Milk-Derived Extracellular Vesicles Suppress Inflammatory Cytokine Expression and Nuclear Factor-κB Activation in Lipopolysaccharide-Stimulated Macrophages
<p>CD81 Western blot of EV isolates. Western blots were conducted on aliquots of pools from the individual EV isolations using a Sephacryl S-500 SEC column (for chromatograms see <a href="#app1-dairy-02-00015" class="html-app">Supplementary Figures S1 and S2</a>). Six µg of each EV isolate were separated on an 18% SDS-PAGE, transferred to a PVDF membrane and probed for CD81, a classical EV surface marker. Eight µg of proteoliposomes (ProLip) were used for the Western blot.</p> "> Figure 2
<p>Bovine extracellular vesicles uptake in RAW264.7 cells. To detect bEV uptake, RAW264.7 cells incubated with either (<b>a</b>) 10 µg/mL or (<b>b</b>) 50 µg/mL of fluorescently labelled bEVs for 1, 4, 7, 18, or 24 h. Afterwards, the fluorescence of the resulting cell lysate was quantified. The values are shown as mean ± SD. Two independent experiments were performed in triplicates. Background fluorescence from the lysis buffer was subtracted from the results. See <a href="#dairy-02-00015-t001" class="html-table">Table 1</a> for sample nomenclature.</p> "> Figure 3
<p>Cell viability after incubation with EV isolates assessed with an MTT assay. RAW264.7 cells incubated for 4 h with 10 µg/mL, 50 µg/mL, or 100 µg/mL of EV protein. The values are calculated from non EV-treated cells grown simultaneously with the EV-treated cells (results not shown). Representative data are presented as means ± SD of four replicas, a minimum of two independent experiments were performed. Differences between control treatment (proteoliposomes, ProLip) and EV treatment were assessed by Student’s <span class="html-italic">t</span>-test. *: <span class="html-italic">p</span>-value < 0.05 vs. the corresponding ProLip treatment. See <a href="#dairy-02-00015-t001" class="html-table">Table 1</a> for sample nomenclature.</p> "> Figure 4
<p>Experimental procedure for cell experiments. Initially, RAW264.7 cells were seeded in a 6-well plate and starved for 24 h to avoid effects of serum constituents before EV treatment for 4 h, followed by 18 h of LPS stimulation. Cell culture supernatant and cell lysate were harvested and used for IκBα Western blots and cytokine assays.</p> "> Figure 5
<p>Evaluation of NF-κB pathway activation. (<b>a</b>) Western blots were carefully loaded with RAW264.7 cell lysates (50 µg protein/well) after treatment with the given EV isolate or control followed by LPS stimulation. β-actin was used as loading control to normalize data. The original Western blots can be seen in <a href="#app1-dairy-02-00015" class="html-app">Figure S3</a>. (<b>b</b>) Quantification of the bands shown in <a href="#dairy-02-00015-f005" class="html-fig">Figure 5</a>a, depicted as a ratio between non-LPS-treated cells/LPS-treated RAW264.7 cells. The bands were quantified using ImageJ. All ratios were compared to the control by a Student’s <span class="html-italic">t</span>-test. * <span class="html-italic">p</span>-value < 0.05 vs. the control. The results were obtained by a minimum of two independent experiments. See <a href="#dairy-02-00015-t001" class="html-table">Table 1</a> for sample nomenclature.</p> "> Figure 6
<p>Cytokine ELISA assay. LPS-stimulated RAW264.7 cells were pre-treated with EV isolates from bovine or human mature milk, bovine colostrum, WPC-PL, or MCI-PL. Afterwards, cell culture supernatants were used for (<b>a</b>) IL-6 ELISA and cell lysates were used for (<b>b</b>) IL-1β ELISA cytokine assay. A minimum of two independent cell experiments were performed for each EV treatment. Cytokine concentration in each sample was calculated from a standard curve made with each individual experiment. Student’s <span class="html-italic">t</span>-test was performed comparing each EV + LPS-treated sample to the LPS-treated control. * <span class="html-italic">p</span>-value < 0.05 vs. the control. See <a href="#dairy-02-00015-t001" class="html-table">Table 1</a> for sample nomenclature.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials and Reagents
2.2. Isolation Procedure for Extracellular Vesicles in Milk
2.3. Formation of Proteoliposomes
2.4. Determination of Protein Concentration
2.5. Cell Culture Maintenance
2.6. EV Labelling and bEV Uptake
2.7. Cell Viability
2.8. Cytokine Assay
2.9. Western Blotting
2.10. Statistics
3. Results
3.1. Detection of CD81 in Extracellular Vesicles Isolates
3.2. Uptake of Extracellular Vesicles in RAW264.7 Cells
3.3. Effect of Extracellular Vesicle Incubation on RAW264.7 Cell Viability
3.4. Immunoregulating Effects of Extracellular Vesicles on RAW264.7 Cells
3.5. Preincubation with EVs Alter Activation of the NF-κB Pathway
3.6. Preincubation with EVs Alter Cytokine Secretion from RAW264.7 Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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EV Source | Abbreviation |
---|---|
Bovine milk | bEV A |
Bovine colostrum | cEV A |
Human breast milk | hEV A |
Whey protein concentrate—Phospholipid enriched | wEV B |
Micellar Casein Isolate—Phospholipid enriched | mcEV B |
Proteoliposomes | ProLip |
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Ascanius, S.R.; Hansen, M.S.; Ostenfeld, M.S.; Rasmussen, J.T. Milk-Derived Extracellular Vesicles Suppress Inflammatory Cytokine Expression and Nuclear Factor-κB Activation in Lipopolysaccharide-Stimulated Macrophages. Dairy 2021, 2, 165-178. https://doi.org/10.3390/dairy2020015
Ascanius SR, Hansen MS, Ostenfeld MS, Rasmussen JT. Milk-Derived Extracellular Vesicles Suppress Inflammatory Cytokine Expression and Nuclear Factor-κB Activation in Lipopolysaccharide-Stimulated Macrophages. Dairy. 2021; 2(2):165-178. https://doi.org/10.3390/dairy2020015
Chicago/Turabian StyleAscanius, Susanne R., Maria S. Hansen, Marie S. Ostenfeld, and Jan T. Rasmussen. 2021. "Milk-Derived Extracellular Vesicles Suppress Inflammatory Cytokine Expression and Nuclear Factor-κB Activation in Lipopolysaccharide-Stimulated Macrophages" Dairy 2, no. 2: 165-178. https://doi.org/10.3390/dairy2020015