The effect of low-level laser therapy conditions on macrophages’ immunomodulatory processes as an example of regeneration process stimulation

The effect of low-level laser therapy conditions on macrophages’ immunomodulatory processes as an example of regeneration process stimulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The effect of low-level laser therapy conditions on macrophages’ immunomodulatory processes as an example of regeneration process stimulation Aleksandra Matuła, Amelia Lizak, Ewa Stodolak-Zych, Aneta Bac, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4620625/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract LLLT (low-level laser therapy) covers a wide range of parameters in terms of laser properties and dosage, which is important for its effects. To obtain the desired therapeutic effect of LLLT on cells, it is important to select optimal irradiation conditions. This article focuses on the selection of biostimulating exposure conditions for LLLT, which are the method of beam application, the radiation power and dose, and then the assessment of the immunomodulatory effect of LLLT on resting macrophages of the RAW 264.7 cell line. Irradiation of cells with a pulsed laser beam with a power of 200 mW and a dose of 5 J/cm 2 results in an increase in the adhesion and viability of macrophages and increase the secretion of protein, NO by macrophages and their TOS, which may suggest the polarization of macrophages towards the M1 phenotype. On the other hand, a decrease in the secretion TNF-α, MCP-1 and MMP-9 by cells may indicate the polarization of macrophages towards the M2 phenotype. It seems that for an optimal response of resting macrophages, they often share common features of the M1 and M2 phenotypes and that their phenotype should be considered as a spectrum of continuous differentiation under the influence of LLLT. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Cell biomodulation offers new possibilities for supporting the treatment of many diseases, including degenerative and periodontal ones, and chronic pain 1;2,3,4 . This method can also be used to initiate regenerative processes in damaged tissues 5,6 . The latter process – regeneration – can be induced or intensified by the synergistic action of inflammatory cells (macrophages) stimulated by low-level laser therapy (LLLT) 7,8 . This approach allows modulation of biochemical and molecular processes in living cells. It has been found that at the cellular level, under the influence of LLLT, changes occur in the production of adenosine triphosphate (ATP), the synthesis of free radicals of reactive oxygen and nitrogen species (RONS) is regulated and transcription factors are induced. Transcription factors influence cell adhesion, proliferation, apoptosis and secretory activity, measured by the synthesis of various types of proteins, among others 9,10–17, 5 . LLLT covers a wide range of parameters in terms of laser properties and dosage, which is important for its effects. To obtain the desired therapeutic effect of LLLT on cells, it is important to select optimal irradiation conditions: radiation wavelength, surface power density, energy supplied and the number of irradiations 6,18,19 . These factors determine what changes will be induced in cells under the influence of LLLT. Most studies on cell biomodulation supported by LLLT mainly assess its impact on skin cells e.g. fibroblasts. It has been shown that LLLT parameters that are too low may result in no cellular response or one that is too weak, while using irradiation parameters that are too strong may have a cytotoxic effect on cells or may not initiate the cellular response at the expected level. An example of such behaviour has been described by (Crisan 2013), among others, who compared the effects of radiation wavelengths of 830 nm and 980 nm on fibroblasts, showing that both significantly stimulated cell proliferation after one-, two- and three-fold irradiations 20 . Ma et al. (2018) have also described the increased proliferation of human fibroblasts and collagen synthesis under irradiation with a wavelength of 830 nm, while irradiation with a wavelength of 635 nm did not increase fibroblast proliferation and collagen synthesis 21 . Mignon et al. (2018) have shown that shorter wavelengths of radiation (530 nm) inhibit the metabolic activity of fibroblasts, while longer wavelengths (550–850 nm) do not produce such an effect 22 . It has been shown that the radiation dose is also a factor that modulates the cellular response. Hawkins and Abrahams (2006) treated human skin fibroblasts with radiation doses of 2.5 J/cm 2 , 5 J/cm 2 and 16 J/cm 2 for two consecutive days. The results showed that a dose of 2.5 J/cm 2 for daily irradiation and 5 J/cm 2 for single irradiation increased cell viability, proliferation and migration. However, exposure at a dose of 16 J/cm 2 inhibited cell proliferation, which had a negative effect on their viability and migration 23 . Another study also showed similar findings that laser irradiation at doses of 3 J/cm 2 and 4 J/cm 2 for six consecutive days resulted in an increase in fibroblast proliferation compared to the control group, while a dose of 5 J/cm 2 did not stimulate cell proliferation 24 . The power of the laser radiation is also a factor influencing the effectiveness of the cellular response. Azevado et al. (2006) showed that the proliferation of fibroblasts increased after two- and six-fold irradiations of cells with a power of 10 mW and 29 mW 6 . Illescas-Montes et al. (2017) observed an increase in the number of cells at an irradiation power of 0.2 W and 0.5 W, while the use of an irradiation power of 1 W did not affect the number of cells 25 . In turn, Chen et al. (2009) showed a decrease in the viability of human fibroblasts after irradiating cells with a laser with a power of 1.0–3.0 W 26 . There is little information in the literature on the impact of LLLT on the response of immune system cells, including macrophages, the activation of which plays a key role in the inflammation process and also during tissue regeneration 27–30 . Macrophages express many receptors and signalling molecules, and their activation depends on local conditions in the tissue. Due to their very diverse, often contradictory functions, macrophages are divided into classically polarized M1 macrophages and alternatively polarized M2 macrophages. M1 macrophages occur mainly in the early stages of inflammation and activate pro-inflammatory signalling pathways. The factors secreted by M1 macrophages include free radicals of RONS, pro-inflammatory cytokines (TNF-α, IL-1 IL-12p70) and many chemokines, such as CCL15, CCL20 and CXCL8. In turn, M2 macrophages are primarily involved in the processes ending inflammation and promoting tissue remodelling 28,29 . The factors secreted by M2 macrophages are cytokines IL10 and TGF-β, chemokines CCL22 and CCL17 and metalloproteinases (MMPs), which are associated with the remodelling of the extracellular matrix (ECM) during regenerative processes in damaged tissues. According to reports on the role and potential of macrophage polarization, the possibility of immunomodulating their behaviour towards the M1 or M2 phenotype with LLLT seems important 31 . The available literature data on the effect of LLLT on macrophages mostly concern the response of macrophages previously stimulated with lipopolysaccharide (LPS) (LPS-stimulated macrophages) 32,33,8 . However, there is no information on the directional effect of LLLT on resting M0 macrophages. This article focuses on the selection of favourable (biostimulating) exposure conditions for LLLT, which are the method of beam application (continuous [C] or pulsed [P] laser beam), the radiation power and dose, and then the assessment of the immunomodulatory effect of LLLT on resting macrophages of the RAW 264.7 cell line. 2. Materials and methods Macrophage culture conditions The RAW 264.7 macrophage cell line (ATCC, USA) was used in the study. The cells were cultured in RPMI 1640 culture medium (Lonza, USA) with the addition of 10% FBS calf serum (Gibco, USA) and a 5% solution of antibiotics, penicillin and streptomycin (Sigma-Aldrich, USA), in an atmosphere of 5% CO 2 at a temperature of 37 o C. Then, 1 ml of cell suspension with a concentration of 1.5 x 10 4 million cells/ml was placed in the wells of a 24-well culture plate (Nest SB, USA) at the bottom of which round culture slides had previously been placed. Low-level laser therapy A PhysioGo 400C device (ASTAR, Poland) was used to irradiate the macrophages. It is a low-level laser (LLL) that generates electromagnetic radiation in the infrared light range with a wavelength of 808 nm, power of 100 mW or 200 mW and radiation doses of 5 J/cm 2 /well with cells or 10 J/cm 2 /well with cells. The laser beam was continuous (C) or pulsed (P) (at a frequency of 100 Hz with a 50% fill factor). Irradiation was performed using a non-contact method, at a distance of 1 cm from the cells, at a right angle to the irradiated surface. Study stages The study was carried out in three stages (Fig. 1). In stage I of the study, laser radiation was applied once a day (at the same time of day) two, four, six, 8 or ten times. On the following days of the experiment: on day 3 (two laser beam applications), on day 5 (four laser beam applications), on day 7 (six laser beam applications), on day 9 (eight laser beam applications) and on day 11 (ten laser beam applications), the macrophage culturing was ended and the cells and cell culture supernatants were assessed for adhesion/proliferation, morphology and the level of adenylate kinase (AK) released from dead cells. Stage I of the study was carried out for the following groups: unirradiated cells (CTR group – control group); cells irradiated with a continuous laser beam (C) with a power of 100 mW or 200 mW and a dose of 5 J/cm 2 or 10 J/cm 2 (100/5/C, 100/10/C, 200/5/C and 200/10/C groups); and cells irradiated with a pulsed laser beam (P) with a power of 100 mW or 200 mW and a dose of 5 J/cm 2 or 10 J/cm 2 (100/5/P, 100/10/P, 200/5/P and 200/10/P groups). The experimental data made it possible to answer the question of how repeated exposure to LLLT with precisely defined parameters (power, intensity and exposure frequency) affects macrophages. In stage II of the study, radiaton doses of a specific power that had a biostimulating effect on cells were used. The cells were irradiated with a continuous (C) or pulsed (P) laser beam with a power of 200 mW and a dose of 5 J/cm 2 . Laser radiation was applied once a day (at the same time of day) two, four or six times. On the following days of the experiment: on day 3 (two laser beam applications), on day 5 (4 laser beam applications), on day 7 (six laser beam applications), the macrophage culture was ended and the cells and cell culture supernatants were assessed for the functional state of macrophages by determining their viability and the level of secreted protein. Stage II of the study was carried out for the following groups: unirradiated cells (CTR group); cells irradiated with a continuous (C) laser beam with a power of 200 mW, a dose of 5 J/cm 2 (200/5/C group); and cells irradiated with a pulsed (P) laser beam with a power of 200 mW, a dose of 5 J/cm 2 (200/5/P group). In stage III of the study, the immunomodulatory effect of LLLT on macrophages was assessed on the supernatants collected from the cell cultures irradiated with a pulsed (P) laser beam with a power of 200 mW and a dose of 5 J/cm 2 (200/5/P group). In this group, the metabolic and immunological activity of the macrophages was examined by determining the levels of: nitric oxide (NO), cytokines (MCP-1), tumour necrosis factor TNF-α, interferon gamma IFN-γ, interleukin 12p70, interleukin 6, interleukin 10, metalloproteinases (MMP-2 and MMP-9) and the total oxidative/capacitive status (TOS/TOC) and total antioxidant/ capacitive status (TAS/TAC) of the cells was determined. Cell adhesion/proliferation: Crystal violet uptake assay Cell adhesion was tested using the crystal violet (CV) uptake assay. Cells adhered to the substrate were fixed for 5 minutes with 2% paraformaldehyde (PFA) (Sigma, USA), stained for 5 minutes with 0.5% CV solution (Sigma, USA) and rinsed with water. Then, the dye absorbed by macrophages was extracted by adding 0.5 ml of 100% methanol (Linegal Chemicals, Poland) to each well. The optical density (OD) of the fluid was read using a FLUOstar Omega reader (BMG Labtech, Germany) at a wavelength of 570 nm. Cell morphology Some of the tested samples (two from each series) were intended for microscopic observation. Adherent cells were stained for 15 seconds with a 0.5% solution of CV in water (Sigma, USA) and then rinsed with water. Cells were observed using a light microscope (Motic AE-2000T, Germany) at 40x magnification. Pictures of cells adhering to the substrate were taken with a Moticam-BTU8 microscope camera (MoticEurope, Spain). Level of released AK (ToxiLight assay) The level of AK was determined quantitatively by the bioluminescence method using a Toxilight reagent set (Lonza, Switzerland). First, 20 µl of supernatant was collected from the cell culture and transferred to a white 96-well plate (Nest SB, USA). Then, 100 µl of AK detection reagent solution was added to each well. After 5 minutes of incubation, luminescence was read using a FLUOstar Omega reader (BMG Labtech, Germany). Cell viability (ViaLight assay) To test the viability of macrophages in the culture, a ready-made assay with a ViaLight reagent set (Lozna, Switzerland) was used. First, 200 µl of cell lysis reagent was added to the wells containing cells and 600 µl of supernatant. After 10 minutes of incubation, 200 µl of the supernatant-lyser mixture was transferred to a white 96-well plate (Nest SB, USA) and 200 µl of AMR PLUS reagent was added. After 2 minutes, the amount of radiation emitted was determined using a FLUOstar Omega reader (BMG Labtech, Germany). Level of secreted protein (BCA assay) First, 10 µl of the tested samples were transferred to each well of a 96-well plate (Nest SB, USA) and 200 µl of a mixture consisting of copper sulfate II (CS (II)) (Sigma-Aldrich, Germany) and bicinchoninic acid (BCA) (Sigma-Aldrich, Germany), mixed in a ratio of 1:50, was added. The plates were then incubated for 30 minutes in the dark. After the designated time, the OD of the liquid was read at a