Probiotic Modulation of Dendritic Cells and Basophils Impacts T Helper Responses

Probiotic stimulation of co-cultures with bone marrow-derived dendritic cells and
basophils modulate T helper responses
MSc Thesis Biotechnology
Course code: CBI-80436
Author: Chantal Deen
Registration nr: 910203 174 010
Specialisation: Biotechnology Cellular/Molecular
Supervisor: Adriaan van Beek
Examinator: Huub Savelkoul
Wageningen University and Research Centre Department Cell Biology and Immunology
Wageningen, May 2014

3
Abstract
Probioticbacteriaexert beneficial effectsonthe immune system.How probioticsestablishthiseffect
needstobe elucidated.Inthisstudy,in vitro culturesof bone marrow-derived dendriticcells
(BMDCs) and basophils (BMB) were stimulatedwithprobioticsandthe modulatoryeffectbythese
cellsonT helpercellswasanalysedbyflow cytometry. Flow cytometrywasusedtomeasure surface
markers,transcriptionfactorsandcytokines. BMBsstimulatedwithprobioticsledto increased
productionof IL-4, whichis involvedinpolarisationtowardsThelper2(Th2) andmodulationof B cell
responses.BMDCsstimulatedwithprobiotics increasinglyproduce TNF,IFN-γ,IL-6andIL-10 which
are importantinpolarisationof naive Thelpercellstoward Thelper1 (Th1),T helper17 (Th17) and
regulatoryT helper(Treg).Co-cultureswith BMDCsandBMBs probablyenhancedTh2immune
responses. The results suggestthatBMBs up-regulate Th2 responses, whentheywere stimulatedin
the presence of BMDCs and CD4+ T cells. WCFS1treatment inducesstrongerTh1polarisation,
comparedto BL23. BMDC andBMB interactwitheachotherduringco-culture influencingthe
immune response uponstimulation.
Introduction
The human bodycontainsmore microbiota thanithas somaticand germcells [1].Notsurprisingly,
thismicrobiotafulfils several importanttasksinthe humanbody.Forexample,microbiotainthe
humanintestine digestandfermentsubstancesthatthe hostisnot able to digestbyitself [2].These
microbiotaalsotrainthe immune systemandpreventgrowthof harmful pathogens [3].The role of
probioticsinthe intestineistosupportthe functionof the microbiotapresentinthe gutandto
supportthe humangut itself subsequently [4].The mostcommonprobioticsare Lactobacilliand
Bifidobacteria [1, 5]. Probioticsare live organismsthathave a beneficial influence onthe intestinal
epithelial andonthe immune systemuponingestion [2,6].There isevidence thatprobioticshave
anti-inflammatoryactivities,compete withpathogensand musttherefore modulate the immune
system[1,3-6]. To modulate the immune systemof the host,probioticsmustbe able togive signals
to immune cellspresentinthe intestine [7].Signalsderivedfromtheseprobiotics canbe recognized
by cellsof the immune system [7].Antigenpresentingcells(APCs)are able tocapture and present
antigensobtainedfromprobiotics atthe surface of theircell toinitiate andactivate immune
responses [8].Althoughitisclearthatprobioticsprovide signalstoimmune cells,the exact
mechanismshowprobioticsestablishanimmune modulatoryresponse isnotclear.
BMDCs are APCsthat are dedicatedtocapture andpresentantigens [9].Ingeneral,antigensgetinto
contact with BMDCs in the mucosal immune areas,suchas the oral cavity,lungsand intestines [3,5,
10]. BMDCs expressmembranereceptors,suchasToll-likereceptors (TLR),C-typelectinsandNOD-
like receptorsthatbindtothese bacteriaanduse these receptorstocapture and endocytose bacteria
and theirantigens [6, 11, 12]. Afterendocytosis,bacteriaare digestedandbacterial antigensare
presentedbyMHC-classIImoleculesonthe surface of BMDCs [13].Activated BMDCs expresshigh
levelsof MHC-IIco-stimulatormolecules andcytokinestoattractand activate naive T helpercells[7,
14].
Dependingonthe availablecytokinesinthe environment, BMDCsare able to triggeran immune
response orto suppressthisresponse byactivatingcertaintypesof T helpercells[5,8, 14].
Productionof IL-12 by BMDCs induces Th1 differentiation [6].IL-10isdescribedasan inhibitory
cytokine andinducesaTreg response [5,15]. BMDCs can alsopolarise the immuneresponsetoward

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Th17 cellsbyproducingIL-6,IL-1β and TGF-β.[16-19]. BMDCs are the major APCstoinitiate Th1,
Th17 and Treg responses, itishoweverunclearhow BMDCsinduce Th2 responsesas BMDCs donot
produce IL-4 [9, 20].
Basophils are granulocytesandare involvedinallergicresponses.Whentheygetinvolvedinallergic
immune responsestheysecretecytokines,like IL-4andIL-6 that are involvedinTh2skewing [21].
Commensal bacteriainthe gutcan influence developmentof allergicreactions [3,22]. AsBMBs are
involvedinallergicresponses,bacteriainthe gutmayinfluence BMBresponsesandpopulations [21].
Studieshave notonlyshownthatbacteriarespondto BMBs, but that BMBs can respondtobacteria
as well [23].Recentstudies alsoshowedantigenpresentingactivitiesfor BMBs[24-27]. The antigenic
capacitiesof BMBs will be testedbystimulating BMBswithprobiotics.AgainCD4+T cells will be used
as a read out system.
Two differentprobioticstrainswere testedinthisstudy. The firstone is Lactobacillusplantarum
WCFS1 (WCFS1), a lactic acidproducingbacteriaandis usedina large varietyof studies [28-36].
Probioticandimmune enhancingpropertieshave beendescribedforWCFS1inliterature.For
example,astudyshowed treatingmice withWCFS1inducesimmunomodulatoryeffects,suchas
reducedproand anti-inflammatoryresponsesandincreasedsplenicBMDCand T cell frequencies
[30]. Anotherstudyhadshownthat consumptionof WCFS1alterstranscriptionprofilesof immune
cellspresentinthe small intestine[37].Genes thatwere involved inimmunetolerance were
upregulated,whilegenes thatwere involvedin amplifyinginflammatoryresponseswere not
modulated [37].Culturesthatcontained bothWCFS1and enteropathogenslike E.coli or S.enteritidis
ledto inhibitedgrowthof the enteropathogensandsuggestedthatWCFS1hadantimicrobial
capacities [38].L. plantarumWCFS1is usedinthisexperimental approachasareference strain.
The secondprobiotictestedinthisstudyisanotherlacticacidproducingbacteria calledLactobacillus
casei BL23 (BL23). This bacterial strainis investigatedinseveral studies[34,39, 40]. For example,
BL23 can reduce oxidative stressduringinflammatoryresponse andtherefore decreasestissue
damage duringimmune responses[41].AnotherstudydemonstratesthatBL23 influencesthe innate
immunitybyupregulatingCD-206and TLR-2 receptors [42].
Both WCFS1 and BL23 have beenshowntointeractwithTLR-2 receptorsonBMDCs [30, 43].
Interactionwith TLR-2receptors leadstoincreasedcytokine expressionlevelsof IL-1α,IL-6and TNF,
IL-10 andIL-12p70 [17, 29, 30, 42, 44, 45]. These cytokinespromoteactivationandproliferationof
effectorCD4+ T cellstowardsTh1, Th17 and Treg [19, 40, 43]. Basedon thisinformationwe expect
that WCFS1 and BL23 promote activationandpolarisationtowardsTh17,Th1 and Treg. BMBs
however, are stronginducersof IL-4and IL-6 productionandcell culturesdone withBMBs will
probably upregulate Th2responses [46,47].
The characteristicsof BMDCs andBMBs as APCs will be usedinthisprojecttoinvestigate the effect
of probioticsonimmune cells. Maturationof BMDCsand stimulationof BMBs and subsequent
polarizationof differenttypesof T-helper(Th) cellsbyBMDCs isassessed.

