Anti-inflammatory effects of mesenchymal stem cell-conditioned media inhibited macrophages activation in vitro

The immunomodulatory effects of mesenchymal stem cells (MSCs) on macrophages have been reported, however, the underlying mechanism remains unknown. Therefore, this study aimed to investigate the anti-inflammatory effects of MSCs on lipopolysaccharide (LPS)-stimulated macrophages and the subsequent downregulation of their inflammatory mediators. Macrophages were treated with conditioned media from MSCs, without a subsequent change of MSCs responding to the inflammation state. This study also evaluated whether the interleukin (IL) 4 stimulation of MSCs can improve their anti-inflammatory effects. Results demonstrated that the MSC-conditioned medium (MSC-CM) stimulated with IL4 significantly inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression of LPS-activated macrophages. MSC-CM treatment inhibited the mRNA transcription of the cytokines IL1β and IL6, the chemokines C–C motif ligand (CCL) 2, CCL3, CCL4, and CCL5, and the chemokine receptors CCR2 and CCR5, in LPS-stimulated macrophages. As revealed through western blot and immunofluorescence analyses, the phosphorylation of p38, JNK, and ERK MAPKs, as well as phosphorylation of NF-κB in stimulated macrophages, were also inhibited by the MSC-CM. Further, more potent anti-inflammatory effects were observed with the IL4-stimulated cells, compared with those observed with the non-stimulated cells. The MSC-CM demonstrated a potent anti-inflammatory effect on LPS-activated macrophages, while the IL4 stimulation improved this effect. These findings indicate that MSCs could exert anti-inflammatory effects on macrophages, and may be considered as a therapeutic agent in inflammation treatment.

Inflammation is a protective response mediated by immune cells and molecular mediators toward invasive pathogens that cause infection and tissue damage. However, inflammation is also related to many pathophysiological processes 1 . Some diseases, such as osteoarthritis (OA) 2 , rheumatoid arthritis 3 , inflammatory bowel disease 4 , diabetes 5 , and acute lung injury 6 have been reported to be related to inflammation induced by immune cells and molecular mediators.
The use of stem cells might offer the most promising cell therapy strategy for the biological treatment of inflammation [15][16][17] . Mesenchymal stem cells (MSCs) currently represent an important immunotherapeutic cell group, and there are several possible applications of MSCs in the treatment of immune function-related diseases [18][19][20] . The regulatory network of the factors inducing the generation of the regulatory immune cells is a characteristic of MSCs participating in immune homeostasis, making them conducive for immunomodulation [21][22][23] . MSCs are circumstance-sensitive cells, as they respond to extracellular stimuli by inducing various interventions that are specifically sensitive to alterations in signals 24,25 . Upon exposure to proinflammatory factors, MSC-mediated immunomodulation is upgraded 17,26 .
MSC-conditioned media (MSC-CM) refer to the supernatants of an MSC culture, containing various cytokines and extracellular vesicles secreted by MSCs which can regulate the immune response 27,28 . Takafuji et al. revealed that a conditioned medium from cultured MSCs downregulated the expression of tumor necrosis factor (TNF)-α and interleukin (IL) 6 in macrophages by suppressing the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) pathways, while also decreasing the expression of M2 markers 27 . MSC-CM was also reported to reduce the expression of MIP-2, a form of chemoattractant, and IL-6 in the tissues of an acute lung injury model 6 . MSC-CM-isolated extracellular vesicles were also reported to downregulate IL6 and CCL2 expression in a skeletal muscle injury model 29 . In addition, Previous studies showed that, as the circumstance sensitive cells, upon exposure to pro-inflammatory factors, MSC-mediated immunomodulation was regulated 17,26 . Therefore, due to the immunomodulatory effects of MSCs, we wondered whether IL4 treatment of MSCs would have less or more anti-inflammatory effects on activated macrophages.
In this study, we hypothesized that several paracrine cytokines exist in stem cell culture media that may facilitate the immunomodulation of macrophages. The IL4 treatment of stem cells could lead to a greater production of those compounds which improve the anti-inflammatory effects of the stem cells on macrophages stimulated by lipopolysaccharide (LPS). In this study, the anti-inflammatory effects of MSC-CM on LPS-stimulated macrophages were investigated, and the signal transduction pathways involved in the immunomodulation of MSC-CM were analyzed.