wavelength of 570 nm using a FLUOstar Omega reader (BMG Labtech, Germany). Level of secreted NO (Griess test) First, 100 µl of cell culture supernatant was transferred to each well of a 96-well plate (Nest SB, USA). Then, 100 µl of reagents (Sigma-Aldrich, Germany): Griess A (1% sulfalamide in 5% phosphate acid) and B (0.1% naphthylenediamine in H 2 O), mixed in a 1:1 ratio, was added. After 5 minutes, the OD of the liquid was read at a wavelength of 540 nm using a FLUOstar Omega reader (BMG Labtech, Germany). Level of secreted cytokines Cytokine levels in the cell culture supernatants were measured by flow cytometry using a Flex Set (CBA, BD Biosciences). The entire assay procedure, as well as all measurements and analyses, were performed following the instructions attached to the cytokine assay kit using a Beckman Coulter flow cytometer (Life Science, USA). The Mouse Inflammation Kit (BD Biosciences, USA) was used, which allows simultaneous determination of the level of six cytokines: interleukin (MCP-1), tumour necrosis factor (TNF-α), interferon gamma (IFN-γ), interleukin 12p70 (IL-12p70), interleukin 6 (IL-6) and interleukin 10 (IL-10). First, 50 µl of a solution containing a mixture of beads coated with antibodies directed against each of the six analysed cytokines was added to each Eppendorf tube (Nest SB, USA) containing the standard, a sample or the control. In the next step of the procedure, standards or samples were added to the Eppendorf tubes and an assay diluent was added to the negative control. Then, 50 µl of phycoerythrin (PE)-conjugated antibodies directed against each of the six cytokines were added to all Eppendorf tubes and the samples were then incubated in the dark for 2 hours at room temperature (RT). After this time, 1 ml of washing buffer was added to each sample and the samples were centrifuged (200 g, 5 min, RT). The supernatant from the pellets was collected using a pipette and 300 µl of washing buffer was added to each sample. Then, the samples were transferred to new Eppendorf tubes (Nest SB, USA), where measurements were performed by a cytometer. The flow cytometer was calibrated using BD CaliBRITE™ beads, which are microspheres coated with fluorescein isothiocyanate (FITC) (FL-1), PE (FL-2) and peridinine-chlorophyll protein complex (PerCp) (FL-3). Next, the device was calibrated using CytExpert Software for the CytoFLEX Platform with the Humane Inflammation Kit using positive controls for FITC and PE detectors. The data were analysed and cytokine concentrations were determined in Microsoft Excel using standard curves based on successive dilutions of the standard. The maximum concentration of the cytokine standard was 5,000 pg/ml. Subsequent serial dilutions were made in the assay diluent at 1:2, 1:4 and 1:8, respectively, ending with a dilution of 1:256, which corresponds to a cytokine concentration of 20 pg/ml. Level of secreted metalloproteinases The level of metalloproteinases secreted by the cells (in the form of an inactive enzyme precursor and active enzyme) was measured using the gelatin zymography method, which is a modified electrophoretic method that allows measuring the proteolytic activity of enzymes whose substrate can be incorporated in a polyacrylamide gel with the addition of sodium dodecyl sulfate (SDS). This method allows detection of the levels of pro-metalloproteinases 9 (pro-MMP-9) and 2 (pro-MMP-2), as well as metalloproteinases 9 (MMP-9) and 2 (MMP-2). First, solutions of separating gel (10%) and thickening gel (4%) were prepared. Polymerization catalysts (TEMED, APS) were added immediately before pouring the gels. The separating gel was poured first and some space left for the thickening gel, in which, after pouring, combs for the wells were placed. After solidifying (about 1 h), the gels were placed in the refrigerator for 12 h at 4°C. To quantitatively analyse the protein levels in the samples, the BCA assay was performed (see section 4.10). Based on the results obtained, protein levels were normalized in the tested samples. Samples with the same protein content were used for further analyses. First, 10 µl of protein standard with a wide range of proteins (coloured with SDS-PAGE; Bio-Rad, USA) was added to the first well and 15 µl of the tested samples added to the remaining wells. Electrophoresis was performed for 45 minutes. After electrophoresis was completed, the gels were collected and then washed (on an Elpan shaker, Poland) twice for 15 minutes in a 2.5% Triton X-100 solution (Bio-Rad, USA) to remove the SDS. After this, the gels were placed in an incubation buffer with the following composition: 5 mM CaCl 2 ; 50 mM Tris-HCl (pH = 8); 0.02% NaN 3 1 µM ZnSO 4 (all from Sigma-Aldrich, Germany) and incubated for 24 hours in a water bath at 37°C. Then, the gels were stained in 0.5% brilliant blue solution (Sigma-Aldrich, Germany) and then destained in an equilibration buffer until light bands were visible. Syngen’s Snaap Gene and GeneTools software were used to analyse the obtained scans. The presence of gelatinases was determined based on the molecular masses of the decolourized bands read from the protein standard. Measurement of the total oxidative/capacitive status (PerOx [TOS/TOC] assay) The TOS of the cells was determined by measuring lipid peroxide levels according to the instructions of the PerOx (TOS/TOC) kit (Immunodiagnostik AG, Bensheim, Germany). Peroxide levels in the tested cell culture supernatant samples were determined based on the reaction of horseradish peroxidase with tetramethylbenzidine dichloride (TMB) in the presence of hydrogen peroxide. The reaction with the enzyme produces a soluble blue product. The addition of 2 M H 2 SO 4 stops the reaction, leading to the solution changing colour from blue to yellow. According to the kit instructions, 10 µl of calibrator (CAL), controls 1 and 2 (CTRL1, CTRL2) (reagents included in the kit) or the tested sample were added to the wells of a 96-well plate (included in the kit). Then, 100 µl of buffer (Reabuf A) included in the kit was added to each well of the plate and the first OD reading was taken at a wavelength of 450 nm, using a FLUOstar Omega reader (BMG Labtech, Germany). Then, 100 µl of the buffer mixture (RBF) (reagent included in the kit) was added to all wells and in the next step, the plate was incubated for 15 min at 37 o C. After this, 50 µl of the reaction-stopping reagent Stop Solution was added to each well and the second OD reading was taken, also at a wavelength of 450 nm, using a FLUOstar Omega reader (BMG Labtech, Germany). Based on the measured optical OD values, the total peroxide level (µmol/l) was determined in the tested cell culture supernatant samples, according to the instructions provided by the kit manufacturer (Immunodiagnostik AG, Bensheim, Germany). Measurement of the total antioxidant/capacitive status (ImAnOx – TAS/TAC assay) The TAS of the cells was determined by reacting antioxidants with a predetermined (known) amount of exogenous hydrogen peroxide (H 2 O 2 ), according to the protocol of the ImAnOx (TAS/TAC) kit (Immunodiagnostik AG, Bensheim, Germany). In this assay, antioxidants react with peroxide and the amount of unreacted H 2 O 2 is measured spectrophotometrically. The difference between the added and measured amount of H 2 O 2 (relative to the calibrator included in the kit) is proportional to the antioxidant activity. First, 10 µl of calibrator (CAL), controls 1 and 2 (CTRL1, CTRL2) (reagents present in the kit) or the test sample were added to the wells of the 96-well plates included in the ImAnOx kit. Then, 100 µl of Reagent 1 was added to all wells of the plates, and then the plates were incubated for 10 min at 37 o C. After the designated incubation time, 100 µl of Reagent 2a was added to all wells of one plate (with enzyme), and Reagent 2b was added to the other plate (without enzyme). Then, both plates were incubated for 5 minutes at RT, and then 50 µl of Stop Solution reagent was added to all wells. The OD reading was performed using a FLUOstar Omega reader (BMG Labtech, Germany) at a wavelength of 450 nm. Based on the measured OD values, the TAS in the cell culture supernatants was determined, expressed in µmol/l, according to the instructions provided by the kit manufacturer (Immunodiagnostik AG, Bensheim, Germany). Statistical analysis Data were presented as mean values ​​and standard error. Differences between the control group and the experimental groups were performed using the Student’s t-test if the assumptions for parametric tests were met. If the assumptions were not met, the non-parametric Mann-Whitney U test was applied. Differences between the control group and the experimental groups were compared using the one-way analysis of variance (ANOVA), Then, the Tukey test was applied for post-hoc evaluation. A significance level of p ≤ 0.05 was adopted in the analyses. 3. Results Selection of optimal LLLT exposure parameters Adhesion/proliferation and morphology of macrophages irradiated with LLL On the following days of the experiment (days 3–9), unirradiated macrophages (CTR group) proliferated, which resulted in an increased number of adherent cells on days 5, 7 and 9, compared to day 3 of macrophage culturing (Table 1). On day 11 of macrophage culturing, the number of adherent cells decreased but was still higher than on day 3 of macrophage culturing (Fig. 2). The analysis of the effect of a continuous laser beam on the adhesion/proliferation and morphology of macrophages allowed the conclusion that the two- and six-fold irradiation of cells (days 3 and 7) with a laser with a power of 200 mW and a dose of 5 J/cm 2 (200/5/C group) resulted in increased adhesion (Fig. 2a) and a higher density of macrophages visible in the morphological picture (Fig. 3) compared to unirradiated cells (CTR group). In this group (200/5/C), further eight-fold irradiation had no effect, and ten-fold irradiation resulted in decreased cell adhesion to the substrate (Fig. 2a). Increased adhesion of macrophages as a result of two applications with a continuous laser beam (day 3) was also observed in the case of cell irradiation with a laser with a power of 100 mW and a dose of 5 J/cm 2 and 10 J/cm 2 (100/5/C and 100/10/C groups) and a power of 200 mW and a dose of 10 J/cm 2 (200/10/C group) (Fig. 2a). In this case, further cell irradiation had no effect or resulted in decreased cell adhesion, particularly on days 9 and 11 of macrophage culturing (100/5/C, 100/10/C, 200/5/C and 200/10/C groups) (Fig. 2a). In the case of pulsed beam treatment, for two- and six-fold irradiation of cells with a laser with a power of 200 mW and a dose of 5 J/cm 2 /well (200/5/P group), it was also found that cell adhesion to the substrate increased compared to the unirradiated cells (CTR group) (Fig. 2b) and the density of macrophages was higher in the microscopic picture (Fig. 3). Further irradiation of macrophages with a laser with these parameters had no effect (day 9) or resulted in decreased cell adhesion to the substrate (day 11) (200/5/P group). An increase in cell adhesion was also found after two-fold irradiation with a pulsed laser beam of 100 mW and a dose of 5 J/cm 2 (100/5/P group), however, further laser irradiation decreased cell adhesion compared to the unirradiated cells (CTR group). In turn, irradiation of cells with a laser with a power of 100 mW or 200 mW and a dose of 10 J/cm 2 (100/10/P and 200/10/P groups) resulted in decreased cell adhesion compared to the unirradiated cells (CTR group) (Fig. 2b). The results of adhesion are confirmed by the microscopic images of cells, where an increase in adhesion was accompanied by an increase in cell density, while a decrease in cell adhesion was accompanied by a decrease in cell density and a cluster arrangement (Fig. 3). Level of AK released by macrophages exposed to LLLT Two-, four- and six-fold irradiation with a continuous laser beam of 200 mW and a dose of 5 J/cm 2 (200/5/C group) did not affect the level of AK released by macrophages (Fig. 4a). In turn, further eight- and ten-times applications of a laser beam with these parameters resulted in an increased AK level, compared to the unirradiated cells (CTR group) (Fig. 4a). Irradiation (regardless of the number of applications) with a continuous laser beam with a power of 100 mW and doses of 5 J/cm 2 and 10 J/cm 2 (100/5/C and 100/10/C groups) and with a power of 200 mW and a dose of 10 J/cm 2 (200/10/C group) increased the AK level released by macrophages in these groups (Fig. 4a) compared to the unirradiated cells (CTR group) (Fig. 4a). The analysis of the effect of a pulsed laser beam with a power of 200 mW and a dose of 5 J/cm 2 /well (200/5/P group) showed that four- and six-fold irradiation reduced the level of AK released by cells compared to the unirradiated macrophages (CTR group) (Fig. 4b), although increasing the number of exposures to 10 increased the level of AK released. Treatment with a pulsed laser beam with a power of 100 mW and doses of 5 and 10 J/cm 2 (100/5/P and 100/10/P groups) and with a power of 200 mW and a dose of 10 J/cm 2 (200/10/P group) induced an increase in the level of AK released (Fig. 4b). The results obtained from stage I of the study allowed us to select the irradiation parameters that had the most beneficial effect on the cells, causing an increase in cell adhesion/proliferation and no effect or a decrease in the release of AK. For stage II of the study, the exposure parameters selected were continuous or pulsed laser beams with a power of 200 mW and a dose of 5 J/cm 2 (200/5/C and 200/5/P groups). Viability of macrophages irradiated with continuous or pulsed laser beams with a power of 200 mW and a dose of 5 J/cm 2 Two-fold irradiation with a continuous laser beam of 200 mW and a dose of 5 J/cm 2 (200/5/C group) resulted in an increase in macrophage viability compared to the unirradiated cells (CTR group) on day 3 of macrophage culturing (Fig. 5a). Increasing the number of laser irradiations (4 or 6 laser beam applications) had no effect on the viability of macrophages on days 5 and 7 of macrophage culturing. In turn, in the case of macrophages irradiated two and six times with a pulsed laser beam of 200 mW and a dose of 5 J/cm 2 (200/5/P group), increased cell viability was observed on days 3 and 7 of macrophage culturing compared to unirradiated cells (CTR group) (Fig. 5b). The level of protein secreted by