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Materialsandmethods
Mice
NMRI mice, both female and male in the age of 5-26 weeks were obtained from the animal facility in
Wageningen (NL). Male C57Bl/6Rjj mice between 8-12 weeks were obtained from Janvier labs. All
mice were housed in the animal facility in Wageningen (NL). All mice used during these experiments
were kept under specific pathogen-free conditions and according to federal Dutch guidelines. Bone
marrow (BM) from femur,tibiaandpelvis,andthe spleenwerecollectedandprocessed.
Probioticcultivation
L. plantarum WCFS1 and L. casei BL23 were obtained from glycerol stocks and transferred to MRS
growth medium and grown overnight at 37°C and 5% CO2, in aerobic conditions. Bacteria were re-
inoculated the next day and grown at 37°C overnight again. CFU were determined by OD600 values,
using a spectrophotometer (U-1500 1390-05 Hitachi). The bacteria were washed twice and diluted
withPBSbefore addedtothe BM cell cultures.
BM isolation
Bones were flushed with sterile RPMI-1640 Glutamax, 25 mM HEPES (RPMI medium) and passed
through a 40 µm cell strainer (BD Falcon) to obtain a single cell suspension. Single cell suspensions
were frozenin90% FCS and10% DMSO and storedinliquidnitrogenuntilusage.
Bonemarrow-derivedBMDC culture
Culture medium contained RPMI-1640 Glutamax, 25 mM HEPES, 10% (vol/vol) heat inactivated FCS,
1% (vol/vol) penicillin/streptomycin (Sigma), 50 µM β-mercaptoethanol, 0.2% (vol/vol) Normocin
(Invivogen) and 20 ng/ml GM-CSF (BioLegend). 2.5*105
cells/ml were cultured in culture medium for
7 days at 37°C and 5% CO2. The cells were stimulated with LPS (100 ng/ml, E. coli 055:B5, Sigma-
Aldrich) orwith5*105
CFU for3 hoursor 20 hours.
Bonemarrow-derivedbasophilculture
Bone marrow cells were cultured in BMB culture medium containing RPMI-1640 Glutamax, 25 mM
HEPES, 10% (vol/vol) heat inactivated FCS, 1% (vol/vol) penicillin/streptomycin (Sigma), 1mMsodium
pyruvate, 50 µMβ-mercaptoethanol, 0.2%(vol/vol) Normocin (Invivogen) and 2 ng/ml IL-3. At day 11-
14, BMBs were purified and separated with the IMagnet System (BD Biosciences) according to
manufacturer instructions. Negative selection was performed for CD11c and CD117 and positive
selection for FcɛRIα. Purity of BMBs was at least 98%. BMBs were stimulated with IL-18 (50 ng/ml)
withIL-33 (100 ng/ml) as positive control orwithprobioticsfor20 hours.
Co-cultureof BMDCsandCD4+T cells
Spleen cells were enriched for CD4+ T cells with the Imagnet (BD Biosciences). Negative selection for
CD11b and positive selectionfor CD4 were performed. Purity of CD4+ T cells was around 80%, within
particular B cells still present. Anti-CD3 (1 µg/ml) and CD4+ T cellswere added to the plate containing
BMDCs in a 10:1 ratio. The cells were cultured in medium containing 1 ml DMEM, 10% (vol/vol) FCS,
1% (vol/vol) Pen/Strep, 50 µM β-mercaptoethanol and 0.2% (vol/vol) Normocin (Invivogen). The
generationof Th1,Th2, Th17 andTreg cellswasdeterminedafter4days.
BMB andBMDC co-culture
A ratio of 1:1 BMDCs and BMBs were added to wells in a plate. Culture medium consisted of RPMI-
1640 Glutamax, 25 mM HEPES, 10% (vol/vol) heat inactivated FCS, 1% (vol/vol)

6
penicillin/streptomycin (Sigma), 1mM sodium pyruvate, 50 µM β-mercaptoethanol, 0.2% (vol/vol)
Normocin (Invivogen), and 2 ng/ml IL-3. After overnight stimulation, cells were co-cultured with CD4+
T cellsat a ratio of 1:10.
Cytokinebeadassay
Cytokine levels in supernatants of BMDC cultures were measured with the CBA Mouse Inflammation
Kit (BD Biosciences), according to the manufacturer’s instructions. Cytokine levels in supernatants of
BMBs andCD4+T cellswere measuredwith the CBA Mouse Th1/Th2/Th17 Kit(BD Biosciences).
FACS analysis
Before incubation with mAbs, cells were blocked with anti-CD16/CD32 (2.4G2) in FACS buffer (PBS
containing 0.25% (vol/vol) BSA, 0.5mM EDTA, 10% FCS). The following mAbs were purchased from
BD Pharmingen: CD3e-APC-Cy7 (145-2C11), CD4-APC-Cy7 (H129.19), CD4-APC (RM4-5), CD4-biotin
(GK1.5), CD8a-PE (53-6.7), CD11b-APC-Cy7 (M1/70), CD11b-BV510 (M1/70), CD11b-BV421 (M1/70),
CD11b-FITC (M1/70), CD16/CD32-FITC (2.4G2), CD44-PerCP-Cy5.5 (IM7), CD45R/B220-FITC (RA3-
6B2), CD62L-APC-Cy7 (MEL-14), CD86-PE-Cy7 (GL1), CD117-biotin (2B8), CD117-PerCP-Cy5.5 (2B8),
CD123-biotin(5B11), FoxP3-AlexaFluor647(MF23), GATA3-PE (L50-823), T-bet-BV421(O4-46).
From eBioscience, the following mAbs were obtained: CD11b-biotin (M1/70), CD11c-PE-Cy7 (N418),
CD19-FITC (1D3), CD86-APC (GL1), CD115-PE (AFS98), FcεRIα-FITC (MAR-1), FcεRIα-biotin (MAR-1),
MHC-II-FITC (M5/114.15.2), MHC-II-APC (M5/114.15), NK1.1-FITC (PK136), Ki-67-PE-Cy7 (SolA15),
streptavidin-APC-Efluor780, streptavidin-FITC, streptavidin-PerCP-Cy5.5, streptavidin-APC and
streptavidin-BV421were obtained.
Dead cell staining was done with 7-AAD (BD Pharmingen) or with Aqua live dead-Efluor506
(eBioscience). Samples were acquired on a FACS Canto II (BD) and analysed with FlowJo vX.0.7
software (Treestar).
Statistical analysis
Student’s t test or two-way analysis of variance was used to determine significant differences
between conditions tested. In figures, p-values are noted as follows: *P<0.05, **P<0.01 and
***P<0.001.