Results
MSC-CM decreased the production of COX-2 and iNOS. The MSC-CM did not exert any toxic effects on macrophages ( Supplementary Fig. 1). The protein expression of COX-2 and iNOS in the LPS-stimulated macrophages was evaluated using western blot analysis. Prior to their stimulation with LPS for 22 h, macrophages were pretreated with the MSC-CM for 2 h. We define the MSC-CM derived from the D1 cell culture (a bone marrow-derived MSC cell line) as D1-M (CON)-D1 cell conditioned media (control); the MSC-CM derived from the IL4-stimulated D1 cell culture is defined as D1-M (IL4)-IL4-stimulated D1 cell conditioned media.
For the immunofluorescence analysis, macrophages were pretreated with MSC-CM for 24 h and then stimulated with LPS (200 ng/mL) for 6 h. The mean gray values were determined and analyzed. The expression of inflammatory cytokines, such as COX-2 and iNOS, was upregulated in response to LPS stimulation, and was inhibited by pretreatment with MSC-CM (Fig. 1D,E). Further, the COX-2 signal was notably inhibited in the D1-M (IL4) group compared to the D1-M (CON) group. Meanwhile, no significant difference was observed in the iNOS signal between the two groups. These results indicate that treatment with MSC-CM inhibited inflammatory marker expression in the activated macrophages.
For the immunofluorescence analysis, RAW264.7 cells were pretreated with MSC-CM for 22 h. Subsequently, the cells were stimulated with 200 ng/mL LPS for 1 h. The mean gray values were determined and analyzed. Phosphorylation of p38 MAPK, JNK, ERK1/2, and NF-κB was upregulated after the LPS stimulation of macrophages. Pretreatment with MSC-CM significantly suppressed the LPS-stimulated phosphorylation of p38

Discussion
In this study, the anti-inflammatory effect of D1 cells was studied in LPS-stimulated RAW264.7 macrophage cells by evaluating the gene and protein expression of pro-inflammatory mediators and factors involved in intracellular signaling pathway activation. The MSC-CM did not exert any toxic effects on macrophages, which was confirmed by treatment with different concentrations. PCR and western blot analyses demonstrated that LPS triggered the inflammatory reaction of macrophages, while the D1 cell media suppressed this response. The findings of this www.nature.com/scientificreports/ www.nature.com/scientificreports/ study indicate that the anti-inflammatory effects of MSC-CM are mediated via the inhibition of the p38 MAPK, JNK, and ERK signaling pathways and the NF-κB signaling pathway.
Macrophages are important cells that participate in inflammatory diseases 5,30,31 . Macrophages also induce the synovial inflammation and pathological changes of the cartilage and bone characteristic of OA 30 . Tissue-resident macrophages perform major roles in the regulation of tissue inflammation, such as in diabetic glomerular sclerosis 5 . In sepsis-induced lung injury, macrophages defend against invading pathogens during sepsis, but also induce injury to alveolar epithelial cells and capillary endothelial cells 31 . Elucidating the exact mechanism of the recruitment and activation of monocytes and macrophages triggered by damage associated molecular patterns may lead to the development of potential therapies [31][32][33] .
MSCs possess strong immunomodulatory potential and can regulate adaptive and innate immunity 19,21 . The pro-inflammatory enzymes COX-2 and iNOS cause the upregulation of inflammatory factors in LPS-stimulated macrophages 16 . Gu et al. demonstrated that COX-2 levels decreased significantly in cigarette-exposed macrophage cells following treatment with MSCs 34 . Transcription of iNOS was reduced in LPS-stimulated MSCmacrophage cocultures 17 . Yang et al. demonstrated that iNOS expression in the LPS-stimulated RAW264.7 cells cultured with the primed canine MSC-CM decreased 35 . In the present study, the MSC-CM pretreatment inhibited the expression of the pro-inflammatory enzymes COX-2 and iNOS.
