Target: IFN-γ, IL-6, IL-17, and TNF-α[1]

In T84 cells, 2'-Fucosyllactose (2'-FL; 0–12 mg/mL; 48 h) attenuates LPS (100 μg/mL) and stimulates IL-8 secretion while suppressing cell-associated CD14 expression[1]. In T84 and HCT8 cells, 2'-Fucosyllactose (2 mg/mL; 48 h) reduces inflammation brought on by bacterial invasion. Inhibiting ETEC invasion and reducing the ensuing IL-8 release are the two effects of 2'-fucosyllactose[1]. In T84 and HCT8 cells, 2'-fucosyllactose (2 mg/mL; 48 h) activates signal pathways for macrophage migration inhibitory factor, which reduces inflammation[1].

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2′-FL/2'-Fucosyllactose suppresses CD14 expression [1]
LPS mediates proinflammatory signalling during UPEC invasion of bladder epithelium and ETEC invasion of T84 cells (not shown). LPS at 100 μg/mL triggered significant release of IL-8 from T84 cells (see online supplementary figure S5) and HMOSs attenuated this induction (figure 1A). In this more facile simplified model, 2′-FL/2'-Fucosyllactose, 3-FL, 6′-SL, 3′-SL, LNFP I and TFiLNO (figure 1B) were tested individually at their concentrations in human milk for ability to attenuate inflammation (figure 1C). Only 2′-FL suppressed LPS-induced induction of IL-8 levels in T84 cells (figure 1B). At 2 mg/mL, 2′-FL decreased the IL-8 secretion induced in LPS-treated T84 cells by 45%, similar to the 50% inhibition by 5 mg/mL HMOSs (figure 1B). Inhibition by 2′-FL is dose-dependent (figure 1D, E) and plateaus at 80% inhibition of IL-8 induction by 4 mg/mL 2′-FL (figure 1E). Cytochalasin D inhibits the intracellular actin machinery in T84 cells that ETEC use for invasion. Incubation with 2 μM cytochalasin D did not affect basal expression of IL-8 in the presence or absence of 2′-FL (figure 1F, bars 5 and 6). Cytochalasin D pretreatment of T84 cells before ETEC infection reduced induction of IL-8 after exposure to ETEC (figure 1F, bars 3 and 7). The inability of 2′-FL to inhibit residual IL-8 expression (figure 1F, bars 7 and 8), indicates that 2′-FL inhibits only the IL-8 induced specifically by ETEC invasion.

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2′-FL/2'-Fucosyllactose ameliorates inflammation induced by bacterial invasion [1]
Consistent with the above effect on LPS, 2 mg/mL 2′-FL/2'-Fucosyllactose inhibited invasion of ETEC in T84 cells and suppressed the associated IL-8 induction (figure 3A) comparably with the 5 mg/mL HMOS seen in online supplementary figure S1. To ensure that the inhibition displayed by 2′-FL is not idiosyncratic to the T84 cell line, 2′-FL activity was tested in HCT8 cells, an IEC human ileal cell line that is especially sensitive to ETEC invasion. In these HCT8 cells, pretreatment for 48 h with 2′-FL at 2 mg/mL inhibited ETEC invasion and attenuated the consequent IL-8 secretion (figure 3B).
To test whether the above observations were general to other type 1 pili organisms, the ability of 2′-FL to inhibit inflammatory signalling was tested in two additional type 1 pili E. coli AIEC (strain LF82) and UPEC (strain CF073). As a control, the activity of 2′-FL was tested on invasion of T84 by Salmonella enterica serovar Typhimurium (strain SL1344), whose invasion is independent of type 1 pili, instead requiring the type III secretion system or Zipper-like or Trigger-like entry processes. 2′-FL inhibited AIEC and UPEC invasion of T84 cells by ∼50% (figure 3C, D) and the IL-8 production by ∼25% and ∼40%, respectively (not shown). Although SL1344 invasion was inhibited by total HMOSs, it was not inhibited by 2′-FL (figure 3E). This suggests that HMOS components other than 2′-FL may inhibit other mechanisms of pathogenesis, but type 1 pili pathogenesis is specifically inhibited by 2′-FL.