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Results
Stimulationof BMBsdidnotleadto upregulation ofsurfacemolecules CD11b andMHC-II
BMBs were stimulated with IL-18, IL-33 and probiotics for 20 hours in vitro. Afterwards was FACS
analysis performed on surface markers CD11b, CD11c, CD62L, CD86, CD117, FcεRIα and MHC-II.
Examples of the gating can be found in supplementary information I. Figure 1 summarizes the data of
the stimulatedBMB.
Figure 1. BMBs stimulated with IL18, IL-33, WCFS1 and BL23. Values were based on average values (+/- SEM) and ten
samples. The experiment was performed twice.
The figure above shows the mean fluorescence intensity of different surface markers. No
upregulation of surface markers MHC-II and CD62L was seen for probiotic stimulation with BMBs.
Expression of CD11b was downregulated for the probiotics compared to the negative control. MHC-II
expression was not affected by probiotic stimulation. CD86 and CD62L expression was upregulatedin
bothprobioticstimulation,butforWCFS1 thisincrease ishighercomparedtoBL23.
BMDCsstimulatedwithprobiotics doesnotleadto maturationofDCs
BMDCs were stimulated with probiotics for 20h in vitro. FACS analysis was performed for CD11b,
CD11c, CD62L, CD86, CD117, FcεRIα and MHC-II. Gating was performed as shown in the
supplementary information I. The data is summarized in Figure 2 and shows the percentage of cells
that were CD86+
MHC-II+
(mature BMDCs), CD86-
MHC-II-
(precursor) and CD86int
MHC-IIint
(immature
BMDCs).

8
Figure 2. BMDCs were stimulated with 105 CFU BL23 or WCFS1 for 20 hours. Graph represents average percentages (+/-
SEM) of each condition tested (n=10). The results are derived from two independent experiments. Figure 2A shows %
mature BMDCs, Figure 2B shows % cells of immature BMDCs and figure 2C shows % cells of precursors. *P<0.05,
***P<0.001.
Compared to the medium control, probiotic stimulation led to significantly decreased maturation of
BMDCs. The results suggest that maturation of BMDCs was downregulated by the probiotic
stimulation with WCFS1 and BL23. Literature does confirm that mice treated with WCFS1 did not
activate BMDCs in vivo [30]. Other articles describe that BL23 induces production of IL-10 Weiss,
2010 #90}[48]. Therefore T regulatory immune responses were upregulated upon stimulation and
prevent maturation of BMDCs [49]. In summary, probiotic stimulation of BMDCs suppresses
activationand inhibits upregulationof co-stimulatory molecules of BMDCs.
Probiotic stimulation of co-cultures does affect cell surface markers of both BMDCs and
BMBs
BMDCs and BMBs togetherina cell culture mayenhance the stimulatoryeffect of BMDCs.BMDCs
and BMBs were stimulated withprobiotics togetherfor20 hours in vitro. FACSanalysisperformedon
CD11b, CD11c, CD62L, CD86, CD117, FcεRIα and MHC-II.Gating isshowninthe supplementary
information I.Figure 3showsthe meanfluorescence intensity(MFI) of FcεRIα+
CD11c-
cells.
FcεRIα+
CD11c-
cellswere identifiedasBMBsand FcεRIα-
CD11c+
cellswere identifiedas(im)mature
BMDCs. FcεRIα-
CD11c+
cellswere giveninfigure 4.

9
Figure 3. The figure above shows mean fluorescent intensity (MFI). The experiment was performed twice and each
experiment contained five replicates. The standard deviation was based on the average values of all samples (+/- SEM).
**P<0.01, ***P<0.001.
Figure 1 showsthe MFI valuesof stimulatedculturesof BMBs.Ingeneral the MFI valuesare lowerof
culturesonlystimulatedwith BMBscomparedwiththe co-cultures.MHC-IIvaluesof BMBsduringco-
culturesare significant higherwhentheyare stimulatedwithprobiotics. Ingeneral, co-cultures
performedwith BMBsand DCs showsignificantdifferencesbetweensome conditionsandthiswas
not observedwiththe BMBcultures. All MFIvaluesof the surface markers show an increase in
surface expressionuponprobioticstimulation.
FcεRIα-
CD11c+
cellswere gatedformature and immature BMDCs andprecursorcells. Gatingcan be
foundinsupplementaryinformationI. Foreachpopulationwasthe MFIof CD11c, CD86 andMHC-II
calculatedandthese graphswere givenbelowinfigure 4.
Figure 4. MFI values of FcεRIα-CD11c+ cells for mature and immature BMDCs. The experiment was performed twice. The
standard deviation (+/- SEM) was based on the average values of ten samples.Figure A shows MFI values of CD86. Figure
B shows MFI values of MHC-II. Figure C shows MFI values of CD11c.
MFI of CD86 of the mature DC population slightlyincreaseduponprobioticstimulation. Nosignificant
differenceswere foundforMFIvaluesof CD86 and MHC-II. The figure below showsMFIvaluesof
DCs stimulatedwithprobioticsfor20 hours.

10
Figure 3. MFI values of CD86, MHC-II and CD11c of BMDCs. BMDCs were separated in three different subpopulations;
mature, immature and precursor. The graph shows average values and the standard deviation (n=10). Figure A shows
MFI values of the co-culture with BMDCs and BMBs. Figure B shows the MFI values of BMDCs.
The expressionof CD86of mature BMDCs infigure 5A is higherinthe controlscomparedto the
probioticstimulated BMDCs.Probioticstimulationdidnotactivate maturationof BMDCs(Fig. 5A).
In summary,probioticsthatstimulatedco-culturesof BMDCsand BMBs induces increasedactivation
of BMBs comparedto the culture containingonly BMDCsor BMBs. Surface markersexpressionof
CD86, CD62L and MHC-IIincreaseduponstimulation.
For BMDCs in general the MFIvalueswere higherinthe co-culture conditioncomparedtothe BMDCs
culture.Maturationof BMDCs duringco-cultureswere upregulated,boththe resultswere not
significant.The resultsdosuggestthat BMB andDCs influence eachotherimmuneresponses.
Cytokine measurementmayreveal more of the mechanismsbehindthe immune response.
ProbioticBMDC stimulationleadsto anincreaseofIL-12, TNF andIL-6 production
BMDCs were stimulated with WCFS1 and BL23 and cytokines were measured in the supernatant of
the culture medium. All cytokines that were measured were given in supplementary information II.
Table 1 belowshowsthe average cytokine valuesmeasuredinpg/ml.
Table 1. Cytokines measured in the supernatant of BMDC cultures. Cytokines were measured in pg/ml. BMDCs were
stimulated with WCFS1 and BL23 for 20 hours. The table displayed average values and were based on 10 samples.
Experiment was performed twice. ND is not detectable.
BMDCs
IL-12p70
(pg/ml)
TNF
(pg/ml)
IFN-γ
(pg/ml)
MCP-1
(pg/ml)
IL-10
(pg/ml)
IL-6
(pg/ml)
- 0.2 22.5 ND 110.8 1.4 0.5
LPS 0.7 2098.8 ND 160.7 1 3822.3
WCFS1 ND 64.5 ND 85.1 ND 7.6
BL23 0.6 40.6 ND 85 2.2 2.2
The table shows that BMDCs stimulated with WCFS1 and with BL23 induces production of TNF, IL-6
and MCP-1. BL23 also produces IL-10. Most cytokines levels were increased compared with the
negative control.

