Multiomics analyses reveal adipose-derived stem cells inhibit the inflammatory response of M1-like macrophages through secreting lactate

doi:10.21203/rs.3.rs-5216382/v1

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Multiomics analyses reveal adipose-derived stem cells inhibit the inflammatory response of M1-like macrophages through secreting lactate

https://doi.org/10.21203/rs.3.rs-5216382/v1

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Background

Adipose-derived stem cells (ADSCs) are widely used in the field of regenerative medicine because of their various functions, including anti-inflammatory effects. ADSCs are considered to exert their anti-inflammatory effects by secreting anti-inflammatory cytokines and extracellular vesicles. Although recent studies have reported that metabolites have a variety of physiological activities, whether those secreted by ADSCs have anti-inflammatory properties remains unclear. Here, we performed multiomics analyses to examine the effect of ADSC-derived metabolites on M1-like macrophages, which play an important role in inflammatory responses.

Methods

The concentration of metabolites in the culture supernatant of ADSCs was quantified using capillary electrophoresis time-of-flight mass spectrometry. To evaluate their effects on inflammatory responses, M1-like macrophages were exposed to the conditioned ADSC medium or their metabolites, and RNA sequencing was used to detect gene expression changes. Immunoblotting was performed to examine how the metabolite suppresses inflammatory processes. To clarify the contribution of the metabolite in the conditioned medium to its anti-inflammatory effects, metabolite uptake was pharmacologically inhibited, and gene expression and the tumor necrosis factor-α concentration were measured by quantitative PCR and enzyme-linked immunosorbent assay (ELISA), respectively.

Results

Metabolomic analysis showed large amounts of lactate in the culture supernatant. The conditioned medium and lactate significantly suppressed or increased the pro-inflammatory and anti-inflammatory gene expressions. However, sequencing and immunoblotting analysis revealed that lactate did not induce polarization from M1- to M2-like macrophages. Based on a recent report that the immunosuppressive effect of lactate depends on epigenetic reprogramming, histone acetylation was investigated, and H3K27ac expression was upregulated. In addition, 7ACC2, which specifically inhibits the monocarboxylate transporter 1, significantly inhibited the anti-inflammatory effect of the conditioned ADSC medium on M1-like macrophages.

Conclusions

Our results showed that ADSCs suppress pro-inflammatory effects of M1-like macrophages by secreting lactate. This study adds to our understanding of the importance of metabolites and is also expected to elucidate new mechanisms of ADSC treatments.

Stem Cell & Developmental Cell Biology

Adipose-derived stem cell

lactate

macrophage

metabolomics

transcriptomics

bioinformatics

In recent years, regenerative medicine using stem cells has become a new treatment strategy [1–4]. Cell therapy can not only alleviate symptoms, but also regenerate tissues, something difficult to accomplish with existing methods. Recently, induced pluripotent stem cells (iPSCs) has been used for regenerative medicine [5]. Already some clinical trials have used iPSCs to regenerate organs (e.g., cardiac muscle and retina), and iPSCs are expected to revolutionize regenerative medicine [6, 7]. However, there are issues to be resolved before iPSCs can be used for clinical applications, such as the risk of tumorigenicity, immunogenicity, financial cost, and unestablished methods of differentiation into the desired cells [8, 9]. On the other hand, adult stem cells like hematopoietic stem cells and neural stem cells, which exist in vivo, have similar properties to iPSCs [10–12]. While having a more limited ability to differentiate than iPSCs, they are more convenient and safer and have been already used clinically, for example, in the treatment of acute myeloid leukemia [13, 14]. Among adult stem cells, mesenchymal stem/stromal cells (MSCs) are particularly promising for clinical applications [15].

MSCs can differentiate into at least three lineages (osteoblasts, chondrocytes and adipocytes) in vitro, being widely distributed throughout the body, including the bone marrow, umbilical cord, placenta and dental pulp [16, 17]. Among them, adipose-derived stem cells (ADSCs), abundant in the adipose tissue, can be harvested with minimal invasiveness by liposuction and have high proliferative potential [18, 19]. MSCs/ADSCs have immunomodulatory effects, which explain their use as treatments for various diseases associated with inflammation like graft-versus-host disease, rheumatoid arthritis, and osteoarthritis (OA) [20, 21]. Previous studies showed that the therapeutic effect of MSCs/ADSCs in these diseases is related to their anti-inflammatory properties, mediated by cytokines such as IL-10 and extracellular vesicles (EVs) [22, 23]. Indeed, these molecules suppress inflammation by polarizing inflammatory M1-like macrophages into anti-inflammatory M2-like macrophages in vitro and in vivo [24–26]. Nuclear factor-kappa B (NF-κB) signaling plays a central role in macrophage activation; these anti-inflammatory molecules have been reported to suppress inflammation partly by reducing the production of inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) by suppressing of NF-κB signaling [27, 28]. Thus, a potential way to enhance the therapeutic effect is to administer cytokines/EVs together with ADSCs; however, it is costly and technically difficult to prepare high-quality cytokines and EVs, and their safety has not been determined yet.

Recent studies revealed a close relationship between macrophage function and intracellular energy metabolism. For instance, lipopolysaccharide (LPS) stimulation of M1-like macrophages increases glucose uptake and enhances the glycolytic system, while inhibition of that system suppresses pro-inflammatory response [29, 30]. Interestingly, these changes have been reported to involve epigenetics, i.e., gene expression regulation. Epigenetic reprogramming includes DNA methylation and histone modifications, which regulate gene expression over long periods. In particular, histones are acetylated by histone acetyltransferase, which loosens the nucleosome structure and enhances gene expression [31, 32]. Noe et al. have shown that histone acetylation contributes to interleukin (IL)-4-induced M2-like macrophage polarization [33]. Furthermore, Shi et al. most recently reported that lactate (LA) exhibits anti-inflammatory effects by suppressing the transcription of pro-inflammatory-related genes via H3K27 acetylation rather than by inducing polarization toward M2-like macrophages or suppressing NF-κB signaling [34].

Metabolites exhibit various physiological activities; for example, the inosine secreted by brown adipocytes stimulates energy expenditure [35]. In addition, certain metabolite combinations derived from apoptotic cells ameliorate inflammation [36]. However, the anti-inflammatory effects of ADSC-derived metabolites remain unknown. This study aimed to identify a novel mechanism of immunosuppressive properties of ADSCs by performing metabolomic and transcriptomic analyses and revealed that they suppress inflammation through LA secretion, which is partially mediated by the monocarboxylate transporter and H3K27 acetylation.

Cell culture

In this study, we used human ADSCs derived from six healthy donors who provided informed consent or were purchased from Lonza (Cat. #PT-5006). Details about the donors are shown in Supplementary Table 1. ADSCs were isolated according to a previous report [37], being cultured in KBM ADSC-1 or KBM ADSC-4 (Kohjin Bio) and expanded within seven passages. Human monocytic THP-1 cells (RIKEN BRC) were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37℃ in 5% CO₂. THP-1 cells were differentiated with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA, FUJIFILM Wako Pure Chemicals) treatment for 24 h. Then, the medium was changed, and cells were polarized toward M1-like macrophages by 24-h exposure to 100 ng/mL of LPS (Sigma-Aldrich).

Metabolome analysis

ADSCs donated by healthy people were cultured in KBM ADSC-4 until approximately 70% confluency; then the conditioned medium (CM) was harvested and transported to Human Metabolome Technologies, Inc. (Yamagata, Japan). A mixture of 80 µL of the CM plus 20 µL of aqueous solution containing internal controls was ultrafiltrated, and the filtrate was used for capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). CE-TOFMS analysis of cationic and anionic metabolites was performed using an Agilent CE-TOFMS system (Agilent Technologies). Signal peaks with a signal-to-noise ratio > 3 were extracted by MasterHands (Keio University); detected peaks were annotated for all substances registered in the HMT metabolite library based on the m/z and migration time, allowing an error of ± 10 ppm and ± 0.5 min, respectively.

Z-score calculation and heatmap creation were performed using the genefilter and pheatmap R packages, respectively. Samples were hierarchically clustered using the “ward.D2” method. Principal component analysis (PCA) was carried out using “prcomp” function and plotted with the ggbiplot R package. Metabolite set enrichment analysis (MSEA) was conducted using MetaboAnalyst 5.0 (https://genap.metaboanalyst.ca/MetaboAnalyst/); a false discovery rate (FDR) < 0.1 was considered significant.

