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In vitro immunomodulatory effects of human milk oligosaccharides on murine macrophage RAW264.7 cells
Carbohydrate Polymers 207 (2019) 230–238
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In vitro immunomodulatory effects of human milk oligosaccharides on murine macrophage RAW264.7 cells
T
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Weiyue Zhanga,1, Jingyu Yanb,1, Lehao Wua,1, Yang Yua, Richard D. Yea,c, Yan Zhanga, , ⁎ Xinmiao Liangb, a
School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China c Institute of Chinese Medical Sciences, University of Macau, Macau, SAR, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Human milk oligosaccharides Immunomodulator Nitric oxide Cytokine NF-κB MAPKs
Human milk plays an important role in the child’s immune system. The human milk oligosaccharides (HMOs) may affect breast-fed infants both locally and systemically. In the present study, HMOs were separated, characterized and investigated for the immunomodulatory effects on RAW264.7 macrophages and its underlying molecular mechanisms. Our results revealed that HMO-7, one of the neutral HMOs fractions can significantly induce the production of nitric oxide (NO) and PGE2 via up-regulating nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression. Additionally, HMO-7 was found to stimulate the release of ROS, TNF-α and cytokines including IL-1β, IL-2, IL-6 and IL-10 in RAW264.7 macrophages. Further study showed that macrophage activated by HMO-7 involved in nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Our study provides additional evidence that HMOs are the functional components in human milk and that HMOs may have the potential application in healthcare industry.
1. Introduction The immune system is divided into inherent immunity and adaptive immunity, which has the function of immune surveillance, defense and regulation. Macrophages are responsible for processes such as antigen processing and presentation to antigen-specific T cells (Sun et al., 2015). They can act as antigen-presenting cells to fight against inflammation and infection, therefore macrophages play an essential role in both innate and adaptive immune responses. Macrophages possess a broad array of cell surface receptors, intracellular mediators and essential secretory molecules (Zhang & Wang, 2014) and they exhibit unique phenotypes and a variety of biological functions in the complex microenvironment of the body, such as phagocytosis, surveillance, chemotaxis, and the destruction of harmful substances (Donovan & Comstock, 2017). Macrophages can defend against external pathogens by releasing various inflammatory mediators and cytokines such as interleukin (IL), interferon (IFN), tumor necrosis factor (TNF) (Lee et al., 2015), nitric oxide (NO) (MacMicking, Xie, & Nathan, 1997) and reactive oxygen species (ROS) (Kohchi, Inagawa, Nishizawa, & Soma, 2009). These defensive mechanisms can be activated when various
external stimulus bind to pattern recognition receptors on the surface of macrophages and trigger several different signaling pathways including the transcription factors nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Altogether, the activation of macrophages is being considered to be a promising strategy to improve the host’s immune capability. Recently, a number of studies have reported that many natural polysaccharides and oligosaccharides show potential as immunotherapeutic agents. For instance, alginate oligosaccharides from a Pseudoalteromonas sp., were found to be capable of stimulating RAW264.7 macrophages to augment the release of various inflammatory mediators, including IL-1α, IL-1β, IL-6 and TNF-α (Iwamoto et al., 2005; Zhou et al., 2015). Alginate-derived guluronate oligosaccharide (GOS) readily activated macrophages through TLR4/ Akt/NF-κB, TLR4/Akt/mTOR and MAPK signaling pathways and exerted impressive immuno-stimulatory activity (Fang et al., 2017). ASKP-1, a novel polysaccharide from Artemisia sphaerocephala Krasch seed was found to activate macrophages via MAPK, PI3K/Akt and NFκB signaling pathways (Ren, Lin, Alim, Zheng, & Yang, 2017). Human milk is a rich source of oligosaccharides. The WHO refers to human milk as the nutritional gold standard for term infants. Human
Abbreviation:HMO, Human milk oligosaccharides ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Zhang), [email protected] (X. Liang). 1 These authors contribute equally to this work. https://doi.org/10.1016/j.carbpol.2018.11.039 Received 8 June 2018; Received in revised form 9 November 2018; Accepted 11 November 2018 Available online 28 November 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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different periods of lactation. After centrifugation at 4 °C the lipid layer was removed and proteins were precipitated from the aqueous phase by addition of ethanol and subsequent centrifugation. HMOs-containing supernatant was concentrated and freeze-dried. Pooled HMOs were fractionated by the degree of polymerization of oligosaccharides using a preparative hydrophilic interaction liquid chromatography (prepHILIC, 250 mm × 50 mm). Lyophilized HMOs powder were dissolved in ethanol/water (1:1) and applied to pre-HILIC system. The mobile phase was ethanol/water with a gradient condition (Supplementary Table S1). The flow rate was 80 ml.min−1. The detection wavelength was 210 nm. Seven fractions containing different HMOs were collected according to their retention time on the chromatogram.
milk compounds are therefore interesting targets for immunomodulatory research. One liter of mature human milk contains ∼5-10 g unbound oligosaccharides, which is similar to the amount of proteins. In contrast, cow’s milk contains only trace amounts of oligosaccharides (Tannock et al., 2013). Over 200 different human milk oligosaccharides (HMOs) have been identified so far and some are unique to human milk (Tao et al., 2012). Five monosaccharides have been found to be major building blocks for HMOs, which include Dglucose (Glc), D-galactose (Gal), N-acetyl-D-glucosamine (GlcNAc), Lfucose (Fuc) and N-acetylneuraminicacid (Neu5Ac) (Chen, 2015; Smilowitz, Lebrilla, Mills, German, & Freeman, 2014). The potential health benefits of HMOs that were uncovered over the years may affect breast-fed infants both locally and systemically. HMOs have been discussed extensively for their role in the development of a specific intestinal flora in breast-fed infants (Underwood et al., 2015). Striking evidences also suggest that HMOs may act as receptor analogs to inhibit pathogen adhesion, thereby protecting breast-fed infants against infections and diarrhea (Newburg, Ruiz-Palacios, & Morrow, 2005). HMOs are partially absorbed intact in the infant’s intestine and appear in the urine of breast-fed, but not formula-fed infants (Kulinich & Liu, 2016), which indicate their presence in the systemic circulation. In fact, it has been observed that HMOs act as immunomodulators by reducing rolling and adhesion of human leukocytes on activated endothelial cells (Bode et al., 2004). Our previous work has successfully profiled sialylated HMOs using online solid phase extraction-hydrophilic interaction chromatography coupled with negative-ion electrospray mass spectrometry (Yan et al., 2018). As our ongoing projects on HMOs, in the present study, we continue to characterize the components of HMOs and examine their immunomodulatory activities on murine macrophage RAW264.7 cells in terms of NO, PGE2, ROS release and cytokines production. And we explore to find out the potential mechanism through identifying the signaling pathways involved in HMOs-induced macrophages activation. To the best of our knowledge, our study provides the first direct evidence on the ability of HMOs to modulate immune responses in macrophages.
2.4. Characterization of HMOs fractions by mass spectrometry (MS) Seven HMO fractions were collected by an off-line method and identified using a Waters Q-TOF Premier mass spectrometer (Waters, Manchester, U.K.) equipped with nano UPLC used for sample introduction. The spray voltage was 2.3 kV and the source temperature at 100 °C. The sampling cone was at 35 V.
2.5. Cytotoxicity assay Cells were plated overnight in 96-well plates (1 × 104 cells/well), different components and concentrations of HMOs were added to these wells. After 24 h, 10 μl MTT solution (5 mg/ml) was added, followed by 4 h incubation at 37 °C; then the culture supernatant was discarded and 100 μl DMSO was added to each well to dissolve the formazan crystal. Absorbance was measured at 570 nm with a microplate reader (FlexStation 3; Molecular Devices, Silicon Valley, CA, USA).