macrophages irradiated with a continuous or pulsed laser beam with a power of 200 mW and a dose of 5 J/cm 2 Four- and six-fold irradiation of macrophages with a continuous laser beam of 200 mW and a dose of 5 J/cm 2 (200/5/C group) resulted in decreased protein secretion on days 5 and 7 of macrophage culturing (Fig. 5c). When cells were treated with a pulsed laser beam of the same power and dose (200/5/P group), an increase in protein secretion was observed after six-fold laser irradiation on day 7 of macrophage culturing, compared to the unirradiated cells (CTR group) (Fig. 5d). Based on the results obtained in stage II of the study (increases in the viability of macrophages and the level of protein secreted by the cells), supernatants from the cell cultures irradiated with a pulsed laser beam with a power of 200 mW and a dose of 5 J/cm 2 (200/5/P group) were selected for stage III of the study to assess the immunomodulatory effect of LLLT on macrophages. The immunomodulatory effect of LLLT on the secretory activity of macrophages irradiated with a pulsed laser beam with a power of 200 mW and a dose of 5 J/cm 2 (200/5/P group) The study showed that six-fold irradiation of macrophages resulted in an increased NO secretion on day 7 of macrophage culturing, compared to the unirradiated cells (Fig. 6a). Four-fold laser irradiation of macrophages reduced the level of MCP-1 and TNF-α secretion (Figs. 6b and 6c). Further six-fold laser irradiation decreased the secretion of TNF-α by macrophages compared to the unirradiated cells (CTR group) (Fig. 6c). For the remaining cytokines measured: IFN-γ, IL-12p70, IL-6 and IL-10, their presence was not detected in any of the tested groups of cells (200/5/P and CTR groups), regardless of the tested time point. The study also showed that four-fold laser irradiation of cells reduced the secretion of MMP-9 by macrophages compared to the unirradiated cells (CTR group) (Table 1). At all the time points examined, there was no secretion or no differences in the secretion of pro-MMP and MMP-2 by macrophages in the 200/5/P group, compared to the unirradiated cells (CTR group) (Table 1). Measurement of the TOS of macrophages showed that six-fold irradiation of macrophages resulted in an increased total oxidative/capacitive status of macrophages (TOS/TOC) compared to unirradiated cells (CTR group) (Fig. 7a). However, for the TAS of irradiated macrophages (TAS/TAC), no differences were found compared to theunirradiated cells (CTR group) (Fig. 7b). 4. Discussion Many scientific studies have shown that LLLT, as a result of contact with cells, may disturb homeostasis. It has been shown that it can affect cell viability and secretory activity 34,12,5 . However, the authors of these works focused primarily on examining the effect of laser radiation on fibroblasts, keratinocytes or osteoblasts 35,10,11,14,16 . However, little is known about the effect of LLL on the response of resting M0 macrophages, the activation of which is crucial in the course of inflammation 28 . In our study, assays based on assessment of the adhesion and morphology of cells were used to identify the impact of laser irradiation. The results showed significant differences in the ability of cells treated with LLLT to adhere/proliferate. The best effectiveness in promoting adhesion/proliferation was achieved when laser irradiation with a power of 200 mW and a dose of 5 J/cm 2 was used (200/5/C and 200/5/P groups). These results are confirmed by the results of observations of the morphology of macrophages in this group, which showed a clear increase in density and a tendency of proliferating cells to form clusters. However, it should be noted that the beneficial effect of LLLT on cells was observed when they were irradiated up to six times. Further irradiation of the cells resulted in decreased adhesion. The observed phenomenon probably resulted from the lack of free surface for cell attachment and/or the lack of nutrients in the culture medium, and not the effect of increasing the number of irradiations. The phenomenon of decreased cell adhesion in the following days of the experiment was also found in the control group. Unfortunately, due to the lack of studies in the available literature on the effect of LLLT on macrophage adhesion/proliferation, it is difficult to compare the results obtained with other studies. However, there are reports discussing the effect of LLLT on the adhesion of other cell types. For example, Li et al. (2020) showed that the use of LLLT at doses of 1.0 J/cm 2 , 2.0 J/cm 2 and 4.0 J/cm 2 increased the proliferation of HUVEC epithelial cells 33 . Similarly, Sperandio et al. (2014) showed increased proliferation of HaCaT keratinocytes at an applied wavelength of 660 nm, power of 100 mW and laser energy densities of 3 J/cm 2 , 6 J/cm 2 and 12 J/cm 2 36 . In turn, Basso et al. (2012) irradiated HGFs (human gingival fibroblasts) with a laser beam with a wavelength of 780 nm, power of 40 mW and doses of 0.5 J/cm 2 , 1.5 J/cm 2 , 3.5 J/cm 2 and 7 J/cm 2 , showing that only two (0.5 J/cm 2 and 3 J/cm 2 ) of the six doses tested increased cell proliferation, while the remaining doses did not affect cell proliferation 37 . The authors also observed no changes in the morphology of cells irradiated with LLLT 37 . In our study, we also checked the level of AK released from dead cells, assessing the possible cytotoxic effect of LLLT on macrophages. Regardless of the method of application of the radiation beam (continuous or pulsed), it was only in the group of cells irradiated with a laser with a power of 200 mW and a dose of 5 J/cm 2 that the level of AK released by the cells did not differ or was lower compared to the control group. Increasing the number of laser irradiations to eight and ten increased the AK released from cells. Our study results indicate a beneficial effect of the irradiation of macrophages with continuous or pulsed laser beams with a power of 200 mW and a dose of 5 J/cm 2 (200/5/C and 200/5/P groups). Therefore, these groups of cells were selected for further tests to assess cell viability and secretory activity measured by the level of secreted protein. Further tests in these groups showed increased viability of macrophages treated with a pulsed laser beam (200/5/P group), while irradiation with a continuous laser beam did not affect the viability of macrophages (200/5/C group). Similar results were obtained by Silva et al. (2016), who irradiated LPS-stimulated RAW 264.7 macrophages with two wavelengths: 660 nm and 880 nm, with a power of 100 mW and a dose of 214 J/cm 2 , showing an increase in viability only when irradiated with a wavelength of 660 nm 38 . Also according to Souza et al. (2014), LPS-stimulated J774 macrophages irradiated with a laser with a wavelength of 660 nm, power of 15 mW and a dose of 7.5 J/cm 2 and a wavelength of 780 nm, power of 70 mW and a dose of 3 J/cm 2 showed increased viability compared to the group of macrophages not exposed to LLLT 32 . In turn, Song et al. (2021) studied the effect of a continuous laser beam with a wavelength of 810 nm and power of 80 mW on LPS-stimulated RAW 264.7 macrophages, using laser radiation doses of 0.4 J/cm 2 , 1.2 J/cm 2 and 2.4 J/cm 2 8 . In this case, onl y the dose of 2.4 J/cm 2 significantly increased the viability of the tested cells. Similarly, Leden et al. (2013) examined the viability of LPS-stimulated BV2 macrophages irradiated with a laser with a wavelength of 808 nm, power of 50 mW and doses of 0.2 J/cm 2 , 4 J/cm 2 , 10 J/cm 2 and 30 J/cm 2 , showing that only irradiation with doses of 4 J/cm 2 and 30 J/cm 2 resulted in viability 39 . The available literature lacks reports assessing the immunomodulatory effect of LLLT on the secretory activity of resting M0 macrophages. In our study, we first checked the level of NO secreted by macrophages exposed to various radiation parameters. We showed that irradiation of macrophages with a continuous laser beam (200/5/C group) resulted in a decrease in NO and protein secretion, while the pulsed beam (200/5/P group) increased secretion, compared to unirradiated cells (CTR group). Additionally, we showed that irradiation of cells with a pulsed laser beam increased the TOS of macrophages, but had no effect on the TAS of cells. According to Silva et al. (2016), monocytes and RAW 264.7 macrophages irradiated with a laser with a wavelength of 808 nm secreted more NO than cells irradiated with a wavelength of 660 nm 38 . An increase in NO secretion by LPS-stimulated BV2 macrophages was also demonstrated by Leden et al. (2013), who irradiated cells with a laser beam of 808 nm, power of 50 mW and doses of 4 J/cm 2 and 30 J/cm 2 39 . These authors also showed that irradiation of cells with doses of 0.2 J/cm 2 and 10 J/cm 2 with the same wavelength and laser power did not affect the secretion of NO by cells. NO is an important factor involved in the inflammatory response, produced and secreted mainly, as already mentioned, by classically polarized M1 macrophages 28 . Most authors agree that a decreased level of NO secreted by cells protects against excessive activation of M1 macrophages and is probably related to the protection of tissues at the inflammation site. In turn, high levels of NO and other pro-inflammatory mediators secreted by M1 macrophages may impair the proper phagocytosis of apoptotic cells and also directly damage host tissues 40 . Reacting with proteins, NO can cause nitrosylation of amino residues, impairing their function and causing a cytotoxic/apoptotic effect on surrounding cells. It can also stimulate the secretion of pro-inflammatory cytokines, stimulating immune system cells and initiating inflammation 41 . Our study results showed a decreased secretion of cytokines TNF - α, MCP-1 and MMP-9 as a result of irradiation with a pulsed laser beam of 200 mW and a dose of 5 J/cm 2 . However, the samples we tested did not contain the other cytokines: IL-10, IL-12, IL-6 and IFN-γ. A decrease in TNF-α secretion with a simultaneous increase in IL-10, IL-4 and IL-13 secretion was noted by Song et al. (2017) who irradiated LPS-stimulated microglia cells with a laser with a wavelength of 810 nm, power of 150 mW and a dose of 4.5 J/cm 2 34 . Similarly, Fernandes et al. (2015) observed a decrease in the secretion of TNF-α by J774 macrophages irradiated with a laser with a wavelength of 780 nm, power of 70 mW and a dose of 2.6 J/cm 2 and 660 nm, 15 mW, 7.5 J/cm 2 , while showing increased secretion of IL-6 by the cells irradiated with a 660 nm laser compared to unirradiated cells 42 . Souza et al. (2014) also examined the effect of laser irradiation with a wavelength of 780 nm, power of 70 mW and a dose of 3 J/cm 2 on the level of TNF - α secreted by LPS-stimulated macrophages. In this case, cell irradiation also reduced TNF - α secretion 32 . In turn, Gavish et al. (2008) showed that laser irradiation with a laser with a wavelength of 780 nm, power of 2 mW and a dose of 2.2 J/cm 2 decreased the secretion of MCP-1 by RAW 264.7 macrophages and had no effect on the secretion of IL-1β 43 . Different results were obtained by Leden et al. (2013). In their study, LPS-stimulated BV2 macrophages, irradiated with a laser with a wavelength of 808 nm, power of 50 mW and a dose of 0.2 J/cm 2 , significantly increased the secretion of MCP-1, while higher doses of 4 J/cm 2 , 10 J/cm 2 and 30 J/cm 2 had no impact on the secretion of this cytokine by cells 39 . Li et al. (2020) measured the effect of laser irradiation with a wavelength of 810 nm, power of 150 mW and doses of 0.4 J/cm 2 , 4 J/cm 2 and 10 J/cm 2 on LPS-stimulated macrophages BMDM and observed that doses of 4 and 10 J/cm 2 , similarly to our study, inhibit the secretion of MCP-1, while a dose of 0.4 J/cm 2 increases the secretion of MCP-1 and reduces the secretion of IL-1β 33 . Unlike in our study, Li et al. (2020) also showed that the irradiation parameters they used did not affect cytokine secretion by resting BMDM macrophages 33 . The results of our study and the other studies presented above confirm the concept that the treatment of macrophages with LLLT may modulate their inflammatory response, and the directions of laser irradiation and macrophage polarization depend on the type of macrophages, their previous stimulation and the irradiation parameters (wavelength, power, dose and method of applying the laser beam). The effect of LLLT on the secretion of MMP-2 and MMP-9 by macrophages has not been described. Studies dealing with this issue mainly focus on assessing its impact on other types of cells, primarily fibroblasts and osteoblasts 44,45 . For example, Ayuk et al. (2018) irradiated WS1 fibroblasts with two wavelengths: 660 nm (with a power of 108 mW) and 830 nm (with a power of 94 mW) and a dose of 5 J/cm 2 , showing that, regardless of the irradiation parameters used, the cells secreted less MMP-9 compared to the control 44 . In turn, Oliviera et al. (2017) irradiated MC3T3 osteoblasts with a laser with a wavelength of 660 nm and 780 nm, power of 20 mW and doses of 1.9 J/cm 2 and 3.8 J/cm 2 , showing no effect of irradiation on the level of secreted MMP-9 and an increase in the secretion of MMP-2 by cells irradiated with a laser with a wavelength of 660 nm and a dose 1.9 J/cm 2 45 . In our study, we have shown that irradiation of cells with a pulsed laser beam with a power of 200 mW and a dose of 5 J/cm 2 increases the secretion of NO by macrophages and their TOS, which may suggest the polarization of macrophages towards the M1 phenotype. On the other hand, a decrease in the secretion of pro-inflammatory cytokines (TNF-α and MCP-1) and MMP-9 by cells may indicate the polarization of macrophages towards the M2 phenotype. Conclusions Our study results suggest that LLLT modulates the biological response of resting RAW 264.7 macrophages and may also influence their polarization. It seems extremely likely that for an optimal response of macrophages, they often share common features of the M1 and M2 phenotypes and that their phenotype should be considered as a spectrum of continuous differentiation under the influence of LLLT. To elucidate the mechanisms underlying such immunomodulation, further studies assessing the effect of LLLT on other markers of resting macrophage polarization are necessary. Declarations Acknowledgements This research project was supported by the statutory activities of the Academy of Physical Education in Krakow (no. 269/BS/INS/21) and the programme ‘Excellence Initiative – Study University’ of the University of Science and Technology (AGH), project no. 4204. Data availability The authors declare that the data supporting the results of this study are available in the article and its supporting documents. If any raw data files are needed in another format, they are available from the corresponding author upon reasonable request References Zhang, Y. & Ji, Q. Current advances of photobiomodulation therapy in treating knee osteoarthritis. Front Cell Dev Biol 16 , 1286025 (2023). Santonocito, S. et al . Impact of Laser Therapy on Periodontal and Peri-Implant Diseases. 