In early life and around weaning, pigs are at risk of developing infectious diseases which compromise animal welfare and have major economic consequences for the pig industry. A promising strategy to enhance resistance against infectious diseases is immunomodulation by feed additives. To assess the immune stimulating potential of feed additives in vitro, bone marrow-derived dendritic cells were used. These cells play a central role in the innate and adaptive immune system and are the first cells encountered by antigens that pass the epithelial barrier. Two different feed additives were tested on dendritic cells cultured from fresh and cryopreserved bone marrow cells; a widely used commercial feed additive based on yeast-derived β-glucans and the gram-negative probiotic strain E. coli Nissle 1917. E. coli Nissle 1917, but not β-glucans, induced a dose-dependent upregulation of the cell maturation marker CD80/86, whereas both feed additives induced a dose-dependent production of pro- and anti-inflammatory cytokines, including TNFα, IL-1β, IL-6 and IL-10. Furthermore, E. coli Nissle 1917 consistently induced higher levels of cytokine production than β-glucans. These immunomodulatory responses could be assessed by fresh as well as cryopreserved in vitro cultured porcine bone marrow-derived dendritic cells. Taken together, these results demonstrate that both β-glucans and E. coli Nissle 1917 are able to enhance dendritic cell maturation, but in a differential manner. A more mature dendritic cell phenotype could contribute to a more efficient response to infections. Moreover, both fresh and cryopreserved bone marrow-derived dendritic cells can be used as in vitro pre-screening tools which enable an evidence based prediction of the potential immune stimulating effects of different feed additives.
S1 Fig. β-glucans does not contain LPS and does not induce hTLR4 mediated NF-κB activation.
(A) 100 μg/mL β-glucans (MacroGard1) was tested for LPS contamination using a
recombinant factor C LAL assay preparation. A 5 EU spiked control was included (n = 1). (B)
Different concentrations (1 mg/mL– 0.1 μg/mL) of commercial β-glucans (MacroGard1) and
LPS (100 pg/mL) were tested for their NF-κB activation via hTLR4 (n = 3).
S2 Fig. Phenotype of cryopreserved cultured porcine mononuclear phagocytes. Gating
strategy following multicolour flow cytometry staining using Abs against CD172a, SLA Class-
II and CD80/86. Cells showing high forward scatter (FSC-A) and side scatter (SSC-A) profiles
were gated, followed by the selection of single cells (FSC-W/H and SSC-W/H) and viable cells
(SSC-A/7-AAD). Among these cells, BMDCs were defined as the CD172a+/high cells (SSC-A/
CD172a) expressing SLA Class-II and CD80/86.
S3 Fig. Phenotype of CD172a+/- (intermediate) cell population. Gating strategy of (A)
frhBMDCs and (B) cryoBMDCs following multicolour flow cytometry staining using Abs
against CD172a, SLA Class-II and CD80/86. The CD172a+/- (intermediate) cell population
(SSC-A/CD172a) does not express SLA Class-II and CD80/86 in both frhBMDC and
cryoBMDC cell cultures.
S4 Fig. FrhBMDCs and cryoBMDCs upregulate SLA Class-II in a dose-dependent manner
upon stimulation with LPS. (A) FrhBMDCs and cryoBMDCs (obtained from the same animal,
n = 1) were stimulated with different concentrations of LPS or unstimulated using cell
culture medium (negative control; Ctrl). After 24 hours, the expression (MFI) of the maturation
markers SLA Class-II were measured using Flow Cytometry. The data are shown as the
means ± the standard error of the mean (SEM) of three technical replicates. A one-way
ANOVA with a Dunnett’s post hoc test was performed, comparing multiple groups to the plots of SLA Class-II expression on LPS stimulated frhBMDCs and cryoBMDCs. The contour
plots are based on forward scatter (y-axis) and SLA Class-II expression (x-axis). The highest
concentration of LPS (10 μg/mL) and cell culture medium (negative control; blue) are presented
in this figure.
S5 Fig. SLA Class-II is not upregulated upon stimulation with EcN, β-glucans or LPS.
Immature (A) frhBMDCs and (B) cryoBMDCs (obtained from the same animal) were stimulated
with different concentrations of E. coli Nissle 1917, β-glucans or LPS. Unstimulated cells
are represented by the white bars (negative control; Ctrl). After 24 hours, the upregulation of
SLA Class-II was measured using Flow Cytometry (n = 4 animals). Relative fold change was
calculated by dividing the MFI of stimulated BMDC/MFI of unstimulated BMDC (Ctrl) of
each animal. The data are shown as the means ± the standard error of the mean (SEM) of 4
animals. A one-way ANOVA with a Dunnett’s post hoc test was performed, comparing multiple
groups to the untreated cells (control): = P<0.001, P<0.01 and P<0.05.