Sarcodia suieae acetyl-xylogalactan regulate RAW 264.7 macrophage NF-kappa B activation and IL-1 beta cytokine production in macrophage polarization

In this study, the effects of acetyl-xylogalactan extracted from Sarcodia suieae on RAW 264.7 macrophage polarisation were evaluated. This extracted acetyl-xylogalactan had a monosaccharide composition of 91% galactose and 9% xylose, with polysaccharide and acetyl contents of 80.6% and 19.3%, respectively. MALDI–TOF mass spectrometry and NMR spectroscopy revealed the molecular weight of the acetyl-xylogalactan to be 88.5 kDa. After acetyl-xylogalactan treatment, RAW 264.7 macrophage polarisation was noted, along with enhanced phagocytic ability. Furthermore, the Cell Counting Kit-8 (CCK-8) assay was performed and the results demonstrated non-significant alteration in lactate dehydrogenase levels in the treated cells. Next, interleukin (IL) 1β, TNF, and Malt-1 expression in RAW 264.7 macrophages treated with the S. suieae acetyl-xylogalactan was investigated through real-time quantitative polymerase chain reaction, and the results demonstrated that S. suieae acetyl-xylogalactan induced IL-1β and Malt-1 expression. RNA sequencing analysis results indicated the S. suieae acetyl-xylogalactan positively regulated cytokine production and secretion, protein secretion, and response to IL-1 activation, based on the observed GO terms. The predicted target genes in the GO enrichment analysis were found to upregulate NF-κB signalling and M0 to M1 macrophage conversion through the observed cytokine production. Thus, acetyl-xylogalactan can positively regulate RAW 264.7 macrophage polarisation.

RAW 264.7 macrophage culture. RAW  Toxicity effect of S. suieae acetyl-xylogalactan on RAW 264.7 macrophages. To examine the toxicity of S. suieae acetyl-xylogalactan to RAW 264.7 macrophages, the experiment groups were classified as control receiving no treatment and treatment groups treated with 10, 20, and 30 μg/mL S. suieae acetyl-xylogalactan.
In brief, RAW 264.7 macrophages (1 × 10 6 cells) were treated with or without S. suieae acetyl-xylogalactan for 12 and 24 h. At the end of the treatment, the cells were observed using the Cell Counting Kit-8 (CCK-8; B34302, Bimake) at an OD of 450 nm.
Effect of S. suieae acetyl-xylogalactan on RAW 264.7 macrophages' phagocytic activity. The phagocytic activity of RAW 264.7 macrophages was determined using pHrodo Green BioParticles Conjugate for Phagocytosis (P35366, Thermo Scientific). In brief, 1 × 10 6 cells were cultured in the medium at 37 °C and 5% CO 2 for 24 h. After incubation, 10, 20, and 30 μg/mL S. suieae acetyl-xylogalactan was added to the cells and then Real-time reverse transcription qPCR for gene expression. Total RNA was extracted using Azol RNA Isolation Reagent (Arrowtech) and quantified through spectrophotometry at 260 nm. Real-time qPCR analysis of the macrophage IL-1β, TNF, and Malt-1 was performed by Biotools Co., Ltd.. GAPDH was used as the reference in the comparative CT to determine the relative alteration. Fluorescence was analysed using the auto CT method to determine the threshold of each gene, and the 2 −ΔΔCT method was used to calculate CT values by using StepOne (version 2.3). Data are presented as fold changes in the mRNA level normalised to the reference gene GAPDH.
The following oligonucleotide sequences were used for creating qPCR primers: Statistical analysis. Scheffé's test and one-way ANOVA were used to analyse the statistical significance between the treatment and control groups. A p-value of <0.05 was considered statistically significant. The results are presented as the means ± SD (p < 0.05 and p < 0.001). For RNA-seq data, DEGseq was used to analyse significant differences between the treatment and control groups. A relative log expression of > 2 and a corrected p-value of < 0.005 were considered statistically significant.