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Table 1 Table 1. The effect of irradiation with a pulsed (P) laser beam on the level of MMPs secreted by macrophages of the RAW 264.7 cell line. Grups Days Number of laser application Tested indicator pro-MMP-9 MMP-9 MMP-2 x̅ SEM Between groups comparison 200/5/P v CTR x̅ SEM Between groups comparison 200/5/P v CTR x̅ SEM Between groups comparison 200/5/P v CTR CTR 3 0 nd day 5 p=0,144 day 7 p=0,143 nd day 3 p=0,05 day 5 p= 0,024 * 4900 1580 day 3 p=0,168 5 0 nd 4498 280 nd 7 0 2513 686 nd nd 200/5/P 3 2 nd 147,06 61,34 1158 466,87 5 4 419,52 286,3 2116,8 574 nd 7 6 16132 9230 nd nd The cells were cultured for a specified number of days (3, 5, 7) and irradiated (2, 4 and 6 times, respectively) with a laser with power of 200 mW and a radiation dose of 5 J/cm 2 /well with cells. The level of metalloproteinases was determined on the following days of the experiment: 3, 5 and 7. Mean values ± SEM; nd - no metalloproteinases were detected. (* for p<0.05) - statistically significant differences between the group of irradiated cells (200/5/P) and the group of non-irradiated cells (CTR group) on individual days of the experiment (3, 5, 7). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4620625","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":324592843,"identity":"4bbdc62f-77e5-4e60-ba40-d82cfac4023a","order_by":0,"name":"Aleksandra Matuła","email":"","orcid":"","institution":"Academy of Physical Education","correspondingAuthor":false,"prefix":"","firstName":"Aleksandra","middleName":"","lastName":"Matuła","suffix":""},{"id":324592847,"identity":"a5a45fe6-ab92-4861-8fd8-cacf9cb49700","order_by":1,"name":"Amelia Lizak","email":"","orcid":"","institution":"Academy of Physical Education","correspondingAuthor":false,"prefix":"","firstName":"Amelia","middleName":"","lastName":"Lizak","suffix":""},{"id":324592848,"identity":"11c3819b-d9c9-432e-84d3-c259ee537011","order_by":2,"name":"Ewa Stodolak-Zych","email":"","orcid":"","institution":"AGH University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ewa","middleName":"","lastName":"Stodolak-Zych","suffix":""},{"id":324592850,"identity":"e5372c92-c862-4af5-892a-50e3795ba20a","order_by":3,"name":"Aneta Bac","email":"","orcid":"","institution":"Academy of Physical Education","correspondingAuthor":false,"prefix":"","firstName":"Aneta","middleName":"","lastName":"Bac","suffix":""},{"id":324592853,"identity":"d7c5c8fa-bce0-411e-b01a-07157ecb020f","order_by":4,"name":"Joanna Homa","email":"","orcid":"","institution":"University of Physical Education and Sport","correspondingAuthor":false,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Homa","suffix":""},{"id":324592855,"identity":"eb30675f-40ab-4922-905e-301f1aec7db7","order_by":5,"name":"Beata Stenka","email":"","orcid":"","institution":"Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Beata","middleName":"","lastName":"Stenka","suffix":""},{"id":324592857,"identity":"5969c29c-59c2-4706-9778-ff2e39dc4580","order_by":6,"name":"Anna Scislowska-Czarnecka","email":"data:image/png;base64,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","orcid":"","institution":"Academy of Physical Education","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"","lastName":"Scislowska-Czarnecka","suffix":""}],"badges":[],"createdAt":"2024-06-22 07:29:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4620625/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4620625/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60493159,"identity":"e9114659-9b4a-4c2e-91ce-9109ce6de2a5","added_by":"auto","created_at":"2024-07-17 11:10:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1228852,"visible":true,"origin":"","legend":"\u003cp\u003eResearch design on the impact of LLLT on macrophage of the RAW 264.7 cell line.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4620625/v1/8d1316cd00f1d2260e9055f7.png"},{"id":60492607,"identity":"9cdbf399-c6a3-4291-a78b-3572322dd4a5","added_by":"auto","created_at":"2024-07-17 11:02:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":143179,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of continuous (C) (Fig. 2a) and pulsed (P) laser beam (Fig. 2b) on macrophage adhesion of the RAW 264.7 cell line. Cells were cultured for a specified number of days and irradiated with a laser with power of 100 or 200 mW, radiation doses of 5 or 10 J/cm\u003csup\u003e2\u003c/sup\u003e/cell well. On subsequent days of the experiment (3, 5, 7, 9, 11), cells were stained with crystal violet. O.D. - optical density was measured at 570 nm. Mean values ± SEM. *, **, *** - differences between cells irradiated with laser of different parameters and cells not irradiated (CTR), (* \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4620625/v1/29d6c412e52140a16e6319a0.png"},{"id":60493162,"identity":"12a0c225-9380-4ad1-b77b-ee1a25690501","added_by":"auto","created_at":"2024-07-17 11:10:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3333136,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of continuous (C) and pulsed (P) laser beam irradiation on the morphology of macrophages of the RAW 264.7 cell line. Cells were cultured for a specified number of days and irradiated with a laser with power of 100 or 200 mW, radiation doses of 5 or 10 J/cm\u003csup\u003e2\u003c/sup\u003e/well with cells. On the following days of the experiment (3, 5, 7, 9, 11), the cells were stained with crystal violet. Analysis under a light microscope, magnification 40x, scale 100 mm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4620625/v1/72b3d38e44404c025f43be80.png"},{"id":60493161,"identity":"5e888f0b-ab10-4a59-8c78-8b821f7497a9","added_by":"auto","created_at":"2024-07-17 11:10:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":146934,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of continuous (C) (Fig. 4a) and pulsed (P) (Fig. 4b) laser beam irradiation on cell adenylyl kinase (AK) release levels by macrophages of the RAW 264.7 cell line. Cells were cultured for a specified number of days and irradiated with a laser with power of 100 or 200 mW, radiation doses of 5 or 10 J/cm\u003csup\u003e2\u003c/sup\u003e/well with cells. AK levels were measured on the next 3, 5, 7, 9 and 11 days of the experiment. RLUs - luminometer flux unit RLUs - luminometer flux unit. Mean values ± SEM. *, **, *** - differences between cells irradiated with laser of different parameters and cells not irradiated (CTR), (* \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4620625/v1/5414e51f0d768edb87e6af8f.png"},{"id":60492613,"identity":"69fe01d4-17e9-40d1-807e-584972163ddb","added_by":"auto","created_at":"2024-07-17 11:02:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98901,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of continuous (C) (Fig. 5a, c) and pulsed (P) (Fig. 5b, d) laser beam irradiation on viability and protein levels secreted by macrophages of the RAW 264.7 cell line. Cells were cultured for a specified number of days and irradiated with a laser with power of 200 mW, a radiation dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e/well with cells. Viability and protein levels were measured on the next 3, 5 and 7 days of the experiment. RLUs - luminometer flux unit. O.D. - optical density measured at 540 nm. Mean values ± SEM. *** - differences between 200/5/C group and control group (CTR), and between 200/5/P and control group (CTR), (* \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4620625/v1/03a7c9039cb32c1a5167b739.png"},{"id":60492608,"identity":"c3908c82-2148-4061-ab96-0d1e0147ab42","added_by":"auto","created_at":"2024-07-17 11:02:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":185595,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pulsed (P) laser beam irradiation on the levels of the nitric oxide (NO) (Fig. 6a) and cytokines MCP-1 (Fig. 6b), TNF-α (Fig. 6c) secreted by macrophages of the RAW 264.7 cell line and example dot plots of cytometric analysis of fluorescently labeled cytokines: MCP-1, TNF-α (Fig. 6d). Cells were cultured for a specified number of days and irradiated with a laser with power of 200 mW, a radiation dose of 5J/cm\u003csup\u003e2\u003c/sup\u003e/well with cells. Cytokine levels were measured on the next 3, 5 and 7 days of the experiment. Mean values ± SM. (* for p\u0026lt;0.05) - statistically significant differences between the group of irradiated cells (200/5/P) and the group of non-irradiated cells (CTR group)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4620625/v1/b86eece9abfbc9f1698c1f8b.png"},{"id":60492610,"identity":"b4764a26-b90e-4306-90fc-87e3dc6fc812","added_by":"auto","created_at":"2024-07-17 11:02:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":54699,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of irradiation with a pulsed laser beam (P) on the oxidative (Fig. 7a) and antioxidant (Fig. 7b) potential of macrophages of the RAW 264.7 cell line. Cells were cultured for a specified number of days and irradiated with a laser with power of 200 mW and a radiation dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e/well with cells. Oxidative potential (TOS/TOC) and antioxidant potential (TAS/TAC) were determined on the following days of the experiment: 3, 5 and 7. Mean values ± SEM. (* for p\u0026lt;0.05) - statistically significant differences between the group of irradiated cells (200/5/P) and the group of non-irradiated cells (CTR group)\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4620625/v1/6a5b17b042f4beac515f7d91.png"},{"id":65642051,"identity":"c41ff024-1217-4fbb-b7d6-b8d9cce38c8d","added_by":"auto","created_at":"2024-09-30 20:16:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7023749,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4620625/v1/78a71001-735a-4ecb-a449-8e4920709688.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effect of low-level laser therapy conditions on macrophages’ immunomodulatory processes as an example of regeneration process stimulation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCell biomodulation offers new possibilities for supporting the treatment of many diseases, including degenerative and periodontal ones, and chronic pain \u003csup\u003e1;2,3,4\u003c/sup\u003e. This method can also be used to initiate regenerative processes in damaged tissues \u003csup\u003e5,6\u003c/sup\u003e. The latter process \u0026ndash; regeneration \u0026ndash; can be induced or intensified by the synergistic action of inflammatory cells (macrophages) stimulated by low-level laser therapy (LLLT) \u003csup\u003e7,8\u003c/sup\u003e. This approach allows modulation of biochemical and molecular processes in living cells. It has been found that at the cellular level, under the influence of LLLT, changes occur in the production of adenosine triphosphate (ATP), the synthesis of free radicals of reactive oxygen and nitrogen species (RONS) is regulated and transcription factors are induced. Transcription factors influence cell adhesion, proliferation, apoptosis and secretory activity, measured by the synthesis of various types of proteins, among others \u003csup\u003e9,10\u0026ndash;17, 5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLLLT covers a wide range of parameters in terms of laser properties and dosage, which is important for its effects. To obtain the desired therapeutic effect of LLLT on cells, it is important to select optimal irradiation conditions: radiation wavelength, surface power density, energy supplied and the number of irradiations \u003csup\u003e6,18,19\u003c/sup\u003e. These factors determine what changes will be induced in cells under the influence of LLLT. Most studies on cell biomodulation supported by LLLT mainly assess its impact on skin cells e.g. fibroblasts. It has been shown that LLLT parameters that are too low may result in no cellular response or one that is too weak, while using irradiation parameters that are too strong may have a cytotoxic effect on cells or may not initiate the cellular response at the expected level. An example of such behaviour has been described by (Crisan 2013), among others, who compared the effects of radiation wavelengths of 830 nm and 980 nm on fibroblasts, showing that both significantly stimulated cell proliferation after one-, two- and three-fold irradiations \u003csup\u003e20\u003c/sup\u003e. Ma et al. (2018) have also described the increased proliferation of human fibroblasts and collagen synthesis under irradiation with a wavelength of 830 nm, while irradiation with a wavelength of 635 nm did not increase fibroblast proliferation and collagen synthesis\u003csup\u003e21\u003c/sup\u003e. Mignon et al. (2018) have shown that shorter wavelengths of radiation (530 nm) inhibit the metabolic activity of fibroblasts, while longer wavelengths (550\u0026ndash;850 nm) do not produce such an effect \u003csup\u003e22\u003c/sup\u003e. It has been shown that the radiation dose is also a factor that modulates the cellular response. Hawkins and Abrahams (2006) treated human skin fibroblasts with radiation doses of 2.5 J/cm\u003csup\u003e2\u003c/sup\u003e, 5 J/cm\u003csup\u003e2\u003c/sup\u003e and 16 J/cm\u003csup\u003e2\u003c/sup\u003e for two consecutive days. The results showed that a dose of 2.5 J/cm\u003csup\u003e2\u003c/sup\u003e for daily irradiation and 5 J/cm\u003csup\u003e2\u003c/sup\u003e for single irradiation increased cell viability, proliferation and migration. However, exposure at a dose of 16 J/cm\u003csup\u003e2\u003c/sup\u003e inhibited cell proliferation, which had a negative effect on their viability and migration\u003csup\u003e23\u003c/sup\u003e. Another study also showed similar findings that laser irradiation at doses of 3 J/cm\u003csup\u003e2\u003c/sup\u003e and 4 J/cm\u003csup\u003e2\u003c/sup\u003e