Flow cytometry

Cultured cells were washed with phosphate buffered saline (PBS) and trypsinized. Detached cells were suspended in Cell Staining Buffer (BioLegend) containing Human TruStain FcX (BioLegend) for Fc receptor blocking, followed by incubation with a cocktail of antibodies. Then, the stained cells were re-suspended in Cell Staining Buffer and analyzed using BD FACSCanto II (BD Biosciences). To detect apoptotic cells, Fc-blocked cells were washed with Cell Staining Buffer and re-suspended in Annexin V Binding Buffer (BioLegend) containing 7-aminoactinomycin D (7-AAD) and an anti-Annexin V antibody. In this study, Annexin V⁺ 7-AAD⁻ cells were defined as apoptotic cells. Details on antibodies are included in Supplementary Table 2.

Preparation of conditioned medium

ADSCs suspended in KBM ADSC-1 were seeded into 10 cm dishes and cultured until cells reached 70% confluency. Then, the medium was changed to RPMI-1640/10% heat-inactivated FBS and cultured for 72 h. Next, the CM was harvested, centrifuged, and stored at -80℃. The LA concentration of the CM was measured using the Lactate Assay Kit-WST (DOJINDO) following the manufacturer’s instructions. The LA concentration in the CM of commercially available and freshly isolated (“in-house”) ADSCs used in the experiments is shown in Fig. S1.

RNA sequencing (RNA-seq)

Total RNA extraction and DNase I treatment were performed using an RNeasy Mini Kit and RNase-Free DNase Set (QIAGEN), respectively. RNA integrity was determined using a 2100 Bioanalyzer and RNA 6000 Nano Kit (Agilent Technologies); all samples having an RNA Integrity Number > 9 were sent to Bioengineering Lab. Co., Ltd. (Kanagawa, Japan). Following quality check using a 5200 Fragment Analyzer System and Agilent HS RNA Kit (Agilent Technologies), DNA libraries were prepared using MGIEasy RNA Directional Library Prep Set (MGI Tech); the quality of the libraries was investigated using a 5200 Fragment Analyzer System and dsDNA 915 Reagent Kit (Agilent Technologies). The libraries were then circularized and prepared as DNA Nanoball using a MGIEasy Circularization Kit and a DNBSEQ-G400RS High-throughput Sequencing Kit (MGI Tech), respectively. Sequencing was performed on the DNBSEQ-G400 sequencer (MGI Tech) (2 × 100 bp).

The quality of sequencing data was checked using FastQC (ver. 0.12.1). Adaptor sequences and low-quality reads (quality score < 30, read length < 30, N > 5) were trimmed using fastp (ver. 0.23.3). Then, clean reads were aligned to the human reference sequence (GRCh38.p13) using STAR (ver. 2.7.10b), and gene counts and transcripts per million (TPM) values were calculated using RSEM (ver. 1.3.1). Differentially expressed genes were determined using a likelihood ratio test with the edgeR R package (“glmLRT” function). PCA was performed as described above. Volcano plots and Venn diagrams were drawn using the EnhancedVolcano and VennDiagram R packages, respectively. Gene ontology (GO) analysis was carried out using the clusterProfiler R package.

Quantitative polymerase chain reaction (qPCR)

Total RNA was extracted as described above, followed by cDNA synthesis using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) with oligo(dT) primers. qPCR was performed using a KAPA SYBR Fast qPCR Kit (Kapa Biosystems) on the QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific). GAPDH was used as loading control. Sequences of gene-specific primers are listed in Supplementary Table 3.

Immunoblotting

Cells were washed with ice-cold PBS and then lysed in RIPA buffer (Thermo Fisher Scientific) containing protease inhibitor cocktail (Roche). Cell lysates treated with Sample Buffer Solution (FUJIFILM Wako Pure Chemicals) were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked with EveryBlot Blocking Buffer (Bio-rad) for 5 min at room temperature and incubated overnight at 4℃ with a primary antibody diluted in the buffer. The next day, membranes were washed with tris-buffered saline with Tween 20 (TBS-T) and incubated for 1 h at room temperature with a secondary antibody diluted in the buffer. After washing with TBS-T, the blots were developed with ECL Prime Western Blotting Detection Reagents (Cytiva), and chemiluminescence was detected by FUSION FX7 (Vilber Lourmat). Protein levels were quantified using ImageJ software. For fractionating nuclear and cytoplasmic proteins, cells were processed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Nacalai Tesque). Details about antibodies are given in Supplementary Table 4, and uncropped blots are shown in Additional File 2.

Cell viability assay

THP-1 cells were seeded into a 96-well plate and differentiated/polarized toward M1-like macrophages as described above. Then, medium was replaced with inhibitor-containing medium and incubated for 24 h. Cell viability was determined using Cell Counting Kit-8 (DOJINDO). Dimethyl sulfoxide (DMSO) and 7ACC2 were purchased from Nacalai Tesque and SellekChem, respectively.

To induce apoptosis, ADSCs were treated with p53-MDM2 inhibitor nutlin-3 (SelleckChem). Trypan blue exclusion assay was used with TC20 Automated Cell Counter (Bio-rad) to measure cell viability.

Measuring intracellular lactate concentration

THP-1 cell-derived M1-like macrophages seeded into a 24-well plate were cultured in medium containing 2 µM of 7ACC2 for 1 h, followed by an additional 23 h incubation in medium containing the same concentration of 7ACC2 and 15 mM of LA (Nacalai Tesque). Thereafter, cells were rinsed with ice-cold PBS and lysed using 0.1% Triton-X (Sigma-Aldrich). Cell lysates were centrifuged, and the supernatant was then ultrafiltrated with an Amicon Ultra-0.5 Centrifugal Filter Unit (Merck). The LA concentration in the filtrate was determined using a Lactate Assay Kit-WST. The intracellular LA concentration was calculated by correcting for total protein content as measured using a Protein Assay BCA Kit (Nacalai Tesque).

Enzyme-linked immunosorbent assay (ELISA)

THP-1 cell-derived M1-like macrophages treated with 7ACC2 for 24 h were washed with PBS, before adding fresh medium. After a 4 h incubation, the supernatant was collected, centrifuged, and stored at -80℃ until further use. The concentration of TNF-α contained in the supernatant was quantified using an LBIS Human TNF-α ELISA Kit (FUJIFILM Wako Pure Chemicals).

In vivo experiments

BALB/cSlc-nu/nu nude mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). The mice were housed under a 12-h light/dark cycle at 22 ± 2℃ and 40–60% relative humidity, with free access to water and food. Twelve-week-old male mice were anesthetized with isoflurane and administered with 2 U/10 µL of type VII collagenase (Sigma-Aldrich) intra-articularly on Days 0 and 2. The same volume of saline was injected as a negative control. Subsequently, on Days 7, 14, 21 and 28, 10 µL of CM from human ADSCs containing DMSO or 7ACC2 (10 µM) was administered intra-articularly. On Day 35, tissue was harvested, fixed overnight in 10% neutral buffered formalin, and decalcified with ethylenediaminetetraacetic acid for 1 week. Sections were prepared from paraffin-embedded tissue and stained with Safranin O and Fast Green.

Statistical analysis

Data are represented as mean ± standard deviation. Statistical analysis was performed using R. Except for RNA-seq analysis, comparisons between two groups and among multiple groups were performed using a Student’s t-test and one- or two-way analysis of variance followed by Dunnett or Tukey-Kramer test, respectively; P < 0.05 was considered statistically significant. For RNA-seq analysis, we performed a generalized linear model likelihood ratio test with edgeR and defined statistical significance as FDR < 0.05.

The culture supernatant of ADSCs contains high concentration of lactate

First, we harvested ADSCs from six healthy people and cultured them in serum-free KBM ADSC-4 medium for 24 h before collecting the supernatant. The concentration of metabolites was quantitatively measured using CE-TOFMS; in this study, we analyzed about 1,100 water-soluble/ionic metabolites and identified 46. A heatmap showed that the composition of the supernatant differed markedly from that of the medium; a difference corroborated by PCA (Fig. 1A, 1B). Compared to the culture medium, the concentration of 33 metabolites was higher and that of 13 metabolites was lower. Particularly, the culture supernatant contained an extremely high LA concentration. To interpret the biological implications of the metabolites secreted by ADSCs, we performed MSEA on metabolites detected at higher concentration than in the culture medium and found that various metabolic pathways were activated, especially involving the Warburg effect (Fig. 1C). The Warburg effect is a phenomenon whereby glycolysis is enhanced, and the production of LA is increased [38].