2.6. NO determination After pre-incubation of RAW264.7 cells (4 × 104 cells/well) for 12 h, HMOs, either with or without LPS (100 ng/ml), were incubated for 24 h. The nitrite accumulation in the cell culture supernatant was measured with Griess method (Zhao et al., 2012). Briefly, 50 μl of cell culture supernatant was mixed with 50 μl of Griess reagent I and 50 μl of Griess reagent Ⅱ in a 96-well plate, incubated at room temperature for 10 min, then measured at 540 nm with a microplate reader.
2. Materials and methods 2.1. Materials RPMI-1640 was purchased from HyClone (Logan, UT). The NO detection kit was obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Mouse TNF-α, IL-1β, IL-6 and IL-10 detecting ELISA kits were obtained from eBioscience (Thermo Fisher Scientific, USA). Anti-ERK, JNK, p38, COX-2, iNOS, p65, β-actin, HDAC1 and phosphoERK, -JNK, -p38 were purchased from Cell Signaling Technology (Danvers, MA). Other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA).
2.7. Real-time PCR Total RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA was reverse-transcribed into cDNA with a Toyobo First Strand cDNA Synthesis Kit. Relative quantification of IL-1β, IL-2, IL-6, IL-10, TNF-α expression were performed with SYBR® Green Real time PCR Master Mix (TOYOBO, Osaka, Japan) and data was presented as 2−△△ct. Primers were used as follow: IL-1β (5′-AGAGCATCCAGCTTCAAAT-3′; 5′-CATCTCGGAGCCTGTA GTG-3′) IL-2 (5′-CTCACCAGGATGCTCACATTTA-3′; 5′-TACAATGGTTGCTG TCTCATCA -3′) IL-6 (5′-GAGGATACCACTCCCAACAGACC-3′; 5′-AAGTGCATCATCG TTGTTCATACA-3′) TNF-α (5′-TTCTCATTCCTGCTTGTGG-3′; 5′-ACTTGGTGGTTTGCT ACG-3′) IL-10 (5′-AGGGTTACTTGGGTTGC-3′; 5′-TGAGGGTCTTCAGC TTC-3′) GAPDH (5′-CCTTCCGTGTTCCTACC-3′; 5 ′CAACCTGGTCCTCAGT GTA-3′)
2.2. Animals and cell culture Male C57BL/6 mice (6–8 weeks of age) were purchased from SLACCAS Laboratory Animal Co., Ltd (Shanghai, China). All animal experiments were performed according to the guidelines for the care and use of animals approved by the Committee on the Ethics of Animal Experiments of Shanghai Jiao Tong University. The RAW264.7 macrophage cell line was obtained from the China Cell Line Bank (Beijing, China). The cells were cultured in RPMI-1640 medium (HyClone, USA) containing 10% FBS (Gibco, USA), penicillin (100 U/ml), streptomycin (100 μg/ml). All cultures were incubated at 37 °C in a humidified atmosphere with 5% CO2. 2.3. Isolation and fractionation of HMOs Human milk samples were collected from 20 healthy mothers at 231
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Table 1 Fractionation and characterization of HMOs. Fraction
RTa (min)
Molecular mass (g/mol)
DPb
Compositionc
Content (%)
HMO-1 HMO-2 HMO-3 HMO-4
2.0-4.0 4.1-21.0 21.1-26.0 26.1-31.0
3-7 2 2-5 2-6
31.1-36.0
HMO-6 HMO-7
36.1-41.0 41.1-55.0
H2A1; H2F1 A1; H3N1 A1; H3N1 F1 A1; H3N1 A2; H4N2A1 lactose lactose; H2F1; H3N1; H3N1 F1 lactose; H2F1; H2F2; H3N1; H3N1 F1; H3N1 F2 H3N1 F1; H3N1 F2; H2F1; H4N2F1 H3N1 F1; H3N1 F2; H4N2F1; H4N2F2 H4N2F1; H4N2F2; H4N2F3; H5N3F1; H5N3F2; H5N3F3
20.5 – 23.0 20.8
HMO-5
633.2; 779.2; 998.3; 1144.8; 1289.9; 1363.9 342.2 342.2; 488.3; 707.5; 853.6 342.2; 488.3; 634.2; 707.5; 853.6; 999.7 853.6; 999.7; 488.3; 1218.9 853.6; 999.7; 1218.9; 1364.9 1218.9; 1364.9; 1511.4; 1584.4; 1730.5; 1876.7
a b c
3-7 5-8 7-11
17.0 12.0 6.7
Retention time. Degree of polymerization. H, Hex; N, HexNAc; F, dHex; A, NeuNAc.
2.8. Measurements of IL-1β, IL-2, IL-6, IL-10, TNF-α and PGE2 production by ELISA
P < 0.001). All statistical analyses were calculated by the GraphPad Prime 7 Software (GraphPad Software, San Diego, CA, USA).
RAW264.7 macrophages were seeded in 6-well plates (1 × 106 cells/well) and pretreated with various concentrations (0∼1 mg/ml) of HMOs for 24 h. LPS (100 ng/ml) was used as a positive control. The levels of IL-Iβ, IL-2, IL-6, TNF-α, and IL-10 were determined with specific ELISA Kits (eBioscience, CA, USA). The level of PGE2 was detected with Mouse PGE2 ELISA Kit (Absamit, China). IL-2 was detected with Mouse IL-2 ELISA Kit (Lianke, China).
3. Results 3.1. Fractionation and characterization of HMOs HMOs contains hundreds of oligosaccharides and it is not feasible to test their activities individually due to the limited availability of isolated and purified HMOs. Therefore, we separated total HMOs into seven fractions based on the electrostatic property and degree of polymerization by preparative HPLC. A zwitterionic matrix with mixedmode action of hydrophilic interaction and cation-exchange was used as the HPLC matrix in the column (Yan et al., 2018). Under the used chromatographic condition, both the matrix and the acidic HMOs carry negative charge, and therefore, the acidic HMOs (aHMOs) elute through the column earlier, while neutral HMOs (nHMOs) retain due to hydrophilic interaction. Therefore, aHMOs were collected as the first fraction (HMO-1), and the nHMOs were separated into six fractions according to different retention time (HMO-2∼HMO-7) (Supplementary Fig. S1). These HMOs fractions were further characterized by mass spectrometry (Supplementary Fig. S2). The predominant components of each fraction were determined and listed in Table 1. HMO-2 was mainly consisted of lactose, which was not used for further study in the following experiments. With the increased retention time, the components in the fractions became more complex, especially in HMO-5, 6 and 7.
2.9. Detection of intracellular ROS The ROS was detected by DCFH method as described earlier (Fan et al., 2017). Cells were plated overnight in 96-well plates (4 × 104 cells/well). After the cells were attached, they were pretreated with different concentrations of HMOs for 24 h. The cells were exposed to DCFH-DA for another 30 min and washed three times with PBS. The fluorescence was measured at 480/530 nm using a microplate reader. 2.10. Western blot analysis The cells were incubated in 6-well plates (1 × 106 cells/well) for 30 min or 24 h with HMOs or LPS, then cells were collected and lysed in loading buffer. Nuclear and cytoplasmic proteins were determined with nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology, China) according to the manufacturer’s protocol. The cell lysates were analyzed by immunoblot using primary antibodies against p38, p-p38, ERK, p-ERK, JNK, p-JNK, iNOS, COX-2, p65, HDAC1and β-actin. Quantification of western blots was performed with Image J soft-ware (NIH, Bethesda, MD).