for six consecutive days resulted in an increase in fibroblast proliferation compared to the control group, while a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e did not stimulate cell proliferation\u003csup\u003e24\u003c/sup\u003e. The power of the laser radiation is also a factor influencing the effectiveness of the cellular response. Azevado et al. (2006) showed that the proliferation of fibroblasts increased after two- and six-fold irradiations of cells with a power of 10 mW and 29 mW\u003csup\u003e6\u003c/sup\u003e. Illescas-Montes et al. (2017) observed an increase in the number of cells at an irradiation power of 0.2 W and 0.5 W, while the use of an irradiation power of 1 W did not affect the number of cells\u003csup\u003e25\u003c/sup\u003e. In turn, Chen et al. (2009) showed a decrease in the viability of human fibroblasts after irradiating cells with a laser with a power of 1.0\u0026ndash;3.0 W\u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere is little information in the literature on the impact of LLLT on the response of immune system cells, including macrophages, the activation of which plays a key role in the inflammation process and also during tissue regeneration \u003csup\u003e27\u0026ndash;30\u003c/sup\u003e. Macrophages express many receptors and signalling molecules, and their activation depends on local conditions in the tissue. Due to their very diverse, often contradictory functions, macrophages are divided into classically polarized M1 macrophages and alternatively polarized M2 macrophages. M1 macrophages occur mainly in the early stages of inflammation and activate pro-inflammatory signalling pathways. The factors secreted by M1 macrophages include free radicals of RONS, pro-inflammatory cytokines (TNF-α, IL-1 IL-12p70) and many chemokines, such as CCL15, CCL20 and CXCL8. In turn, M2 macrophages are primarily involved in the processes ending inflammation and promoting tissue remodelling \u003csup\u003e28,29\u003c/sup\u003e. The factors secreted by M2 macrophages are cytokines IL10 and TGF-β, chemokines CCL22 and CCL17 and metalloproteinases (MMPs), which are associated with the remodelling of the extracellular matrix (ECM) during regenerative processes in damaged tissues. According to reports on the role and potential of macrophage polarization, the possibility of immunomodulating their behaviour towards the M1 or M2 phenotype with LLLT seems important \u003csup\u003e31\u003c/sup\u003e. The available literature data on the effect of LLLT on macrophages mostly concern the response of macrophages previously stimulated with lipopolysaccharide (LPS) (LPS-stimulated macrophages) \u003csup\u003e32,33,8\u003c/sup\u003e. However, there is no information on the directional effect of LLLT on resting M0 macrophages.\u003c/p\u003e \u003cp\u003eThis article focuses on the selection of favourable (biostimulating) exposure conditions for LLLT, which are the method of beam application (continuous [C] or pulsed [P] laser beam), the radiation power and dose, and then the assessment of the immunomodulatory effect of LLLT on resting macrophages of the RAW 264.7 cell line.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e \u003cb\u003eMacrophage culture conditions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe RAW 264.7 macrophage cell line (ATCC, USA) was used in the study. The cells were cultured in RPMI 1640 culture medium (Lonza, USA) with the addition of 10% FBS calf serum (Gibco, USA) and a 5% solution of antibiotics, penicillin and streptomycin (Sigma-Aldrich, USA), in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at a temperature of 37\u003csup\u003eo\u003c/sup\u003eC. Then, 1 ml of cell suspension with a concentration of 1.5 x 10\u003csup\u003e4\u003c/sup\u003e million cells/ml was placed in the wells of a 24-well culture plate (Nest SB, USA) at the bottom of which round culture slides had previously been placed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLow-level laser therapy\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA PhysioGo 400C device (ASTAR, Poland) was used to irradiate the macrophages. It is a low-level laser (LLL) that generates electromagnetic radiation in the infrared light range with a wavelength of 808 nm, power of 100 mW or 200 mW and radiation doses of 5 J/cm\u003csup\u003e2\u003c/sup\u003e/well with cells or 10 J/cm\u003csup\u003e2\u003c/sup\u003e/well with cells. The laser beam was continuous (C) or pulsed (P) (at a frequency of 100 Hz with a 50% fill factor). Irradiation was performed using a non-contact method, at a distance of 1 cm from the cells, at a right angle to the irradiated surface.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStudy stages\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe study was carried out in three stages (Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eIn stage I of the study, laser radiation was applied once a day (at the same time of day) two, four, six, 8 or ten times. On the following days of the experiment: on day 3 (two laser beam applications), on day 5 (four laser beam applications), on day 7 (six laser beam applications), on day 9 (eight laser beam applications) and on day 11 (ten laser beam applications), the macrophage culturing was ended and the cells and cell culture supernatants were assessed for adhesion/proliferation, morphology and the level of adenylate kinase (AK) released from dead cells.\u003c/p\u003e \u003cp\u003eStage I of the study was carried out for the following groups:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eunirradiated cells (CTR group \u0026ndash; control group);\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ecells irradiated with a continuous laser beam (C) with a power of 100 mW or 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e or 10 J/cm\u003csup\u003e2\u003c/sup\u003e (100/5/C, 100/10/C, 200/5/C and 200/10/C groups); and\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ecells irradiated with a pulsed laser beam (P) with a power of 100 mW or 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e or 10 J/cm\u003csup\u003e2\u003c/sup\u003e (100/5/P, 100/10/P, 200/5/P and 200/10/P groups).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe experimental data made it possible to answer the question of how repeated exposure to LLLT with precisely defined parameters (power, intensity and exposure frequency) affects macrophages.\u003c/p\u003e \u003cp\u003eIn stage II of the study, radiaton doses of a specific power that had a biostimulating effect on cells were used. The cells were irradiated with a continuous (C) or pulsed (P) laser beam with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e. Laser radiation was applied once a day (at the same time of day) two, four or six times. On the following days of the experiment: on day 3 (two laser beam applications), on day 5 (4 laser beam applications), on day 7 (six laser beam applications), the macrophage culture was ended and the cells and cell culture supernatants were assessed for the functional state of macrophages by determining their viability and the level of secreted protein.\u003c/p\u003e \u003cp\u003eStage II of the study was carried out for the following groups:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eunirradiated cells (CTR group);\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ecells irradiated with a continuous (C) laser beam with a power of 200 mW, a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (200/5/C group); and\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ecells irradiated with a pulsed (P) laser beam with a power of 200 mW, a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (200/5/P group).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eIn stage III of the study, the immunomodulatory effect of LLLT on macrophages was assessed on the supernatants collected from the cell cultures irradiated with a pulsed (P) laser beam with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (200/5/P group). In this group, the metabolic and immunological activity of the macrophages was examined by determining the levels of: nitric oxide (NO), cytokines (MCP-1), tumour necrosis factor TNF-α, interferon gamma IFN-γ, interleukin 12p70, interleukin 6, interleukin 10, metalloproteinases (MMP-2 and MMP-9) and the total oxidative/capacitive status (TOS/TOC) and total antioxidant/ capacitive status (TAS/TAC) of the cells was determined.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell adhesion/proliferation: Crystal violet uptake assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCell adhesion was tested using the crystal violet (CV) uptake assay. Cells adhered to the substrate were fixed for 5 minutes with 2% paraformaldehyde (PFA) (Sigma, USA), stained for 5 minutes with 0.5% CV solution (Sigma, USA) and rinsed with water. Then, the dye absorbed by macrophages was extracted by adding 0.5 ml of 100% methanol (Linegal Chemicals, Poland) to each well. The optical density (OD) of the fluid was read using a FLUOstar Omega reader (BMG Labtech, Germany) at a wavelength of 570 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell morphology\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSome of the tested samples (two from each series) were intended for microscopic observation. Adherent cells were stained for 15 seconds with a 0.5% solution of CV in water (Sigma, USA) and then rinsed with water. Cells were observed using a light microscope (Motic AE-2000T, Germany) at 40x magnification. Pictures of cells adhering to the substrate were taken with a Moticam-BTU8 microscope camera (MoticEurope, Spain).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLevel of released AK (ToxiLight assay)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe level of AK was determined quantitatively by the bioluminescence method using a Toxilight reagent set (Lonza, Switzerland). First, 20 \u0026micro;l of supernatant was collected from the cell culture and transferred to a white 96-well plate (Nest SB, USA). Then, 100 \u0026micro;l of AK detection reagent solution was added to each well. After 5 minutes of incubation, luminescence was read using a FLUOstar Omega reader (BMG Labtech, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell viability (ViaLight assay)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo test the viability of macrophages in the culture, a ready-made assay with a ViaLight reagent set (Lozna, Switzerland) was used. First, 200 \u0026micro;l of cell lysis reagent was added to the wells containing cells and 600 \u0026micro;l of supernatant. After 10 minutes of incubation, 200 \u0026micro;l of the supernatant-lyser mixture was transferred to a white 96-well plate (Nest SB, USA) and 200 \u0026micro;l of AMR PLUS reagent was added. After 2 minutes, the amount of radiation emitted was determined using a FLUOstar Omega reader (BMG Labtech, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLevel of secreted protein (BCA assay)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFirst, 10 \u0026micro;l of the tested samples were transferred to each well of a 96-well plate (Nest SB, USA) and 200 \u0026micro;l of a mixture consisting of copper sulfate II (CS (II)) (Sigma-Aldrich, Germany) and bicinchoninic acid (BCA) (Sigma-Aldrich, Germany), mixed in a ratio of 1:50, was added. The plates were then incubated for 30 minutes in the dark. After the designated time, the OD of the liquid was read at a wavelength of 570 nm using a FLUOstar Omega reader (BMG Labtech, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLevel of secreted NO (Griess test)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFirst, 100 \u0026micro;l of cell culture supernatant was transferred to each well of a 96-well plate (Nest SB, USA). Then, 100 \u0026micro;l of reagents (Sigma-Aldrich, Germany): Griess A (1% sulfalamide in 5% phosphate acid) and B (0.1% naphthylenediamine in H\u003csub\u003e2\u003c/sub\u003eO), mixed in a 1:1 ratio, was added. After 5 minutes, the OD of the liquid was read at a wavelength of 540 nm using a FLUOstar Omega reader (BMG Labtech, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLevel of secreted cytokines\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCytokine levels in the cell culture supernatants were measured by flow cytometry using a Flex Set (CBA, BD Biosciences). The entire assay procedure, as well as all measurements and analyses, were performed following the instructions attached to the cytokine assay kit using a Beckman Coulter flow cytometer (Life Science, USA). The Mouse Inflammation Kit (BD Biosciences, USA) was used, which allows simultaneous determination of the level of six cytokines: interleukin (MCP-1), tumour necrosis factor (TNF-α), interferon gamma (IFN-γ), interleukin 12p70 (IL-12p70), interleukin 6 (IL-6) and interleukin 10 (IL-10). First, 50 \u0026micro;l of a solution containing a mixture of beads coated with antibodies directed against each of the six analysed cytokines was added to each Eppendorf tube (Nest SB, USA) containing the standard, a sample or the control. In the next step of the procedure, standards or samples were added to the Eppendorf tubes and an assay diluent was added to the negative control. Then, 50 \u0026micro;l of phycoerythrin (PE)-conjugated antibodies directed against each of the six cytokines were added to all Eppendorf tubes and the samples were then incubated in the dark for 2 hours at room temperature (RT). After this time, 1 ml of washing buffer was added to each sample and the samples were centrifuged (200 g, 5 min, RT). The supernatant from the pellets was collected using a pipette and 300 \u0026micro;l of washing buffer was added to each sample. Then, the samples were transferred to new Eppendorf tubes (Nest SB, USA), where measurements were performed by a cytometer. The flow cytometer was calibrated using BD CaliBRITE\u0026trade; beads, which are microspheres coated with fluorescein isothiocyanate (FITC) (FL-1), PE (FL-2) and peridinine-chlorophyll protein complex (PerCp) (FL-3). Next, the device was calibrated using CytExpert Software for the CytoFLEX Platform with the Humane Inflammation Kit using positive controls for FITC and PE detectors. The data were analysed and cytokine concentrations were determined in Microsoft Excel using standard curves based on successive dilutions of the standard. The maximum concentration of the cytokine standard was 5,000 pg/ml. Subsequent serial dilutions were made in the assay diluent at 1:2, 1:4 and 1:8, respectively, ending with a dilution of 1:256, which corresponds to a cytokine concentration of 20 pg/ml.