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Cytokines produced during BMB stimulation show an increase in TNF, IL-6 and IL-4
production
BMBs were stimulated with probiotics and cytokines were measured to validate the immune
response. The average valuesof everycytokinemeasuredinpg/ml wasgivenintable 2.
Table 2. Cytokines measured in the supernatant of cell cultures with BMBs. BMBs were stimulated for 20 hours with
probiotics. Cytokines were measured in pg/ml. Values in the table were based on averages and on ten samples. The
experiment was performed twice. ND is not detectable.
BMB
IL-10
(pg/ml)
IL-17
(pg/ml)
TNF
(pg/ml)
IFN-γ
(pg/ml)
IL-6
(pg/ml)
IL-4
(pg/ml)
IL-2
(pg/ml)
- ND 0.2 ND ND 4.9 3.8 0.5
IL-18+IL-33 0.8 0.2 ND ND 22 1.9 0.5
WCFS1 1.8 0.2 0.9 ND 5.9 4.3 0.4
BL23 ND 0.2 0.3 ND 7.1 3.4 0.4
For WCFS1 were the cytokine values of IL-10, TNF and IL-4 higher compared to BL23. For BMBs
stimulatedwithBL23were cytokineslevelsof IL-6higher.
Probiotic stimulation of co-cultures leads to increase production of cytokine involved in
inflammatoryresponses
For co-cultures with BMDC and BMBs were also cytokines measured. Co-cultures of BMBs and
BMDCs were performed in vitro and stimulated for 20 hours with WCFS1 or with BL23. The cytokines
measuredwere summarizedintable 3.
Table 3. Co-cultures of BMDCs and BMBs were stimulated with probiotics. Supernatant was analysed for inflammatory
cytokines and cytokines were measured in pg/ml. The table shows average values based on ten samples. The experiment
was performed twice.
BMDCs and
BMB
IL-12p70
(pg/ml)
TNF
(pg/ml)
IFN-γ
(pg/ml)
MCP-1
(pg/ml)
IL-10
(pg/ml)
IL-6
(pg/ml)
IL-17
(pg/ml)
IL-4
(pg/ml)
IL-2
(pg/ml)
- ND 14.8 ND 85.3 2 5.5 0.2 0.2 0.4
LPS/IL-18+IL-
33 ND 632.9 0.1 117.4 1.7 1766.8 0.9 1.7 0.3
WCFS1 0.8 94.7 0.4 102.4 2.1 15.1 0.4 ND 0.1
BL23 1.3 36 0.1 97.4 3.3 8.5 0.2 0.2 0.2
Co-cultures that were stimulated with probiotics show in both cases production of IL-12, TNF, MCP-1,
IL-10 and IL-6. WCFS1 had higher cytokine levels of TNF, MCP-1 and IL-6 compared to BL23. BL23 had
highervalues measuredforcytokinesIL-12andIL-10 comparedtoWCFS1.
In summary, TNF values measured in both probiotics conditions tested showed an increase in
production compared to the negative control. This is shown in all tables(Table 1-4). This was also the
case for MCP-1 and IL-6. These cytokines are involved in pro-inflammatory immune response
towards Th1 and Th17 [19, 40, 43]. The results suggest that the probiotics regulate the pro-
inflammatory immune response,butregulatoryimmune response was notinducedeither.
Co-cultureexperimentofstimulated BMDCs,BMBsandCD4+Tcells
BMDCs and BMBs were stimulated in vitro for 3h and co-cultured with CD4+ T cells for 4 days. After 3
hours was FACS analysis performed on the surface markers of BMDCs and BMBs. No differences in
up- or down regulation of surface markers of BMDCs and BMBs were found. After 4 days of co-

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culture with CD4+ T cells was FACS analysis performed on extra- and intracellular markers of these T
cells. Gating was performed to determine the amount of FoxP3, T-bet and GATA3 positive cells. The
gating can be found in the supplementary information I. Proliferation of CD4+ T cells were measured
withKi-67and cell viabilitywasmeasuredwithaqualive-deadstaining.
CD4+ T cells were purified with magnetic cell sorting, but still contained cells that were not CD4
positive. Therefore, the percentage of CD4- cells was determined. The percentage of CD19+ cells (B
cells) varied between 15 and 25%. These cells had proliferated and had high Ki-67 values at the time
of measurement. Thispopulationhastobe keptinmindand mayhave influencedourresults.
The CD4+ T cells were gated for FoxP3, T-bet and GATA3 positive populations. Figure 11 shows a bar
graph, showing the quantity of positive values of FoxP3, T-bet and GATA3 cells after 4 days of co-
culture. FoxP3 values were measured to determine the polarization towards regulatory T cells, T-bet
positive cells were measured to determine polarization towards Th1 cells and GATA3 was measured
to determine polarizationtowards Th2cells.
Figure 4. Percentage of cells those were positive for expression markers of CD4+ T cells. CD4+ T cells were cultured for 4
days with BMDCs. Average values ( +/- SEM) were based on ten sample and given in the graph. Figure A shows
percentage of cells positive for FoxP3. Figure B shows percentage of cells positive for GATA3. Figure C shows percentage
of cells positive for T-bet.
Probioticstimulationof BMDCsupregulate FoxP3expression,while GATA3expressionwas
downregulated.
Figure 5. Percentage of cells those were positive for expression markers of CD4+ T cells. CD4+ T cells were cultured for 4
days with BMBs. Average values ( +/- SEM) were based on ten sample and given in the graph. Figure A shows percentage
of cells positive for FoxP3. Figure B shows percentage of cells positive for GATA3. Figure C shows percentage of cells
positive for T-bet.

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Probioticstimulationof BMBsand CD4+ T cellsleadstoupregulationof GATA3.FoxP3andT-bet
expressionwasupregulatedfor BMBsstimulatedwithBL23,but downregulatedforWCFS1.
Figure 6. FoxP3, GATA3 and T-bet measurements of stimulated CD4+ T cells. BMDCs and BMBs were stimulated for 3
hours, CD4+ T cells were added and 4 days later analysis was performed. The experiment was only performed once. The
bars in the graph show average values (+/- SEM) based on three samples. Figure A shows percentage of cells positive for
FoxP3. Figure B shows percentage of cells positive for GATA3. Figure C shows percentage of cells positive for T-bet.
In figure 8B, probiotic stimulation does decrease GATA3 compared to the other conditions tested.
WCFS1 increases expression of T-bet, but BL23 decreases T-bet expression. Expression of FoxP3 was
increased forbothprobiotics.
In summary, BMBs increase GATA3 expression and BMDCs increase FoxP3 expression of CD4+ T cells
upon probiotic stimulation. Remarkable is the difference in T-bet expression between the two
probiotics we tested. T-bet expression was higher for BMDCs stimulated with WCFS1 compared to
BL23 stimulation, butfor BMBs this seemed to be the opposite. Although for BMBs the deviation was
larger.
Discussion
The purpose of this research was to investigate the effect of probiotics on immune cells. Cell cultures
of BMDCs and BMBs were cultured under in vitro conditions and stimulated with L. plantarum WCFS1
and L. casei BL23. CD4+ T cells were used as a read out system to investigate polarisation of the
immune response bystimulated BMDCsandBMBs.
BMDCsstimulatedinduceastrongerimmuneresponsewithWCFS1comparedto BL23
BMDCs that were stimulated with probiotics induce immune responses towards Th1, Th17 and Treg.
For WCFS1 this was clearer than for BL23. The cytokine levels of TNF and IL-6 were measured in
higher levels and T-bet expression was upregulated for WCFS1, but not for BL23. We found that BL23
induces IL-10 production. Studies were performed earlier whereby BMDCs were stimulated with
BL23 and WCFS1 [29, 45, 50]. These studies found that BL23 can induce high levels of IL-10 and can
induce regulatory immune responses [5, 29, 40]. However, one of the studies was performed in vitro
with immune cells obtained from human blood, but the results were similar [29]. WCFS1 can induce
inflammatory responses towards Th1 and Th17 by production of TNF and IL-12 in vitro [30, 37, 51].
The studies also consist of in vivo experiments in mice and human and these studies suggested that
lactobacillus induce anti-inflammatory responses [30, 37]. For WCFS1 the results in relation to
regulatory immune responses were contradictory as FoxP3 expression was observed during the
BMDC and CD4+ T cell co-culture, but IL-10 was not detected during cytokine measurements of the
BMDC culture. Maybe IL-10 production needs more time before it is released in the supernatant. For