3.2. HMO-7 induced NO and PGE2 production in RAW264.7 cells by induction of iNOS and COX-2 expression The amount of released NO was measured using the Griess reagent when RAW264.7 cells were exposed to HMOs fractions. As shown in Fig. 1A, HMO-7 significantly enhanced the NO production. To further investigate the immune stimulating effect of HMO-7 on NO and PGE2 release by activated macrophages, RAW264.7 cells were treated with different doses of HMO-7 (0.1 mg/ml∼1 mg/ml). As shown in Fig. 1B and C, HMO-7 stimulated the NO and PGE2 production in a dose-dependent manner. To investigate the effects of HMO-7 on the growth of RAW264.7 cells, cells were detected for the viability using MTT assay after treatment with HMO-7 (0.1 mg/ml∼1 mg/ml) for 24 h. As shown in Fig. 1D, HMO-7 did not affect the growth of RAW264.7 cells up to the concentration of 1 mg/ml. Therefore, the stimulating effects of HMO-7 on NO and PGE2 production were not due to the growth effects. To investigate whether the effects of HMO-7 on NO and PGE2 production were mediated through the corresponding iNOS and COX-2 modulation, the protein expression of iNOS and COX-2 was determined by immunoblotting, respectively. As show in Fig. 1E and F, HMO-7
2.11. Isolation of peritoneal macrophages The mice were intraperitoneally injected with 4% thioglycolate solution (2 ml) and sacrificed on the fourth day after injection. Their peritoneal cavities were rinsed with RPMI-1640 (10 ml). The collected cells were washed twice with RPMI-1640 and then cultured in RPMI1640 containing 10% heat-inactivated FBS, 1 mM glutamine, 100 of IU/ ml penicillin and 0.1 mg/ml of streptomycin at a density 1 × 105 cells/ well in 96-well plate. In all experiments, cells were allowed to acclimate for 24 h before any treatment. 2.12. Statistical analysis The results were expressed as mean ± S.E.M (n = 3). Statistical differences were analyzed by student’s t-test. P values below 0.05 were considered to be statistically significant (*P < 0.05, **P < 0.01, *** 232
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Fig. 1. Effects of HMO-7 on the production of NO and PGE2 through up-regulation of iNOS and COX-2 expression in RAW264.7 macrophage. RAW264.7 cells (4 × 104 cells/well in 96-well plates) were incubated with HMOs fractions (A) or different concentrations of HMO-7 (B). After 24 h, the nitrite levels in culture medium were measured by Griess reagent. LPS (100 ng/ml) was used as a positive control. (C) Cells (1 × 106 cells/well in 6-well plates) were treated with HMO-7 for 24 h. The level of PGE2 was determined by ELISA kits. (D) RAW264.7 cells (2 × 104 cells /well in 96-well plates) were treated with different concentrations of HMO7 for 24 h. The cell viability was assessed by MTT reduction assays. The protein levels of iNOS (E) and COX-2 (F) in HMO-7 treated RAW264.7 cells (1 × 106 cells/ well in 6-well plates) were detected by Western blot and normalized to the β-actin signal. The data were obtained from three independent experiments and expressed as the mean ± S.E.M. Drugs vs control: * p < 0.05, ** p < 0.01, ***p < 0.001.
3.3. HMO-7 stimulated the expression of cytokines in RAW264.7 macrophages
treatment markedly up-regulated the levels of iNOS and COX-2 protein expression in RAW264.7 cells in a dose-dependent manner (0.1 mg/ ml∼1 mg/ml). These results suggested that HMO-7 was able to stimulate the production of NO and PGE2 via up-regulating iNOS and COX-2, respectively.
Cytokines are low molecular weight soluble protein, which are mainly produced through transcriptional and translational regulation upon stimulating by immunogen, mitogen or other stimulants. RAW264.7 were treated with HMO-7 for 24 h, and the mRNA and 233
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Fig. 2. Effects of HMO-7 on cytokines release in RAW264.7 cells. (A–E) The mRNA expression of cytokines. Cells were treated with two concentrations of HMO-7 for 4 h, and LPS (100 ng/ml) was used as a positive control. The transcription levels of IL-1β, IL-6, IL-2, IL-10, TNF-α were determined by quantitative real-time PCR. (FJ) The protein expression of cytokines. Cells were treated with two concentrations of HMO-7 for 24 h. The protein expression levels of IL-1β, IL-6, IL-2, IL-10 and TNF-α were measured by ELISA kits. The data were obtained from three independent experiments and expressed as the mean ± S.E.M. Drugs vs control: *P < 0.05, **P < 0.01, ***P < 0.001.
3.4. HMO-7 promoted ROS production in RAW264.7 cells
protein levels of cytokines were evaluated using RT-PCR and ELISA kit, respectively. As shown in Fig. 2A–E, the addition of HMO-7 resulted in remarked increase in the mRNA expression of IL-1β, IL-2, IL-6, IL-10 and TNF-α in a dose-dependent manner. Similar results were observed when the effects of HMO-7 on the protein levels of IL-1β, IL-2, IL-6, IL-10 and TNF-α were investigated (Fig. 2F–J).
The effect of HMO-7 on ROS generation was examined in RAW264.7 cells. The content of ROS was measured by ROS-sensitive fluorophore DCFH-DA. As shown in Fig. 3, the intensity of the DCF fluorescence was significantly increased in HMO-7-stimulated RAW264.7 compared to control cells, indicating that HMO-7 promoted intracellular ROS 234
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3.7. HMO-7 induced NO production in mouse peritoneal macrophages To determine whether the immunological activity of HMO-7 occur in primary cells as well, we further examined the effects on HMO-7induced NO production in peritoneal macrophages isolated from mice. As shown in Fig. 6A, HMO-7 was found to significantly induce NO production. Furthermore, HMO-7 did not exert growth effects on peritoneal macrophages as determined by MTT reduction assay (Fig. 6B). 4. Discussion Human milk is often the only dietary source for the first few months of life. It contains all the nutrients needed for the healthy growth of infants. Despite the fact that HMOs are complex glycans and the third most abundant components in milk (after lactose and lipids) (Seppo, Autran, Bode, & Jarvinen, 2017), they were long thought to have no biological significance. It is now known that HMOs are also playing an important role for the healthy growth of infants, which comprise part of the functional ingredients in human milk. Some studies show that HMOs shape the composition of the infant intestinal microbiota through selective consumption by commensal bacteria (Underwood et al., 2015). Certain HMOs may serve as receptors for pathogens due to the similar structural motifs with glycans on the infant’s intestinal epithelia (Zivkovic, German, Lebrilla, & Mills, 2011). A systematic method to characterize the HMOs structures are developed in this study. The HMOs are first separated by pre-HPLC into several fractions, and each fraction is then examined by MS to determine the number of structures in each fraction as well as their chromatographic retention time. Using this process, a library of structures found in each fraction of HMOs is being constructed. The combination of retention time and accurate masses provide the degree of polymerization and composition of monosaccharides. The HMOs structures are often divided into two main categories: acidic and neutral oligosaccharides. In our study, the acidic oligosaccharides (mainly derived from HMO-1) contain sialic acids, are present in human milk in relatively low amounts (20%). Fractions of HMO 3∼7 are neutral oligosaccharides. With the increasing retention time, the polymerizations become bigger and fraction compositions become more complex. There are many isomers in each fraction. H3N1 in HMO-3 consist of two isomers including lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). The neutral fraction of HMOs seems to be a key factor for the development of the intestinal microbiota typical for breast-fed infants and hence for the prebiotic effect (Intanon et al., 2014). We further evaluate the immunomodulatory effects of each fraction
Fig. 3. Effects of HMO-7 on ROS production in RAW264.7 cells. RAW264.7 cells (4 × 104 cells/well in 96-well plates) were incubated with HMO-7 (0.01 mg/ml - 1 mg/ml) at 37℃. After 24 h, intracellular ROS was measured by flow cytometry utilizing an oxidation-sensitive fluorescent probe, DCFH-DA. LPS (100 ng/ml) was used as a positive control. Each value represents mean ± S.E.M of triplicate measurements. Drugs vs control: **P < 0.01, ***P < 0.001.
generation.