Effect of lactate and conditioned ADSC medium on M1-like macrophages

In diseases including OA, ADSCs have been reported to suppress the pro-inflammatory response of M1-like macrophages [39, 40]. Moreover, some research groups have indicated that LA induces macrophage polarization toward an M2 phenotype [41]. Therefore, in vitro experiments were carried out using human monocytic THP-1 cells to test whether ADSC-derived LA could induce anti-inflammatory effects. Previous reports showed that THP-1 cells differentiated into M0 macrophages and polarized toward M1-like macrophages using PMA and LPS, respectively; we confirmed that these treatments successfully induced differentiation/polarization by detecting CD11b (a pan-macrophage marker) and CD80 (a M1-like macrophage marker) expression using flow cytometry (Fig. S2A–D) [42, 43]. We next conducted an Annexin V assay to assess the effect of LA on macrophages; no change in the percentage of apoptotic cells was observed up to 15 mM (Fig. 2A, 2B). Therefore, we used 15 mM LA in subsequent experiments.

Next, we performed a transcriptomic analysis to investigate how the conditioned ADSC medium and LA affect gene expression in macrophages. PCA revealed a distinct difference among the controls (medium only), CM prepared from commercially available ADSCs, and 15 mM LA-treated cells (Fig. 3A). Compared to the controls, 2,814 and 3,621 genes were significantly upregulated by CM and LA treatment, respectively (Fig. 3B–D). Conversely, 3,070 genes in CM-treated cells and 3,561 genes in LA-treated cells were significantly downregulated (Fig. 3B, 3C and 3E). Next, we performed GO analysis for co-upregulated and co-downregulated genes (877 and 983, respectively) in CM and LA as shown in Fig. 3F and 3G. Differentially expressed genes were highly clustered some ontologies, e.g., “wound healing” is associated with an anti-inflammatory property of macrophages [44]. These results suggested that both the CM and LA dramatically alter the properties of M1-like macrophages.

Lactate has an anti-inflammatory effect but probably does not induce M2 polarization

Considering previous reports describing that ADSCs polarize macrophages from an M1 to an M2 phenotype, we next examined whether this function is mediated by secreted LA [26, 45]. Differential expression analysis of our RNA-seq data revealed a significantly increased expression of M2-like macrophage markers like CD209 and VEGFA in CM- and LA-treated macrophages. On the contrary, the expression levels of TNF, which codes TNF-α (a typical inflammatory cytokine) and PTGS2, which encodes cyclooxygenase 2, an enzyme important in inflammatory responses, were significantly decreased by CM and LA treatment (Fig. 4A, 4B) [34, 46–50]. In addition, CM treatment significantly suppressed the expression of inducible nitric oxide synthase (iNOS coded by NOS2), a major functional marker of M1-like macrophages, and increased that of CD163, a representative cell surface marker of M2-like macrophages, at both the gene and protein levels (Fig. 4A–G). Consistent with these results, CM harvested from in-house ADSCs also secreted comparable LA concentration to commercially available ADSCs and showed similar gene expression profiles (Fig. S1, S3A–D). The expression of arginase-1 (coded by ARG1), which is used as a representative functional marker for M2-like macrophages, was undetectable at mRNA (Fig. 4B) and protein (Additional File 2) levels in all samples.

Previous reports have shown that apoptotic cells regulate the function of surrounding cells via metabolites, and we tested the anti-inflammatory effect of apoptotic ADSCs on M1-like macrophages [35, 36]. In this study, the p53-MDM2 inhibitor nutlin-3 was used to induce apoptosis [51, 52]. The percentage of Annexin V⁺ 7-AAD⁻ apoptotic cells was significantly increased at 24 and 48 h after applying 10 µM and 20 µM of nutlin-3 (Fig. S4A, S4B). Furthermore, trypan blue exclusion assay showed that 20 µM nutlin-3 significantly induced a decrease in cell viability at both 24 and 48 h time points (Fig. S4C, S4D). Nutlin-3-induced apoptotic ADSCs secreted comparable LA concentration compared to control cells, and no significant differences were observed in the effects of culture supernatant on the gene expression in M1-like macrophages (Fig. S3E–J).

Despite the reduced expression of pro-inflammatory factors, as noted above, no significant changes in the key marker expression were observed in the LA-treated cells; rather, the protein level of CD80 (a major M1-like macrophage markers) was upregulated (Fig. 4A–G). These results suggested that LA has an anti-inflammatory effect but may not be mediated by polarization from M1- to M2-like macrophages.

Lactate induces histone acetylation rather than NF-κB signaling suppression

We then examined how LA suppresses the pro-inflammatory effect of M1-like macrophages. Macrophage stimulation by pathogens and/or cytokines like LPS and INF-γ activates NF-κB signaling, a master regulator of inflammatory responses, followed by activating the transcription of genes encoding pro-inflammatory cytokines [53]. Therefore, we hypothesized that LA might suppress NF-κB signaling. As already known, LPS treatment of M0 macrophages induced nuclear translocation of NF-κB p65 subunit and degradation of cytoplasmic IκBα in a time-dependent manner (Fig. S5A) [54]. However, no remarkable difference was observed in p65 or IκB phosphorylation levels after the LA treatment (Fig. S5B). In other words, LA had little effect on the NF-κB signaling activity, at least under the conditions of the present experiment.

A new study recently reported that LA suppresses the inflammatory response of M1-like macrophages, which showed that LA does not suppress NF-κB signaling but rather exerts its anti-inflammatory effects through transcriptional regulation via H3K27 acetylation [34]. GO analysis of our RNA-seq data suggested that both LA and CM treatment altered gene transcription (Fig. 5A, 5B, S6A and S6B). Interestingly, consistent with these results, LA treatment for 6 h increased H3K27ac expression (Fig. 5D), indicating that the anti-inflammatory effect of LA may be associated with transcription changes.

Inhibition of a lactate transporter cancels the effect of the conditioned medium

In our experimental conditions, CM and LA had similar effects on M1-like macrophages in terms of suppressing inflammatory profiles. However, the presence of other compounds than LA in the CM (Fig. 1A) might influence the observed results. Therefore, we examined whether the effect of CM could be inhibited by pharmacologically inhibiting LA uptake in M1-like macrophages. Since LA influx and efflux in macrophages are mainly carried out by monocarboxylate transporter 1 (MCT1) and MCT4, respectively, we used the selective MCT1 inhibitor 7ACC2 [55–57]. Previous reports have shown that 7ACC2 specifically inhibits LA uptake in cells expressing both MCT1 and MCT4; using RNA-seq, we confirmed that THP-1 cells highly express SLC16A1 (MCT1) and SLC16A4 (MCT4) compared to other MCTs (SLC16A7 and SLC16A8, coding MCT2 and MCT3, respectively; Fig. 6A) [57]. A cell viability assay showed no significant change in survival rates at 24 h after 7ACC2 treatment within the 1–20 µM range (Fig. 6B). Intracellular LA levels significantly decreased when LA was added in the presence of the inhibitor, indicating that the LA uptake was inhibited by 7ACC2 (Fig. 6C). As expected, the expression of CD209, GPNMB, PPARG, VEGFA, and VSIG4, which were increased by CM treatment, significantly decreased with 7ACC2 (Fig. 6D). In addition, TNF mRNA expression and TNF-α production decreased by CM stimulation; an effect inhibited by 7ACC2 administration (Fig. 6E, 6F).

Finally, to evaluate the therapeutic effect of LA in CM, in vivo experiments were performed using a mouse model of OA, a representative inflammatory disease. We employed the widely used type VII collagenase-induced OA model (Fig. S7A) [58, 59]. This model induces OA by deteriorating tendons and ligaments rather than directly degrading articular cartilage [60]. Histological analysis showed that articular cartilage was dramatically reduced by collagenase treatment (top panels in Fig. S7B). In addition, while CM partially rescued collagenase-induced cartilage lesions, the application of 7ACC2 slightly diminished the effect of CM (bottom panels in Fig. S7B). These results indicate that LA in CM may potentially contribute to the effectiveness of OA treatment.