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2′-FL/2'-Fucosyllactose induces macrophage migration inhibitory factor signal pathways that suppress inflammation [1]
Changes in intracellular signalling associated with 2′-FL-induced changes in CD14 expression were studied in T84 cells. Signalling molecules were measured via an array of 512 antibodies to signalling proteins. Cy5/Cy3 fluorescent signal ratios were analysed using GenePix Pro array analysis software. Filtering criteria were set as internally normalised ratios of >1.3 or <0.77 based on statistical significance with correction for multiple comparisons.
By these criteria, 2′-FL/2'-Fucosyllactose treatment of cells significantly modulated 28 signalling molecules (table 1). Functional analysis of these microarray data was conducted using integrated software from Metacore (GeneGo, http://trials.genego.com). Subsets of the macrophage migration inhibitory factor (MIF) inflammatory signalling network exhibited 2′-FL-induced changes that matched the 2′-FL-induced anti-inflammatory outcomes (see online supplementary figure S6). These changes in antibody microarray ligands induced by 2′-FL treatment were confirmed by western blot of LPS/TLR4 signal pathway mediators: 2′-FL depressed expression of CD14 and NF-κB, while inducing expression of iκB, a negative regulator of the NF-κB signal pathway (figure 4A). TLR4 and MyD88 expression was not changed (figure 4A). Erk phosphorylation decreased while p38 and Akt phosphorylation increased (figure 4A). Among the suppressors of cytokine signalling (SOCSs), 2′-FL increased expression of SOCS2 but not SOCS1 or SOCS3 (figure 4A). 2′-FL increased the phosphorylation (activation) of signal transducer and activator of transcription 3 (STAT3), a downstream signalling molecule shared by several SOCS pathways, but not of STAT1 (figure 4A). Changes in western blot intensity for all measured signalling molecules are shown in figure 4B. In H4 cells, (immature enterocyte model), 2′-FL modulated similar signal molecules: CD14 and NF-κB induction was repressed, while iκB and SOCS2 expression and STAT3 phosphorylation were induced (figure 4C). Thus, 2′-FL modulated the same signalling pathways in models of immature and mature enterocytes.

2'-Fucosyllactose (2'-FL; 100 mg (200 μL); ig; daily, for 4 d; C57BL/6 mice with AIEC infection) reduces inflammation and AIEC infection in vivo[1].

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2′-FL/2'-fucosyllactose inhibits AIEC infection and inflammation in vivo [1]
Three days of 0.25% DSS (a low dose) with streptomycin on the 4th day disrupts mouse microbiota. AIEC inoculation on the 5th day resulted in overt infection, manifesting as body weight loss (∼10%) 3 days and 4 days post AIEC. 2'-Fucosyllactose/2′-FL by gavage once per day for the 4 days preceding inoculation prevented the body weight loss (figure 5A). AIEC infected mice had shortened colons, but the lengths were closer to normal in AIEC inoculated mice pretreated with 2′-FL (figure 5B). Antibody against O83-antigen (expressed on LPS-positive AIEC LF82) revealed less colonisation in the 2′-FL pretreated group (figure 5C). Cultures from fresh faeces (not shown) and colon confirmed that mice in the 2′-FL pretreated group were colonised less by inoculated AIEC (figure 5D). There was less AIEC-induced CD14 expression in colonic crypts of 2′-FL treated mice, (figure 5E), fewer CD14 positive cells in muscularis mucosa (not shown), and lower CD14-mRNA levels (figure 5F). H&E staining in colon tissue of AIEC-infected mice revealed epithelial cell sloughing, immune cell infiltration, and muscularis mucosa hyperplasia, while colons of 2′-FL pretreated mice exhibited fewer of these manifestations of inflammation (see online supplementary figure S7). AIEC infection was accompanied by elevated IL-6, IL-17 and TNF-α, major inflammatory cytokines of mouse mucosa, and 2′-FL pretreatment inhibited this induction (figure 5G). IL-1β, IFN-γ and IL-10 were not significantly affected by AIEC infection nor by 2′-FL (not shown).

LPS stimulation in vitro [1]
IECs were cultured at 5×104 cells per well (subconfluent) in 24-well plates for 48 h in 500 μL media containing HMOS approximating physiological levels (HMOSs, 5 mg/mL; 2'-Fucosyllactose/2′-FL, 2 mg/mL; 3-FL, 0.4 mg/mL; 6′-SL, 0.5 mg/mL; 3′-SL, 0.5 mg/mL; LNFP I, 2.5 mg/mL; TFiLNO, 3 pg/mL), followed by LPS (E. coli) stimulation (T84 cells 100 μg/mL; H4 cells 200 ng/mL) for 16 h. Supernatants were stored at −20°C until analysis.

