Salmonella enhances osteogenic differentiation in adipose-derived mesenchymal stem cells

Abstract

The potential of mesenchymal stem cells (MSCs) for tissue repair and regeneration has garnered great attention. While MSCs are likely to interact with microbes at sites of tissue damage and inflammation, like in the gastrointestinal system, the consequences of pathogenic association on MSC activities have yet to be elucidated. This study investigated the effects of pathogenic interaction on MSC trilineage differentiation paths and mechanisms using model intracellular pathogen Salmonella enterica ssp enterica serotype Typhimurium. The examination of key markers of differentiation, apoptosis, and immunomodulation demonstrated that Salmonella altered osteogenic and chondrogenic differentiation pathways in human and goat adipose-derived MSCs. Anti-apoptotic and pro-proliferative responses were also significantly upregulated (p < 0.05) in MSCs during Salmonella challenge. These results together indicate that Salmonella, and potentially other pathogenic bacteria, can induce pathways that influence both apoptotic response and functional differentiation trajectories in MSCs, highlighting that microbes have a potentially significant role as influencers of MSC physiology and immune activity.

Full text

Salmonella enhances osteogenic
differentiation in adipose-derived
mesenchymal stem cells
Nuradilla Mohamad-Fauzi
1†‡
, Claire Shaw
1‡
, Soraya H. Foutouhi
2‡
,
Matthias Hess
1
, Nguyet Kong
2
, Amir Kol
3
, Dylan Bobby Storey
2‡
,
Prerak T. Desai
2
, Jigna Shah
2
, Dori Borjesson
3
,
James D. Murray
1
,
2
* and Bart C. Weimer
2
*
1
Department of Animal Science, College of Agricultural and Environmental Sciences, University of
California, Davis, Davis, CA, United States,
2
Department of Population Health and Reproduction, 100K
Pathogen Genome Project, Davis, CA, United States,
3
Department of Pathology, Microbiology and
Immunology, University of California, Davis, Davis, CA, United States
The potential of mesenchymal stem cells (MSCs) for tissue repair and regeneration
has garnered great attention. While MSCs are likely to interact with microbes at
sites of tissue damage and inflammation, like in the gastrointestinal system, the
consequences of pathogenic association on MSC activities have yet to be
elucidated. This study investigated the effects of pathogenic interaction on
MSC trilineage differentiation paths and mechanisms using model intracellular
pathogen Salmonella enterica ssp enterica serotype Typhimurium. The
examination of key markers of differentiation, apoptosis, and
immunomodulation demonstrated that Salmonella altered osteogenic and
chondrogenic differentiation pathways in human and goat adipose-derived
MSCs. Anti-apoptotic and pro-proliferative responses were also significantly
upregulated (p<0.05) in MSCs during Salmonella challenge. These results
together indicate that Salmonella, and potentially other pathogenic bacteria,
can induce pathways that influence both apoptotic response and functional
differentiation trajectories in MSCs, highlighting that microbes have a
potentially significant role as influencers of MSC physiology and immune activity.
KEYWORDS
pathogenic and infectious disease, apoptosis, cell death, host/bacteria interactions, MSC
Introduction
Mesenchymal stem cells (MSCs) have a known capacity for self-renewal and
differentiation into cartilage, bone, and adipose tissue (Dominici et al., 2006), making
these cells of great interest in regenerative medicine research (Fossett and Khan, 2012;Marra
and Rubin, 2012;Merimi et al., 2021). In addition to their renewal abilities, MSCs are also
recruited via the secretion of paracrine factors to areas of inflammation where they exhibit
immunomodulatory functions (Ortiz et al., 2003;Balaji et al., 2012). In cooperation with
recruited immune cells, MSCs moderate inflammation via expression of anti-inflammatory
cytokines (Romieu-Mourez et al., 2009;Galindo et al., 2011;Roddy et al., 2011), inhibit
T-lymphocyte activation, and alter macrophages to express a regulatory anti-inflammatory
phenotype toward increased phagocytic activity (Balaji et al., 2012;Eggenhofer and
Hoogduijn, 2012).
OPEN ACCESS
EDITED BY
Anna Olivieri,
National Institute of Health (ISS), Italy
REVIEWED BY
Tomoyuki Iwata,
Hiroshima University, Japan
Elaheh Ferdosi-Shahandashti,
Babol University of Medical Sciences, Iran
*CORRESPONDENCE
James D. Murray,
jdmurray@ucdavis.edu
Bart C. Weimer,
bcweimer@ucdavis.edu
†
PRESENT ADDRESS
Nuradilla Mohamad-Fauzi,
Institute of Biological Sciences, Faculty of
Science, Universiti Malaya,
Kuala Lumpur
Institute of Ocean and Earth Sciences,
Universiti Malaya,
Kuala Lumpur
‡
These authors have contributed equally
to this work
SPECIALTY SECTION
This article was submitted
to Membrane Traffic,
a section of the journal
Frontiers in Cell and
Developmental Biology
RECEIVED 22 October 2022
ACCEPTED 17 February 2023
PUBLISHED 15 March 2023
CITATION
Mohamad-Fauzi N, Shaw C, Foutouhi SH,
Hess M, Kong N, Kol A, Storey DB,
Desai PT, Shah J, Borjesson D, Murray JD
and Weimer BC (2023), Salmonella
enhances osteogenic differentiation in
adipose-derived mesenchymal
stem cells.