Overall, LA secreted from ADSCs is incorporated into M1-like macrophages via MCTs (partially MCT1) and exhibits anti-inflammatory effects through transcriptional regulation via histone acetylation (Fig. 6G).

In this study, we performed a comprehensive metabolomic analysis on culture supernatants of ADSCs isolated from humans; although some studies have performed metabolomic analysis on ADSCs’ secretion products, they were performed on mouse or rat ADSCs [61, 62]; to the best of our knowledge, this is the first study to reveal the detailed metabolite profiles of human ADSCs.

Despite the existence of previous reports on the LA secreted from ADSCs, most are related to the energy metabolism of ADSCs themselves; in other words, LA is considered as a “waste” produced by altered metabolic systems, and the functional role of secreted LA was not clear, thus far [62–64]. In this study, LA secreted by ADSCs suppresses the pro-inflammatory activity of M1-like macrophages. Since inflammatory responses are mainly elicited by M1-like macrophages, strategies to suppress the pro-inflammatory cytokine production would be effective against inflammatory diseases; in this regard, enhancing LA secretion from ADSCs may be clinically beneficial. LA has been shown to be secreted in large amounts in previous metabolomic analysis of mouse ADSCs [62], suggesting that this might be a common phenomenon across species. Previous studies revealed that LA production from ADSCs increased under hypoxia [63–65]. Interestingly, oxygen levels in the normal knee joint are maintained in hypoxic conditions but increase with OA progression [66]. Treatment with ADSCs has weak effects in patients with severe OA, possibly by suppressing LA secretion from ADSCs due to oxygen concentration changes within the knee joint [67]. Further studies are needed to determine whether LA contributes to the therapeutic effects against various inflammatory diseases (e.g., arthritis, inflammatory bowel disease and sepsis) by in vivo experiments. Referring to OA, where ADSCs are frequently used during therapy, the conditioned ADSC medium alleviated OA symptoms, and it would be worthwhile to investigate whether LA also contributes to the therapeutic effect of ADSCs in vivo [21, 68, 69]. However, LA may yield positive and negative effects on OA. For example, LA is expected to exert its therapeutic effects by suppressing the expression of ADAMTS5, a typical extracellular matrix-degrading protease, by increasing the expression of COL2A1, a significant component of the articular cartilage, or by suppressing the migration and IL-6 production of synovial macrophages [70, 71]. Conversely, the acidic pH caused by lactic acid may exacerbate symptoms by inhibiting chondrocyte proliferation and matrix synthesis and by promoting IL-6 production from synovial fibroblasts [70, 71]. In this study, the therapeutic effect of LA on OA was investigated using a collagenase-induced mice model, with LA uptake blocked using 7ACC2. Histological analysis showed that 7ACC2 treatment partially impaired the reduction of cartilage loss by CM (Fig. S7). Although M1-like macrophages are reportedly accumulated in collagenase-induced OA, 7ACC2 could not completely cancel the therapeutic effect of CM [72]. This might be because (1) 7ACC2 administered to the knee joint affects not only macrophages but also other cell types such as chondrocytes and synoviocytes and (2) ADSCs secrete various cytokines and EVs in addition to LA, which may also contribute to their therapeutic effect [22, 23]. Therefore, alternative methods (e.g., use of macrophage-specific MCT1-deficient mice and administration of ADSCs with suppressed LA production) to accurately validate the anti-inflammatory effects of LA in vivo need to be explored.

There are several reports on the effects of LA on monocytes and/or macrophages, with tumor cell-derived LA inducing M2 polarization of tumor-associated macrophages and bone marrow-derived MSC-derived LA affecting the differentiation of monocytes to dendritic cells [73, 74]. Further, activation of NF-κB signaling is important for macrophage activation; however, whether LA affects the NF-κB signaling is controversial. For example, Samuvel et al. reported that LA activates NF-κB pathway by boosting TLR4 signaling [75]. On the contrary, Yang et al. and other research groups indicated that LA suppresses pro-inflammatory phenotypes [76–78]. Moreover, Hée et al. demonstrated that LA does not activate NF-κB signaling under certain conditions [79]. Although the effect of LA on NF-κB pathway is not entirely clear, some studies indicate that LA activates NF-κB signaling in some cell types but not in oxidative cells [79–81]. As previous reports showed that THP-1 cells depend on oxidative phosphorylation for energy production, we do not see any inconsistency between these studies [82, 83] and the present report. It should be noted that a major M2-like macrophage marker ARG1 expression was not observed in this study, possibly because the phenotype of THP-1 cells is biased toward the M1 type, and the expression of IL10 and ARG1, which are characteristic of M2-like macrophages, is relatively low compared to human monocytic U937 cell-derived macrophages [84]. In addition, ARG1 expression varies among species and cell origins, and it is debatable whether ARG1 can be a marker for human M2-like macrophages [85].

Recently, Shi et al. have reported that LA exerts an anti-inflammatory effect through transcriptional alterations via histone acetylation rather than suppressing NF-κB signaling or inducing polarization toward M2-like macrophages [34]. In our experiments, LA increased or decreased the expression of different M1- and M2-like macrophage markers, making it unlikely to induced polarization toward M2-like macrophages. Mechanistically, LA incorporated into macrophages via MCTs is metabolized to citrate as fuel for the TCA cycle, followed by acetylation of histone H3K27, and this histone modification triggers transcriptional program shift toward immunosuppression. They showed that treatment with LPS and LA increased H3K27ac expression in murine macrophages, and trichostatin A, an HDAC inhibitor also promoted H3K27 acetylation. Consistent with their reports, we demonstrated that LA increased H3K27ac expression in human-derived M1-like macrophages (Fig. 5). Furthermore, previous studies have shown that epigenetic changes, especially histone modifications, play an important role in the long-term memory of innate immunity [86, 87]. As discussed by Shi et al., LA-treated macrophages exhibit long-lasting immunosuppressive phenotype. Results of randomized controlled trials have shown that ADSC treatment for OA relieved symptoms even at 12 months postoperatively [88]. These results suggest that ADSCs may, of course, differentiate into chondrocytes and repair tissues. Still, LA secreted from administered ADSCs may cause epigenetic reprogramming of macrophages and induce a long-term immunosuppressive state, resulting in prolonged alleviation of symptoms. Although analyses of detailed molecular mechanisms and experiments using CM have not been performed, the results suggest that high LA concentrations secreted from ADSCs can modify the transcriptional program of M1-like macrophages.

In this study, the recently developed MCT1 inhibitor 7ACC2 was used to block LA uptake. The effect of 7ACC2 varies by cell type, with 7ACC2 effectively inhibiting LA uptake in cells expressing both MCT1 and MCT4. Indeed, transcriptome analysis showed that THP-1 cells highly express both MCT1 and MCT4. However, since LA uptake could not be completely inhibited by 7ACC2, other approaches, such as alternative inhibitors or genetic inhibition, are needed to more accurately examine the impact on macrophages of the LA secreted by ADSCs.

Overall, we demonstrated that ADSCs exert anti-inflammatory effects via LA secretion. As shown in the metabolome analysis, ADSCs secrete other metabolites aside from LA. Medina et al. reported that the combination of multiple metabolites released from apoptotic cells suppress the inflammatory response of myeloid cells [36]. In our study, the percentage of apoptotic macrophages did not change at 24 h of LA exposure. Still, metabolites secreted from apoptotic macrophages present earlier and/or certain metabolite combinations may enhance the observed anti-inflammatory effects. Also, we investigated the effect of apoptotic ADSCs. In the present study, apoptosis was induced in ADSCs using nutlin-3, which was used in the study by Niemann et al., analyzing metabolites secreted by apoptotic brown adipocytes [35]. Nutlin-3 is a small molecule compound that activates the p53 pathway by inhibiting the interaction between MDM2 and p53, thereby inducing apoptosis [51, 52]. Annexin V assay revealed that nutlin-3 successfully induced apoptosis in ADSCs. Inducing apoptosis in ADSCs did not change the amount of LA secreted, and the CM of apoptotic ADSCs induced gene expression comparable to that of normal cells, indicating that, at least under the present conditions, no notable features of apoptotic ADSCs were observed. However, since nutlin-3 mildly induced apoptosis, it would be interesting to investigate the anti-inflammatory effects of apoptotic ADSCs under more severe conditions, such as ultraviolet irradiation.