14
future experiments we can try to detect IL-10 intracellular, or measure at a later time point (after 24
hoursof stimulationforexample).
BMBinduceTh2responsesafterBL23andWCFS1stimulation
Probiotic stimulation was also performed on BMBs and on co-cultures with BMBs and CD4+ T cells.
BMBs induced Th2 responses after BL23 and WCFS1 stimulation. BMBs are known to induce Th2
responses upon stimulation, because BMBs are efficient producers of IL-4 [21, 46, 47]. However we
found different IL-4 levels in our different probiotic conditions tested. Previous studies shown that
the ratio of BMDCs and BMBs influence the quantity of IL-4 produced, but this does not explain why
we measure different IL-4 levels for our different probiotics [21]. Apart from the IL-4 production it
seems that the stimulation of BMBs with BL23 induces Th1 and Th17 polarization, while this was not
the case for WCFS1. In general, our measurements make it difficult to distinguish if the immune
response was polarised towards Th1 or towards Th17. IL-17A was not detected and suggest the
immune response was not towards Th17, but for future experiments expression of RORγt can be
measured withflowcytometry tohave more reliability.
In case of surface marker expression, basophils have been shown to express MHC-II in both in vivo
and in vitro settings in studies. Both studies mention that MHC-II expression of basophils respond to
th2 immune responses [22, 52]. This does not explain why MHC-II expression was not affected in our
culture. However the other surface markers were upregulated, suggesting that BMBs do respond
uponprobioticstimulation.
BMBs stimulated with BL23 induce Treg responses, but IL-10 was not measured for this condition.
The lack of IL-10 production is contradictory with the high expression of FoxP3 we found when BMBs
were co-cultured with CD4+ T cells and stimulated with BL23. One study were human PBMCs where
basophils activated and stimulated with L.casei [53]. They found that L.casei inhibits activation of
basophils in an IgE dependent way [53]. This may explain the lower levels of cytokines we measured
for BL23 and the upregulation of FoxP3, but not the upregulation of GATA3 and T-bet. However, in
the experimenttheydidnotperformco-cultureswithT-cells.
Most of the studies in which BMBs were stimulated with probiotics concluded that the production of
cytokines was regulated by the MyD88-dependent pathway [23, 46, 54]. This signalling pathway plays
an important role for induction of T cell proliferation [55]. MyD88 is also needed for TLR signalling
and TLR receptors are often involved in recognizing antigens, like bacterial compounds [11]. The
signallingbetweenprobiotics, BMDCsand BMBs is likelytobe influencedbythispathway.
BMBsenhanceCD4+T cell responsestowardTh2and BMDCsenhancetheimmune
responsetowardTh1andTh17uponprobioticstimulation
Co-culturesdone with BMDCs, BMBs andCD4+ T cellswere stimulatedwithprobioticsandanalysed.
Co-cultureswith BMBsskew CD4+ T cell towardsTh2. Treatmentwith WCFS1increasesthe Th1
polarisation,whileBL23 treatmentdecreasesthe skewingtowards Th1.Studiesthatwere performed
earlierfound thatwhenBMBs were co-culturedwithBMDCsandCD4+ T cells, BMBs induce Th2
differentiation,buttheyare alsoable to suppressTh1differentiation [21,56]. One of the studies
illustratesthatBMBsinfluence CD4+T cellsandsplenicDCsby supportingTh2immune responses.
However,thisstudyobtainedbasophilsbyimplantingmice withIL-3andharvestcellsfrombone
marrow andliver.CD4+ T cell were obtainedfromthe lymphnodes andDCsfromthe spleen [21].

15
BMDCs and BMBs influence eachotherwhentheyare culturedtogether.Surface expressionof BMB
were notaltereduponprobioticstimulation.ButwhencoculturedwithDCsthe surface expression of
BMBs increases.ThisincreaseishigherforWCFS1comparedto BL23 and suggesta strongerimmune
response towardsWCFS1,butit also suggestthatDCs enhance the immune responseof BMBs.
Duringcoculture were cytokinesMCP-1,IL-10and IL-17a upregulatedwhileIL-4andIL-2 were
downregulatedinthe co-culturescomparedtocell culturesthatonlycontainedBMDCsor BMBs. But
additionof CD4+ T cellsenhances GATA3expression inthe co-culture.Expressionof FoxP3andT-bet
was generallylowerinthe co-culture comparedtothe BMDCs and BMB culture.Ourresultssuggest
that BMDCs induce inflammatoryresponsestowardsTh1and Th17 andBMB induce immune
responsestowardsTh2.WCFS1 inducesastrongerT-betexpressionandthusmayinduce stronger
inflammatoryresponsescomparedtoBL23.
The results mightindicate thatBMBs and DCs communicate witheachother.Surface markersof
BMBs were upregulatedunderinfluence of DCsincombinationwithprobiotics.FoxP3andTbet
expressionwere downregulatedduringcoculturesunderinfluence of BMBs,while GATA-3expression
was upregulated.We hypothesisedthat BMBsupregulate Th2polarisation andthis isinline withour
results.We alsohypothesizedthatprobioticstimulationof BMDCswouldleadtoTh1, Th17 and Treg
polarisation. Thisisthe case forBMDCs cell cultures,butco-cultureswithbothBMDCsand BMBs this
isnot the case.
Future recommendations
Finally, we have several recommendations for the future. First, for all experiments we performed,
the T cell responses were not very strong. This can be caused by the fact the T cells are part of the
adaptive immune system. No immunization was done for our immune cells, so T memory cells were
not developed. In some experiments T cells were restimulated to induce a stronger CD4+ T cells
response [57].
Second, the cell amounts we used were not tested beforehand. The cell amounts and ratios could be
tested to obtain optimal immune responses between BMDCs or BMBs and CD4+ T cells. Several
studies mentioned that cell ratios may influence cell viability and an efficient immune response [21,
57].
Another future recommendation is to test more probiotics since we only tested two different
probiotic strains, which are both gram-positive and aerobes. The majority of the microbiota in the
human intestine and probiotics are anaerobes [1, 3]. To get a better understanding of the
mechanisms of probiotics on immune cells we recommend testing probiotics that are gram negative
and/or anaerobe,like Akkermansia muciniphila andBifidobacteriumbreve.
Also, we only tested the effect of probiotics on co-cultures of CD4+ T cells, but BMDCs respond to
more types of immune cells, like CD8+ T cells and B cells. For example B cells can be used to
investigate what antibodieswill be produced (or not) upon stimulation with probiotics. And last, BMB
responses on CD8+ T cells can be tested in future experiments as BMBs may be able to act as APCs
for CD8+ T cells [56].BMBs may alsoplaya role ingenerationof FoxP3Treg cellsandB cells [57, 58].