Inflammation is triggered by the activation of pro-inflammatory cytokines IL1β and IL6 20,36,37 as well as the chemokines CCL2, CCL3, CCL4, and CCL5 38,39 . These cytokines and chemokines are involved in inflammation progression, tissue repair, and immune responses via the recruitment of immune cells, as well as the regulation of autocrine/paracrine activities 39 . Therefore, a novel therapeutic strategy involving the development of an antiinflammatory agent should target the inhibition of pro-inflammatory cytokines expressed by macrophages 40 . The expression of the pro-inflammatory macrophage marker IL1β is decreased in LPS-stimulated and MSC-treated macrophages compared to the controls 17 . Xu et al. reported that IL-6 expression decreased in macrophages due to LPS-stimulated MSC-derived exosomes 41 . In the current study, the MSC-CM reduced the expression of pro-inflammatory cytokines, such as IL1β and IL6, in LPS-stimulated macrophages. The inhibition effect on LPS-induced IL1β and IL6 production was more pronounced in the D1-M (IL4) group compared with the D1-M (CON) group. The mechanism involving the chemokine alterations, the anti-inflammatory effects, and the activated macrophages has not yet been thoroughly investigated 42  The MAPK and NF-κB signaling pathways lead to the activation of macrophages [48][49][50][51] , which may be inhibited by MSC-CM. A previous study demonstrated that phosphorylation of MAPKs was activated by LPS and was involved in LPS-induced NO production and pro-inflammatory cytokine expression 52 . Zheng et al. determined that in LPS-stimulated RAW264.7 cells, an MSC-conditioned medium might reduce NF-κB signaling pathway activation 49 . The MAPK and NF-κB signaling pathways play important roles in mediating the anti-inflammatory effects of MSCs on activated macrophages [53][54][55] . The results obtained in the present study were consistent with those of previous studies. JNK expression remained unchanged in all groups across several replicate experiments. This might be attributed to the use of an incompatible antibody for immunofluorescence analysis. These results demonstrated that LPS treatment stimulated the phosphorylation of p38 MAPK, JNK, and ERK1/2 as well as that of NF-κB, which is consistent with the results of previous studies. Thus, our findings demonstrate that the MSC-CM exerts anti-inflammatory effects by mediating the MAPK and NF-κB signaling pathways (Fig. 4). In addition, the MSC-CM with IL4 stimulation showed more potent anti-inflammatory effects than that without IL4 stimulation.
This study had some limitations. First, the exact components primarily affecting the macrophages were not identified. However, as reported by previous studies, MSCs could express the anti-inflammatory cytokines IL4, IL10, and IL13 that might suppress macrophage activation [15][16][17] . Second, several methods for studying the effects of MSCs on macrophages have been reported in literature, including co-culture techniques with or without Transwell, utilization of MSC-CM, and that of the MSC-CM-derived exosomes 17,41,49,56 . In this study, we only used the MSC-CM method; thus, additional methods should be used in further research to confirm the antiinflammatory effects of MSCs. Third, the stimulation of MSCs by the cytokines IL10, IL13, TNF-α, and IL1β should be considered in order to determine whether these factors also enhance the anti-inflammatory effects of MSCs along with IL4. Finally, we did not include a comparison group of macrophages treated directly with IL4. Some IL4 could have remained despite considering the possibility that IL4 in MSC-CM may have affected macrophages and changed the media before MSC-CM collection. www.nature.com/scientificreports/