As many researchers have pointed out, there is no doubt that anti-inflammatory cytokines and EVs contribute to the therapeutic effect of ADSCs [22, 23]. Some studies have also shown that the administered ADSCs regenerate tissues by differentiation [89, 90]. Therefore, we believe that the therapeutic effect of ADSCs is exerted not only by LA but also by various secretomes and differentiation capacities. In this regard, it would be interesting to assess whether the therapeutic effect is enhanced by combining multiple secretomes, as reported by Medina et al. [36]. Considering the clinical application of this study, the establishment of a new culture method (e.g., developing better culture medium and/or culture dishes) in such a way as to increase the secretory capacity of LA may help to improve the therapeutic outcome of ADSC. Furthermore, LA secretion of cultured ADSCs may be used as a novel biomarker to predict therapeutic effects. Although we have demonstrated a functional role of the metabolite secreted by ADSCs prepared from a limited number and background of people, it is not clear whether ADSCs from humans with different backgrounds (e.g., race, age, medical history, sex) also secrete large amounts of LA, which should be investigated in the future.

ADSCs derived from healthy individuals showed LA hypersecretion. Further, ADSC-derived LA suppresses the pro-inflammatory effects of M1-like macrophages, presumably through transcriptional modulation via histone acetylation. Accordingly, metabolites, especially LA, could be “new players” with therapeutic potential, aside from cytokines and EVs.

7-AAD

7-aminoactinomycin D

ADSC

Adipose-derived stem cell

CE-TOFMS

Capillary electrophoresis time-of-flight mass spectrometry

Conditioned medium

CPM

Counts per million

DMSO

Dimethyl sulfoxide

ELISA

Enzyme-linked immunosorbent assay

Extracellular vesicle

FBS

Fetal bovine serum

FDR

False discovery rate

Gene ontology

HDAC

Histone deacetylase

Interleukin

iNOS

Inducible nitric oxide synthase

iPSC

Induced pluripotent stem cell

Lactate

LPS

Lipopolysaccharide

MCT

Monocarboxylate transporter

MFI

Mean fluorescence intensity

MSC

Mesenchymal stem/stromal cell

MSEA

Metabolite set enrichment analysis

NES

Normalized enrichment score

NF-κB

Nuclear factor-kappa B

Osteoarthritis

PBS

Phosphate buffered saline

PCA

Principal component analysis

PMA

Phorbol 12-myristate 13-acetate

qPCR

Quantitative polymerase chain reaction

RNA-seq

RNA sequencing

RPMI-1640

Roswell Park Memorial Institute 1640

TBP

TATA-binding protein

TBS-T

Tris buffered saline with Tween 20

TNF-α

Tumor necrosis factor-alpha

TPM

Transcripts per million

Ethics approval and consent to participate

This study was conducted in accordance with the Declaration of Helsinki and approved by the Kanazawa Medical University Specified Certified Regenerative Medicine Committee, the Institutional Review Board for Genetic Analysis Research according to the following two protocols: (1) Investigation of the regenerative effects of adipose-derived stem cells on various organ failures and gene expression (approval number: G129, date of approval: April 17, 2017); (2) Omics analysis of autologous adipose-derived stem cells for treatment of knee osteoarthritis (approval number: G173, date of approval: June 25, 2021). Informed consent was obtained from all subjects involved in this study.

The animal protocol titled "Investigation of therapeutic effects of adipose-derived stem cells on knee osteoarthritis" was approved by the Institutional Animal Care and Use Committee of Kanazawa Medical University (approval number: 2022-32, date of approval: July 27, 2022).

Consent for publication

Not applicable.

Availability of data and materials

Raw fastq files generated by our RNA-seq were deposited in DNA Data Bank of Japan (accession number: DRA016915), and other data generated in this study are available from the corresponding authors upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by JSPS KAKENHI (JP21K16666 and JP22K20726), a Grant for Collaborative Research from Kanazawa Medical University (C2021-3 and C2022-1), grants-in-aid from The Nakatomi Foundation and Shibuya Science Culture and Sports Foundation.

Authors’ contributions

HH, YS, YI, TI and KI contributed to the conceptualization of the work; TH, HH, YI and TI designed the study; TH, TS and YN performed the experiments; IT, HS and YS reviewed methodology; TH, HH, HK, AF, YT, YI, NY and TI interpreted the data; YI, TI, AK, SO and NK supervised the study; TH, HH, YI and TI wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank Ms. Ayane Kuwano (Kanazawa Institute of Technology) for technical assistance with the experiments on the early stages of this work and Ms. Yuka Hiramatsu (Kanazawa Medical University) for her help with the histological analysis. Metabolomic analysis and RNA-seq were performed by Human Metabolome Technologies, Inc. (Yamagata, Japan) and Bioengineering Lab. Co., Ltd. (Kanagawa, Japan), respectively. The super-computing resource was provided by Human Genome Center, the Institute of Medical Science, the University of Tokyo.