16
Acknowledgements
I wouldlike tothank AdriaanvanBeekforsupervisingme and Joanne Hoogerlandforhersupport
and forthe time we spenttogetherinandoutside the lab.Ialsowouldlike tothankBenMeijerfor
hishelpforthe flow cytometer.ThankstoHuub SavelkoulandEdwinTijhaarfortheir advice during
my projectandeverybody presentatthe Wednesdaymorningmeetings.
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20
Supplementaryinformation I-Gating
GatingofBMDC cultures
The figure belowshowsgatingperformedonall FACSdatacontaining BMDCs.Gating wasperformed
inthe followingorder:Lymphocytesandsinglets.Singletswere gatedforlivinganddeadcellsandfor
CD86+MHC-II+ cells.
Figure 7. Example how Gating of BMDC cultures was performed is showed above. Mature BMDCs were identified as
CD86+MHC-II+, immature BMDCs as intermediate CD86 MHC-II and precursor BMDCs were identified as CD86-MHC-II-.
GatingofBMBcultures
The figure belowshowsgatingperformedonall FACSdatacontainingBMBs.On all sampleswas
gatingperformedinthe followingorder:Lymphocytes,Singlets,CD117- 7-AAD- cells,FcεRIα+cells.
Singletswere notshown,butsame wasdone asshownforgating of BMDCs.
Figure 8. FACS profiles and gating performed on BMB cells. Figure above functions as an example. Gating performed for
singlets was not shown, but was similaras shown in figure 1. BMB cells were identified as FceRIa positive.
Gatingofco-culturesof BMDC andBMB
Gating of co-culturescontainingboth BMDCsandBMBs was done asshownin the figure below.
Everysample wasgatedfor lymphocytesandsinglets.Singletsweregatedforlivinganddeadcells
and forFcεRIα+CD11c+ cells.FcεRIα positive cellswere identified asBMBs, CD11c positive cellswere
identifiedas BMDCs.CD11c positive cellswere gatedformature BMDCs,immature BMDCs and
precursorcells.

21
Figure 9. Example of gating of co-cultures containing BMDCs and BMBs.Gating was performed in the following order;
lymphocytes and singlets.Singlets were gated for living and dead cells, for FceRIa positive and CD11c positive cells.
GatingofCD4+Tcells
Gating of the stimulatedCD4+T cellsisshowninthe figure below.All sampleswere gatedfor
lymphocytes,andsinglets.SingletsweregatedforlivinganddeadcellsandforCD4+CD19+ cells.
CD4+ cellswere gatedforT-bet+FoxP3+andGATA3+ cells.Ki-67wasusedto determine the amount
of proliferatingcellsandforusedaftergatingforliving/deadcells,forCD4+ cellsandfor CD19+ cells.
Figure 10. FACS profiles and gating performed on CD4+ T cells. All samples were gated for Lymphocytes, singlets,
live/dead and CD4 positive cells. Remained population were gated separately for T-bet, GATA3 and FoxP3 positive cells.
Ki-67 gating was performed for live/dead cells and for CD4 positive and CD19 positive cells.

22
SupplementaryinformationII-Cytokinemeasurements
SupernatantfromBMDCsandBMBs20hours
Co-cultures with BMDCs and BMBs that were stimulated for 20 hours were measured. Also cultures
with CD4+T cells were measured. The first set of figures represent BMBs stimulated for 20 hours with
probiotics.
Figure 11. Cytokine levels of IL-2 measured in the
supernatant of BMB cell cultures. Average values and
the standard deviation is shown in the graph. Average
values were based on ten samples and the experiment
Figure 13. Cytokine levels of IL-17A measured in the

CytokinemeasurementsBMDCs
Cytokine were also measured for the cell cultures containing BMDCs. The second set of figures shows
cytokineslevels(pg/ml) measuredinthe supernatantof BMDCs.
Figure 15. Cytokine levels of TNF measured in
supernatant of BMDC cell cultures. The average values
and the standard deviation was shown in the figure.
This value was based on ten samples obtained in from
two experiments.
Figure 16. Cytokine levels of MCP-1 measured in
two experiments.
Figure 17.Cytokine levels of IL-10 measured in
two experiments.
Figure 18.Cytokine levels of IL-6 measured in
two experiments.

24
Supernatantofco-culturesofBMDCsandBMBs
Co-cultures of BMDCs and BMBs were stimulated for 20 hours. Supernatant was collected to
measure cytokine levels. The next set of figures represents the results of the cytokines measured.
Figure 19. Cytokines levels measured of TNF in the
supernatant from cell cultures of BMDCs and BMBs.
Each values measured was given in the figure as well as
the average values and the standard deviation.
Figure 20.Cytokines levels measured of MCP-1 in the
Figure 21. Cytokines levels measured of IL-10 in the

25
Figure 23.Cytokines levels measured of IL-17A in the
Figure 24.Cytokines levels measured of IL-10 in the
Figure 25.Cytokines levels measured of IL-4 in the

26
SupernatantofstimulatedCD4+T cells
Co-cultures of BMBs, BMDCs and CD4+ T cells were performed. Surface markers were measured but
alsocytokines.Below are all cytokinesmeasuredwithTcell polarizationcontrol experiment.
Figure 27. Cytokine levels of IL-10 were measured with
the mouse Th1/Th2/Th17 Kit. CD4+ Tcells were
stimulated four days with interleukins. Two samples
were measured per condition tested and the
experiment was performed only once.
Figure 28. Levels of IFN-γ were measured with the
mouse Th1/Th2/Th17 Kit. CD4+ Tcells were stimulated
four days with interleukins. The figure shows the
cytokine level of every sample that was measured
(N=2).
Figure 29. IL-17A cytokine levels were measured with
stimulated four days with interleukins. The figure
shows the cytokine level of every sample that was
measured (N=2).
Figure 30. Levels of IL-2 were measured with mouse
Th1/Th2/Th17 Kit CD4+ Tcells were stimulated for four
days with interleukins. The figure shows the cytokine
level of every sample that was measured. Figure shows
the cytokine level of every sample measured (N=2).
Figure 31. IL-4 cytokine levels were measured with the
mouse Th1/Th2/Th17 Kit. CD4+ Tcells were stimulated
four days with interleukins. The figure shows the

27
cytokine level of every sample that was measured
(N=2).
Figure 32. Cytokine levels of IL-6 were measured with
measured (N=2).
Figure 33. Cytokine levels of TNF were measured with
measured (N=2).