Front. Cell Dev. Biol. 11:1077350.
doi: 10.3389/fcell.2023.1077350
COPYRIGHT
© 2023 Mohamad-Fauzi, Shaw, Foutouhi,
Hess, Kong, Kol, Storey, Desai, Shah,
Borjesson, Murray and Weimer. This is an
open-access article distributed under the
terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication
in this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
Frontiers in Cell and Developmental Biology frontiersin.org01
TYPE Original Research
PUBLISHED 15 March 2023
DOI 10.3389/fcell.2023.1077350

MSCs are recruited to and subsequently secrete anti-microbial
peptides at sites of bacterial infection due to localized inflammation
(Krasnodembskaya et al., 2010). Pathogenic and non-pathogenic
microbes interact with MSCs at mucus membranes, where tissue
turnover is high and immune-responsive cells infiltrate to control
pathogens (Xu et al., 2014). The outer mucosal layer matrix of the
gut lumen is one such interface that is rich with both microbes and
host cells and an area where MSCs come into frequent contact with
microbial inhabitants and invaders (Nagashima et al., 2017;
Schroeder, 2019). Gut inflammation and the subsequent
destruction of intestinal epithelial cells induces MSC recruitment
to facilitate tissue recovery in the intestinal tract (Semont et al.,
2006). While this apoptotic epithelial cell response to infection is
well characterized (Kim et al., 1998;Wemyss and Pearson, 2019),
little is known about the consequences of bacterial association on the
behavior of mesenchymal stem cells.
The long-term effects of MSCs and pathogen interactions remain
understudied, but there are aspects of this host cell-pathogen
relationship that have been previously explored. Treatment of MSCs
with the immunostimulant membrane component lipopolysaccharide
(LPS) and with Gram-negative Escherichia coli increases osteogenesis
and decreases adipogenesis, while stimulation with Gram-positive
Staphylococcus aureus decreases osteogenesis and adipogenesis
(Fiedler et al., 2013). Immune functionality of MSCs can also be
altered by bacterial association, as evidenced by previous work done
using invasive pathogen Salmonella Typhimurium (Kol et al., 2014).
Intracellular association of Salmonella in MSCs increased transcription
and secretion of immunomodulatory products IL6 and IL8,butalso
reduced MSCs’ability to inhibit T-cell proliferation (Kol et al., 2014).
These changes in viability, immunomodulatory functions, and
differentiation paths of MSCs due to bacterial association confirms
that MSC-bacterial interactions not only follow a different course than
epithelial cells, but also illustratesthatMSCsmaintainsuchaltered
functions beyond acute infection and association.
Microbial invaders, like Salmonella, are in part recognized by
host cells via Toll-like receptors (TLRs). TLRs are present on many
host cells and recognize surface-expressed bacterial components like
LPS, alerting the triggered cell to the presence of a pathogen
(Kawasaki and Kawai, 2014). MSCs, along with gut epithelial
cells, express TLRs but it is unclear how pathogens regulate and
interact with these receptors on MSCs as compared to epithelial cells
(Hwa Cho et al., 2006;Raicevic et al., 2010). Tomchuck et al. (2008)
reported the promotion of MSC migratory abilities as a result of TLR
activation, whereas a study by Pevsner-Fischer et al. (2007) found
TLR activation shifts lineage commitment to proliferation. TLRs
play a central role in the detection of and defense against pathogens,
and though important, there are also other pathogen-specific
activating signals, like the type three secretion system (T3SS) in
Salmonella.
Salmonella pathogenesis in epithelial cells is primarily mediated
via the T3SS, which injects effector proteins that ultimately lead to
apoptosis (Pilar et al., 2013;Rivera-Chavez et al., 2013). T3SS
proteins target a variety of host cell regulators, including the
NFκB pathway, creating an inflammatory environment which
increases microbial internalization (Thiennimitr et al., 2012;Pilar
et al., 2013). MSCs have early transcription factors, such as
peroxisome proliferator-activated receptor gamma (PPARG)
(Pascual et al., 2005) and secreted phosphoprotein 1 (SPP1)
(Sabo-Attwood et al., 2011), that are involved in inflammatory
responses but which also have a role in MSC differentiation.
These transcription factors may be a bridge between microbe-
induced inflammation and the cellular response of MSCs. The
different responses of epithelial cells and stem cells to invasion
by Salmonella suggest stem cells undergo distinct conditioning that
results in lasting immunomodulatory changes. The microbial
modulation of the immune system is an area of much interest,
but the unique abilities of MSCs coupled to their physical
positioning at pathogenic interfaces makes these stem cells of
interest given the potential widespread impact cellular
reprogramming of such a fundamental cell type could have. It is
possible that microbial modulation of the immune system in MSCs
provides pathogens with an uncharted method of tissue infiltration,
resulting in yet undescribed biological impact.
Testing the impact of microbial association on the proliferation and
differentiation of stem cells in vitro is resource intensive, as human
sampling can be both costly and difficult to arrange. Livestock models
present an invaluable option to the problem of acquiring human-
derived stem cells, as livestock MSCs are typically more readily
accessible than human subjects, provide larger sample quantities
than small animal models, and livestock stem cells have been shown
to accurately reflect the behavior of their human counterparts in many
instances (Harness et al., 2022). One source of livestock-derived stem
cells is goats, who share many physiological similarities with humans
and whose adipose-derived MSCs have been previously confirmed as
appropriate for modeling human adipose-derived MSCs (Harness et al.,
2022). The goal of understanding if goat cells can be used as a
translational model human studies is a goal of this strudy. In this
work, both human and goat MSCs were utilized to explicate the effect of
pathogen association on trilineage behavior, to assess similarities and
differences in their general response to bacterial invasion/association
between the species, and to further highlight the usefulness of livestock
stem cells for human research with translational perspectives prior to
clinical trials.