Clarke G, Harley P, Hubber EL, Manea T, Manuelli L, Read E et al (2018) Bench to bedside: Current advances in regenerative medicine. Curr Opin Cell Biol 55:59–66
McKinley KL, Longaker MT, Naik S (1979) Emerging frontiers in regenerative medicine. Science [Internet]. 2023 [cited 2023 Oct 11];380:796–8. https://www.science.org/doi/10.1126/science.add6492
Hoang DM, Pham PT, Bach TQ, Ngo ATL, Nguyen QT, Phan TTK et al Stem cell-based therapy for human diseases. Signal Transduction and Targeted Therapy 2022 7:1 [Internet]. 2022 [cited 2023 Oct 11];7:1–41. https://www.nature.com/articles/s41392-022-01134-4
Zakrzewski W, Dobrzyński M, Szymonowicz M, Rybak Z Stem cells: Past, present, and future. Stem Cell Res Ther [Internet]. 2019 [cited 2023 Oct 11];10:1–22. https://stemcellres.biomedcentral.com/articles/10.1186/s13287-019-1165-5
Kim JY, Nam Y, Rim YA, Ju JH (2022) Review of the Current Trends in Clinical Trials Involving Induced Pluripotent Stem Cells. Stem Cell Rev Rep [Internet]. [cited 2023 Oct 11];18:142. /pmc/articles/PMC8445612/
Ylä-Herttuala S (2018) iPSC-Derived Cardiomyocytes Taken to Rescue Infarcted Heart Muscle in Coronary Heart Disease Patients. Mol Ther 26:2077
Ahmed I, Robert J, Johnston J, Singh MS (2021) Pluripotent stem cell therapy for retinal diseases. Ann Transl Med [Internet] 9:1279–1279 Available from: /pmc/articles/PMC8421932/
Doss MX, Sachinidis A (2019) Current Challenges of iPSC-Based Disease Modeling and Therapeutic Implications. Cells [Internet]. [cited 2023 Oct 11];8. Available from: /pmc/articles/PMC6562607/
Yamanaka S (2020) Pluripotent Stem Cell-Based Cell Therapy—Promise and Challenges. Cell Stem Cell 27:523–531
Orkin SH, Zon LI, Hematopoiesis An Evolving Paradigm for Stem Cell Biology. Cell [Internet]. 2008 [cited 2023 Oct 11];132:631–44. http://www.cell.com/article/S0092867408001256/fulltext
Cheng H, Zheng Z, Cheng T (2020) New paradigms on hematopoietic stem cell differentiation. Protein Cell [Internet]. [cited 2023 Oct 11];11:34–44. https://link.springer.com/article/10.1007/s13238-019-0633-0
Zhao X, Moore DL (2018) Neural stem cells: developmental mechanisms and disease modeling. Cell Tissue Res [Internet]. [cited 2023 Oct 11];371:1. /pmc/articles/PMC5963504/
Hamilton BK, Copelan EA, Concise, Review (2012) The Role of Hematopoietic Stem Cell Transplantation in the Treatment of Acute Myeloid Leukemia. Stem Cells [Internet]. [cited 2023 Oct 11];30:1581–6. https://dx.doi.org/10.1002/stem.1140
Kassim AA, Savani BN Hematopoietic stem cell transplantation for acute myeloid leukemia: A review. Hematol Oncol Stem Cell Ther [Internet]. 2017 [cited 2023 Oct 11];10:245–51. https://pubmed.ncbi.nlm.nih.gov/28666104/
Ntege EH, Sunami H, Shimizu Y (2020) Advances in regenerative therapy: A review of the literature and future directions. Regen Ther 14:136–153
Pittenger MF, Discher DE, Péault BM, Phinney DG, Hare JM, Caplan AI Mesenchymal stem cell perspective: cell biology to clinical progress. npj Regenerative Medicine 2019 4:1 [Internet]. 2019 [cited 2023 Oct 11];4:1–15. https://www.nature.com/articles/s41536-019-0083-6
Ledesma-Martínez E, Mendoza-Núñez VM, Santiago-Osorio E (2016) Mesenchymal Stem Cells Derived from Dental Pulp: A Review. Stem Cells Int [Internet]. [cited 2023 Oct 11];2016. /pmc/articles/PMC4686712/
Schneider S, Unger M, Van Griensven M, Balmayor ER Adipose-derived mesenchymal stem cells from liposuction and resected fat are feasible sources for regenerative medicine. Eur J Med Res [Internet]. 2017 [cited 2023 Oct 11];22:1–11. https://eurjmedres.biomedcentral.com/articles/10.1186/s40001-017-0258-9
Si Z, Wang X, Sun C, Kang Y, Xu J, Wang X et al (2019) Adipose-derived stem cells: Sources, potency, and implications for regenerative therapies. Biomed Pharmacother 114:108765
Gao F, Chiu SM, Motan DAL, Zhang Z, Chen L, Ji HL et al (2016) Mesenchymal stem cells and immunomodulation: current status and future prospects. Cell Death Dis [Internet]. [cited 2023 Oct 11];7:e2062. /pmc/articles/PMC4816164/
Ceccarelli S, Pontecorvi P, Anastasiadou E, Napoli C, Marchese C (2020) Immunomodulatory Effect of Adipose-Derived Stem Cells: The Cutting Edge of Clinical Application. Front Cell Dev Biol 8:531513
Ferreira JR, Teixeira GQ, Santos SG, Barbosa MA, Almeida-Porada G, Gonçalves RM (2018) Mesenchymal Stromal Cell Secretome: Influencing Therapeutic Potential by Cellular Pre-conditioning. Front Immunol [Internet]. [cited 2023 Oct 11];9:2837. /pmc/articles/PMC6288292/
Zhao C, Chen JY, Peng WM, Yuan B, Bi Q, Xu YJ Exosomes from adipose-derived stem cells promote chondrogenesis and suppress inflammation by upregulating miR-145 and miR-221. Mol Med Rep [Internet]. 2020 [cited 2023 Oct 11];21:1881. /pmc/articles/PMC7057766/
Heo JS, Choi Y, Kim HO, Matta C (2019) Adipose-Derived Mesenchymal Stem Cells Promote M2 Macrophage Phenotype through Exosomes. Stem Cells Int [Internet]. [cited 2023 Oct 11];2019. https://pubmed.ncbi.nlm.nih.gov/31781246/
Liu J, Qiu P, Qin J, Wu X, Wang X, Yang X et al (2020) Allogeneic adipose-derived stem cells promote ischemic muscle repair by inducing M2 macrophage polarization via the HIF-1α/IL-10 pathway. Stem Cells [Internet]. [cited 2023 Oct 11];38:1307–20. https://pubmed.ncbi.nlm.nih.gov/32627897/
Kamada K, Matsushita T, Yamashita T, Matsumoto T, Iwaguro H, Sobajima S et al (2077) Attenuation of Knee Osteoarthritis Progression in Mice through Polarization of M2 Macrophages by Intra-Articular Transplantation of Non-Cultured Human Adipose-Derived Regenerative Cells. Journal of Clinical Medicine 2021, Vol 10, Page 4309 [Internet]. 2021 [cited 2023 Oct 11];10:4309. https://www.mdpi.com/-0383/10/19/4309/htm
Guillén MI, Platas J, Pérez del Caz MD, Mirabet V, Alcaraz MJ (2018) Paracrine anti-inflammatory effects of adipose tissue-derived mesenchymal stem cells in human monocytes. Front Physiol 9:341918
Bai X, Li J, Li L, Liu M, Liu Y, Cao M et al Extracellular Vesicles From Adipose Tissue-Derived Stem Cells Affect Notch-miR148a-3p Axis to Regulate Polarization of Macrophages and Alleviate Sepsis in Mice. Front Immunol [Internet]. 2020 [cited 2023 Oct 11];11. https://pubmed.ncbi.nlm.nih.gov/32719678/
Fukuzumi M, Shinomiya H, Shimizu Y, Ohishi K, Utsumi S (1996) Endotoxin-induced enhancement of glucose influx into murine peritoneal macrophages via GLUT1. Infect Immun [Internet]. [cited 2024 Feb 20];64:108–12. https://journals.asm.org/doi/10.1128/iai.64.1.108-112.1996
Freemerman AJ, Johnson AR, Sacks GN, Milner JJ, Kirk EL, Troester MA et al (2014) Metabolic reprogramming of macrophages: Glucose transporter 1 (GLUT1)-mediated glucose metabolism drives a proinflammatory phenotype. Journal of Biological Chemistry [Internet]. [cited 2024 Feb 20];289:7884–96. http://www.jbc.org/article/S0021925820442917/fulltext
Greenberg MVC, Bourc’his D The diverse roles of DNA methylation in mammalian development and disease. Nature Reviews Molecular Cell Biology 2019 20:10 [Internet]. 2019 [cited 2024 Feb 20];20:590–607. https://www.nature.com/articles/s41580-019-0159-6
Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Research. 21:3 [Internet]. 2011 [cited 2024 Feb 20];21:381–95. https://www.nature.com/articles/cr201122
Noe JT, Rendon BE, Geller AE, Conroy LR, Morrissey SM, Young LEA et al Lactate supports a metabolic-epigenetic link in macrophage polarization. Sci Adv [Internet]. 2021 [cited 2024 Feb 20];7:8602. /pmc/articles/PMC8589316/
Shi W, Cassmann TJ, Bhagwate AV, Hitosugi T, Ip WKE (2024) Lactic acid induces transcriptional repression of macrophage inflammatory response via histone acetylation. Cell Rep [Internet]. [cited 2024 Feb 21];43:113746. http://www.ncbi.nlm.nih.gov/pubmed/38329873
