in collection

and assembly of data. JG participated in its design, coordination as well as helped draft the

manuscript. ASF contributed to the critical revision of the article for important intellectual

content, statistical expertise and manuscript drafting. All authors have contributed to critically

revising the manuscript for important intellectual content, read and approved the manuscript for

publication.

Conflict of interest

No competing financial interest exist.

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Graphical abstract

LLLT is extensively used in Rheumatoid Arthritis treatment, due to its anti-

inflammatory properties; however, little is known about immune response modifications

induced by this treatment. This study brings novelties for understanding LLLT immunological

modulation mechanisms, demonstrating changes lymph node cell populations which drains joint

inflammation, especially increase in Treg cells populations and cytokine reduction in inflamed

ankle after irradiation.


, Flávia de Paoli and Jacy Gameiro Accepted Article · 2018. 11. 6. · 2 Departamento de...

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