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Inhibition of ETEC infection by cytochalasin D [1]
Cytochalasin D, an inhibitor of actin polymerisation, inactivates host cell machinery needed for invasion by bacteria. Cytochalasin D (2 μM) was added to media of T84 cells for 30 min before ETEC inoculation (MOI=20) to inhibit invasion, and after exposure to ETEC, the ability of 2'-Fucosyllactose/2′-FL to inhibit IL-8 expression was measured in six replicate experiments.

Western Blot Analysis[1]
Cell Types: T84 cells
Tested Concentrations: 0, 2, and 4 mg/mL
Incubation Duration: 48 hrs (hours)
Experimental Results: Suppressed CD14 mRNA and decreased cell-associated CD14 protein expression.

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Western Blot Analysis[1]
Cell Types: T84 and HCT8 cellsT84 and HCT8 cells
Tested Concentrations: 2 mg/mL
Incubation Duration: 48 hrs (hours)
Experimental Results: Suppressed CD14 mRNA and decreased cell-associated CD14 protein expression. Inhibited CD14 and NF-κB induction. Induced iκB and SOCS2 expression and STAT3 phosphorylation.

Animal/Disease Models: C57BL/6 mice (8 weeks) with AIEC (uropathogenic E. coli, and adherent-invasive E. coli) infection[1]
Doses: 100 mg (200 μL)
Route of Administration: po (oral gavage); daily, for 4 days
Experimental Results: Had colons lengths were closer to normal. Inhibited the colonization of the colonic mucosa by O83-positive bacteria. diminished CD14 expression, CD14 mRNA levels, IL-6, IL-17 and TNF -α levels in colonic.

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In vivo study: A murine model of AIEC infection.37 ,38 was adapted and validated. Eight-week-old female C57BL/6 mice received 0.25% dextran sodium sulfate (DSS) in their drinking water for 3 days, and were given 20 mg of streptomycin by gavage on day 4; half also received 100 mg of 2'-Fucosyllactose/2′-FL in 200 μL by gavage for each of the 4 days. On the 5th day, the two groups of experimental mice were inoculated with 109 colony forming unit (CFU) AIEC via 200 μL gavage and sacrificed after an additional 4 days; a control group received DSS and antibiotic, but only a sham PBS inoculation. Body weight was monitored daily. AIEC in faeces and colonic tissue were quantified as CFU on erythromycimpicillin LB plates.37 Formalin (4%) fixed, paraffin-embedded 5 μm sections of mouse colon were stained with H&E. Cryosections (5 μm) of mouse colons stained with CD14 or O83 antibodies were studied by confocal microscopy. Total RNA was extracted from other colonic samples with Trizol for real-time quantitative PCR of CD14 mRNA levels, and protein was extracted for ELISA analysis of proinflammatory cytokines.

[1]. The human milk oligosaccharide 2'-fucosyllactose modulates CD14 expression in human enterocytes, thereby attenuating LPS-induced inflammation. Gut. 2016 Jan;65(1):33-46.

[2]. Human milk--derived oligosaccharides and plant-derived oligosaccharides stimulate cytokine production of cord blood T-cells in vitro. Pediatr Res. 2004 Oct;56(4):536-40.