Niemann B, Haufs-Brusberg S, Puetz L, Feickert M, Jaeckstein MY, Hoffmann A et al Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine. Nature 2022 609:7926 [Internet]. 2022 [cited 2023 May 20];609:361–8. https://www.nature.com/articles/s41586-022-05041-0
Medina CB, Mehrotra P, Arandjelovic S, Perry JSA, Guo Y, Morioka S et al Metabolites released from apoptotic cells act as tissue messengers. Nature 2020 580:7801 [Internet]. 2020 [cited 2023 May 20];580:130–5. https://www.nature.com/articles/s41586-020-2121-3
Fuku A, Taki Y, Nakamura Y, Kitajima H, Takaki T, Koya T et al (2073) Evaluation of the Usefulness of Human Adipose-Derived Stem Cell Spheroids Formed Using SphereRing® and the Lethal Damage Sensitivity to Synovial Fluid In Vitro. Cells 2022, Vol 11, Page 337 [Internet]. 2022 [cited 2023 Sep 25];11:337. https://www.mdpi.com/-4409/11/3/337/htm
Liberti MV, Locasale JW (2016) The Warburg Effect: How Does it Benefit Cancer Cells? Trends Biochem Sci [Internet]. [cited 2023 Oct 16];41:211. /pmc/articles/PMC4783224/
Chang TH, Wu CS, Chiou SH, Chang CH, Liao HJ (2022) Adipose-Derived Stem Cell Exosomes as a Novel Anti-Inflammatory Agent and the Current Therapeutic Targets for Rheumatoid Arthritis. Biomedicines [Internet]. [cited 2024 Feb 25];10. https://pubmed.ncbi.nlm.nih.gov/35885030/
Jia Q, Zhao H, Wang Y, Cen Y, Zhang Z (2023) Mechanisms and applications of adipose-derived stem cell-extracellular vesicles in the inflammation of wound healing. Front Immunol 14:1214757
Zhou Hcun, yan YX, Yu Y, wen W, Liang X, qin, Du X, yan, Liu Z et al (2022) chang,. Lactic acid in macrophage polarization: The significant role in inflammation and cancer. Int Rev Immunol [Internet]. [cited 2023 Oct 16];41:4–18. https://www.tandfonline.com/doi/abs/10.1080/08830185.2021.1955876
Lin F, Yin H, Bin, Li XY, Zhu GM, He WY, Gou X (2020) Bladder cancer cell-secreted exosomal miR-21 activates the PI3K/AKT pathway in macrophages to promote cancer progression. Int J Oncol [Internet]. [cited 2023 Oct 16];56:151. /pmc/articles/PMC6910194/
Deng Y, Govers C, Beest E ter, van Dijk AJ, Hettinga K, Wichers HJ (2021) A THP-1 Cell Line-Based Exploration of Immune Responses Toward Heat-Treated BLG. Front Nutr. ;7:612397
Krzyszczyk P, Schloss R, Palmer A, Berthiaume F (2018) The Role of Macrophages in Acute and Chronic Wound Healing and Interventions to Promote Pro-wound Healing Phenotypes. Front Physiol [Internet]. [cited 2023 Oct 17];9:419. /pmc/articles/PMC5938667/
Manferdini C, Paolella F, Gabusi E, Gambari L, Piacentini A, Filardo G et al (2017) Adipose stromal cells mediated switching of the pro-inflammatory profile of M1-like macrophages is facilitated by PGE2: in vitro evaluation. Osteoarthritis Cartilage [Internet]. [cited 2023 Oct 16];25:1161–71. http://www.oarsijournal.com/article/S1063458417300419/fulltext
Yao Y, Xu XH, Jin L (2019) Macrophage polarization in physiological and pathological pregnancy. Front Immunol 10:434399
Zhou L, Zhuo H, Ouyang H, Liu Y, Yuan F, Sun L et al (2017) Glycoprotein non-metastatic melanoma protein b (Gpnmb) is highly expressed in macrophages of acute injured kidney and promotes M2 macrophages polarization. Cell Immunol [Internet]. [cited 2023 Oct 17];316:53–60. https://pubmed.ncbi.nlm.nih.gov/28433199/
Wang Y, Zhang Y, Li J, Li C, Zhao R, Shen C et al Hypoxia Induces M2 Macrophages to Express VSIG4 and Mediate Cardiac Fibrosis After Myocardial Infarction. Theranostics [Internet]. 2023 [cited 2023 Oct 17];13:2192. /pmc/articles/PMC10157727/
Zizzo G, Hilliard BA, Monestier M, Cohen PL (2012) Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction. J Immunol [Internet]. [cited 2023 Oct 17];189:3508–20. https://pubmed.ncbi.nlm.nih.gov/22942426/
Xie Y, Chen Z, Zhong Q, Zheng Z, Chen Y, Shangguan W et al M2 macrophages secrete CXCL13 to promote renal cell carcinoma migration, invasion, and EMT. Cancer Cell Int [Internet]. 2021 [cited 2023 Oct 17];21:1–13. https://cancerci.biomedcentral.com/articles/10.1186/s12935-021-02381-1
Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z et al (1979) In Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2. Science [Internet]. 2004 [cited 2024 Feb 26];303:844–8. https://www.science.org/doi/10.1126/science.1092472
Vassilev LT (2004) Cell Cycle Small-Molecule Antagonists of p53-MDM2 Binding: Research Tools and Potential Therapeutics. [cited 2024 Feb 26]; https://www.tandfonline.com/action/journalInformation?journalCode=kccy20
Dorrington MG, Fraser IDC (2019) NF-κB signaling in macrophages: Dynamics, crosstalk, and signal integration. Front Immunol 10:443978
Liu T, Zhang L, Joo D, Sun SC NF-κB signaling in inflammation. Signal Transduction and Targeted Therapy 2017 2:1 [Internet]. 2017 [cited 2024 Feb 25];2:1–9. https://www.nature.com/articles/sigtrans201723
Jha MK, Passero JV, Rawat A, Ament XH, Yang F, Vidensky S et al (2021) Macrophage monocarboxylate transporter 1 promotes peripheral nerve regeneration after injury in mice. J Clin Invest [Internet]. [cited 2023 Oct 18];131. https://doi.org/10.1172/JCI141964
Tan Z, Xie N, Banerjee S, Cui H, Fu M, Thannickal VJ et al (2015) The Monocarboxylate Transporter 4 Is Required for Glycolytic Reprogramming and Inflammatory Response in Macrophages. J Biol Chem [Internet]. [cited 2023 Oct 18];290:46. /pmc/articles/PMC4281748/
Draoui N, Schicke O, Seront E, Bouzin C, Sonveaux P, Riant O et al (2014) Antitumor activity of 7-aminocarboxycoumarin derivatives, a new class of potent inhibitors of lactate influx but not efflux. Mol Cancer Ther [Internet]. [cited 2023 Sep 29];13:1410–8. https://dx.doi.org/10.1158/1535-7163.MCT-13-0653
Maumus M, Roussignol G, Toupet K, Penarier G, Bentz I, Teixeira S et al (2016) Utility of a mouse model of osteoarthritis to demonstrate cartilage protection by IFNγ-primed equine mesenchymal stem cells. Front Immunol [Internet]. [cited 2024 Jul 24];7:218572. Available from: www.frontiersin.org
Lee MC, Saleh R, Achuthan A, Fleetwood AJ, Förster I, Hamilton JA et al CCL17 blockade as a therapy for osteoarthritis pain and disease. Arthritis Res Ther [Internet]. 2018 [cited 2024 Jul 24];20:1–10. https://arthritis-research.biomedcentral.com/articles/10.1186/s13075-018-1560-9
Alves-Simões M (2022) Rodent models of knee osteoarthritis for pain research. Osteoarthritis Cartilage 30:802–814
Yang C, Zhang J, Wu T, Zhao K, Wu X, Shi J et al (2021) Multi-Omics Analysis to Examine Gene Expression and Metabolites From Multisite Adipose-Derived Mesenchymal Stem Cells. Front Genet 12:627347
Lefevre C, Panthu B, Naville D, Guibert S, Pinteur C, Elena-Herrmann B et al (2019) Metabolic Phenotyping of Adipose-Derived Stem Cells Reveals a Unique Signature and Intrinsic Differences between Fat Pads. Stem Cells Int [Internet]. [cited 2023 Sep 25];2019. /pmc/articles/PMC6541987/
Mischen BT, Follmar KE, Moyer KE, Buehrer B, Olbrich KC, Levin LS et al Metabolic and functional characterization of human adipose-derived stem cells in tissue engineering? Plast Reconstr Surg [Internet]. 2008 [cited 2023 Sep 28];122:725–38. https://journals.lww.com/plasreconsurg/fulltext/2008/09000/metabolic_and_functional_characterization_of_human.7.aspx
Park HS, Kim JH, Sun BK, Song SU, Suh W, Sung JH (2016) Hypoxia induces glucose uptake and metabolism of adipose-derived stem cells. Mol Med Rep [Internet]. [cited 2023 Sep 27];14:4706–14. http://www.spandidos-publications.com/10.3892/mmr.2016.5796/abstract
Roemeling-Van Rhijn M, Mensah FKF, Korevaar SS, Leijs MJ, Van Osch GJVM, IJzermans JNM et al (2013) Effects of hypoxia on the immunomodulatory properties of adipose tissue-derived mesenchymal stem cells. Front Immunol 4:56061