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLevel of secreted metalloproteinases\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe level of metalloproteinases secreted by the cells (in the form of an inactive enzyme precursor and active enzyme) was measured using the gelatin zymography method, which is a modified electrophoretic method that allows measuring the proteolytic activity of enzymes whose substrate can be incorporated in a polyacrylamide gel with the addition of sodium dodecyl sulfate (SDS). This method allows detection of the levels of pro-metalloproteinases 9 (pro-MMP-9) and 2 (pro-MMP-2), as well as metalloproteinases 9 (MMP-9) and 2 (MMP-2). First, solutions of separating gel (10%) and thickening gel (4%) were prepared. Polymerization catalysts (TEMED, APS) were added immediately before pouring the gels. The separating gel was poured first and some space left for the thickening gel, in which, after pouring, combs for the wells were placed. After solidifying (about 1 h), the gels were placed in the refrigerator for 12 h at 4\u0026deg;C. To quantitatively analyse the protein levels in the samples, the BCA assay was performed (see section 4.10). Based on the results obtained, protein levels were normalized in the tested samples. Samples with the same protein content were used for further analyses. First, 10 \u0026micro;l of protein standard with a wide range of proteins (coloured with SDS-PAGE; Bio-Rad, USA) was added to the first well and 15 \u0026micro;l of the tested samples added to the remaining wells. Electrophoresis was performed for 45 minutes. After electrophoresis was completed, the gels were collected and then washed (on an Elpan shaker, Poland) twice for 15 minutes in a 2.5% Triton X-100 solution (Bio-Rad, USA) to remove the SDS. After this, the gels were placed in an incubation buffer with the following composition: 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e; 50 mM Tris-HCl (pH\u0026thinsp;=\u0026thinsp;8); 0.02% NaN\u003csub\u003e3\u003c/sub\u003e 1 \u0026micro;M ZnSO\u003csub\u003e4\u003c/sub\u003e (all from Sigma-Aldrich, Germany) and incubated for 24 hours in a water bath at 37\u0026deg;C. Then, the gels were stained in 0.5% brilliant blue solution (Sigma-Aldrich, Germany) and then destained in an equilibration buffer until light bands were visible. Syngen\u0026rsquo;s Snaap Gene and GeneTools software were used to analyse the obtained scans. The presence of gelatinases was determined based on the molecular masses of the decolourized bands read from the protein standard.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasurement of the total oxidative/capacitive status (PerOx [TOS/TOC] assay)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe TOS of the cells was determined by measuring lipid peroxide levels according to the instructions of the PerOx (TOS/TOC) kit (Immunodiagnostik AG, Bensheim, Germany). Peroxide levels in the tested cell culture supernatant samples were determined based on the reaction of horseradish peroxidase with tetramethylbenzidine dichloride (TMB) in the presence of hydrogen peroxide. The reaction with the enzyme produces a soluble blue product. The addition of 2 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e stops the reaction, leading to the solution changing colour from blue to yellow. According to the kit instructions, 10 \u0026micro;l of calibrator (CAL), controls 1 and 2 (CTRL1, CTRL2) (reagents included in the kit) or the tested sample were added to the wells of a 96-well plate (included in the kit). Then, 100 \u0026micro;l of buffer (Reabuf A) included in the kit was added to each well of the plate and the first OD reading was taken at a wavelength of 450 nm, using a FLUOstar Omega reader (BMG Labtech, Germany). Then, 100 \u0026micro;l of the buffer mixture (RBF) (reagent included in the kit) was added to all wells and in the next step, the plate was incubated for 15 min at 37\u003csup\u003eo\u003c/sup\u003eC. After this, 50 \u0026micro;l of the reaction-stopping reagent Stop Solution was added to each well and the second OD reading was taken, also at a wavelength of 450 nm, using a FLUOstar Omega reader (BMG Labtech, Germany). Based on the measured optical OD values, the total peroxide level (\u0026micro;mol/l) was determined in the tested cell culture supernatant samples, according to the instructions provided by the kit manufacturer (Immunodiagnostik AG, Bensheim, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasurement of the total antioxidant/capacitive status (ImAnOx \u0026ndash; TAS/TAC assay)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe TAS of the cells was determined by reacting antioxidants with a predetermined (known) amount of exogenous hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), according to the protocol of the ImAnOx (TAS/TAC) kit (Immunodiagnostik AG, Bensheim, Germany). In this assay, antioxidants react with peroxide and the amount of unreacted H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is measured spectrophotometrically. The difference between the added and measured amount of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (relative to the calibrator included in the kit) is proportional to the antioxidant activity. First, 10 \u0026micro;l of calibrator (CAL), controls 1 and 2 (CTRL1, CTRL2) (reagents present in the kit) or the test sample were added to the wells of the 96-well plates included in the ImAnOx kit. Then, 100 \u0026micro;l of Reagent 1 was added to all wells of the plates, and then the plates were incubated for 10 min at 37\u003csup\u003eo\u003c/sup\u003eC. After the designated incubation time, 100 \u0026micro;l of Reagent 2a was added to all wells of one plate (with enzyme), and Reagent 2b was added to the other plate (without enzyme). Then, both plates were incubated for 5 minutes at RT, and then 50 \u0026micro;l of Stop Solution reagent was added to all wells. The OD reading was performed using a FLUOstar Omega reader (BMG Labtech, Germany) at a wavelength of 450 nm. Based on the measured OD values, the TAS in the cell culture supernatants was determined, expressed in \u0026micro;mol/l, according to the instructions provided by the kit manufacturer (Immunodiagnostik AG, Bensheim, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eData were presented as mean values ​​and standard error. Differences between the control group and the experimental groups were performed using the Student\u0026rsquo;s t-test if the assumptions for parametric tests were met. If the assumptions were not met, the non-parametric Mann-Whitney U test was applied. Differences between the control group and the experimental groups were compared using the one-way analysis of variance (ANOVA), Then, the Tukey test was applied for post-hoc evaluation. A significance level of p\u0026thinsp;\u0026le;\u0026thinsp;0.05 was adopted in the analyses.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003eSelection of optimal LLLT exposure parameters\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAdhesion/proliferation and morphology of macrophages irradiated with LLL\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOn the following days of the experiment (days 3\u0026ndash;9), unirradiated macrophages (CTR group) proliferated, which resulted in an increased number of adherent cells on days 5, 7 and 9, compared to day 3 of macrophage culturing (Table\u0026nbsp;1). On day 11 of macrophage culturing, the number of adherent cells decreased but was still higher than on day 3 of macrophage culturing (Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eThe analysis of the effect of a continuous laser beam on the adhesion/proliferation and morphology of macrophages allowed the conclusion that the two- and six-fold irradiation of cells (days 3 and 7) with a laser with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (200/5/C group) resulted in increased adhesion (Fig.\u0026nbsp;2a) and a higher density of macrophages visible in the morphological picture (Fig.\u0026nbsp;3) compared to unirradiated cells (CTR group). In this group (200/5/C), further eight-fold irradiation had no effect, and ten-fold irradiation resulted in decreased cell adhesion to the substrate (Fig.\u0026nbsp;2a). Increased adhesion of macrophages as a result of two applications with a continuous laser beam (day 3) was also observed in the case of cell irradiation with a laser with a power of 100 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e and 10 J/cm\u003csup\u003e2\u003c/sup\u003e (100/5/C and 100/10/C groups) and a power of 200 mW and a dose of 10 J/cm\u003csup\u003e2\u003c/sup\u003e (200/10/C group) (Fig.\u0026nbsp;2a). In this case, further cell irradiation had no effect or resulted in decreased cell adhesion, particularly on days 9 and 11 of macrophage culturing (100/5/C, 100/10/C, 200/5/C and 200/10/C groups) (Fig.\u0026nbsp;2a).\u003c/p\u003e \u003cp\u003eIn the case of pulsed beam treatment, for two- and six-fold irradiation of cells with a laser with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e/well (200/5/P group), it was also found that cell adhesion to the substrate increased compared to the unirradiated cells (CTR group) (Fig.\u0026nbsp;2b) and the density of macrophages was higher in the microscopic picture (Fig.\u0026nbsp;3). Further irradiation of macrophages with a laser with these parameters had no effect (day 9) or resulted in decreased cell adhesion to the substrate (day 11) (200/5/P group). An increase in cell adhesion was also found after two-fold irradiation with a pulsed laser beam of 100 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (100/5/P group), however, further laser irradiation decreased cell adhesion compared to the unirradiated cells (CTR group). In turn, irradiation of cells with a laser with a power of 100 mW or 200 mW and a dose of 10 J/cm\u003csup\u003e2\u003c/sup\u003e (100/10/P and 200/10/P groups) resulted in decreased cell adhesion compared to the unirradiated cells (CTR group) (Fig.\u0026nbsp;2b). The results of adhesion are confirmed by the microscopic images of cells, where an increase in adhesion was accompanied by an increase in cell density, while a decrease in cell adhesion was accompanied by a decrease in cell density and a cluster arrangement (Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLevel of AK released by macrophages exposed to LLLT\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTwo-, four- and six-fold irradiation with a continuous laser beam of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (200/5/C group) did not affect the level of AK released by macrophages (Fig.\u0026nbsp;4a). In turn, further eight- and ten-times applications of a laser beam with these parameters resulted in an increased AK level, compared to the unirradiated cells (CTR group) (Fig.\u0026nbsp;4a). Irradiation (regardless of the number of applications) with a continuous laser beam with a power of 100 mW and doses of 5 J/cm\u003csup\u003e2\u003c/sup\u003e and 10 J/cm\u003csup\u003e2\u003c/sup\u003e (100/5/C and 100/10/C groups) and with a power of 200 mW and a dose of 10 J/cm\u003csup\u003e2\u003c/sup\u003e (200/10/C group) increased the AK level released by macrophages in these groups (Fig.\u0026nbsp;4a) compared to the unirradiated cells (CTR group) (Fig.\u0026nbsp;4a). The analysis of the effect of a pulsed laser beam with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e/well (200/5/P group) showed that four- and six-fold irradiation reduced the level of AK released by cells compared to the unirradiated macrophages (CTR group) (Fig.\u0026nbsp;4b), although increasing the number of exposures to 10 increased the level of AK released. Treatment with a pulsed laser beam with a power of 100 mW and doses of 5 and 10 J/cm\u003csup\u003e2\u003c/sup\u003e (100/5/P and 100/10/P groups) and with a power of 200 mW and a dose of 10 J/cm\u003csup\u003e2\u003c/sup\u003e (200/10/P group) induced an increase in the level of AK released (Fig.\u0026nbsp;4b).\u003c/p\u003e \u003cp\u003eThe results obtained from stage I of the study allowed us to select the irradiation parameters that had the most beneficial effect on the cells, causing an increase in cell adhesion/proliferation and no effect or a decrease in the release of AK. For stage II of the study, the exposure parameters selected were continuous or pulsed laser beams with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (200/5/C and 200/5/P groups).\u003c/p\u003e \u003cp\u003e \u003cb\u003eViability of macrophages irradiated with continuous or pulsed laser beams with a power of 200 mW and a dose of 5 J/cm\u003c/b\u003e \u003csup\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sup\u003e \u003c/p\u003e \u003cp\u003eTwo-fold irradiation with a continuous laser beam of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (200/5/C group) resulted in an increase in macrophage viability compared to the unirradiated cells (CTR group) on day 3 of macrophage culturing (Fig.\u0026nbsp;5a). Increasing the number of laser irradiations (4 or 6 laser beam applications) had no effect on the viability of macrophages on days 5 and 7 of macrophage culturing. In turn, in the case of macrophages irradiated two and six times with a pulsed laser beam of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (200/5/P group), increased cell viability was observed on days 3 and 7 of macrophage culturing compared to unirradiated cells (CTR group) (Fig.\u0026nbsp;5b).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe level of protein secreted by macrophages irradiated with a continuous or pulsed laser beam with a power of 200 mW and a dose of 5 J/cm\u003c/b\u003e \u003csup\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sup\u003e \u003c/p\u003e \u003cp\u003eFour- and six-fold irradiation of macrophages with a continuous laser beam of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (200/5/C group) resulted in decreased protein secretion on days 5 and 7 of macrophage culturing (Fig.\u0026nbsp;5c). When cells were treated with a pulsed laser beam of the same power and dose (200/5/P group), an increase in protein secretion was observed after six-fold laser irradiation on day 7 of macrophage culturing, compared to the unirradiated cells (CTR group) (Fig.