This study evaluated the effect of Salmonella association on the
immunomodulatory behavior of human and goat MSCs. It was
hypothesized that MSCs would internalize Salmonella,resultingin
altered MSC trajectories toward pro-osteogenic commitment in
conjunction with the induction of an anti-inflammatory, and altered
immunological phenotypes. Mechanisms indicative of MSC survival,
proliferation, and immune regulation in response to Salmonella
interaction were evaluated to address this hypothesis. The data from
this study indicates stem cells altered their therapeutic phenotypes and
behavior in response to association with an intracellular pathogen,
suggesting that microbial-specific alterations in MSC differentiation and
inflammatory status can influence broader stem cell fate and ultimately
stem cell functionality.
Materials and methods
Cell culture
Human adipose-derived mesenchymal stem cells (hASCs) were
isolated in the laboratory of Dr. Dori Borjesson (University of
California, Davis) and cultured in Minimum Essential Medium
Alpha Modification (MEM-α, HyClone Laboratories, Logan, UT)
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with 20% fetal bovine serum (FBS, HyClone) and 1% penicillin-
streptomycin (P/S, Gibco Life Technologies). Goat adipose-derived
mesenchymal stem cells (gASCs) were isolated in the laboratory of
Dr. Matthew Wheeler (University of Illinois, Urbana-Champaign),
as described by Monaco et al. (2009), and expanded on as described
by Mohamad-Fauzi et al. (2015). ASCs were cultured in 5% CO
2
/
37°C, and used at passage six. Colonic epithelial cells (Caco-2; ATCC
HTB-37) were obtained from American Type Culture Collection
(Manassas, VA) and grown according the method defined by Shah
et al. (2014).
Bacteria culture
Salmonella enterica ssp enterica serotype Typhimurium LT2 (ST),
14028S, serotype Enteritidis (BCW_4673), serotype Saint Paul (BCW_
88) and serotype Newport (BCW_1378) were grown in Luria-Bertani
(LB) broth (Teknova, Holister, CA) and incubated with shaking
(200 rpm) at 37°C. Bacterial cultures were grown according to the
method in Kol et al. (2014) for this study. Multiple serotypes were used
to account for sero-diversity and assess if these organisms induced
different responses as previously observed (Kol et al., 2014).
Quantification of microbe association
Association was determined using the gentamicin protection
assay (Elsinghorst, 1994) and modified from Kol et al. (2014) as
follows: ASCs were plated (4 × 10
4
) in a 96-well plate and incubated
overnight; bacteria were suspended in serum-free medium
(10
8
CFU/mL) and added to the ASCs with a multiplicity of
infection (MOI) 1:100 (MSC:bacteria).
Transmission electron microscopy
hASCs were plated on glass slides (Nalge Nuc International,
Naperville, IL) and incubated for 2 h with ST (Kol et al., 2014).
Preparation and completion of transmission electron microscopy
(TEM) was conducted as outlined in Kol et al. (2014).
Differentiation
Adipogenic and osteogenic differentiation was done using ASCs
in 6-well plates at 2.5 × 10
5
ASCs/well and incubated with ST for 1 h
as described above. Chondrogenic differentiation was done in T-25
flasks at 3 × 10
5
ASCs/flask and incubated with ST for 1 h as
described above. Following treatment with gentamicin, ASCs
were washed with PBS to remove bacteria from the suspension
and subsequently cultured for 48 h in expansion medium to 70%–
80% confluence, after which differentiation medium was added.
Osteogenic differentiation assay
ASCs were cultured in osteogenic medium, fixed, rinsed and
visualized under light microscopy as described in Mohamad-Fauzi
et al. (2015). hASCs were cultured for 14 days, whereas gASCs were
cultured for 21 days. Control non-induced cells were cultured in
expansion medium.
Chondrogenic differentiation assay
Chondrogenic differentiation was carried out as described by
Zuk et al. (2001). Following ST incubation, 70%–80% confluent cells
were trypsinized and suspended in expansion medium for 14 days,
then processed and visualized as described by Mohamad-Fauzi et al.
(2015).
Adipogenic differentiation assay
Cells were cultured for 21 days in adipogenic induction medium,
fixed, stained, and visualized according to the methods described in
Mohamad-Fauzi et al. (2015).
RNA extraction and cDNA synthesis
ASCs were flash frozen prior to RNA extraction. For analysis of
immunomodulatory factors, ASCs were plated in 6-well plates (3 ×
10
5
cells/well) and incubated with ST as described above. LPS
(Sigma) was added at 10 ng/mL. MSCs were washed with PBS,
and immediately lysed with TRIzol Reagent (Life Technologies) as
described previously (Chen et al., 2017) Total RNA was extracted as
described by Mohamad-Fauzi et al. (2015). Total RNA (1 µg) was
used for first-strand cDNA synthesis using SuperScript II Reverse
Transcriptase (Life Technologies) and oligo-dT primers according
to the manufacturer.