Supplementaryinformation III-Cryopreservation
Theeffect ofcryopreservationondifferentiationandmaturationof BMDCs
For practical reasons bone marrow cells obtained from mice were frozen in cryovials. Fresh bone
marrow (fBM) and cryopreserved bone marrow (cBM) cells were cultured with GM-CSF and
stimulated with LPS and after 8 days the development of the different BMDC populations was
analyzed by flow cytometry. Examples of the gating performed on FACS profiles are shown in figure
7. Every sample was gated for singlets (FCS-A vs FCS-H) before gating the different BMDC populations
inthe figure.
Figure 42A shows the cell amounts of the mature, immature and precursor BMDC populations of
NMRI mice. Figure 42B shows the percentages of the different BMDC populations of Black 6 mice. In
both figures stimulated fBM shows a decrease in the percentage of precursor BMDCs, compared to
the unstimulated fBM. Stimulated fBM also shows an increase in the percentage of immature
BMDCs, compared to the unstimulated fBM. All stimulated BM cells were able to form mature
BMDCs, but only for Black6 fBM the mature BMDC population increases significantly. Another
remarkable difference between cBM from both mice strains was the percentages of precursor
BMDCs. In NMRI mice thispercentage ismuchhighercomparedtoBlack 6.
Figure 34. BMDCs derived from freshly isolated BM and from cryopreserved BM. NMRI and B6 mice strains were tested.
BMDC populations were measured with FACS and populations were gated for singlets and gating of precursor, immature
and mature BMDCs as shown in figure 1. The different BMDC populations in the graph represent the average values
(n=3). The experiment was performed once. A) BM isolated freshly from C57Bl/6Rjj mice and cryopreserved at liquid
nitrogen. B) BMisolated freshly from NMRI mice and cryopreserved at liquid nitrogen.
Freezingofbonemarrowleadsto increasedpopulationofprecursorcells
When comparing both mice strains it was remarkable that cBM cell cultures induce larger mature
BMDC populations upon stimulation compared to the fBM. fBMcontains a larger variety of cell types
compared to the cBM. Freezing of cellular tissue affect cells that are not robust, either by ice crystals
formed or by using DMSO [50, 59]. In case of bone marrow, hematopoietic stem cells are the most
robust cells, while cells like granulocytes are not and will eventually die upon freezing [60]. All our
BMDC cultures did start with the same amount of cells per well. In the cBMthe portion of stem cells
that will develop into BMDCs was larger compared to the freshly harvested cell tissues and this did
leadto a largermature BMDC population.

29
Another remarkable result was the quantity of precursor BMDCs between the different mice strains.
One of the explanations could be that our experiment was performed once, with only three
replicates. Also the age between the two mice differs; Black6 mice were 8-12 weeks and NMRI mice
were 26 weeks old. But there is not a clear explanation for this finding, literature we found did not
underpinourfindings.
Most importantly, bone marrow cells stored in cryovials were able to form mature BMDCs. F.M.
Marim [59] used bone-marrow derived cells and froze them in cryovials using DMSO. They concluded
that fBM and cBM cells equally respond to LPS and also maintain differentiation competence over
time. Another article also confirms that cryopreservation of human iBMDCs does not affect
expression of maturation markers [50]. Concluded; cryopreservation does not affect differentiation
and maturationof BMDCs.
Supplementary information IV- IL-3 stimulation of BMDCs
Effect ofIL-3 onBMDCs
To ensure survival of BMBs after stimulation and during co-culture with BMDCs and CD4+ T cells, IL-3
needs to be added to the medium. We tested if IL-3 may affect the immune response of BMDCs by
adding IL-3 to BMDCs after 7 days of in vitro culture. The results in figure 43 summarize the data. The
data suggest that BMDCs that were not stimulated and BMDCs that were stimulated with IL-3 show
similarquantitiesof the different BMDCpopulations.
Figure 353. BMDCs derived from freshly isolated BM and from male B6 mice (n=5). Populations were gated for singlets.
The figure represents average values of the different BMDC populations. Gating was performed as shown in figure 1.
Every condition was tested in duplicate and the experiment was performed once.
IL-3 has no effect onmaturationof BMDCs
The cell quantity of the different BMDC populations of the negative control was virtually equal
compared with the IL-3 stimulated BMDCs. In other words, IL-3 does not induce maturation of
BMDCs. Literature confirms that generation of BMDCs was not affected by IL-3 [61]. However, in BM
cultures with IL-3, BMDC develop together with mast cells [62]. Also protocols exist using IL-3 to
generate BMDC precursors from murine hematopoietic progenitor cells [63]. IL-3 may induce
differentiation of BMDCs, but activated T cells also produce IL-3 which may enhance BMDC
differentiation [10, 46]. To prevent development of mast cells, but ensure survival of BMBs IL-3 will
be addedto the co-culture with BMDCs, BMBs and CD4+ T cellswhenwe startwithstimulation.

30
SupplementaryinformationV-Salmonellastimulationof BMDCs
Stimulationof BMDC withsalmonella
To investigate if there are differences in immune responses between probiotic bacteria and non-
probiotic bacteria, BMDCs are stimulated with salmonella strain SL3261. According to literature, this
salmonella strain is able to rapidly kill BMDCs [64-66]. To solve this problem we will also use a
salmonella mutant called ΔsipB, which is used in literature before[65]. In this strain the sipB gene is
replacedbya kanamycinresistance gene.
Cultivationofsalmonella
Strain SL3261 and SL3261 ΔsipB were obtained from a glycerol stock at -80°C and streaked out on
Brilliant Green plates. The plates were put overnight in an incubator at 37°C. The next day a single
colony from the plates was picked and put in liquid LB for SL3261 and liquid LB containing 15 µg/ml
kanamycin for SL3261 ΔsipB and put back overnight at 37°C. The day after, BMDCs were washed with
RPMI medium without pen/strep and 5*10^7, 5*10^6 or 5*10^5, bacteria (CFU) were added to wells
containing BMDCs. After one hour at 37 °C the bacteria were washed away with RPMI medium with
50 ng/ml gentamicin. Another hour at 37°C and the medium was replaced with complete culture
medium containing 20 µg/ml gentamicin. Analysis was done after overnight culture with flow
cytometry.
StrainSL3261allowsless BMDCsto maturecomparedto SL3261ΔSipB
Extracellular membrane molecules were measured via flow cytometry. Figure 44 below shows an
example of the gating used of the conditions tested and the response of BMDCs on the different
salmonellastrains.
Figure 36. Flow cytometer figures shows gating performed and stimulation of BMDCs with A) salmonella mutant strain
SL3261ΔsipB (5*105 CFU) and with B) salmonella strain SL3261 (5*105 CFU). BMDC populations were divided into
precursor, immature and mature BMDCs.
The figure above shows that SL3261 strain lead to less mature BMDCs compared to the mutant
strain. Most notable is the difference in quantity of mature BMDCs. The quantity of mature BMDCs is
smaller when BMDCs were treated with SL3261, compared to ΔSipB. Figure 45 shows an overview of
all differentconditionstested.