Conclusion
The MSC-CM suppressed the production of pro-inflammatory compounds in the LPS-stimulated macrophages.
The inhibition of pro-inflammatory cytokine and chemokine production was associated with the inhibition of the MAPK and NF-κB pathways in LPS-stimulated macrophages. The IL4-treated MSCs demonstrated a more potent anti-inflammatory effect on LPS-induced macrophage activation. These findings may have implications for potential therapeutic strategies for inflammatory disease.

Materials and methods
Cell culture and treatment. RAW264.7 cells are murine macrophages that were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). In this study, RAW264.7 cells were cultured with 5% CO 2 at 37 °C in DMEM supplemented with 10% FBS, 200 μg/mL streptomycin, 200 IU/mL penicillin, 4 mM L-glutamine, and 1 mM sodium pyruvate (complete medium). LPS was diluted to obtain the final concentration of 200 ng/mL in the medium (Fig. 5).
A bone marrow-derived MSC cell line, D1 ORL UVA (D1) (ATCC #CRL-12424), was purchased from the ATCC. Primary mouse D1 cells were purchased from the ATCC and incubated with or without 20 ng/mL IL4 for stimulation for 24 h, then incubated for an additional 24 h following media change. The D1 cell supernatant was collected and classified into two groups; D1-M (IL4) and D1-M (CON) (Fig. 5).
Cytotoxicity assay. Cytotoxicity was evaluated via colorimetric MTT assay. RAW264.7 cells were incubated in a 96-well cell culture plate and 100 µl complete medium overnight. Different ratios of D1-M and DMEM media (1:100 to 1) were added to each well, and the plate was then incubated for 24 h at 37 °C in an incubator. After D1-M treatment, the supernatant was discarded, and MTT solution (0.5 mg/mL) was pipetted into each well. Following incubation for 2 h at 37 ℃, the MTT solution was carefully discarded, and the formazan deposit was dissolved with dimethyl sulfoxide. A purple-colored solution was obtained. Optical density (OD) of the formazan solution was estimated by measuring the absorbance at 570 nm using the Synergy™ HTX Multi-Mode Microplate Reader.
Reverse transcription-PCR. RAW264.7 were pretreated by a 1:1 ratio of D1-M and media for 18 h. The cells were then incubated with or without LPS for 6 h. After 24 h of incubation, the supernatant was discarded via centrifugation, and a total RNA extraction reagent (RNAiso Plus. Takara, Japan) was used to isolate total RNA from the cells according to the manufacturer's instructions. Subsequently, 1 μg of isolated total RNA was carefully reverse transcribed with oligo dT (Bioneer, Daejeon, Korea), Taq DNA polymerase, dNTP, and reaction buffer (AccuPower® PCR PreMix kit, Bioneer, Daejeon, Korea) to synthesize cDNA. To evaluate mRNA transcription regarding target inflammatory cytokines and chemokines, the primer sequences specific for mouse target genes were designed (Supplement Table 1). The MyGenie 96 gradient thermal cycler system, AccuPower® RT PreMix kit (Bioneer, Daejeon, Korea), and the primers were used to amplify the cDNA. The PCR products were visualized using NEOgreen DNA staining reagent (NEO Science, Daejeon, Korea) and a NaBI Gel-doc system.  www.nature.com/scientificreports/ (for COX-2 and iNOS) of incubation. Subsequently, the cells were rinsed twice with cold phosphate-buffered saline (PBS). The total cells were lysed with radio-immunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, MO, USA) and treated with protease inhibitor cocktail (GenDEPOT, Katy, TX, USA) at 4 °C in an icebox for 30 min. The lysates were concentrated through the centrifuge, which was conducted at 12,000 × g and 4 °C for 15 min. After centrifugation, using the BCA assay kit (Thermofisher, Rockford, IL, USA) to measure the protein concentration of cell lysates according to the manufacturer's instructions. The protein was divided using 10% or 15% sodium dodecyl SDS-PAGE with equivalent amounts of protein samples, and a polyvinylidene difluoride (PVDF) membrane was used to transfer proteins. The 5% skim milk solution was used to block membranes for 1 h at room temperature and the blot was cut prior to hybridization with antibodies during blotting. Subsequently, with the following primary antibodies: anti-COX-2 (Abcam, Cambridge, UK), anti-iNOS (Abcam, Cambridge, UK), anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-p38, anti-p-p38, anti-SAPK/ JNK, anti-p-SAPK/JNK, anti-ERK1/2, and anti-p-ERK1/2 (from Cell Signaling Technology, Danvers, MA, USA), PVDF membranes were incubated overnight at 4 °C (Supplement Table 2). Secondary antibodies used the horseradish peroxidase (HRP) conjugated anti-rabbit, anti-mouse, and anti-American hamster antibodies (Santa Cruz Biotechnology). Bands were visualized with the Cooled CCD Gel Imaging System. Pre-stained protein markers (HiQ™ Blueye Prestained Protein Marker, Bio-D) were used for the determination of molecular weight.

Western blot analysis.
Immunofluorescence. Cells were seeded on coverslips in 12 well plates, and were incubated in the absence or presence of a 1:1 ratio of D1-M and DMEM media for 24 h. This was followed by LPS (200 ng/mL) treatment during the last 1 h (for MAPKs and NF-κB) or 6 h (for COX-2 and iNOS) of incubation at 37 °C. After fixation with paraformaldehyde (4%) and permeabilization with Triton X-100 along with washing with 1 × PBS, cells were blocked with the 1% BSA for 1 h at 37 °C incubation and stained overnight with primary antibodies at 4 °C (Supplement Table 3). The cells were then stained with Alexa 488 dye-labeled secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) (Supplement Table 3). All primary antibodies were used at a ratio of 1:400. All secondary antibodies were used at a ratio of 1:2000 for cultured RAW264.7. Nuclei of the cells were stained with 4′,6 -diamidino-2-phenylindole (DAPI)-containing mounting solution (VECTASHIELD® Antifade Mounting Medium with DAPI, Vector, Burlingame, CA, USA), and the images were obtained using an augmented microscopy system with a microplate reader and imager software. For semi-quantitative analysis of the fluorescence, the mean gray values were detected and compared between each group with Image J (Collins, 2007). Under 200 × magnification imaging, three fields of each cell slide were selected, and the positively stained macrophages were calculated to obtain a mean value (Supplement Table 4). Sections were randomly coded and scored by a blinded observer with three sections per group. www.nature.com/scientificreports/ Statistical analysis. The data obtained in this study were assessed via SPSS, and were analyzed using a one-way analysis of variance (ANOVA) and post-hoc Tukey's test. The data were expressed as mean ± standard deviation (mean ± SD). The alpha level for all tests was 0.05, with two-tailed, and statistically significant was considered as p < 0.05. www.nature.com/scientificreports/