Quantitative RT-PCR
Primers (Supplementary Tables S1, S2) were designed using
Primer3 when not directly obtained from references. All primers
spanned exon junctions or included introns. mRNA expression was
quantified using Fast SYBR Green reagent (Life Technologies) on
the Bio-Rad CFX96 platform (95°C for 20 s, 40 cycles of 95°C for 3 s,
and 60°C for 30 s), followed by melt curve analysis. Gene expression
was normalized to GAPDH using 2
−ΔΔCT
(Livak and Schmittgen,
2001;Schmittgen and Livak, 2008). Differences in differentiation
gene expression were calculated as fold-changes relative to cells
cultured in expansion medium (non-induced) and not treated with
bacteria (non-treated). Inflammatory gene expression was calculated
as fold-changes relative to non-treated control cells. Treatments
were analyzed in pairwise comparisons using the Student’st-test on
JMP software (SAS Institute) (p≤0.05). Data are presented as
mean ± SEM with three biological and technical replicates.
GeneChip expression analyses
Caco-2 infection samples with Salmonella LT2 were conducted
using Affymetrix HGU133Plus2 GeneChip. Custom arrays
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containing all annotated coding and intergenic sequences of S.
enterica spp. enterica sv Typhimurium LT2 (Marcobal et al.,
2011;Ng et al., 2013;Shah et al., 2013). Data were normalized
using MS-RMA (Stevens, 2008) and analyzed using Significance
Analysis of Microarrays (SAM) (Champine et al., 2007;Ng et al.,
2013).
FIGURE 1
Microbial Association with human and goat ASCs (A–F). ASCs presented a uniform pattern of Salmonella enterica ssp enterica serotype
Typhimurium LT2 (ST) infection, the total associated bacteria were invaded, gASC show significantly higher invasion compared to human cells (A). ASCs
susceptibility to invasion was not exclusive to ST, association patterns were microbe specific; 35%, 12% Salmonella enterica ssp enterica serotype
Typhimurium 14,028, and 25%, 100% Salmonella enterica ssp enterica serotype Enteritidis (BCW_4673) were invaded in goat and human ASCs
respectively (B). In gASCs, 35% Salmonella enterica ssp enterica serotype Newport (BCW_1378) and in hASCs, 7% of Salmonella enterica ssp enterica
serotype Saint Paul (BCW_88) were invaded (B). Intracellular ST was observed by TEM 2 h post MSC co-incubation (D–F), consistent with control non-
treated hASCs (C), ST infected cells showed no signs of cellular toxicity (D–F). ST adherence to hASC was observed at various sites (E–F).
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hASC RNA sequencing
Total RNA (1 µg) from hASCs was used to construct sequencing
libraries with the Truseq Stranded Total RNA LT Kit (Illumina) as
described previously (Chen et al., 2017;Haiminen et al., 2019;Beck
et al., 2021). Quality of RNA and constructed libraries was
determined via 2100 Bioanalyzer. Libraries sequenced using an
Illumina HiSeq 2000 (BGI@UC Davis, Sacramento, CA) with
single-end 50 bp. Reads were aligned using the UCSC
hg19 human reference genome (ftp://igenome:G3nom3s4u@ussd-
ftp.illumina.com/Homo_sapiens/UCSC/hg19/Homo_sapiens_
UCSC_hg19.tar.gz) and annotated using “-a 10 –b2-very-sensitive
-G”. Read count and normalization was done using Cufflinks
package (version 2.2.0) with flags “-u -G”. Tables from cuffnorm
and cuffdiff imported into Ingenuity Pathway Analysis (IPA;
Ingenuity Systems, version spring 2014). Sequence quality was
examined using Phred.
Ingenuity Pathway Analysis
IPA (version 01-20-04) was used to determine biological
pathways associated with gene expression profiles. Networks
represent molecular interaction based on the IPA knowledge
database. Estimation of probable pathway association was
determined Fisher’s exact test, and predicted direction change
was decided by the IPA regulation z-score algorithm (z-score ≥
2 and ≤2 means a function is significantly increased or decreased,
respectively) (St-Pierre et al., 2013).
Results
Microbial association with adipose-derived
mesenchymal stem cells
Human and goat adipose-derived mesenchymal stem cells
(ASCs) were susceptible to in vitro infection with S. enterica ssp
enterica Typhimurium LT2 (ST) (Figure 1), corroborating
previous observations regarding canine ASC susceptibility (Kol
et al., 2014). Though both human ASCs (hASC) and goat ASCs
(gASC) were vulnerable to and displayed intracellular invasion
by ST, gASCs showed significantly increased ST invasion
compared to human cells (p= 0.006) (Figure 1A). In addition
to ST, the other Salmonella serotypeswerealsoabletoinvade
ASCs, though to a lesser degree as compared to ST (Figure 1B).
The ability of multiple Salmonella serotypes to exist
intracellularly in ASCs further supports previous work by our
group demonstrating this ability in multiple serotypes (Kol et al.,
2014), suggesting a consistent trend of ASC vulnerability to
common pathogens (Figures 1A, B).
Invasion and adhesion of hASCs by ST was further evaluated
utilizing TEM 2 h post hASC-microbe co-incubation (Figures 1C,
F). Intracellularly infected hASCs did not display visual markers of
morphological distress or signs of apoptosis. In addition to invasion,
adherence of ST to hASC cell surface was also observed; an intimate
host-microbe association which is consistent with what has been
seen with other non-pathogenic bacteria (Kol et al., 2014).
