marine drugs Article Transcriptome Analysis Reveals Possible Immunomodulatory Activity Mechanism of Chlorella sp. Exopolysaccharides on RAW264.7 Macrophages Siwei Wu, Hongquan Liu * , Siyu Li, Han Sun, Xiumiao He, Ying Huang and Han Long Guangxi Key Laboratory for Polysaccharide Materials and Modifications, School of Marine and Biotechnology, GuangXi University for Nationalities, Nanning 530006, China; [email protected] (S.W.); [email protected] (S.L.); [email protected] (H.S.); [email protected] (X.H.); [email protected] (Y.H.); [email protected] (H.L.) * Correspondence: [email protected] Abstract: In this study, the exopolysaccharides of Chlorella sp. (CEP) were isolated to obtain the purified fraction CEP4. Characterization results showed that CEP4 was a sulfated heteropolysaccha- ride. The main monosaccharide components of CEP4 are glucosamine hydrochloride (40.8%) and glucuronic acid (21.0%). The impact of CEP4 on the immune activity of RAW264.7 macrophage cy- tokines was detected, and the results showed that CEP4 induced the production of nitric oxide (NO), TNF-α, and IL-6 in a dose-dependent pattern within a range of 6 µg/mL. A total of 4824 differentially expressed genes (DEGs) were obtained from the results of RNA-seq. Gene enrichment analysis showed that immune-related genes such as NFKB1, IL-6, and IL-1b were significantly upregulated, while the genes RIPK1 and TLR4 were significantly downregulated. KEGG pathway enrichment analysis showed that DEGs were significantly enriched in immune-related biological processes, Citation: Wu, S.; Liu, H.; Li, S.; including toll-like receptor (TLR) signaling pathway, cytosolic DNA-sensing pathway, and C-type Sun, H.; He, X.; Huang, Y.; Long, H. Transcriptome Analysis Reveals lectin receptor signaling pathway. Protein–protein interaction (PPI) network analysis showed that Possible Immunomodulatory Activity HSP90AB1, Rbx1, ISG15, Psmb6, Psmb3, Psmb8, PSMA7, Polr2f, Rpsa, and NEDD8 were the hub genes Mechanism of Chlorella sp. with an essential role in the immune activity of CEP4. The preliminary results of the present study Exopolysaccharides on RAW264.7 revealed the potential mechanism of CEP4 in the immune regulation of RAW264.7 macrophages, Macrophages. Mar. Drugs 2021, 19, suggesting that CEP4 is a promising immunoregulatory agent. 217. https://doi.org/10.3390/ md19040217 Keywords: Chlorella; polysaccharide; immune activity; immune mechanism; transcriptome Academic Editor: Hitoshi Sashiwa Received: 25 February 2021 1. Introduction Accepted: 8 April 2021 Published: 14 April 2021 Immune system disorders can affect the immune response of the body and cause immune diseases. It has become increasingly important to use immunomodulators to treat Publisher’s Note: MDPI stays neutral and prevent human diseases caused by the destruction of the immune system [1,2]. Over the with regard to jurisdictional claims in years, polysaccharides from natural sources have attracted increasing attention due to their published maps and institutional affil- antitumor, immunomodulatory activities, and low toxicity. Polysaccharides from plants [3], iations. bacteria [4,5] and fungi [6] have been reported to have immunoregulatory abilities. Many microalgae polysaccharides have exhibited immunomodulatory abilities [7–10]. In recent years, bioactive natural products extracted from microalgae have become a research hotspot; their extracellular products have particularly attracted significant atten- tion. It has been reported that polysaccharides from the green microalga Chlorella have Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. a variety of biological activities, such as anti-asthma [11], antioxidation [12], blood lipid- This article is an open access article decreasing capacity [13], anticancer, and immunoregulatory activities [14]. Studies have distributed under the terms and shown that polysaccharides from Cyanobacteria spirulina can enhance the immune function conditions of the Creative Commons in mice [15,16]. Qi et al. extracted the intracellular polysaccharides from Chloroidium Attribution (CC BY) license (https:// ellipsoidea and studied its potential immune mechanism on macrophages [17]. However, creativecommons.org/licenses/by/ the immunomodulatory activity mechanism of extracellular polysaccharides from Chlorella 4.0/). remains to be elucidated. Mar. Drugs 2021, 19, 217. https://doi.org/10.3390/md19040217 https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2021, 19, x 2 of 19 Mar. Drugs 2021, 19, 217 from Chloroidium ellipsoidea and studied its potential immune mechanism on macro-2 of 17 phages [17]. However, the immunomodulatory activity mechanism of extracellular pol- ysaccharides from Chlorella remains to be elucidated. To clarify the immunomodulatory