Results
S. suieae acetyl-xylogalactan extraction and analysis. Acetyl-xylogalactan was extracted from S. suieae by using hot water and then extracted using ethanol (Fig. 1). In brief, the water-soluble materials were extracted from freeze-dried S. suieae powder by using hot water (60 °C) for 6 h, and then, acetyl-xylogalactan was obtained from the aqueous supernatant through 99.8% ethanol extraction. The polysaccharide analysis results are presented in Table 1. The recovery rate for water extraction was 14%, followed by a polysaccharide recovery rate was 9% at 60 °C for 6 h. Although we performed polysaccharide extraction at 30 °C, 60 °C, and 90 °C, only extraction at 60 °C for 6 h afforded a desirable monosaccharide composition of 91% galactose and 9% xylose (Table 2). Furthermore, its polysaccharide and acetyl contents were nearly 80.6% and 19.3%, respectively, and its molecular weight was 88.5 kDa. Thus, this relative pure acetyl-xylogalactan was selected for the further investigation. www.nature.com/scientificreports www.nature.com/scientificreports/ Toxicity of S. suieae acetyl-xylogalactan to RAW 264.7 macrophages. RAW 264.7 macrophages were treated with 10, 20, and 30 μg/mL acetyl-xylogalactan for 12 and 24 h and compared with untreated cells. The results indicated RAW 264.7 macrophages toxicity was not reduced in the experiment. After 12 h, the 20and 30-μg/mL treatment groups demonstrated significant difference compared with the control group (p < 0.05; Fig. 2a), which diminished after 24 h (p > 0.05). Moreover, at 24 h, NGS detection of the fold changes in gene expression demonstrated significant induction of lactate dehydrogenase expression (p < 0.05). Thus, S. suieae acetyl-xylogalactan was not considered to be acutely toxic to RAW 264.7 macrophages.

Effect of S. suieae acetyl-xylogalactan on RAW 264.7 macrophages' phagocytic activity.
The effects of the acetyl-xylogalactan on RAW 264.7 macrophages' phagocytic activity are presented in Fig. 3a. At 12 h, compared with control, 30 μg/mL acetyl-xylogalactan treatment significantly increased RAW 264.7 macrophages' phagocytic ability (p < 0.05), but the effect of 10 and 20 μg/mL acetyl-xylogalactan was non-significant (p > 0.05). After 24 h, these changes in the phagocytic ability became non-significant in all groups (p > 0.05). Moreover, acetyl-xylogalactan treatment led to morphology alterations in RAW 264.7 macrophages: RAW 264.7 macrophages polarised to a fusiform shape was noted (Fig. 3b).
At 24 h, RNA-seq for detection of the fold change in IL6 expression also demonstrated nonsignificant differences for all groups (p > 0.05), but RNA-seq analysis for IL17A was not performed.
Thus, the extracted S. suieae acetyl-xylogalactan can regulate the production of macrophage cytokines, responsible for macrophage polarisation.   www.nature.com/scientificreports www.nature.com/scientificreports/ RNA-seq (transcriptome) and real-time reverse transcription qPCR. RNA was extracted from RAW 264.7 macrophages and first mapped to the reference genome (Table 3). Multiple mapped reference genome counts were <10%, and the unique mapped counts to the reference genome were nearly 88%. In other words, the isolated RNA could be accurately mapped to the mouse reference genome.      www.nature.com/scientificreports www.nature.com/scientificreports/ RNA-seq analysis further revealed that S. suieae acetyl-xylogalactan treatment significantly altered the RNA gene expression. The log2 fold change in gene expression is presented in Fig. 5a. Acetyl-xylogalactan treatment increased RNA gene expression of TNF, IL1B, MALT1, and other genes in RAW 264.7 macrophages, as presented in the heatmap in Fig. 5a.
A volcano map (Fig. 5b) of the biological effects (log2 fold change) and their statistical significance (−log10 p-value) was used to compare the alteration in gene expression in the treatments with control. In the volcano map, the red spots represent the differentially expressed genes significantly upregulated by the treatment (p < 0.005,  www.nature.com/scientificreports www.nature.com/scientificreports/ log2 fold change > 2). Real-time qPCR results for the IL-1β, TNF and Malt-1 terms are presented in Fig. 5c. M1 macrophage conversion was observed in RAW 264.7 macrophages treated with S. suieae acetyl-xylogalactan: 20 and 30 μg/mL acetyl-xylogalactan treatments significantly increased IL-1β expression nearly 400-and 600-fold, respectively (both p < 0.01). For Malt-1 expression, the relative transcript level increased approximately sixfold in the 20 and 30 μg/mL acetyl-xylogalactan treatment groups (p < 0.05). However, TNF expression did not significantly differ between the control and treatment groups (p > 0.05).
Taken together, these findings indicate that S. suieae acetyl-xylogalactan aided the M0 to M1 macrophage conversion. Pathway analyses revealed significantly upregulation of NF-kappa B signalling pathway components. Moreover, S. suieae acetyl-xylogalactan induced the expression of IL1B, TNF, and MALT1, which are involved in signalling transduction processes, based on KEGG observations presented in Fig. 7.