3.5. HMO-7 activated the NF-κB signaling pathway in RAW264.7 cells Since NF-κB is the key regulator and leading transcription factor of modulating the expression of various cytokines, immunoblotting was performed to investigate whether HMO-7 can induce nuclear translocation of the NF-κB p65 subunit in RAW264.7 macrophages. As illustrated in Fig. 4A, the nuclear level of p65 protein increased in HMO-7treated cells. Accordingly, the cytoplasmic level of p65 protein decreased in a dose-dependent manner (Fig. 4B).
3.6. HMO-7 activated the MAPKs signaling pathway in RAW264.7 cells In order to further explore the mechanism of the immunomodulatory effects of HMO-7, RAW264.7 cells were treated with different doses of HMO-7 and the phosphorylation levels of MAPKs signaling proteins were analyzed by immunoblotting. As shown in Fig. 5A–D, in HMO-7-treated RAW264.7 cells, the phosphorylation levels of the p38 kinase (p38), extracellular signal-related kinases (ERK1/ 2), and the c-Jun N-terminal kinases (JNK) were significantly increased in a dose-dependent manner, with up-regulated ERK1/2 being the most pronounced.
Fig. 4. Effects of HMO-7 on activation of NF-κB signaling pathway. RAW264.7 cells were stimulated with HMO-7 for 30 min. LPS (100 ng/ml) was used as a positive control. The protein expression levels of nucleus p65 (A), cytoplasm p65 (B) were determined by Western blot and normalized to the HDAC1, β-actin signal, respectively. The data were obtained from three independent experiments and expressed as the mean ± S.E.M. Drugs vs control, *P < 0.05, **P < 0.01, ***P < 0.001. 235
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Fig. 5. Effects of HMO-7 on the activation of MAPKs signaling pathway in RAW264.7 cells. Cells were treated with HMO-7 for 30 min. The protein expression levels of phosphorylated p38 (p-p38), ERK (p-ERK), and JNK (p-JNK) were determined by Western blot (A) and normalized to the total p38, ERK and JNK proteins, respectively (B-D). The data were obtained from three independent experiments and expressed as the mean ± S.E.M. Drug vs control: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 6. Effects of HMO-7 on production of NO in isolated mouse peritoneal macrophages. (A) Effects of HMO-7 on production of NO in isolated mouse peritoneal macrophages. (B)MTT assay for cell viability of HMO-7 in mouse peritoneal macrophages. Date was expressed as the mean ± S.E.M. Drugs vs control: **P < 0.01.
HMO-7. Polymyxin B binds to LPS, thus neutralizing its activity (Lu et al., 2017; Saito et al., 2014). As shown in Supplementary Fig. S3, the attenuation of NO production was observed for LPS, but not for HMO-7, indicating that NO production in RAW264.7 macrophages was induced by HMO-7 instead of endotoxins LPS. Moreover, HMO-7 significantly increased the production of IL-1β, IL-6, IL-2, IL-10 and TNF-α at both mRNA and protein levels, further indicating that HMO-7 effectively
in macrophages. Macrophage activation is one of the most important steps in the immune response. During activation, macrophages produce inflammatory mediators and cytokines including NO, PGE2, ROS, TNFα, and some certain interleukins. Our data show that one of the fractions, HMO-7 is able to promote NO, PGE2 and ROS generation and cytokines production in RAW264.7 macrophages. Polymyxin B was used in this study to exclude the possibility of LPS contamination of 236
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activates RAW264.7 macrophages. Simultaneous stimulation that TNFα, IL-1β and IL-6 are as pro-inflammatory cytokines and growth factors for B cells; IL-2 is as a key cytokine for the differentiation of T cells; IL10 is as an anti-inflammatory cytokine, indicates the role of HMO-7 in the maintenance of the immune homeostasis. Compared to other neutral HMOs, HMO-7 contains complex oligosaccharides with high-order structures, which may mainly contribute to the immune response of HMOs. To further insight into the molecular mechanism on immunomodulatory action of HMO-7, several signaling pathways closely related to cellular immunological process including NF-κB and MAPK pathways are investigated. The transcription factor NF-κB proteins bind with its inhibitor, IκB-α and reside in the cytoplasm in unstimulated cells. Its activation is regulated by the degradation of IκB-α that frees NF-κB and allows it to be translocated to the nucleus (Bonizzi & Karin, 2004). In the present study, HMO-7 remarkably promoted nuclear levels of the NF-κB p65 subunit and decreased cytoplasmic NF-κB p65 accordingly in RAW264.7, indicating that NF-κB pathway is involved in HMO-7-induced macrophage activation. The MAPK cascades, consisting of the ERK1/2, JNK and p38 (Junttila, Li, & Westermarck, 2007; Medeiros et al., 2014), play an important role in intracellular signal transduction pathways and regulating cytokines release. The phosphorylation of MAPKs is known to be part of the upstream signaling pathway for the NF-κB activation. Consistent with this observation, HMO-7 significantly altered the phosphorylation of ERK1/2 and JNK, and relatively less altered the phosphorylation of p38 pathway. Our results suggest that HMO-7 may activate macrophages through ERK1/ 2/JNK- NF-κB signaling pathways. In conclusion, we successfully characterized a fraction derived from HMOs, termed HMO-7, and evaluated its immunomodulatory activities in RAW264.7 macrophage cells. HMO-7 was found to possess significant immunomodulation activities by upregulating the release of TNF-α, IL-1β, IL-6, IL-2, ROS, PGE2 and NO from macrophages. Furthermore, we also found that MAPKs, NF-kB signaling pathways were involved in the macrophage activation induced by HMO-7. These in vitro data can be translated into ex vivo models, taking into consideration the activation of mouse peritoneal macrophages upon HMO7 pretreatment. Altogether, these results provide us a better understanding of the molecular mechanisms of murine macrophage activation induced by HMO-7. Further isolation and purification of HMO-7 remains to be performed. The individual active components derived from HMO-7 need to be compared to known HMOs for the assessment of their activity. Our findings suggest that HMOs are closely related to the health of the baby. Some certain oligosaccharides derived from HMO-7 may have the potential application in healthcare industry. Human milk-derived specific oligosaccharides may be of great commercial importance to be added into baby food such as infant formula for the better development of immune systems in growing infants.
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Kemp, M. W. (2014). Polymyxin B agonist capture therapy for intrauterine inflammation: Proof-ofprinciple in a fetal ovine model. Reproductive Sciences, 21(5), 623–631. https://doi. org/10.1177/1933719113508820. Seppo, A. E., Autran, C. A., Bode, L., & Jarvinen, K. M. (2017). Human milk oligosaccharides and development of cow’s milk allergy in infants. The Journal of Allergy and Clinical Immunology, 139(2), 708–711.e5. https://doi.org/10.1016/j.jaci.2016. 08.031. Smilowitz, J., Lebrilla, C., Mills, D., German, J., & Freeman, S. (2014). Breast milk oligosaccharides: Structure-function relationships in the neonate. Annual Review of Nutrition, 34, 143–169. https://doi.org/10.1146/annurev-nutr-071813-105721. Breast. Sun, H., Zhang, J., Chen, F., Chen, X., Zhou, Z., & Wang, H. (2015). Activation of RAW264.7 macrophages by the polysaccharide from the roots of Actinidia eriantha and its molecular mechanisms. Carbohydrate Polymers, 121, 388–402. https://doi. org/10.1016/j.carbpol.2014.12.023. 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Acknowledgements This work was supported by the grant from National Natural Science Foundation of China (grant number: 81402806). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2018.11.039. References Bode, L., Kunz, C., Muhly-Reinholz, M., Mayer, K., Seeger, W., & Rudloff, S. (2004). Inhibition of monocyte, lymphocyte, and neutrophil adhesion to endothelial cells by human milk oligosaccharides. Thrombosisand Haemostasis, 92(6), 1402–1410. https:// doi.org/10.1160/TH04-01-0055. Bonizzi, G., & Karin, M. (2004). The two NF-κB activation pathways and their role in innate and adaptive immunity. Trends in Immunology, 25(6), 280–288. https://doi.