31
Figure 37. Average populations of BMDCs stimulated with strain sl3261 or sl3261 ΔsipB. Average values (± SD) are based
on samples amounts varying 2-5 and on three experiments. Figure A) Populations of BMDCs stimulated with SL3261.
Figure B) Populations of BMDCs stimulated with SL3261 ΔsipB.
Figure 44 shows the mature, immature and precursor populations of BMDCs. Stimulation was done
with 100 ng/ml LPS, 0 ng/ml LPS and different CFU of SL3261 or with SL3261 ΔSipB. As been indicated
in the FACS profile in figure 12, the amount of mature BMDCs treated with ΔSipB is larger compared
to BMDCs treated with SL3261. This difference was in all concentrations tested significant (P<0.05)
except SL3261 105
CFU. If we compared same CFU of SL3261 and ΔSipB with each other, the P-values
of the mature BMDCs testedwere all significant(P<0.05).
SalmonellaSL3261Δ SipBisnotableto kill BMDCsrapidly
When salmonella get into contact with immature BMDC, the BMDCs quickly respond by taking up the
antigen and presenting it on the surface. Maturation of BMDCs is characterized by upregulation of
surface markers including CD80, CD86, MHC-I and MHC-II occurs, which is also shown in figure 45
[64, 66]. The results in figure 13 above show that salmonella strain SL3261 does not cause
maturation of BMDCs. This is caused by the ability of SL3261 to kill BMDCs within a few hours and is
also confirmed by literature [65, 66]. Literature also describes a mutation in SL3261 that prevents
salmonella from killing BMDCs [65]. Our results show that when BMDCs are stimulated with the
mutant strain SL3261 ΔsipB, 15-20% of the entire BMDC population becomes mature. Concluding,
the mutation in salmonella strain ΔSipB does not kill BMDCs and allows BMDCs to reach a mature
stage. Salmonella mutant Δ sipB may be used in future experiments where BMDCs are stimulated
with this mutant and co-cultured with CD4+ T cells. In this way it may become clearer if there are
differencesinimmuneresponse betweenprobioticbacteriaandnon-probioticbacteria.

32
SupplementaryInformationVI-CD4+T cell differentiation
Testingthe effectof CD4+T cell differentiation
To test the proliferation and differentiation of the CD4+ T cells a test experiment with CD4+ T cells
only was performed. All CD4+ T cells were cultured with anti-CD3 (0.5 µg/ml) and anti-CD28 (1
µg/ml) and tested for their proliferation capacity by adding different mixture of antibodies. For Th1
polarization IFN-γ (50 ng/ml) anti-IL4 (10 µg/ml); for Th2 skewing IL-4 (50 ng/ml) and anti-INF-γ (5
µg/ml); for Th17 skewing IL-10 (1 µg/ml), anti-IFN-γ (5 µg/ml), anti-IL4 (10 µg/ml) were added to test
differentiation towards Th2, Th17 and Th1 cells. After 4 days, FACS analysis was performed on
extracellular and intracellular markers. Each sample obtained was gated for singlets, lymphocytes,
and CD4+ T cells.Figure 10 showsan example of the gatingthatwasapplied.
Figure 38. The graph shows the polarization of CD4+ T cells. Values in the graph were based on mean of two samples.
Experiment was performed once. Of every condition tested the quantity of FoxP3, T-bet and GATA3 positive cells were
given.
Figure 46 above shows different conditions tested for T cell polarization. T cells stimulated with CD28
and CD3 were considered as a positive control and T cells only stimulated with CD3 were considered
as negative control, although this is not entirely true. The quantity of FoxP3 positive cells is high
compared to the T-bet and GATA3 positive cells. This is seen in all conditions tested. For Th2 and Treg
polarization the FoxP3 population is significantly higher compared to both controls added (P<0.05).
GATA3 positive and T-bet positive cell quantity different compared to the controls and values were
alsonot significant.
Mostcytokinesweremeasuredat CD4+T cellsusedforTh1polarization
Cytokine assays may show if cytokines were released in the supernatant. Cytokines of the CD4+ T
cells were measured. Based on the results above, we do not expect to see upregulation of cytokines
involved in inflammatory responses (IL-12, TNF, IL-6), but expect to measure cytokines involved in
regulatory immune responses (IL-10). All cytokines measured were given in supplementary
information.

33
Table 4. Average levels of cytokines measured. Values were based on two samples and the experiment was performed
only once.
T cell
polarization
IL-10
(pg/ml)
IL-2
(pg/ml)
IFN
(pg/ml)
IL-6
(pg/ml)
IL-17A
(pg/ml)
IL-4
(pg/ml)
TNF
(pg/ml)
Th1 49.0 424.9 6604.3 ND 36.6 ND 159.4
Th2 ND 4.5 1.0 ND 0.5 3282.4 18.4
Treg 3209.1 3.1 0.4 0.2 0.9 0.3 13.2
anti-CD3+CD28 228.0 2718.3 1129.2 4.9 96.1 10.5 327.3
anti-CD3 83.0 12.7 87.4 3.3 58.5 2.6 46.3
For Th1 polarization was the following cytokines measured: TNF (170 pg/ml), IFN-γ, IL-17A (40 pg/ml)
and IL-2 (500 pg/ml). IFN-γ was added by us to the wells. The production of other cytokines suggests
that the polarization towards Th1 was successful. The production of IL-6 and IL-2 indicated that also
Treg cells were developed. For Th2 was only IL-4 measured (3300 pg/ml). This cytokine was added by
us. Active Th2 cells also produce IL-5 and IL-13, we did not measure these cytokines and this makes it
difficult to say that Th2 polarization succeeded or failed. For Treg were cytokine levels of IL-10
measured. This cytokine was added by us. Adding of IL-10 should leads to Th17 and Treg
differentiation. IL-2 and TGF-β are needed for Treg development and IL-17A for Th17 development.
IL-2 and IL-17A were not detected in the supernatant. For the positive control were levels of TNF
(350 pg/ml), IFN-γ (1800 pg/ml), IL-17A (100 pg/ml), IL-2 (3000 pg/ml) and IL-6 (5 pg/ml) measured.
Adding anti-CD3 and CD28 leads to aninflammatory response of Tcells and thisis clearly visible in the
positive control. For the negative control were levels of IL-17A (70 pg/ml) and IL-6 (3.5 pg/ml)
measured. These cytokines were involved in Treg responses. Most upregulation of cytokines were
foundforthe positive control andinlesseramountforthe negative control.
Th1and Tregpolarizationdidoccur,butnotTh2polarization
The results of the surface markers did not show a strong Th polarization. Only upregulation of FoxP3
was convincing and significant. For GATA3 and T-bet populations no upregulation was observed. But
the cytokine bead assay does show that Th1 polarization took place as TNF, IFN-γ, IL-17A and IL-2
cytokines was measured. For the Treg population was only IL-10 measured and for Th2 was only IL-4
measured. Concluded, polarization for Th1 and Treg cells did occur and does prove that our CD4+ T
cells were functioning properly. Polarization for Th2 was not very convincing. An explanation could
be that literature describes that BMDCs are able to polarize CD4+ T cells [61]. The lack of BMDCs
couldexplain the poor polarization for th2. Another explanation of the poor polarization could be the
lack of a true negative control. Our negative control received α-CD3, which can also activate CD4+ T
cells[67].
Also Ki-67 values were low,indicating the T-cells were not proliferating at the moment we measured.
This was caused by the fact that most T cells were dead at the time of measurement. The high death
rate could be caused by the lack of (anti- apoptotic) signals T cells normally get from other immune
cells that prevent T cell apoptosis. The ratio of T cells we used was not tested beforehand. It could be
the medium volume was too high and the cell density was too low for T cells to receive proper signals
neededtokeepthemalive.

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