Impact of Yeast-Derived β-Glucans on the Porcine Gut Microbiota and Immune System in Early Life
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethical Statement
2.2. Study Design
2.3. Experimental Procedures
2.3.1. Dietary Intervention and Oral Vaccination
2.3.2. Blood and Faecal Sampling
2.3.3. Dissection
2.4. Measurements
2.4.1. Microbiota Analysis
2.4.2. Serology
2.4.3. Cell Isolation
2.4.4. Stimulation Assay
2.4.5. Flow Cytometry
2.5. Statistical Analysis
2.5.1. Microbiota Analysis
2.5.2. Immunological Analysis
2.5.3. Correlation Analysis
3. Results
3.1. Microbial Colonization
3.1.1. Alpha Diversity
3.1.2. Microbiota Composition over Time
3.1.3. Beta Diversity
3.1.4. Differentially Abundant Genera between Treatment Groups
3.2. Immunological Analysis
3.2.1. Immunological Response to Oral Vaccination
3.2.2. Ex Vivo Stimulation of MLN Cells and PBMCs
3.2.3. Analysis of Immune Cells in Blood and the MLN
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Antibody | Host/Isotype | Clone | Fluorochrome | Company | Dilution |
---|---|---|---|---|---|
CD14 | Mouse, IgG2b | MIL-2 | FITC | Bio-Rad | 1:50 |
CD172a | Mouse, IgG2b | 74-22-15A | PE | BD Biosciences | 1:40 |
CD4a | Mouse, IgG2b | 74-12-4 | PerCP-Cy5.5 | BD PharmingenTM | 1:320 |
CADM1 | Chicken, IgY | 3E1 | Biotin | MBL | 1:200 |
Streptav. | n/a | n/a | BV421 | BD HorizonTM | 1:50 |
CD152 * | Mouse, IgG2a | n/a | APC | Ancell | 1:320 |
CD3ε | Mouse, IgG2a | BB23-8E6-8C8 | PE-CyTM7 | BD PharmingenTM | 1:160 |
CD4a | Mouse, IgG2b | 74-12-4 | PerCP-Cy5.5 | BD PharmingenTM | 1:320 |
CD8a | Mouse, IgG2a | 76-2-11 | FITC | BD PharmingenTM | 1:10 |
FoxP3 | Rat, IgG2a | FJK-16s | Alexa Fluor® 700 ** | eBioscienceTM | 1:20 |
Ki67 | Mouse, IgG1 | B56 | BV421 ** | BD HorizonTM | 1:80 |
CD25 | Mouse, IgG1 | K231.3B2 | Purified *** | Bio-Rad | 1:200 |
γδ T cells | Rat, IgG2a | MAC320 | PE | BD PharmingenTM | 1:20 |
Unpaired T-Test | Two-Way ANOVA | |||||
---|---|---|---|---|---|---|
Subset | Phenotype | D14 | D26 | D43 | D69 | |
p-value [CI] | p-value [CI] | p-value [CI] | p-value [CI] | |||
pDCs (%) | CD14−CD4+CD172 a+CADM1− | ns | ns | ns | 0.060 ↓ [−0.66 to 34.14] | T (<0.001), I (0.020) |
pDCs (mat.) | CD14−CD4+CD172 a+CADM1−CTLA4-Ig+ | ns | ns | ns | ns | T (<0.001) |
cDC1 (%) | CD14−CD4−CD172 alowCADM1+ | ns | ns | ns | ns | T (<0.001) |
cDC1 (mat.) | CD14−CD4−CD172 alowCADM1+ CTLA4-Ig+ | ns | ns | ns | ns | T (<0.001) |
cDC2 (%) | CD14−CD4−CD172 a+CADM1+ | ns | ns | ns | ns | T (0.092) |
cDC2 (mat.) | CD14−CD4−CD172 a+CADM1+ CTLA4-Ig+ | ns | ns | ns | ns | T (<0.001) |
NK cells (%) | CD3−CD8α+ | ns | 0.060 ↓ [−0.41 to 15.68] | ns | ns | G (0.080), T (0.063) |
NK cells (act.) | CD3−CD8α+CD25+ | ns | ns | ns | ns | T (<0.001) |
NK cells (Ki67%) | CD3−CD8α+Ki67+ | ns | 0.009 ↓ [5.15 to 28.53] | ns | ns | G (0.010), T (<0.001) |
γδ T cells (%) | CD3+TCR-γδ+ | ns | ns | ns | ns | T (0.005) |
γδ T cells (Ki67%) | CD3+TCR-γδ+Ki67+ | ns | 0.049 ↓ [0.02 to 12.75] | ns | ns | G (0.034), T (<0.001) |
CTLs (%) | CD3+TCR-γδ−CD8α+ | ns | 0.090 ↓ [−0.53 to 6.13] | ns | ns | G (0.008), T (<0.001) |
CTLs (Ki67%) | CD3+TCR-γδ−CD8α+Ki67+ | ns | ns | ns | ns | T (<0.001) |
T helper (%) | CD3+TCR-γδ−CD4+ | ns | ns | ns | ns | T (<0.001) |