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Zhou, R., Shi, X., Gao, Y., Cai, N., Jiang, Z., & Xu, X. (2015). Anti-inflammatory activity of guluronate oligosaccharides obtained by oxidative degradation from alginate in lipopolysaccharide-activated murine macrophage RAW 264.7 cells. Journal of Agricultural and Food Chemistry, 63(1), 160–168. https://doi.org/10.1021/jf503548a. Zivkovic, A. M., German, J. B., Lebrilla, C. B., & Mills, D. A. (2011). Human milk glycobiome and its impact on the infant gastrointestinal microbiota. Proceedings of the National Academy of Sciences, 108(Supplement 1), 4653–4658. https://doi.org/10. 1073/pnas.1000083107.
analchem.7b04468. Zhang, L., & Wang, C. C. (2014). Inflammatory response of macrophages in infection. Hepatobiliary and Pancreatic Diseases International, 13(2), 138–152. https://doi.org/ 10.1016/s1499-3872(14)60024-2. Zhao, J. H., Shen, T., Yang, X., Zhao, H., Li, X., & Xie, W. D. (2012). Sesquiterpenoids from Farfugium japonicum and their inhibitory activity on NO production in RAW264. 7 cells. Archives of Pharmacal Research, 35(7), 1153–1158. https://doi.org/10.1007/ s12272-012-0705-7.
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In vitro immunomodulatory effects of human milk oligosaccharides on murine macrophage RAW264.7 cells
T
⁎
Weiyue Zhanga,1, Jingyu Yanb,1, Lehao Wua,1, Yang Yua, Richard D. Yea,c, Yan Zhanga, , ⁎ Xinmiao Liangb, a
School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China c Institute of Chinese Medical Sciences, University of Macau, Macau, SAR, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Human milk oligosaccharides Immunomodulator Nitric oxide Cytokine NF-κB MAPKs
Human milk plays an important role in the child’s immune system. The human milk oligosaccharides (HMOs) may affect breast-fed infants both locally and systemically. In the present study, HMOs were separated, characterized and investigated for the immunomodulatory effects on RAW264.7 macrophages and its underlying molecular mechanisms. Our results revealed that HMO-7, one of the neutral HMOs fractions can significantly induce the production of nitric oxide (NO) and PGE2 via up-regulating nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression. Additionally, HMO-7 was found to stimulate the release of ROS, TNF-α and cytokines including IL-1β, IL-2, IL-6 and IL-10 in RAW264.7 macrophages. Further study showed that macrophage activated by HMO-7 involved in nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Our study provides additional evidence that HMOs are the functional components in human milk and that HMOs may have the potential application in healthcare industry.
1. Introduction The immune system is divided into inherent immunity and adaptive immunity, which has the function of immune surveillance, defense and regulation. Macrophages are responsible for processes such as antigen processing and presentation to antigen-specific T cells (Sun et al., 2015). They can act as antigen-presenting cells to fight against inflammation and infection, therefore macrophages play an essential role in both innate and adaptive immune responses. Macrophages possess a broad array of cell surface receptors, intracellular mediators and essential secretory molecules (Zhang & Wang, 2014) and they exhibit unique phenotypes and a variety of biological functions in the complex microenvironment of the body, such as phagocytosis, surveillance, chemotaxis, and the destruction of harmful substances (Donovan & Comstock, 2017). Macrophages can defend against external pathogens by releasing various inflammatory mediators and cytokines such as interleukin (IL), interferon (IFN), tumor necrosis factor (TNF) (Lee et al., 2015), nitric oxide (NO) (MacMicking, Xie, & Nathan, 1997) and reactive oxygen species (ROS) (Kohchi, Inagawa, Nishizawa, & Soma, 2009). These defensive mechanisms can be activated when various
external stimulus bind to pattern recognition receptors on the surface of macrophages and trigger several different signaling pathways including the transcription factors nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Altogether, the activation of macrophages is being considered to be a promising strategy to improve the host’s immune capability. Recently, a number of studies have reported that many natural polysaccharides and oligosaccharides show potential as immunotherapeutic agents. For instance, alginate oligosaccharides from a Pseudoalteromonas sp., were found to be capable of stimulating RAW264.7 macrophages to augment the release of various inflammatory mediators, including IL-1α, IL-1β, IL-6 and TNF-α (Iwamoto et al., 2005; Zhou et al., 2015). Alginate-derived guluronate oligosaccharide (GOS) readily activated macrophages through TLR4/ Akt/NF-κB, TLR4/Akt/mTOR and MAPK signaling pathways and exerted impressive immuno-stimulatory activity (Fang et al., 2017). ASKP-1, a novel polysaccharide from Artemisia sphaerocephala Krasch seed was found to activate macrophages via MAPK, PI3K/Akt and NFκB signaling pathways (Ren, Lin, Alim, Zheng, & Yang, 2017). Human milk is a rich source of oligosaccharides. The WHO refers to human milk as the nutritional gold standard for term infants. Human
Abbreviation:HMO, Human milk oligosaccharides ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Zhang), [email protected] (X. Liang). 1 These authors contribute equally to this work. https://doi.org/10.1016/j.carbpol.2018.11.039 Received 8 June 2018; Received in revised form 9 November 2018; Accepted 11 November 2018 Available online 28 November 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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different periods of lactation. After centrifugation at 4 °C the lipid layer was removed and proteins were precipitated from the aqueous phase by addition of ethanol and subsequent centrifugation. HMOs-containing supernatant was concentrated and freeze-dried. Pooled HMOs were fractionated by the degree of polymerization of oligosaccharides using a preparative hydrophilic interaction liquid chromatography (prepHILIC, 250 mm × 50 mm). Lyophilized HMOs powder were dissolved in ethanol/water (1:1) and applied to pre-HILIC system. The mobile phase was ethanol/water with a gradient condition (Supplementary Table S1). The flow rate was 80 ml.min−1. The detection wavelength was 210 nm. Seven fractions containing different HMOs were collected according to their retention time on the chromatogram.
milk compounds are therefore interesting targets for immunomodulatory research. One liter of mature human milk contains ∼5-10 g unbound oligosaccharides, which is similar to the amount of proteins. In contrast, cow’s milk contains only trace amounts of oligosaccharides (Tannock et al., 2013). Over 200 different human milk oligosaccharides (HMOs) have been identified so far and some are unique to human milk (Tao et al., 2012). Five monosaccharides have been found to be major building blocks for HMOs, which include Dglucose (Glc), D-galactose (Gal), N-acetyl-D-glucosamine (GlcNAc), Lfucose (Fuc) and N-acetylneuraminicacid (Neu5Ac) (Chen, 2015; Smilowitz, Lebrilla, Mills, German, & Freeman, 2014). The potential health benefits of HMOs that were uncovered over the years may affect breast-fed infants both locally and systemically. HMOs have been discussed extensively for their role in the development of a specific intestinal flora in breast-fed infants (Underwood et al., 2015). Striking evidences also suggest that HMOs may act as receptor analogs to inhibit pathogen adhesion, thereby protecting breast-fed infants against infections and diarrhea (Newburg, Ruiz-Palacios, & Morrow, 2005). HMOs are partially absorbed intact in the infant’s intestine and appear in the urine of breast-fed, but not formula-fed infants (Kulinich & Liu, 2016), which indicate their presence in the systemic circulation. In fact, it has been observed that HMOs act as immunomodulators by reducing rolling and adhesion of human leukocytes on activated endothelial cells (Bode et al., 2004). Our previous work has successfully profiled sialylated HMOs using online solid phase extraction-hydrophilic interaction chromatography coupled with negative-ion electrospray mass spectrometry (Yan et al., 2018). As our ongoing projects on HMOs, in the present study, we continue to characterize the components of HMOs and examine their immunomodulatory activities on murine macrophage RAW264.7 cells in terms of NO, PGE2, ROS release and cytokines production. And we explore to find out the potential mechanism through identifying the signaling pathways involved in HMOs-induced macrophages activation. To the best of our knowledge, our study provides the first direct evidence on the ability of HMOs to modulate immune responses in macrophages.