\u0026nbsp;5d).\u003c/p\u003e \u003cp\u003eBased on the results obtained in stage II of the study (increases in the viability of macrophages and the level of protein secreted by the cells), supernatants from the cell cultures irradiated with a pulsed laser beam with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (200/5/P group) were selected for stage III of the study to assess the immunomodulatory effect of LLLT on macrophages.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe immunomodulatory effect of LLLT on the secretory activity of macrophages irradiated with a pulsed laser beam with a power of 200 mW and a dose of 5 J/cm\u003c/b\u003e \u003csup\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sup\u003e \u003cb\u003e(200/5/P group)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe study showed that six-fold irradiation of macrophages resulted in an increased NO secretion on day 7 of macrophage culturing, compared to the unirradiated cells (Fig.\u0026nbsp;6a).\u003c/p\u003e \u003cp\u003eFour-fold laser irradiation of macrophages reduced the level of MCP-1 and TNF-α secretion (Figs.\u0026nbsp;6b and 6c). Further six-fold laser irradiation decreased the secretion of TNF-α by macrophages compared to the unirradiated cells (CTR group) (Fig.\u0026nbsp;6c). For the remaining cytokines measured: IFN-γ, IL-12p70, IL-6 and IL-10, their presence was not detected in any of the tested groups of cells (200/5/P and CTR groups), regardless of the tested time point.\u003c/p\u003e \u003cp\u003eThe study also showed that four-fold laser irradiation of cells reduced the secretion of MMP-9 by macrophages compared to the unirradiated cells (CTR group) (Table\u0026nbsp;1). At all the time points examined, there was no secretion or no differences in the secretion of pro-MMP and MMP-2 by macrophages in the 200/5/P group, compared to the unirradiated cells (CTR group) (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eMeasurement of the TOS of macrophages showed that six-fold irradiation of macrophages resulted in an increased total oxidative/capacitive status of macrophages (TOS/TOC) compared to unirradiated cells (CTR group) (Fig.\u0026nbsp;7a). However, for the TAS of irradiated macrophages (TAS/TAC), no differences were found compared to theunirradiated cells (CTR group) (Fig.\u0026nbsp;7b).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMany scientific studies have shown that LLLT, as a result of contact with cells, may disturb homeostasis. It has been shown that it can affect cell viability and secretory activity \u003csup\u003e34,12,5\u003c/sup\u003e. However, the authors of these works focused primarily on examining the effect of laser radiation on fibroblasts, keratinocytes or osteoblasts \u003csup\u003e35,10,11,14,16\u003c/sup\u003e. However, little is known about the effect of LLL on the response of resting M0 macrophages, the activation of which is crucial in the course of inflammation \u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our study, assays based on assessment of the adhesion and morphology of cells were used to identify the impact of laser irradiation. The results showed significant differences in the ability of cells treated with LLLT to adhere/proliferate. The best effectiveness in promoting adhesion/proliferation was achieved when laser irradiation with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e was used (200/5/C and 200/5/P groups). These results are confirmed by the results of observations of the morphology of macrophages in this group, which showed a clear increase in density and a tendency of proliferating cells to form clusters. However, it should be noted that the beneficial effect of LLLT on cells was observed when they were irradiated up to six times. Further irradiation of the cells resulted in decreased adhesion. The observed phenomenon probably resulted from the lack of free surface for cell attachment and/or the lack of nutrients in the culture medium, and not the effect of increasing the number of irradiations. The phenomenon of decreased cell adhesion in the following days of the experiment was also found in the control group. Unfortunately, due to the lack of studies in the available literature on the effect of LLLT on macrophage adhesion/proliferation, it is difficult to compare the results obtained with other studies. However, there are reports discussing the effect of LLLT on the adhesion of other cell types. For example, Li et al. (2020) showed that the use of LLLT at doses of 1.0 J/cm\u003csup\u003e2\u003c/sup\u003e, 2.0 J/cm\u003csup\u003e2\u003c/sup\u003e and 4.0 J/cm\u003csup\u003e2\u003c/sup\u003e increased the proliferation of HUVEC epithelial cells \u003csup\u003e33\u003c/sup\u003e. Similarly, Sperandio et al. (2014) showed increased proliferation of HaCaT keratinocytes at an applied wavelength of 660 nm, power of 100 mW and laser energy densities of 3 J/cm\u003csup\u003e2\u003c/sup\u003e, 6 J/cm\u003csup\u003e2\u003c/sup\u003e and 12 J/cm\u003csup\u003e2 36\u003c/sup\u003e. In turn, Basso et al. (2012) irradiated HGFs (human gingival fibroblasts) with a laser beam with a wavelength of 780 nm, power of 40 mW and doses of 0.5 J/cm\u003csup\u003e2\u003c/sup\u003e, 1.5 J/cm\u003csup\u003e2\u003c/sup\u003e, 3.5 J/cm\u003csup\u003e2\u003c/sup\u003e and 7 J/cm\u003csup\u003e2\u003c/sup\u003e, showing that only two (0.5 J/cm\u003csup\u003e2\u003c/sup\u003e and 3 J/cm\u003csup\u003e2\u003c/sup\u003e) of the six doses tested increased cell proliferation, while the remaining doses did not affect cell proliferation \u003csup\u003e37\u003c/sup\u003e. The authors also observed no changes in the morphology of cells irradiated with LLLT \u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our study, we also checked the level of AK released from dead cells, assessing the possible cytotoxic effect of LLLT on macrophages. Regardless of the method of application of the radiation beam (continuous or pulsed), it was only in the group of cells irradiated with a laser with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e that the level of AK released by the cells did not differ or was lower compared to the control group. Increasing the number of laser irradiations to eight and ten increased the AK released from cells.\u003c/p\u003e \u003cp\u003eOur study results indicate a beneficial effect of the irradiation of macrophages with continuous or pulsed laser beams with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e (200/5/C and 200/5/P groups). Therefore, these groups of cells were selected for further tests to assess cell viability and secretory activity measured by the level of secreted protein. Further tests in these groups showed increased viability of macrophages treated with a pulsed laser beam (200/5/P group), while irradiation with a continuous laser beam did not affect the viability of macrophages (200/5/C group). Similar results were obtained by Silva et al. (2016), who irradiated LPS-stimulated RAW 264.7 macrophages with two wavelengths: 660 nm and 880 nm, with a power of 100 mW and a dose of 214 J/cm\u003csup\u003e2\u003c/sup\u003e, showing an increase in viability only when irradiated with a wavelength of 660 nm \u003csup\u003e38\u003c/sup\u003e. Also according to Souza et al. (2014), LPS-stimulated J774 macrophages irradiated with a laser with a wavelength of 660 nm, power of 15 mW and a dose of 7.5 J/cm\u003csup\u003e2\u003c/sup\u003e and a wavelength of 780 nm, power of 70 mW and a dose of 3 J/cm\u003csup\u003e2\u003c/sup\u003e showed increased viability compared to the group of macrophages not exposed to LLLT \u003csup\u003e32\u003c/sup\u003e. In turn, Song et al. (2021) studied the effect of a continuous laser beam with a wavelength of 810 nm and power of 80 mW on LPS-stimulated RAW 264.7 macrophages, using laser radiation doses of 0.4 J/cm\u003csup\u003e2\u003c/sup\u003e, 1.2 J/cm\u003csup\u003e2\u003c/sup\u003e and 2.4 J/cm\u003csup\u003e2 8\u003c/sup\u003e. In this case, onl y the dose of 2.4 J/cm\u003csup\u003e2\u003c/sup\u003e significantly increased the viability of the tested cells. Similarly, Leden et al. (2013) examined the viability of LPS-stimulated BV2 macrophages irradiated with a laser with a wavelength of 808 nm, power of 50 mW and doses of 0.2 J/cm\u003csup\u003e2\u003c/sup\u003e, 4 J/cm\u003csup\u003e2\u003c/sup\u003e, 10 J/cm\u003csup\u003e2\u003c/sup\u003e and 30 J/cm\u003csup\u003e2\u003c/sup\u003e, showing that only irradiation with doses of 4 J/cm\u003csup\u003e2\u003c/sup\u003e and 30 J/cm\u003csup\u003e2\u003c/sup\u003e resulted in viability \u003csup\u003e39\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe available literature lacks reports assessing the immunomodulatory effect of LLLT on the secretory activity of resting M0 macrophages. In our study, we first checked the level of NO secreted by macrophages exposed to various radiation parameters. We showed that irradiation of macrophages with a continuous laser beam (200/5/C group) resulted in a decrease in NO and protein secretion, while the pulsed beam (200/5/P group) increased secretion, compared to unirradiated cells (CTR group). Additionally, we showed that irradiation of cells with a pulsed laser beam increased the TOS of macrophages, but had no effect on the TAS of cells. According to Silva et al. (2016), monocytes and RAW 264.7 macrophages irradiated with a laser with a wavelength of 808 nm secreted more NO than cells irradiated with a wavelength of 660 nm \u003csup\u003e38\u003c/sup\u003e. An increase in NO secretion by LPS-stimulated BV2 macrophages was also demonstrated by Leden et al. (2013), who irradiated cells with a laser beam of 808 nm, power of 50 mW and doses of 4 J/cm\u003csup\u003e2\u003c/sup\u003e and 30 J/cm\u003csup\u003e2 39\u003c/sup\u003e. These authors also showed that irradiation of cells with doses of 0.2 J/cm\u003csup\u003e2\u003c/sup\u003e and 10 J/cm\u003csup\u003e2\u003c/sup\u003e with the same wavelength and laser power did not affect the secretion of NO by cells. NO is an important factor involved in the inflammatory response, produced and secreted mainly, as already mentioned, by classically polarized M1 macrophages \u003csup\u003e28\u003c/sup\u003e. Most authors agree that a decreased level of NO secreted by cells protects against excessive activation of M1 macrophages and is probably related to the protection of tissues at the inflammation site. In turn, high levels of NO and other pro-inflammatory mediators secreted by M1 macrophages may impair the proper phagocytosis of apoptotic cells and also directly damage host tissues \u003csup\u003e40\u003c/sup\u003e. Reacting with proteins, NO can cause nitrosylation of amino residues, impairing their function and causing a cytotoxic/apoptotic effect on surrounding cells. It can also stimulate the secretion of pro-inflammatory cytokines, stimulating immune system cells and initiating inflammation \u003csup\u003e41\u003c/sup\u003e. Our study results showed a decreased secretion of cytokines TNF\u003cb\u003e-\u003c/b\u003eα, MCP-1 and MMP-9 as a result of irradiation with a pulsed laser beam of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e. However, the samples we tested did not contain the other cytokines: IL-10, IL-12, IL-6 and IFN-γ.\u003c/p\u003e \u003cp\u003eA decrease in TNF-α secretion with a simultaneous increase in IL-10, IL-4 and IL-13 secretion was noted by Song et al. (2017) who irradiated LPS-stimulated microglia cells with a laser with a wavelength of 810 nm, power of 150 mW and a dose of 4.5 J/cm\u003csup\u003e2 34\u003c/sup\u003e. Similarly, Fernandes et al. (2015) observed a decrease in the secretion of TNF-α by J774 macrophages irradiated with a laser with a wavelength of 780 nm, power of 70 mW and a dose of 2.6 J/cm\u003csup\u003e2\u003c/sup\u003e and 660 nm, 15 mW, 7.5 J/cm\u003csup\u003e2\u003c/sup\u003e, while showing increased secretion of IL-6 by the cells irradiated with a 660 nm laser compared to unirradiated cells \u003csup\u003e42\u003c/sup\u003e. Souza et al. (2014) also examined the effect of laser irradiation with a wavelength of 780 nm, power of 70 mW and a dose of 3 J/cm\u003csup\u003e2\u003c/sup\u003e on the level of TNF\u003cb\u003e-\u003c/b\u003eα secreted by LPS-stimulated macrophages. In this case, cell irradiation also reduced TNF\u003cb\u003e-\u003c/b\u003eα secretion \u003csup\u003e32\u003c/sup\u003e. In turn, Gavish et al. (2008) showed that laser irradiation with a laser with a wavelength of 780 nm, power of 2 mW and a dose of 2.2 J/cm\u003csup\u003e2\u003c/sup\u003e decreased the secretion of MCP-1 by RAW 264.7 macrophages and had no effect on the secretion of IL-1β \u003csup\u003e43\u003c/sup\u003e. Different results were obtained by Leden et al. (2013). In their study, LPS-stimulated BV2 macrophages, irradiated with a laser with a wavelength of 808 nm, power of 50 mW and a dose of 0.2 J/cm\u003csup\u003e2\u003c/sup\u003e, significantly increased the secretion of MCP-1, while higher doses of 4 J/cm\u003csup\u003e2\u003c/sup\u003e, 10 J/cm\u003csup\u003e2\u003c/sup\u003e and 30 J/cm\u003csup\u003e2\u003c/sup\u003e had no impact on the secretion of this cytokine by cells \u003csup\u003e39\u003c/sup\u003e. Li et al. (2020) measured the effect of laser irradiation with a wavelength of 810 nm, power of 150 mW and doses of 0.4 J/cm\u003csup\u003e2\u003c/sup\u003e, 4 J/cm\u003csup\u003e2\u003c/sup\u003e and 10 J/cm\u003csup\u003e2\u003c/sup\u003e on LPS-stimulated macrophages BMDM and observed that doses of 4 and 10 J/cm\u003csup\u003e2\u003c/sup\u003e, similarly to our study, inhibit the secretion of MCP-1, while a dose of 0.4 J/cm\u003csup\u003e2\u003c/sup\u003e increases the secretion of MCP-1 and reduces the secretion of IL-1β \u003csup\u003e33\u003c/sup\u003e. Unlike in our study, Li et al. (2020) also showed that the irradiation parameters they used did not affect cytokine secretion by resting BMDM macrophages \u003csup\u003e33\u003c/sup\u003e. The results of our study and the other studies presented above confirm the concept that the treatment of macrophages with LLLT may modulate their inflammatory response, and the directions of laser irradiation and macrophage polarization depend on the type of macrophages, their previous stimulation and the irradiation parameters (wavelength, power, dose and method of applying the laser beam).