ASC immunomodulation activity is altered
by infection
Following co-incubation with ST, the expression of several key
immunomodulatory genes in ASCs was evaluated via qPCR
(Figure 2). Interleukin 8 and 6 (IL8,IL6), prostaglandin-
endoperoxide synthase 2 (PTGS2), nuclear factor of kappa light
polypeptide gene enhancer in B-cells 1 (NFΚB1), transforming
growth factor beta 1 (TGFB1), PPARG and SPP1 were selected as
markers of immunomodulatory activity in human and goat ASCs.
As compared to non-infected cells, ST treated gASCs (Figure 2) and
hASCs (Figure 2) both significantly increased IL8 expression (p≤
0.033, p≤0.005, respectively). No other surveyed ASC
immunomodulatory markers displayed significant differences in
expression as compared to the controls.
The effect of LPS treatment on these immunomodulatory genes
was also evaluated as a positive control for MSC response to
inflammatory markers. IL8 expression was increased in both
hASCs and gASCs in response to LPS treatment, but this
increase was only significant in gASCs (p≤0.0001). LPS
treatment of gASCs also induced a significant increase in the
expression of IL6 (p=0.0001), PTGS2 (p=0.0009), NFKB1 (p=
0.0002), PPARG (p= 0.0204), SPP1 (p=0.037), as well as IL8 (p≤
0.0001) (Figure 2). LPS treatment of hASCs also resulted in
increased expression of the evaluated immunomodulatory genes,
but none were significant compared to the control hASCs (Figure 2).
Effect of ST association on the genetic
activity of ASCs
Broad analysis of gene expression in hASCs co-incubated with
ST found 118 significantly differentially expressed genes (p≤0.05,
FDR = 0.1). Further canonical pathway analysis of this expression
data revealed infected hASCs repressed gene pathways associated
with cell-to-cell signaling, cell death, and apoptosis (Figure 3).
The repression of genes related to cell survival and death, as well
as promotion of proliferation and multipotency in infected hASCs
(Figure 4) is consistent with the observation of continued hASC
viability post ST association. Expression of single genes related to
cell death and viability confirms the importance of these pathways in
cell persistence post-ST association. The gene for survival-related
heat shock protein B6 (HSPB6) was upregulated and there was
MAP-predicted association of v-akt murine thymoma viral
oncogene homolog 1 (AKT1) with the generated genetic network
in ST treated hASCs. HSPB6 inhibits apoptosis of murine tumor
cells and protects against oxidative damage (Ghulmiyyah et al., 2011;
Chen et al., 2014), while AKT1 helps mediate cell survival and
clonogenic potential (Song et al., 2005;Xu et al., 2012;Gharibi et al.,
2014;Liu et al., 2014). Supporting observations in ASCs, epithelial
Caco-2 cells likewise show downregulation of apoptosis-related
genes in response to treatment with ST. Comparison of the
apoptosis-regulating TNF/FasL pathway in ST-infected hASCs
and Caco-2 cells reveals similarities in cellular responses
regarding survival pathways across the two distinct cell types
[Supplementary Figure S2].
Upregulated in ST-treated hASCs were genes involved in
molecular organization, differentiation, and proliferation
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(Figure 5). Epidermal differentiation influencer patched 2 (PTCH2)
displayed increased levels of expression in ST-treated hASCs. The
upregulation of PTCH2 in conjunction with the connection to Hh
signaling in the network suggests promotion of hASC proliferation
pathways in response to co-incubation with ST (Adolphe et al.,
2014). Immunomodulatory regulator, central regulator superoxide
dismutase 3 (SOD3), was induced in treated hASCs (Figure 3),
aligning with previous observations in INFγ/LPS-activated
microglial cells (Kemp et al., 2010). Extracellular superoxide
dismutase (EC-SOD) facilitates bacterial clearance and an anti-
inflammatory response by promoting phagocytosis (Bowler et al.,
2004;Kim et al., 2011;Manni et al., 2011). Upstream regulators of
SOD3,SOX10 and heparin sulfate (HS), were predicted via MAP to
have increased activity in treated hASCs. Both SOX10 and heparin
sulfate (HS), are known to play a role in the maintenance of
multipotency and self-renewal (Shakhova and Sommer, 2008;
Helledie et al., 2012). Other regulators of SOD3, interferon
gamma (IFNG), IKBKB, and NOS3, also had a predicted increase
in activity for infected hASCs. INFG activation is of particular
interest given an INFG-activated MSC suppresses T-cells and
provide the necessary signal for MSC immunosuppression
(Sivanathan et al., 2014).
As with the apoptotic pathways mentioned above, the
immunomodulatory response of epithelial Caco-2 cells was
evaluated in comparison to that of hASCs. Interestingly, while
Caco-2 cells strongly induced TLR signaling in response to
FIGURE 2
Expression of immunomodulatory factors in ASCs post-microbial association. Quanti tative PCR analysis of IL6, PTGS2, NFKB1, TGFB1, PPARG, SPP1,
and IL8 expression in goat and human ASCs treated with ST or LPS. Data is presented as fold change (± SEM) in relative to expression levels in non-treated
cells (“C”) (fold change ~1, indicated by the dotted line). Statistical significance of p<0.05 is denoted by an asterisk (*), and p<0.01 denoted by two
asterisks (**). Goat cell data is presented in gray and human cell data is presented in maroon.
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exposure to Salmonella, which facilitates downstream cellular
response to pathogenic challenges, this observation was not seen
in the challenged hASCs [Supplementary Figure S2]. Interestingly,
this indicates that hASCs do not use TLR signaling in response to
Salmonella association, suggesting an alternative mechanism for
Salmonella internalization that is cell death independent.