2.4. Characterization of HMOs fractions by mass spectrometry (MS) Seven HMO fractions were collected by an off-line method and identified using a Waters Q-TOF Premier mass spectrometer (Waters, Manchester, U.K.) equipped with nano UPLC used for sample introduction. The spray voltage was 2.3 kV and the source temperature at 100 °C. The sampling cone was at 35 V.
2.5. Cytotoxicity assay Cells were plated overnight in 96-well plates (1 × 104 cells/well), different components and concentrations of HMOs were added to these wells. After 24 h, 10 μl MTT solution (5 mg/ml) was added, followed by 4 h incubation at 37 °C; then the culture supernatant was discarded and 100 μl DMSO was added to each well to dissolve the formazan crystal. Absorbance was measured at 570 nm with a microplate reader (FlexStation 3; Molecular Devices, Silicon Valley, CA, USA).
2.6. NO determination After pre-incubation of RAW264.7 cells (4 × 104 cells/well) for 12 h, HMOs, either with or without LPS (100 ng/ml), were incubated for 24 h. The nitrite accumulation in the cell culture supernatant was measured with Griess method (Zhao et al., 2012). Briefly, 50 μl of cell culture supernatant was mixed with 50 μl of Griess reagent I and 50 μl of Griess reagent Ⅱ in a 96-well plate, incubated at room temperature for 10 min, then measured at 540 nm with a microplate reader.
2. Materials and methods 2.1. Materials RPMI-1640 was purchased from HyClone (Logan, UT). The NO detection kit was obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Mouse TNF-α, IL-1β, IL-6 and IL-10 detecting ELISA kits were obtained from eBioscience (Thermo Fisher Scientific, USA). Anti-ERK, JNK, p38, COX-2, iNOS, p65, β-actin, HDAC1 and phosphoERK, -JNK, -p38 were purchased from Cell Signaling Technology (Danvers, MA). Other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA).
2.7. Real-time PCR Total RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA was reverse-transcribed into cDNA with a Toyobo First Strand cDNA Synthesis Kit. Relative quantification of IL-1β, IL-2, IL-6, IL-10, TNF-α expression were performed with SYBR® Green Real time PCR Master Mix (TOYOBO, Osaka, Japan) and data was presented as 2−△△ct. Primers were used as follow: IL-1β (5′-AGAGCATCCAGCTTCAAAT-3′; 5′-CATCTCGGAGCCTGTA GTG-3′) IL-2 (5′-CTCACCAGGATGCTCACATTTA-3′; 5′-TACAATGGTTGCTG TCTCATCA -3′) IL-6 (5′-GAGGATACCACTCCCAACAGACC-3′; 5′-AAGTGCATCATCG TTGTTCATACA-3′) TNF-α (5′-TTCTCATTCCTGCTTGTGG-3′; 5′-ACTTGGTGGTTTGCT ACG-3′) IL-10 (5′-AGGGTTACTTGGGTTGC-3′; 5′-TGAGGGTCTTCAGC TTC-3′) GAPDH (5′-CCTTCCGTGTTCCTACC-3′; 5 ′CAACCTGGTCCTCAGT GTA-3′)
2.2. Animals and cell culture Male C57BL/6 mice (6–8 weeks of age) were purchased from SLACCAS Laboratory Animal Co., Ltd (Shanghai, China). All animal experiments were performed according to the guidelines for the care and use of animals approved by the Committee on the Ethics of Animal Experiments of Shanghai Jiao Tong University. The RAW264.7 macrophage cell line was obtained from the China Cell Line Bank (Beijing, China). The cells were cultured in RPMI-1640 medium (HyClone, USA) containing 10% FBS (Gibco, USA), penicillin (100 U/ml), streptomycin (100 μg/ml). All cultures were incubated at 37 °C in a humidified atmosphere with 5% CO2. 2.3. Isolation and fractionation of HMOs Human milk samples were collected from 20 healthy mothers at 231
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Table 1 Fractionation and characterization of HMOs. Fraction
RTa (min)
Molecular mass (g/mol)
DPb
Compositionc
Content (%)
HMO-1 HMO-2 HMO-3 HMO-4
2.0-4.0 4.1-21.0 21.1-26.0 26.1-31.0
3-7 2 2-5 2-6
31.1-36.0
HMO-6 HMO-7
36.1-41.0 41.1-55.0
H2A1; H2F1 A1; H3N1 A1; H3N1 F1 A1; H3N1 A2; H4N2A1 lactose lactose; H2F1; H3N1; H3N1 F1 lactose; H2F1; H2F2; H3N1; H3N1 F1; H3N1 F2 H3N1 F1; H3N1 F2; H2F1; H4N2F1 H3N1 F1; H3N1 F2; H4N2F1; H4N2F2 H4N2F1; H4N2F2; H4N2F3; H5N3F1; H5N3F2; H5N3F3
20.5 – 23.0 20.8
HMO-5
633.2; 779.2; 998.3; 1144.8; 1289.9; 1363.9 342.2 342.2; 488.3; 707.5; 853.6 342.2; 488.3; 634.2; 707.5; 853.6; 999.7 853.6; 999.7; 488.3; 1218.9 853.6; 999.7; 1218.9; 1364.9 1218.9; 1364.9; 1511.4; 1584.4; 1730.5; 1876.7
a b c
3-7 5-8 7-11
17.0 12.0 6.7
Retention time. Degree of polymerization. H, Hex; N, HexNAc; F, dHex; A, NeuNAc.
2.8. Measurements of IL-1β, IL-2, IL-6, IL-10, TNF-α and PGE2 production by ELISA
P < 0.001). All statistical analyses were calculated by the GraphPad Prime 7 Software (GraphPad Software, San Diego, CA, USA).
RAW264.7 macrophages were seeded in 6-well plates (1 × 106 cells/well) and pretreated with various concentrations (0∼1 mg/ml) of HMOs for 24 h. LPS (100 ng/ml) was used as a positive control. The levels of IL-Iβ, IL-2, IL-6, TNF-α, and IL-10 were determined with specific ELISA Kits (eBioscience, CA, USA). The level of PGE2 was detected with Mouse PGE2 ELISA Kit (Absamit, China). IL-2 was detected with Mouse IL-2 ELISA Kit (Lianke, China).
3. Results 3.1. Fractionation and characterization of HMOs HMOs contains hundreds of oligosaccharides and it is not feasible to test their activities individually due to the limited availability of isolated and purified HMOs. Therefore, we separated total HMOs into seven fractions based on the electrostatic property and degree of polymerization by preparative HPLC. A zwitterionic matrix with mixedmode action of hydrophilic interaction and cation-exchange was used as the HPLC matrix in the column (Yan et al., 2018). Under the used chromatographic condition, both the matrix and the acidic HMOs carry negative charge, and therefore, the acidic HMOs (aHMOs) elute through the column earlier, while neutral HMOs (nHMOs) retain due to hydrophilic interaction. Therefore, aHMOs were collected as the first fraction (HMO-1), and the nHMOs were separated into six fractions according to different retention time (HMO-2∼HMO-7) (Supplementary Fig. S1). These HMOs fractions were further characterized by mass spectrometry (Supplementary Fig. S2). The predominant components of each fraction were determined and listed in Table 1. HMO-2 was mainly consisted of lactose, which was not used for further study in the following experiments. With the increased retention time, the components in the fractions became more complex, especially in HMO-5, 6 and 7.