Okada K, Mori D, Makii Y, Nakamoto H, Murahashi Y, Yano F et al Hypoxia-inducible factor-1 alpha maintains mouse articular cartilage through suppression of NF-κB signaling. Scientific Reports 2020 10:1 [Internet]. 2020 [cited 2023 Sep 27];10:1–11. https://www.nature.com/articles/s41598-020-62463-4
Yokota N, Hattori M, Ohtsuru T, Otsuji M, Lyman S, Shimomura K et al (2019) Comparative Clinical Outcomes After Intra-articular Injection With Adipose-Derived Cultured Stem Cells or Noncultured Stromal Vascular Fraction for the Treatment of Knee Osteoarthritis. Am J Sports Med 47:2577–2583
Cheng JH, Hsu CC, Hsu SL, Chou WY, Wu YN, Kuo CEA et al Adipose-Derived Mesenchymal Stem Cells-Conditioned Medium Modulates the Expression of Inflammation Induced Bone Morphogenetic Protein-2, -5 and – 6 as Well as Compared with Shockwave Therapy on Rat Knee Osteoarthritis. Biomedicines 2021, Vol 9, Page 1399 [Internet]. 2021 [cited 2024 Feb 28];9:1399. https://www.mdpi.com/2227-9059/9/10/1399/htm
Amodeo G, Niada S, Moschetti G, Franchi S, Savadori P, Brini AT et al (2021) Secretome of human adipose-derived mesenchymal stem cell relieves pain and neuroinflammation independently of the route of administration in experimental osteoarthritis. Brain Behav Immun 94:29–40
Zhang X, Wu Y, Pan Z, Sun H, Wang J, Yu D et al (2016) The effects of lactate and acid on articular chondrocytes function: Implications for polymeric cartilage scaffold design. Acta Biomater 42:329–340
Pucino V, Nefla M, Gauthier V, Alsaleh G, Clayton SA, Marshall J et al (2023) Differential effect of lactate on synovial fibroblast and macrophage effector functions. Front Immunol 14:1183825
Zhu X, Lee CW, Xu H, Wang YF, Yung PSH, Jiang Y et al (2021) Phenotypic alteration of macrophages during osteoarthritis: a systematic review. Arthritis Res Ther [Internet]. [cited 2024 Jul 24];23:1–13. https://arthritis-research.biomedcentral.com/articles/10.1186/s13075-021-02457-3
Selleri S, Bifsha P, Civini S, Pacelli C, Dieng MM, Lemieux W et al (2016) Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming. Oncotarget [Internet]. [cited 2023 Sep 28];7:30193–210. https://www.oncotarget.com/article/8623/text/
Colegio OR, Chu NQ, Szabo AL, Chu T, Rhebergen AM, Jairam V et al (2014) Functional polarization of tumour-associated macrophages by tumour-derived lactic acid. Nature 2014 513:7519 [Internet]. [cited 2023 Sep 28];513:559–63. https://www.nature.com/articles/nature13490
Samuvel DJ, Sundararaj KP, Nareika A, Lopes-Virella MF, Huang Y (2009) Lactate Boosts TLR4 Signaling and NF-κB Pathway-Mediated Gene Transcription in Macrophages via Monocarboxylate Transporters and MD-2 Up-Regulation. J Immunol [Internet]. [cited 2024 Feb 26];182:2476. /pmc/articles/PMC2673542/
Yang K, Xu J, Fan M, Tu F, Wang X, Ha T et al (2020) Lactate Suppresses Macrophage Pro-Inflammatory Response to LPS Stimulation by Inhibition of YAP and NF-κB Activation via GPR81-Mediated Signaling. Front Immunol 11:587913
Zhou HC, Yu WW, Yan XY, Liang XQ, Ma XF, Long JP et al (2022) Lactate-driven macrophage polarization in the inflammatory microenvironment alleviates intestinal inflammation. Front Immunol 13:1013686
Stone SC, Rossetti RAM, Alvarez KLF, Carvalho JP, Margarido PFR, Baracat EC et al (2019) Lactate secreted by cervical cancer cells modulates macrophage phenotype. J Leukoc Biol [Internet]. [cited 2024 Feb 26];105:1041–54. https://onlinelibrary.wiley.com/doi/full/10.1002/JLB.3A0718-274RR
Van Hée VF, Pérez-Escuredo J, Cacace A, Copetti T, Sonveaux P (2015) Lactate does not activate NF-κB in oxidative tumor cells. Front Pharmacol [Internet]. [cited 2023 Oct 18];6:228. /pmc/articles/PMC4602127/
Végran F, Boidot R, Michiels C, Sonveaux P, Feron O (2011) Lactate influx through the endothelial cell monocarboxylate transporter MCT1 supports an NF-kB/IL-8 pathway that drives tumor angiogenesis. Cancer Res [Internet]. [cited 2023 Oct 18];71:2550–60. https://dx.doi.org/10.1158/0008-5472.CAN-10-2828
Miller AM, Nolan MJ, Choi J, Koga T, Shen X, Yue BYJT et al (2007) Lactate Treatment Causes NF-κB Activation and CD44 Shedding in Cultured Trabecular Meshwork Cells. Invest Ophthalmol Vis Sci 48:1615–1621
Suganuma K, Miwa H, Imai N, Shikami M, Gotou M, Goto M et al (2010) Energy metabolism of leukemia cells: glycolysis versus oxidative phosphorylation. Leuk Lymphoma [Internet]. [cited 2023 Oct 18];51:2112–9. https://pubmed.ncbi.nlm.nih.gov/20860495/
Griessinger E, Pereira-Martins D, Nebout M, Bosc C, Saland E, Boet E et al (2023) Oxidative Phosphorylation Fueled by Fatty Acid Oxidation Sensitizes Leukemic Stem Cells to Cold. Cancer Res [Internet]. [cited 2023 Oct 18];83:2461–70. https://dx.doi.org/10.1158/0008-5472.CAN-23-1006
Nascimento CR, Rodrigues Fernandes NA, Gonzalez Maldonado LA, Rossa Junior C (2022) Comparison of monocytic cell lines U937 and THP-1 as macrophage models for in vitro studies. Biochem Biophys Rep 32:101383
Thomas AC, Mattila JT (2014) Of Mice and Men: Arginine Metabolism in Macrophages. Front Immunol [Internet]. [cited 2024 Feb 27];5. Available from: /pmc/articles/PMC4188127/
Chen S, Yang J, Wei Y, Wei X (2019) Epigenetic regulation of macrophages: from homeostasis maintenance to host defense. Cellular & Molecular Immunology 2019 17:1 [Internet]. [cited 2024 Feb 27];17:36–49. https://www.nature.com/articles/s41423-019-0315-0
Netea MG, Domínguez-Andrés J, Barreiro LB, Chavakis T, Divangahi M, Fuchs E et al (2020) Defining trained immunity and its role in health and disease. Nature Reviews Immunology 2020 20:6 [Internet]. [cited 2024 Feb 27];20:375–88. https://www.nature.com/articles/s41577-020-0285-6
Issa MR, Naja AS, Bouji NZ, Sagherian BH The role of adipose-derived mesenchymal stem cells in knee osteoarthritis: a meta-analysis of randomized controlled trials. Ther Adv Musculoskelet Dis [Internet]. 2022 [cited 2024 Feb 27];14. Available from: /pmc/articles/PMC9806366/
Yang WT, Ke CY, Yeh KT, Huang SG, Lin ZY, Wu WT et al (2022) Stromal-vascular fraction and adipose-derived stem cell therapies improve cartilage regeneration in osteoarthritis-induced rats. Scientific Reports. 12:1 [Internet]. 2022 [cited 2024 Feb 27];12:1–11. https://www.nature.com/articles/s41598-022-06892-3
Sadri B, Hassanzadeh M, Bagherifard A, Mohammadi J, Alikhani M, Moeinabadi-Bidgoli K et al Cartilage regeneration and inflammation modulation in knee osteoarthritis following injection of allogeneic adipose-derived mesenchymal stromal cells: a phase II, triple-blinded, placebo controlled, randomized trial. Stem Cell Res Ther [Internet]. 2023 [cited 2024 Feb 27];14:1–21. https://stemcellres.biomedcentral.com/articles/10.1186/s13287-023-03359-8

The authors declare no competing interests.

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Multiomics analyses reveal adipose-derived stem cells inhibit the inflammatory response of M1-like macrophages through secreting lactate

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Cell culture

Metabolome analysis

Flow cytometry

Preparation of conditioned medium

RNA sequencing (RNA-seq)

Quantitative polymerase chain reaction (qPCR)

Immunoblotting

Cell viability assay

Measuring intracellular lactate concentration

Enzyme-linked immunosorbent assay (ELISA)

Statistical analysis

Results

The culture supernatant of ADSCs contains high concentration of lactate

Effect of lactate and conditioned ADSC medium on M1-like macrophages

Lactate has an anti-inflammatory effect but probably does not induce M2 polarization

Lactate induces histone acetylation rather than NF-κB signaling suppression

Inhibition of a lactate transporter cancels the effect of the conditioned medium

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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