Analysis of trilineage differentiation
post-microbial association
Chondrogenic differentiation
Co-incubation with ST did not decrease the ability of ASCs to
undergo chondrogenesis. Differentiated ST-treated hASCs migrated
to form ridges that stained with Alcian Blue (Figure 5A).
Differentiated ST-treated gASCs displayed more advanced
morphological changes compared to hASCs (Figure 5A). After
ridge formation, gASCs aggregated and formed clumps that also
stained with Alcian Blue. For comparison, uninfected and
uninduced control cells remained in a monolayer and exhibited
minimal background staining.
Essential for this cartilage formation in ASCs is SRY (sex
determining region Y)-box 9 (SOX9)(Bi et al., 1999), which
encodes a transcription factor that promotes cartilage-specific
extracellular matrix components (Bell et al., 1997;Han and
Lefebvre, 2008). Expression of SOX9 in infected hASCs and
gASCs was measured by qPCR 14 days post chondrogenic
induction. Infected and induced hASCs had decreased SOX9
expression (p= 0.04) compared to non-infected but induced
hASCs, while non-induced hASCs showed no significant change
in SOX9 expression (Figure 5B). Chondrogenic induction of non-
infected hASCs increased SOX9 expression compared to hASCs
treated with non-inducing control medium (p= 0.034). gASCs
displayed different expression patterns of SOX9 across treatment
types as compared to hASCs (Figure 5C). Induced gASCs, both
FIGURE 3
Downstream trends analysis of differentially expressed genes in hASCs post microbial challenge. The IPA regulation z-score algorithm was used to
identify biological functions expected to increase or decrease based on the gene expression changes described in our dataset. Predictions base on
p-value and z-score; positive z-score implies an increase in the predicted function, a negative z-score a decrease (z-score ≥2or≤−2 represented by
black dotted lines). p-values ≤0.05 (orange dots determined by Fischer’s exact test), illustrate a significant associati on between a given biological
function and genes differentially expressed in our dataset (p-value ≤0.05). p-values are presented as log-transformed. Shapes associated with each gene
name and broad category indicate the general classification of the gene product, enzyme, growth factor, etc.
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FIGURE 4
Network displays interactions between genes regulating cell signaling, cellular function and maintenance, and vitamin and mineral metabolism that
were differentially expressed in hASCs treated for 60 min with S.T compared with untreated control. Upregulated genes are colored in shades of red,
downregulated in shades of green (p-value ≤0.05). IPA inserted Genes in white because they are connected to this network; dashed and solid lines
denote indirect and direct relationships between molecules. The IPA molecule activity predictor assessed the activity of molecules strongly
connected to this network; blue and orange colored molecules are predicted to have decreased and increased activity, respectively. Shapes associated
with each gene name and broad category indicate the general classification of the gene product, enzyme, growth factor, etc.
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FIGURE 5
Chondrogenic induction. Representative images are shown in phase contrast at ×40 magnification. (A) Alcian Blue staining of chondrogenic
differentiation in ASCs post-microbial association. Human and goat ASCs were cultured in chondrogenic differentiation medium for 14 days, and
subsequently stained with Alcian Blue. Cellular condensation, as well ridge and micromass formations that stain positive were observed in human and
goat ASCs induced for chondrogenesis, independent of S.T treatment. Some background staining was observed in S.T-treated and non-treated cells
cultured in control medium, but cells remained in monolayer. Expression of chondrogenic markers post-microbial association was analyzed via
quantitative PCR analysis of SOX9 expression in (B) human and (C) goat ASCs induced with chondrogenic differen tiation medium and/or treated with S.T.
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FIGURE 6
Adipogenic induction. Representative images are shown in bright field at ×200 magnification. (A) Oil Red O staining of adipogenic differentiation in
ASCs post-microbial association. hASCs and gASCs were cultured in adipogenic induction medium for 21 days and stained with Oil Red O. Accumulation
of cytoplasmic lipid droplets were observed in ASCs induced for adipogenesis, independent of S.T treatment. S.T-treated and non-treated ASCs cultured
in control medium did not yield lipid-positive cells. Expression of adipogenic markers in ASCs post-microbial association was analyzed via
quantitative PCR analysis of PPARγand FABP4 expression in (B) human and (C) goat ASCs induced with adipogenic induction medium and/or treated with
S.T. Data is presented as fold change (±SEM) relative to expression levels in non-treated, non-induced cells (fold change ~1, indicated by the dotted line).
Statistical significance of p<0.05 is denoted by an asterisk (*), and p<0.01 denoted by two asterisks (**).
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infected and non, displayed decreased SOX9 expression as compared
to non-induced controls (p= 0.012). ST-treated gASCs, both
induced and non, also had decreased SOX9 expression as
compared to non-induced and non-treated gASCs (p= 0.027,
p= 0.039 respectively). Overall, hASCs had increased expression
of SOX9 as compared to gASCs, but no clear pattern of effect of
infection status or induction status on SOX9 expression across the
two cell types emerged during the treatment.
Adipogenic differentiation
Infected and non-infected ASCs cultured in adipogenic medium
accumulated lipid-filled vacuoles that stained with Oil Red O
(Figure 6A). Cells not cultured in adipogenic medium, uninduced
ASCs, did not form these lipid-filled adipocytes and did not stain
with Oil Red O. No visual morphological differences were observed
between non-induced ST-treated cells and non-induced control
cells.