2'-Fucosyllactose is being investigated in the clinical trial NCT03847467 (a preliminary feasibility study of 2'-FL as a dietary supplement for IBD patients receiving stable maintenance anti-TNF therapy). Background: Gram-negative pathogens are a major cause of intestinal infections, activating mucosal inflammation through the binding of lipopolysaccharide (LPS) to the intestinal Toll-like receptor 4 (TLR4). Breastfeeding reduces disease risk, and human milk has a modulatory effect on inflammation. Objective: This study aims to investigate whether human milk oligosaccharides (HMOSs) affect the release of interleukin (IL)-8 from pathogenic Escherichia coli-induced intestinal epithelial cells (IECs), identify specific pro-inflammatory signaling molecules regulated by HMOSs, and clarify the active HMOSs and their mechanisms of action. Methods: An inflammatory model was constructed using an in vitro infection model of IECs infected with enterotoxigenic Escherichia coli type 1 (ETEC): T84 cells represented mature IECs, and H4 cells represented immature IECs. Identify lipopolysaccharide (LPS)-induced signaling molecules associated with IL-8 release in the presence or absence of HMOSs. Validate signaling pathway mediators by gene knockdown and overexpression. Oligosaccharides leading to altered signaling pathways were identified. Results: HMOSs attenuated LPS-dependent IL-8 induction induced by enterotoxigenic Escherichia coli (ETEC), uropathogenic Escherichia coli, and adhesion-invasive Escherichia coli (AIEC) infections, and inhibited CD14 transcription and translation. CD14 knockdown reproduced the attenuation induced by HMOSs. CD14 overexpression enhanced the ETEC-induced inflammatory response and increased its sensitivity to HMOS inhibition. 2'-fucosylated lactose (2'-FL), at milk concentrations, exhibited comparable inhibitory activity against CD14 expression as total HMOSs and protected AIEC-infected mice. Conclusion: HMOSs and 2'-FL directly inhibited LPS-mediated inflammatory responses during ETEC invasion of T84 and H4 intestinal epithelial cells by attenuating CD14 induction. CD14 expression mediates partial activity of the LPS-TLR4-stimulated macrophage migration inhibitory factor (MIF) inflammatory pathway, which functions through cytokine signaling inhibitor 2/signal transducer and transcription activator 3/NF-κB. The direct inhibitory effect of human milk oligosaccharides (HMOs) on inflammation supports their function as an innate immune system, enabling mothers to protect vulnerable newborns through breast milk. 2'-Fucogran (2'-FL) is a major HMO that inhibits inflammatory signaling. [1]

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Human milk contains a large amount of free oligosaccharides (HMOs). HMOs have been shown to have anti-inflammatory properties, and there is increasing evidence that they have immunomodulatory effects. This study aimed to evaluate the effects of two human milk-derived oligosaccharide samples (neutral and acidic oligosaccharides) and a low molecular weight fucogran on cytokine production and umbilical cord blood monocyte activation. Monocytes were isolated from umbilical cord blood of randomly selected healthy newborns and co-cultured with oligosaccharide samples. Intracellular cytokine production (day 20) and T cell surface marker expression (day 5) were detected by flow cytometry. In vitro induced immunoglobulin levels were quantified in cell culture supernatants by turbidimetric assay (total IgG1) and ELISA (total IgE). The acidic oligosaccharide fraction increased the proportion of interferon-γ-producing CD3+CD4+ and CD3+CD8+ cells (p < 0.05) and increased IL-13 production in CD3+CD8+ cells (p < 0.05). In acidic oligosaccharide cultures, CD25 expression on CD3+CD4+ cells was significantly increased (p < 0.05). Low molecular weight fucoidan induced IL-4 production from CD3+CD4+ T cells (p < 0.05) and IL-13 production from CD3+CD8+ T cells (p < 0.05), while interferon-γ production was not affected in either T cell population. Immunoglobulin production (total IgE and total IgG1) was also unaffected. Oligosaccharides derived from human milk and plants can affect cytokine production and activation in umbilical cord blood-derived T cells in vitro. Therefore, oligosaccharides, especially acidic oligosaccharides, may affect lymphocyte maturation in breastfed newborns. [2]

C18H32O15

488.44

488.174

C, 44.26; H, 6.60; O, 49.13

41263-94-9

170484

White to off-white solid powder

1.68g/cm3

902.2ºC at 760 mmHg

230-231 °C

311.9ºC

0mmHg at 25°C

1.631

-6.1

10

15

10

33

609

14

C[C@H]1[C@H]([C@H]([C@@H]([C@@H](O1)O[C@@H]2[C@H]([C@H]([C@H](O[C@H]2O[C@H]([C@@H](CO)O)[C@@H]([C@H](C=O)O)O)CO)O)O)O)O)O

HWHQUWQCBPAQQH-BWRPKUOHSA-N

InChI=1S/C18H32O15/c1-5-9(24)12(27)14(29)17(30-5)33-16-13(28)11(26)8(4-21)31-18(16)32-15(7(23)3-20)10(25)6(22)2-19/h2,5-18,20-29H,3-4H2,1H3/t5-,6-,7+,8+,9+,10+,11-,12+,13-,14-,15+,16+,17-,18-/m0/s1

(2R,3R,4R,5R)-4-[(2S,3R,4S,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxy-2,3,5,6-tetrahydroxyhexanal

2'-Fucosyllactose; 41263-94-9; 2'-O-fucosyllactose; Lactose, 2'-o-fucosyl-; 2'-o-l-fucosyl-d-lactose; XO2533XO8R; UNII-XO2533XO8R; DTXSID40194179;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : 50 mg/mL (102.37 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)