2.9. Detection of intracellular ROS The ROS was detected by DCFH method as described earlier (Fan et al., 2017). Cells were plated overnight in 96-well plates (4 × 104 cells/well). After the cells were attached, they were pretreated with different concentrations of HMOs for 24 h. The cells were exposed to DCFH-DA for another 30 min and washed three times with PBS. The fluorescence was measured at 480/530 nm using a microplate reader. 2.10. Western blot analysis The cells were incubated in 6-well plates (1 × 106 cells/well) for 30 min or 24 h with HMOs or LPS, then cells were collected and lysed in loading buffer. Nuclear and cytoplasmic proteins were determined with nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology, China) according to the manufacturer’s protocol. The cell lysates were analyzed by immunoblot using primary antibodies against p38, p-p38, ERK, p-ERK, JNK, p-JNK, iNOS, COX-2, p65, HDAC1and β-actin. Quantification of western blots was performed with Image J soft-ware (NIH, Bethesda, MD).
3.2. HMO-7 induced NO and PGE2 production in RAW264.7 cells by induction of iNOS and COX-2 expression The amount of released NO was measured using the Griess reagent when RAW264.7 cells were exposed to HMOs fractions. As shown in Fig. 1A, HMO-7 significantly enhanced the NO production. To further investigate the immune stimulating effect of HMO-7 on NO and PGE2 release by activated macrophages, RAW264.7 cells were treated with different doses of HMO-7 (0.1 mg/ml∼1 mg/ml). As shown in Fig. 1B and C, HMO-7 stimulated the NO and PGE2 production in a dose-dependent manner. To investigate the effects of HMO-7 on the growth of RAW264.7 cells, cells were detected for the viability using MTT assay after treatment with HMO-7 (0.1 mg/ml∼1 mg/ml) for 24 h. As shown in Fig. 1D, HMO-7 did not affect the growth of RAW264.7 cells up to the concentration of 1 mg/ml. Therefore, the stimulating effects of HMO-7 on NO and PGE2 production were not due to the growth effects. To investigate whether the effects of HMO-7 on NO and PGE2 production were mediated through the corresponding iNOS and COX-2 modulation, the protein expression of iNOS and COX-2 was determined by immunoblotting, respectively. As show in Fig. 1E and F, HMO-7
2.11. Isolation of peritoneal macrophages The mice were intraperitoneally injected with 4% thioglycolate solution (2 ml) and sacrificed on the fourth day after injection. Their peritoneal cavities were rinsed with RPMI-1640 (10 ml). The collected cells were washed twice with RPMI-1640 and then cultured in RPMI1640 containing 10% heat-inactivated FBS, 1 mM glutamine, 100 of IU/ ml penicillin and 0.1 mg/ml of streptomycin at a density 1 × 105 cells/ well in 96-well plate. In all experiments, cells were allowed to acclimate for 24 h before any treatment. 2.12. Statistical analysis The results were expressed as mean ± S.E.M (n = 3). Statistical differences were analyzed by student’s t-test. P values below 0.05 were considered to be statistically significant (*P < 0.05, **P < 0.01, *** 232
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Fig. 1. Effects of HMO-7 on the production of NO and PGE2 through up-regulation of iNOS and COX-2 expression in RAW264.7 macrophage. RAW264.7 cells (4 × 104 cells/well in 96-well plates) were incubated with HMOs fractions (A) or different concentrations of HMO-7 (B). After 24 h, the nitrite levels in culture medium were measured by Griess reagent. LPS (100 ng/ml) was used as a positive control. (C) Cells (1 × 106 cells/well in 6-well plates) were treated with HMO-7 for 24 h. The level of PGE2 was determined by ELISA kits. (D) RAW264.7 cells (2 × 104 cells /well in 96-well plates) were treated with different concentrations of HMO7 for 24 h. The cell viability was assessed by MTT reduction assays. The protein levels of iNOS (E) and COX-2 (F) in HMO-7 treated RAW264.7 cells (1 × 106 cells/ well in 6-well plates) were detected by Western blot and normalized to the β-actin signal. The data were obtained from three independent experiments and expressed as the mean ± S.E.M. Drugs vs control: * p < 0.05, ** p < 0.01, ***p < 0.001.
3.3. HMO-7 stimulated the expression of cytokines in RAW264.7 macrophages
treatment markedly up-regulated the levels of iNOS and COX-2 protein expression in RAW264.7 cells in a dose-dependent manner (0.1 mg/ ml∼1 mg/ml). These results suggested that HMO-7 was able to stimulate the production of NO and PGE2 via up-regulating iNOS and COX-2, respectively.
Cytokines are low molecular weight soluble protein, which are mainly produced through transcriptional and translational regulation upon stimulating by immunogen, mitogen or other stimulants. RAW264.7 were treated with HMO-7 for 24 h, and the mRNA and 233
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Fig. 2. Effects of HMO-7 on cytokines release in RAW264.7 cells. (A–E) The mRNA expression of cytokines. Cells were treated with two concentrations of HMO-7 for 4 h, and LPS (100 ng/ml) was used as a positive control. The transcription levels of IL-1β, IL-6, IL-2, IL-10, TNF-α were determined by quantitative real-time PCR. (FJ) The protein expression of cytokines. Cells were treated with two concentrations of HMO-7 for 24 h. The protein expression levels of IL-1β, IL-6, IL-2, IL-10 and TNF-α were measured by ELISA kits. The data were obtained from three independent experiments and expressed as the mean ± S.E.M. Drugs vs control: *P < 0.05, **P < 0.01, ***P < 0.001.
3.4. HMO-7 promoted ROS production in RAW264.7 cells
protein levels of cytokines were evaluated using RT-PCR and ELISA kit, respectively. As shown in Fig. 2A–E, the addition of HMO-7 resulted in remarked increase in the mRNA expression of IL-1β, IL-2, IL-6, IL-10 and TNF-α in a dose-dependent manner. Similar results were observed when the effects of HMO-7 on the protein levels of IL-1β, IL-2, IL-6, IL-10 and TNF-α were investigated (Fig. 2F–J).