The lack of observed visual differentiation was further explored
via the measurement of PPARG and fatty acid binding protein 4
(FABP4) expression, which together indicated induction of early
events within adipogenesis (Shin et al., 2009;Shi et al., 2013).
PPARG and FABP4 expression was measured by qPCR in cells
21 days post induction. ST treatment alone did not have a marked
effect on PPARG or FABP4 expression in either hASCs or gASCs,
instead, induction status had a more distinct effect on expression
(Figures 6B, C). Significant increases in PPARG expression were
observed in both human and goat cells grown in induction medium
as compared to uninduced controls (p≤0.0001, p≤0.0001,
respectively).
The expression of FABP4, a fatty acid binding protein specificto
mammalian adipose tissue (Bernlohr et al., 1985;Bakhtiarizadeh
et al., 2013), followed the same trend as PPARG across both hASCs
and gASCs. No significant difference was observed between induced
ASCs treated with ST and uninfected control cells. Non-induced
hASCs, but not gASCs, treated with ST has a significant increase in
FABP4 expression (p=0.019). Both species had increased FABP4
expression in cells cultured in adipogenic induction medium
(p=0.029, p≤0.0001) (Figures 6B, C).
Osteogenic differentiation
Osteogenic induction of infected ASCs resulted in the formation
and accumulation of mineralized calcium deposits within the
monolayer, as confirmed by Alizarin Red S staining [Figure 6A].
There was no readily apparent visual difference between induced
cells treated or not treated prior with ST. ASC controls grown in
expansion medium did not display the same calcium mineralization
as their induced counterparts and did not stain with Alizarin Red S,
independent of ST treatment (Figure 7A).
The expression of osteogenic-related genes, collagen type I alpha
1(COL1A1), alkaline phosphatase (ALPL) and SPP1, was evaluated
by qPCR at the termination of differentiation. COL1A1 encodes for a
major component of the most abundant collagen found in bone
matrix (Viguet-Carrin et al., 2006) and so gene expression of
COL1A1 was used as a marker of osteogenic differentiation in
ASCs. COL1A1 expression was significantly decreased in induced
cells as compared to non-induced cells, regardless of ST treatment
status (p≤0.0001) (Figures 7B, C). Across induced hASCs, COL1A1
expression was significantly higher in ST-treated cells compared to
un-infected controls (p= 0.025). No difference in expression was
detected between non-induced infected and non-infected control
hASCs. gASCs displayed no significant change in COL1A1
expression across induced cells that were either treated with ST
or not, though there was a significant decrease in COL1A1
expression by induced gASCs as compared to uninduced controls
(p= 0.018) (Figure 7C).
ALPL expression, which is responsible for the availability of
phosphate ions during the production of bone mineral during
matrix maturation (Tenenbaum and Heersche, 1982;de Bernard
et al., 1986;Choi et al., 1996), was measured across both cell types in
response to induction and infection status. Expression of ALPL was
significantly higher in induced hASCs than in non-induced cells,
regardless of ST treatment (p= 0.02) (Figure 7B). ST-treated induced
hASCs displayed 3.3-fold higher ALPL expression than non-ST
treated but induced hASCs (p= 0.03). Non-induced hASCs
showed no significant difference in expression patterns by ST-
treatment status. ALPL expression was not detected in any
gASCs across all treatment types.
Expression of a third osteogenic-related gene, SPP1, was
evaluated in ASCs in response to induction and ST-treatment.
SPP1 is a non-collagenous bone protein expressed during the
mineralization phase late in osteogenesis (Nagata et al., 1991).
SPP1 expression was repressed in both ST-treated and non-
treated induced hASCs (p≤0.0001) (Figure 7B). ST-treated and
induced hASCs displayed 4.3-fold higher expression of SPP1 than
induced but not infected cells (p= 0.002). Non-induced hASCs
showed no difference in expression between infected and non-
infected cells, though expression of SPP1 was generally increased
in comparison to the induced hASCs. No significant difference was
detected between ST-treated and non-treated induced gASCs,
however; there was a significant change in SPP1 expression
observed in non-induced cells across ST-treatment status
(Figure 7C). A 7.2-fold increase in SPP1 expression was observed
in non-induced but ST-treated gASCs as compared to non-induced,
non-infected cells (p≤0.05).
In addition to the specific markers of osteogenesis laid out above,
a broader survey of gene activity of hASCs in response to osteogenic
induction and ST treatment was done utilizing RNAseq.
Pretreatment of hASCs with ST, followed by 14 days of
osteogenic induction, resulted in the differential expression of
1,060 genes (data not shown). Downstream biological functions
of these differentially expressed genes were determined using
RNAseq z-score algorithm in IPA. These expression data
predicted the repression of genes associated with cell-to-cell
signaling, inflammation, and the response to infectious disease
(p-value ≤0.05, z-score ≥2) (Figure 8). Genes involved in cellular
communication, migration, and lineage commitment were also
differentially expressed in ST-treated and induced hASCs.
Differentially expressed genes related to the pathways mentioned
above include stanniocalcin 1 (STC1) and mesenchyme homeobox 2
(MEOX2)(Figure 9). STC1 expression, a response to apoptotic
signals (Kim et al., 2013) that is involved in inflammation
suppression and mineral homeostasis (Block et al., 2009;Kim
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FIGURE 7
Osteogenic induction. Representative images are shown in phase contrast at ×40 magnification. (A) Alizarin Red S staining of osteogenic
differentiation in ASCs post-microbial association. hASCs were cultured in osteogenic differentiation medium for 14 days, whereas gASCs for 21 days and
stained with Alizarin Red S. ASCs cultured in osteoinductive medium stained positive for calcium but did not stain when cultured in control medium,
regardless of S.T treatment. Expression of osteogenic markers in ASCs post-microbial association were analyzed via quantitative PCR analysis of
COL1A1, ALP and OPN gene expression in (B) human and (C) goat ASCs induced with osteogenic differentiation medium and/or treated.