Discussion
In this study, the regulatory effects of S. suieae acetyl-xylogalactan were investigated in RAW 264.7 macrophages. RAW 264.7 macrophages were treated with various concentrations of the S. suieae acetyl-xylogalactan and then the cellular response was analysed using microscopic observation and RNA-seq methods. The findings revealed that S. suieae acetyl-xylogalactan positively regulates cytokine production and activates the NF-kappa B signalling pathway.
Studies have linked the signalling mechanisms to inflammation regulation: NF-κB signalling regulates expression of cytokines (e.g., IL-1, IL-6, IL-8, and TNF) and chemokines and modulates adhesion molecules and cell-cycle regulatory molecules 20 . NF-κB, a transcription factor, has several functions in macrophages 21 , categorised into M1 and M2 macrophages: M1 macrophages release pro-inflammatory cytokines, such as IL-1, IL-6, IL-12, and TNF, which regulate inflammation response 22. On stimulation, transforming growth factor-β-activated kinase 1 (TAK1) is first activated to induce the downstream kinase multisubunit IκB kinase complex (IKK) response 23 . Activated IKK can phosphorylate and degrade the NF-κB inhibitor, IκBα, and cause NF-κB activation 24 . Activated NF-κB is responsible for the conversion of M0 macrophages to M1 macrophages and for cytokine production.
A Lycium barbarum polysaccharide could promote TNF-α and IL-1β production 25 . A signalling pathway analysis revealed that it enhanced p38-MAPK phosphorylation and reduced JNK and ERK1/2 MAPK phosphorylation 26 . Moreover, a study on the effects of a purified Laminaria japonica polysaccharide on the cytokine production in RAW 264.7 macrophages demonstrated that TNF and IL-1β increased with sample concentration 26 . Additionally, NF-κB p65 levels significantly increased after the Laminaria japonica polysaccharide treatment 27 . According to our findings in RAW 264.7 macrophages treated with S. suieae acetyl-xylogalactan for 24  h, real-time PCR results revealed increased TNF, IL-1β, and Malt-1 levels, ELISA demonstrated reduced IL-6 and IL-17 levels. Other studies have reported that IL-6 production is typically involved in the host defence observed in the infection or wounded tissues during the acute-phase response. In the current study, S. suieae acetyl-xylogalactan treatment did not cause toxicity to RAW 264.7 macrophages; hence, IL-6 and IL-17A must have not been produced. Thus, S. suieae acetyl-xylogalactan possibly increases the production of inflammation cytokines, such as TNF, IL-1β, and Malt-1, but inhibits that of acute proinflammatory cytokines, such as IL-6 and IL-17A. Acetylated Bletilla striata polysaccharide modulates macrophage activation and wound healing 28 . Addition of methyl, acetyl, sulphate, and phosphate groups to polysaccharides increases the complexity of their primary structure and enhances their biological functions 29 . Compared with the nonacetyl polysaccharide, acetyl polysaccharides have antioxidant abilities and can inhibit the β-carotene-linoleic acid system; they also increase TNF-α expression by approximately 25%. The proposed underlying immunomodulatory mechanisms of these acetyl groups involve their interaction with the specific receptors and stimulation of the macrophage activation 30 . In this study, S. suieae acetyl-xylogalactan contained 19.3% acetyl groups and had a molecular weight of 88.5 kDa. Regarding the relationship between the S. suieae acetyl-xylogalactan and immunomodulation activation, the acetyl groups possibly facilitates the maintenance of the polysaccharide structure and their interaction with the cell-specific receptors to finally activate RAW 264.7 macrophages.
This study investigated the cellular functions of RAW 264.7 macrophages treated with S. suieae acetyl-xylogalactan. In the RNA-seq analysis, the S. suieae acetyl-xylogalactan was demonstrated to have positively regulated cytokine production and secretion, protein secretion, and response to the IL-1 activation, based on the observed GO terms. Of the predicted target genes in the GO enrichment analysis, CCL3, CD36, LPL, TNF, IL1RL1, IL1RN, IL1Β, PTGS2, and MALT1, all involved in the NF-κB signalling pathway, were upregulated. Taken together, S. suieae acetyl-xylogalactan induced the NF-κB signalling pathway in macrophages by the KEGG database, thus eliciting an immune response 31 .
In conclusion, polysaccharide from the animals, plants, microorganisms and macro-algae was known with a functional biological activities 32 , such as the anti-virus 33 , anti-tumor 34 , and anti-oxidation 35 effects. Summarization of our findings, we observed that the extracted S. suieae acetyl-xylogalactan might directly induce TNF, IL-1, and Malt1 production but reduces IL-6 and IL-17A production, resulting in regulating inflammation response via the NF-κB pathway. Here, RAW 264.7 macrophages treated with S. suieae acetyl-xylogalactan had increased phagocytic ability. Thus, S. suieae acetyl-xylogalactan potentially modulates RAW 264.7 macrophage activation and polarisation to M1 macrophages.