T helper (Ki67%) | CD3+TCR-γδ−CD4+Ki67+ | ns | ns | ns | ns | T (<0.001) |
Mem./Act. (%) | CD3+TCR-γδ−CD4+CD8α+ | ns | ns | ns | ns | T (<0.001) |
Mem./Act. (Ki67%) | CD3+TCR-γδ−CD4+CD8α+ki67+ | ns | ns | ns | ns | T (<0.001) |
Tregs (%) | CD3+TCR-γδ−CD4+CD25 highFoxp3+ | ns | ns | ns | ns | T (0.003) |
Tregs (Ki67%) | CD3+ TCR-γδ−CD4+CD25 highFoxp3+Ki67+ | ns | ns | ns | ns | T (<0.001) |
Unpaired T-Test | Two-Way ANOVA | ||||
---|---|---|---|---|---|
Subset | Phenotype | D27 | D44 | D70 | |
p-value [CI] | p-value [CI] | p-value [CI] | |||
pDCs (%) | CD14−CD4+CD172 a+CADM1− | ns | ns | ns | T (<0.001) |
pDCs (mat.) | CD14−CD4+CD172 a+CADM1−CTLA4-Ig+ | 0.034 [3.21 to 67.89] ↓ | ns | 0.024 [7.75 to 92.70] ↓ | G (0.007), T (0.001), I (0.08) |
cDC1 (%) | CD14−CD4−CD172 alowCADM1+ | ns | 0.011 [−0.11 to −0.02] ↑ | ns | T (<0.001), I (0.028) |
cDC1 (mat.) | CD14−CD4−CD172 alowCADM1+CTLA4-Ig+ | ns | ns | ns | T (<0.001) |
cDC2 (%) | CD14−CD4−CD172 a+CADM1+ | ns | ns | ns | T (0.001) |
cDC2 (mat.) | CD14−CD4−CD172 a+CADM1+CTLA4-Ig+ | 0.084 [−62.71 to 4.50] ↑ | ns | ns | ns |
NK cells (%) | CD3−CD8 a+ | n/a | n/a | n/a | n/a |
NK cells (act.) | CD3−CD8α+CD25+ | n/a | n/a | n/a | n/a |
NK cells (Ki67%) | CD3−CD8α+Ki67+ | n/a | n/a | n/a | n/a |
γδ T cells (%) | CD3+TCR-γδ+ | ns | ns | ns | T (<0.001) |
γδ T cells (Ki67%) | CD3+TCR-γδ+Ki67+ | 0.089 [−15.45 to 1.26] ↑ | ns | ns | T (<0.001), I (0.086) |
CTLs (%) | CD3+TCR-γδ−CD8α+ | ns | 0.035 [−13.03 to −0.60] ↑ | ns | T (0.076) |
CTLs (Ki67%) | CD3+TCR-γδ−CD8α+Ki67+ | ns | ns | ns | T (<0.001) |
T helper (%) | CD3+TCR-γδ−CD4+ | ns | ns | ns | T (<0.001) |
T helper (Ki67%) | CD3+TCR-γδ−CD4+Ki67+ | 0.079 [−12.53 to 0.77] ↑ | ns | ns | T (<0.001), I (0.019) |
Mem./Act. (%) | CD3+CD4+CD8α+ | ns | ns | ns | T (<0.001) |
CD3+CD4 highCD8αlow | ns | ns | ns | T (0.002) | |
CD3+CD4 high CD8αlowki67+ | ns | ns | ns | T (<0.001) | |
CD3+CD4 lowCD8αhigh | ns | ns | ns | ns | |
CD3+CD4 low CD8αhighki67+ | ns | ns | ns | T (<0.001) | |
Tregs (%) | CD3+TCR-γδ−CD4+CD25 highFoxp3+ | ns | ns | ns | T (<0.001) |
Tregs (Ki67%) | CD3+ TCR-γδ−CD4+CD25 highFoxp3+Ki67+ | n/a | n/a | n/a | n/a |
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de Vries, H.; Geervliet, M.; Jansen, C.A.; Rutten, V.P.M.G.; van Hees, H.; Groothuis, N.; Wells, J.M.; Savelkoul, H.F.J.; Tijhaar, E.; Smidt, H. Impact of Yeast-Derived β-Glucans on the Porcine Gut Microbiota and Immune System in Early Life. Microorganisms 2020, 8, 1573. https://doi.org/10.3390/microorganisms8101573
de Vries H, Geervliet M, Jansen CA, Rutten VPMG, van Hees H, Groothuis N, Wells JM, Savelkoul HFJ, Tijhaar E, Smidt H. Impact of Yeast-Derived β-Glucans on the Porcine Gut Microbiota and Immune System in Early Life. Microorganisms. 2020; 8(10):1573. https://doi.org/10.3390/microorganisms8101573
Chicago/Turabian Stylede Vries, Hugo, Mirelle Geervliet, Christine A. Jansen, Victor P. M. G. Rutten, Hubèrt van Hees, Natalie Groothuis, Jerry M. Wells, Huub F. J. Savelkoul, Edwin Tijhaar, and Hauke Smidt. 2020. "Impact of Yeast-Derived β-Glucans on the Porcine Gut Microbiota and Immune System in Early Life" Microorganisms 8, no. 10: 1573. https://doi.org/10.3390/microorganisms8101573