activity mechanism of CEP, the impact of CEP4 on theTo immune clarify the activity immunomodulatory of RAW264.7 macrophage activity mechanism cytokines of was CEP, detected, the impact and of the CEP4 rela- on tionshipthe immune between activity immune of RAW264.7 activity macrophage and related cytokines gene expression was detected, was and studied the relationship through transcriptomicsbetween immune analysis activity to and develop related a genen experimental expression basis was studied for using through CEP in transcriptomics treating hu- mananalysis disease to develops caused an by experimental immune system basis compromise for using CEP. in treating human diseases caused by immune system compromise. 2.2. Results Results 2.1.2.1. Isolation Isolation and and Purification Purification of of Crude Crude CEP CEP TheThe crude crude CEP CEP was was purified purified by by DEAE DEAE-cellulose-cellulose (DEAE (DEAE-52)-52) anion anion-exchange-exchange chro- chro- matographymatography column, andand fivefive main main independent independent peaks, peaks, namely namely CEP1 CEP1 (eluted (eluted by ultrapure by ul- trapurewater), water), CEP2 (eluted CEP2 (eluted by 0.2M by NaCl), 0.2 M NaCl), CEP3 (eluted CEP3 (eluted by 0.5 Mby NaCl), 0.5 M NaCl), CEP4 (eluted CEP4 (eluted by 1 M byNaCl), 1 M NaCl), and CEP5 and (eluted CEP5 (eluted by 2 M by NaCl), 2 M NaCl), were obtained were obtained (Figure 1(Figure). This 1 study). This mainly study mainlyfocused focused on CEP4, on andCEP4, the and other the fractions other fractions will be will studied be studied in future in research.future research. FigureFigure 1. 1. TheThe elution elution profile profile of crude of crude polysaccharide polysaccharide from fromChlorellaChlorella on DEAEon DEAE-cellulose-cellulose ani- anion- onexchange-exchange chromatography chromatography column. column. 2.2.2.2. Characterization Characterization of of CEP4 CEP4 −1 FigureFigure 22 showsshows the the FT-IR FT-IR spectrum. spectrum A. wideA wide and and intense intense absorption absorption peak peak at 3425 at cm3425 −1 cmand−1 and a small a small signal signal at 2919 at 2919 cm cmare−1 theare characteristicsthe characteristic of thes of stretchingthe stretching vibration vibration of –OH of −1 –andOH C–H,and C respectively–H, respectively [18,19 [18,19]. Meanwhile,]. Meanwhile, FT-IR FT bands-IR band at 1623s at and1623 1415 and cm1415 cmarise−1 arise from fromasymmetric asymmetric and symmetricand symmetric stretching stretching vibrations vibrations of carboxylate of carboxylate [20,21 [20,21]. Consistent]. Consistent with −1 withother oth sulfateder sulfated polysaccharides, polysaccharides, the peaks the peaks were recordedwere recorded at 1245 at and 1245 867 and cm 867, correspond- cm−1, cor- respondinging to stretching to stretching vibration vibration of S–O of and S–O bending and bending vibration vibration of C–O–S of C of–O sulfate–S of sulfate [22]. More[22]. −1 β Moreimportantly, importantly, the band the band at 898 at cm 898 cmis− characteristic1 is characteristic of of-polysaccharide β-polysaccharide [20 ].[20 Overall,]. Overall, the theFT-IR FT- resultsIR results indicated indicated that that CEP4 CEP4 is a is sulfated a sulfated polysaccharide. polysaccharide. Based on the IC analysis (Table1 and Figure3), the monosaccharide composition of Based on the IC analysis (Table 1 and Figure 3), the monosaccharide composition of CEP4 was determined to be GlcN (40.8%), GlcA (21%), Xyl (8.6%), Ara (8.3%), GlcNAc CEP4 was determined to be GlcN (40.8%), GlcA (21%), Xyl (8.6%), Ara (8.3%), GlcNAc (7.7%), Rha (3.7%), and Gal (2.6%). The results suggested that CEP4 is a heteropolysaccha- (7.7%), Rha (3.7%), and Gal (2.6%). The results suggested that CEP4 is a heteropolysac- ride mainly consist of glucose and glucuronic acid. charide mainly consist of glucose and glucuronic acid. Mar. Drugs 2021, 19, x 3 of 19 Table 1. Monosaccharide composition. Name RT Ratio (%) Rha 12.667 3.7 Ara 14.125 8.3 GlcN 16.134 40.8 Mar. Drugs 2021, 19, x Gal 18.342 2.6 3 of 19 GlcNAc 21.55 7.7 Mar. Drugs 2021, 19, 217 Xyl 22.65 8.6 3 of 17 GlcATable 1. Monosaccharide composition48.259 . 21.0 Name RT Ratio (%) Rha 12.667 3.7 Ara 14.125 8.3 GlcN 16.134 40.8 Gal 18.342 2.6 GlcNAc 21.55 7.7 Xyl 22.65 8.6 GlcA 48.259 21.0 FigureFigure 2. 2. FTFT-IR-IR spectrum spectrum of of CEP4 CEP4 from from CChlorellahlorella spsp.. Table 1. Monosaccharide composition. Name RT Ratio (%) Rha 12.667 3.7 Ara 14.125 8.3 GlcN 16.134 40.8 Gal 18.342 2.6 GlcNAc 21.55 7.7 Xyl 22.65 8.6 GlcAFigure 2. FT-IR spectrum of CEP4 48.259 from Chlorella sp. 21.0 Figure 3. IC chromatograms of monosaccharide composition of CEP4. Figure 3. IC chromatogramsFigure 3. IC chromatograms of monosaccharide of monosaccharide