The effect of HMO-7 on ROS generation was examined in RAW264.7 cells. The content of ROS was measured by ROS-sensitive fluorophore DCFH-DA. As shown in Fig. 3, the intensity of the DCF fluorescence was significantly increased in HMO-7-stimulated RAW264.7 compared to control cells, indicating that HMO-7 promoted intracellular ROS 234
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3.7. HMO-7 induced NO production in mouse peritoneal macrophages To determine whether the immunological activity of HMO-7 occur in primary cells as well, we further examined the effects on HMO-7induced NO production in peritoneal macrophages isolated from mice. As shown in Fig. 6A, HMO-7 was found to significantly induce NO production. Furthermore, HMO-7 did not exert growth effects on peritoneal macrophages as determined by MTT reduction assay (Fig. 6B). 4. Discussion Human milk is often the only dietary source for the first few months of life. It contains all the nutrients needed for the healthy growth of infants. Despite the fact that HMOs are complex glycans and the third most abundant components in milk (after lactose and lipids) (Seppo, Autran, Bode, & Jarvinen, 2017), they were long thought to have no biological significance. It is now known that HMOs are also playing an important role for the healthy growth of infants, which comprise part of the functional ingredients in human milk. Some studies show that HMOs shape the composition of the infant intestinal microbiota through selective consumption by commensal bacteria (Underwood et al., 2015). Certain HMOs may serve as receptors for pathogens due to the similar structural motifs with glycans on the infant’s intestinal epithelia (Zivkovic, German, Lebrilla, & Mills, 2011). A systematic method to characterize the HMOs structures are developed in this study. The HMOs are first separated by pre-HPLC into several fractions, and each fraction is then examined by MS to determine the number of structures in each fraction as well as their chromatographic retention time. Using this process, a library of structures found in each fraction of HMOs is being constructed. The combination of retention time and accurate masses provide the degree of polymerization and composition of monosaccharides. The HMOs structures are often divided into two main categories: acidic and neutral oligosaccharides. In our study, the acidic oligosaccharides (mainly derived from HMO-1) contain sialic acids, are present in human milk in relatively low amounts (20%). Fractions of HMO 3∼7 are neutral oligosaccharides. With the increasing retention time, the polymerizations become bigger and fraction compositions become more complex. There are many isomers in each fraction. H3N1 in HMO-3 consist of two isomers including lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). The neutral fraction of HMOs seems to be a key factor for the development of the intestinal microbiota typical for breast-fed infants and hence for the prebiotic effect (Intanon et al., 2014). We further evaluate the immunomodulatory effects of each fraction
Fig. 3. Effects of HMO-7 on ROS production in RAW264.7 cells. RAW264.7 cells (4 × 104 cells/well in 96-well plates) were incubated with HMO-7 (0.01 mg/ml - 1 mg/ml) at 37℃. After 24 h, intracellular ROS was measured by flow cytometry utilizing an oxidation-sensitive fluorescent probe, DCFH-DA. LPS (100 ng/ml) was used as a positive control. Each value represents mean ± S.E.M of triplicate measurements. Drugs vs control: **P < 0.01, ***P < 0.001.
generation.
3.5. HMO-7 activated the NF-κB signaling pathway in RAW264.7 cells Since NF-κB is the key regulator and leading transcription factor of modulating the expression of various cytokines, immunoblotting was performed to investigate whether HMO-7 can induce nuclear translocation of the NF-κB p65 subunit in RAW264.7 macrophages. As illustrated in Fig. 4A, the nuclear level of p65 protein increased in HMO-7treated cells. Accordingly, the cytoplasmic level of p65 protein decreased in a dose-dependent manner (Fig. 4B).
3.6. HMO-7 activated the MAPKs signaling pathway in RAW264.7 cells In order to further explore the mechanism of the immunomodulatory effects of HMO-7, RAW264.7 cells were treated with different doses of HMO-7 and the phosphorylation levels of MAPKs signaling proteins were analyzed by immunoblotting. As shown in Fig. 5A–D, in HMO-7-treated RAW264.7 cells, the phosphorylation levels of the p38 kinase (p38), extracellular signal-related kinases (ERK1/ 2), and the c-Jun N-terminal kinases (JNK) were significantly increased in a dose-dependent manner, with up-regulated ERK1/2 being the most pronounced.
Fig. 4. Effects of HMO-7 on activation of NF-κB signaling pathway. RAW264.7 cells were stimulated with HMO-7 for 30 min. LPS (100 ng/ml) was used as a positive control. The protein expression levels of nucleus p65 (A), cytoplasm p65 (B) were determined by Western blot and normalized to the HDAC1, β-actin signal, respectively. The data were obtained from three independent experiments and expressed as the mean ± S.E.M. Drugs vs control, *P < 0.05, **P < 0.01, ***P < 0.001. 235
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Fig. 5. Effects of HMO-7 on the activation of MAPKs signaling pathway in RAW264.7 cells. Cells were treated with HMO-7 for 30 min. The protein expression levels of phosphorylated p38 (p-p38), ERK (p-ERK), and JNK (p-JNK) were determined by Western blot (A) and normalized to the total p38, ERK and JNK proteins, respectively (B-D). The data were obtained from three independent experiments and expressed as the mean ± S.E.M. Drug vs control: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 6. Effects of HMO-7 on production of NO in isolated mouse peritoneal macrophages. (A) Effects of HMO-7 on production of NO in isolated mouse peritoneal macrophages. (B)MTT assay for cell viability of HMO-7 in mouse peritoneal macrophages. Date was expressed as the mean ± S.E.M. Drugs vs control: **P < 0.01.
HMO-7. Polymyxin B binds to LPS, thus neutralizing its activity (Lu et al., 2017; Saito et al., 2014). As shown in Supplementary Fig. S3, the attenuation of NO production was observed for LPS, but not for HMO-7, indicating that NO production in RAW264.7 macrophages was induced by HMO-7 instead of endotoxins LPS. Moreover, HMO-7 significantly increased the production of IL-1β, IL-6, IL-2, IL-10 and TNF-α at both mRNA and protein levels, further indicating that HMO-7 effectively
in macrophages. Macrophage activation is one of the most important steps in the immune response. During activation, macrophages produce inflammatory mediators and cytokines including NO, PGE2, ROS, TNFα, and some certain interleukins. Our data show that one of the fractions, HMO-7 is able to promote NO, PGE2 and ROS generation and cytokines production in RAW264.7 macrophages. Polymyxin B was used in this study to exclude the possibility of LPS contamination of 236
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activates RAW264.7 macrophages. Simultaneous stimulation that TNFα, IL-1β and IL-6 are as pro-inflammatory cytokines and growth factors for B cells; IL-2 is as a key cytokine for the differentiation of T cells; IL10 is as an anti-inflammatory cytokine, indicates the role of HMO-7 in the maintenance of the immune homeostasis. Compared to other neutral HMOs, HMO-7 contains complex oligosaccharides with high-order structures, which may mainly contribute to the immune response of HMOs. To further insight into the molecular mechanism on immunomodulatory action of HMO-7, several signaling pathways closely related to cellular immunological process including NF-κB and MAPK pathways are investigated. The transcription factor NF-κB proteins bind with its inhibitor, IκB-α and reside in the cytoplasm in unstimulated cells. Its activation is regulated by the degradation of IκB-α that frees NF-κB and allows it to be translocated to the nucleus (Bonizzi & Karin, 2004). In the present study, HMO-7 remarkably promoted nuclear levels of the NF-κB p65 subunit and decreased cytoplasmic NF-κB p65 accordingly in RAW264.7, indicating that NF-κB pathway is involved in HMO-7-induced macrophage activation. The MAPK cascades, consisting of the ERK1/2, JNK and p38 (Junttila, Li, & Westermarck, 2007; Medeiros et al., 2014), play an important role in intracellular signal transduction pathways and regulating cytokines release. The phosphorylation of MAPKs is known to be part of the upstream signaling pathway for the NF-κB activation. Consistent with this observation, HMO-7 significantly altered the phosphorylation of ERK1/2 and JNK, and relatively less altered the phosphorylation of p38 pathway. Our results suggest that HMO-7 may activate macrophages through ERK1/ 2/JNK- NF-κB signaling pathways. In conclusion, we successfully characterized a fraction derived from HMOs, termed HMO-7, and evaluated its immunomodulatory activities in RAW264.7 macrophage cells. HMO-7 was found to possess significant immunomodulation activities by upregulating the release of TNF-α, IL-1β, IL-6, IL-2, ROS, PGE2 and NO from macrophages. Furthermore, we also found that MAPKs, NF-kB signaling pathways were involved in the macrophage activation induced by HMO-7. These in vitro data can be translated into ex vivo models, taking into consideration the activation of mouse peritoneal macrophages upon HMO7 pretreatment. Altogether, these results provide us a better understanding of the molecular mechanisms of murine macrophage activation induced by HMO-7. Further isolation and purification of HMO-7 remains to be performed. The individual active components derived from HMO-7 need to be compared to known HMOs for the assessment of their activity. Our findings suggest that HMOs are closely related to the health of the baby. Some certain oligosaccharides derived from HMO-7 may have the potential application in healthcare industry. Human milk-derived specific oligosaccharides may be of great commercial importance to be added into baby food such as infant formula for the better development of immune systems in growing infants.
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