\u003c/p\u003e \u003cp\u003eThe effect of LLLT on the secretion of MMP-2 and MMP-9 by macrophages has not been described. Studies dealing with this issue mainly focus on assessing its impact on other types of cells, primarily fibroblasts and osteoblasts \u003csup\u003e44,45\u003c/sup\u003e. For example, Ayuk et al. (2018) irradiated WS1 fibroblasts with two wavelengths: 660 nm (with a power of 108 mW) and 830 nm (with a power of 94 mW) and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e, showing that, regardless of the irradiation parameters used, the cells secreted less MMP-9 compared to the control \u003csup\u003e44\u003c/sup\u003e. In turn, Oliviera et al. (2017) irradiated MC3T3 osteoblasts with a laser with a wavelength of 660 nm and 780 nm, power of 20 mW and doses of 1.9 J/cm\u003csup\u003e2\u003c/sup\u003e and 3.8 J/cm\u003csup\u003e2\u003c/sup\u003e, showing no effect of irradiation on the level of secreted MMP-9 and an increase in the secretion of MMP-2 by cells irradiated with a laser with a wavelength of 660 nm and a dose 1.9 J/cm\u003csup\u003e2 45\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our study, we have shown that irradiation of cells with a pulsed laser beam with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e increases the secretion of NO by macrophages and their TOS, which may suggest the polarization of macrophages towards the M1 phenotype. On the other hand, a decrease in the secretion of pro-inflammatory cytokines (TNF-α and MCP-1) and MMP-9 by cells may indicate the polarization of macrophages towards the M2 phenotype.\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eOur study results suggest that LLLT modulates the biological response of resting RAW 264.7 macrophages and may also influence their polarization. It seems extremely likely that for an optimal response of macrophages, they often share common features of the M1 and M2 phenotypes and that their phenotype should be considered as a spectrum of continuous differentiation under the influence of LLLT. To elucidate the mechanisms underlying such immunomodulation, further studies assessing the effect of LLLT on other markers of resting macrophage polarization are necessary.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis research project was supported by the\u0026nbsp;statutory activities of the Academy of Physical Education in Krakow\u0026nbsp;(no. 269/BS/INS/21) and the programme \u0026lsquo;Excellence Initiative \u0026ndash; Study University\u0026rsquo; of the University of Science and Technology (AGH), project no. 4204.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe authors declare that the data supporting the results of this study are available in the article and its supporting documents. If any raw data files are needed in another format, they are available from the corresponding author upon reasonable request\u003c/strong\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang, Y. \u0026amp; Ji, Q. Current advances of photobiomodulation therapy in treating knee osteoarthritis. \u003cem\u003eFront Cell Dev Biol \u003c/em\u003e\u003cstrong\u003e16\u003c/strong\u003e,\u003cem\u003e \u003c/em\u003e1286025 (2023).\u003c/li\u003e\n\u003cli\u003eSantonocito, S. \u003cem\u003eet al\u003c/em\u003e. Impact of Laser Therapy on Periodontal and Peri-Implant Diseases. \u003cem\u003ePhotobiomodul Photomed Laser Surg\u003c/em\u003e\u003cstrong\u003e40\u003c/strong\u003e, 454-462 (2022).\u003c/li\u003e\n\u003cli\u003eSourvanos, D. \u003cem\u003eet al\u003c/em\u003e. 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Irradiation With 780 nm Diode Laser Attenuates Inflammatory Cytokines but Upregulates Nitric Oxide in Lipopolysaccharide-Stimulated Macrophages: Implications for the Prevention of Aneurysm Progression. \u003cem\u003eLasers Surg Med\u003c/em\u003e\u003cstrong\u003e 40\u003c/strong\u003e, 371-378 (2008).\u003c/li\u003e\n\u003cli\u003eOliveira, F.A. \u003cem\u003eet al.\u003c/em\u003e Low level laser therapy modulates viability, alkaline phosphatase and matrix metalloproteinase-2 activities of osteoblasts. \u003cem\u003eJ Photoch Photobio B\u003c/em\u003e\u003cstrong\u003e 169\u003c/strong\u003e, 35-40 (2017).\u003c/li\u003e\n\u003cli\u003eAyuk, S.M., Houreld, N.N. \u0026amp; Abrahamse, H. Effect of 660 nm visible red light on cell proliferation and viability in diabetic models in vitro under stressed conditions. \u003cem\u003eLasers Med Sci\u003c/em\u003e\u003cstrong\u003e33\u003c/strong\u003e, 1085-1093 (2018).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eThe effect of irradiation with a pulsed (P) laser beam on the level of MMPs secreted by macrophages of the RAW 264.7 cell line. \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"left\" width=\"738\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.723577235772358%\" rowspan=\"3\"\u003e\n \u003cp\u003eGrups\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.097560975609756%\" rowspan=\"3\"\u003e\n \u003cp\u003eDays\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.70460704607046%\" rowspan=\"3\"\u003e\n \u003cp\u003eNumber\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eof laser application\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.47425474254743%\" colspan=\"10\"\u003e\n \u003cp\u003eTested indicator\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.31597845601436%\" colspan=\"3\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; pro-MMP-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.21364452423698%\" colspan=\"3\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;MMP-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.47037701974865%\" colspan=\"4\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; MMP-2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.874326750448834%\"\u003e\n \u003cp\u003ex̅\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.43806104129264%\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.00359066427289%\"\u003e\n \u003cp\u003eBetween groups comparison\u003c/p\u003e\n \u003cp\u003e200/5/P v CTR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.59245960502693%\"\u003e\n \u003cp\u003ex̅\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.797127468581687%\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.00359066427289%\"\u003e\n \u003cp\u003eBetween groups comparison\u003c/p\u003e\n \u003cp\u003e200/5/P v CTR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.797127468581687%\"\u003e\n \u003cp\u003ex̅\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.8491921005386%\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.464991023339318%\"\u003e\n \u003cp\u003eBetween groups comparison\u003c/p\u003e\n \u003cp\u003e200/5/P\u003c/p\u003e\n \u003cp\u003ev CTR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.17953321364452424%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.723577235772358%\" rowspan=\"3\"\u003e\n \u003cp\u003eCTR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.097560975609756%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.70460704607046%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.452574525745257%\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.368563685636857%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\" rowspan=\"6\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eday 5\u003c/p\u003e\n \u003cp\u003ep=0,144\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eday 7\u003c/p\u003e\n \u003cp\u003ep=0,143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.994579945799458%\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.639566395663957%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\" rowspan=\"6\"\u003e\n \u003cp\u003eday 3\u003c/p\u003e\n \u003cp\u003ep=0,05\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eday 5\u003c/p\u003e\n \u003cp\u003ep=\u003cstrong\u003e0,024 *\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.639566395663957%\"\u003e\n \u003cp\u003e4900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.94308943089431%\"\u003e\n \u003cp\u003e1580\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.16260162601626%\" rowspan=\"6\"\u003e\n \u003cp\u003eday 3\u003c/p\u003e\n \u003cp\u003ep=0,168\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.13550135501355012%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.555555555555557%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.222222222222221%\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.444444444444445%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.11111111111111%\"\u003e\n \u003cp\u003e4498\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.88888888888889%\"\u003e\n \u003cp\u003e280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.88888888888889%\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.666666666666666%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.2222222222222222%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.555555555555557%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.222222222222221%\"\u003e\n \u003cp\u003e2513\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.444444444444445%\"\u003e\n \u003cp\u003e686\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.11111111111111%\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.88888888888889%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.88888888888889%\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.666666666666666%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.2222222222222222%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.242603550295858%\" rowspan=\"3\"\u003e\n \u003cp\u003e200/5/P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.875739644970414%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.581854043392505%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.848126232741617%\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.270216962524655%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.637080867850099%\"\u003e\n \u003cp\u003e147,06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.664694280078896%\"\u003e\n \u003cp\u003e61,34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.664694280078896%\"\u003e\n \u003cp\u003e1158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.017751479289942%\"\u003e\n \u003cp\u003e466,87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.19723865877712032%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.555555555555557%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.222222222222221%\"\u003e\n \u003cp\u003e419,52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.444444444444445%\"\u003e\n \u003cp\u003e286,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.11111111111111%\"\u003e\n \u003cp\u003e2116,8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.88888888888889%\"\u003e\n \u003cp\u003e574\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.88888888888889%\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.666666666666666%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.2222222222222222%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.555555555555557%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.222222222222221%\"\u003e\n \u003cp\u003e16132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.444444444444445%\"\u003e\n \u003cp\u003e9230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.11111111111111%\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.88888888888889%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.88888888888889%\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.666666666666666%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.2222222222222222%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;The cells were cultured for a specified number of days (3, 5, 7) and irradiated (2, 4 and 6 times, respectively) with a laser with power of 200 mW and a radiation dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e/well with cells. The level of metalloproteinases was determined on the following days of the experiment: 3, 5 and 7. Mean values \u0026plusmn; SEM; nd - no metalloproteinases were detected. (* for p\u0026lt;0.05) - statistically significant differences between the group of irradiated cells (200/5/P) and the group of non-irradiated cells (CTR group) on individual days of the experiment (3, 5, 7).\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4620625/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4620625/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLLLT (low-level laser therapy) covers a wide range of parameters in terms of laser properties and dosage, which is important for its effects. To obtain the desired therapeutic effect of LLLT on cells, it is important to select optimal irradiation conditions. This article focuses on the selection of biostimulating exposure conditions for LLLT, which are the method of beam application, the radiation power and dose, and then the assessment of the immunomodulatory effect of LLLT on resting macrophages of the RAW 264.7 cell line. Irradiation of cells with a pulsed laser beam with a power of 200 mW and a dose of 5 J/cm\u003csup\u003e2\u003c/sup\u003e results in an increase in the adhesion and viability of macrophages and increase the secretion of protein, NO by macrophages and their TOS, which may suggest the polarization of macrophages towards the M1 phenotype. On the other hand, a decrease in the secretion TNF-α, MCP-1 and MMP-9 by cells may indicate the polarization of macrophages towards the M2 phenotype. It seems that for an optimal response of resting macrophages, they often share common features of the M1 and M2 phenotypes and that their phenotype should be considered as a spectrum of continuous differentiation under the influence of LLLT.\u003c/p\u003e","manuscriptTitle":"The effect of low-level laser therapy conditions on macrophages’ immunomodulatory processes as an example of regeneration process stimulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-17 11:02:43","doi":"10.21203/rs.3.rs-4620625/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1a6191f-733e-489c-8061-13624b38e8a5","owner":[],"postedDate":"July 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T20:08:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-17 11:02:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4620625","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4620625","identity":"rs-4620625","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}