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et al., 2013), was downregulated in differentiated and ST-treated
hASCs. In contrast, MEOX2 was upregulated in the infected and
induced hASCs. Also upregulated in ST-treated osteogenic
differentiated hASCs (Figure 9), was chloride intracellular
channel 4 (CLIC4), which is induced during cellular stress and
influences cell cycle arrest and apoptosis (Suh et al., 2005).
Intracellular chloride regulates cation transport and may be
involved in cellular signaling and CLIC4 expression has
reportedly been associated with Ca
2+
-induced differentiation of
keratinocytes (Suh et al., 2005;Suh et al., 2007).
Genes related to bone anabolism were also differentially
expressed in ST-treated hASCs. Bone anabolism, an important
part of osteogenic differentiation, is in part regulated by the
Wnt/β-catenin signaling cascade. The Wnt/β-catenin signaling
cascade is influenced by extracellular factors, including heparin
sulfate proteoglycans (Ling et al., 2009), and regulated in part by
periostin (POSTN)(Bonnet et al., 2012;Cho et al., 2012). Increased
induction of POSTN following ST challenge and osteogenic
differentiation was observed as compared to cells not challenged
with ST (Figure 9). Another Wnt activator, secreted frizzled-related
protein 1 (SFRP1), had predicted activation in the ST-treated hASCs,
and related transcription factors JUN and AXIN2 were upregulated
in differentiated and challenged ASCs [Supplementary Figure S3].
The Wnt/β-catenin canonical pathway overall appears to be
activated by ST treatment and drives MSCs towards pluripotency
[Supplementary Figure S3].
Osteogenic commitment is regulated by multiple genes in MSCs.
Upregulated in this dataset were the ephrin-B2 ligand (EFNB2) and
EPHB4 receptor (Figure 9). EFNB2 is involved in osteogenic
commitment and is required for the differentiation of osteoclasts
and osteoblasts in vivo (Tierney et al., 2013). Both the EPHB4
receptor and EFNB2 ligand are reportedly expressed on the
surface of MSCs (Tierney et al., 2013). Also involved in
proliferation and osteogenesis is COL1A2, the increased
expression of which was also detected in ST treated hASCs
(Figure 9). COL1A2 promotes cellular proliferation and
osteogenesis, a response in part regulated by ERK/AKT1 pathway
activation (Tsai et al., 2010).
The progression of osteoblasts is driven by activation of ERK
mitogen-activated protein kinase family (MAPK), which
phosphorylates related transcription factors (Salasznyk et al.,
2004). Gene expression data from ST challenged hASCs indicated
there was upregulation of integrins involved in MAPK1 activation,
as well as activation of intracellular signal transducer,
FIGURE 8
Downstream trends analysis of differentially expressed genes in hASCs induced towards osteogenesis post microbial challenge. The IPA regulation
z-score algorithm was used to identify biological functions expected to increase or decrease based on the gene expression changes observed in our
dataset. Predictions base on p-value and z-score; positive z-score implies an increase in the predicted function, a negative z-score a decrease (z-score ≥
2or≤−2 represented by black dotted lines). p-values ≤0.05 (orange dots determined by Fischer’s exact test), illustrate a significant association
between a given biological function and genes differentially expresse d in our dataset (p-value ≤0.05). Shapes associated with each gene name and broad
category indicate the general classification of the gene product, enzyme, growth factor, etc.
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FIGURE 9
Network displays interactions between genes involved in cellular movement, hematological system development and function, and inflammatory
response that were differentially expressed in hASCs induced towards an osteogenic lineage following S.T challenge. Upregulated genes are colored in
shades of red, downregulated in shades of green. Genes in white were inserted by IPA because they are connected to this network; dashed and solid lines
denote indirect and direct relationships between molecules. The IPA molecule activity predictor assessed the activity of molecules strongly
connected to this network; blue and orange colored molecules are predicted to have decreased and increased activity, respectively. Shapes associated
with each gene name and broad category indicate the general classification of the gene product, enzyme, growth factor, etc.
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phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)
[Supplementary Figure S4].
Genes related to immunomodulatory behaviors were also
detected as differentially expressed in this dataset of ST-treated
and differentiated hASCs (Figure 9). One immunomodulatory
related MSC gene, cadherin 11 (CDH11), showed increased
expression in treated hASCs. CDH11 expression is known to be
upregulated by TGFB and subsequently, increases calcium-
dependent cell-to-cell interactions in MSCs (Park et al., 2014).
Though CDH11 engagement on fibroblast-like synoyiocytes (FLS)
has been reported to produce inflammatory mediators IL6 and IL8
(Park et al., 2014), differential expression of these cytokines was not
detected in this transcriptomic dataset. The association with ST did
overall alter the genetic activity of MSCs in osteogenically induced
cells, diverging from the lack of visual cues indicating ST treatment
had no effect on differentiation. At the level of gene expression, ST
treatment did alter the behavior of induced MSCs towards pro-
proliferation pathways.
Discussion
Host-microbe interactions are important for the immune system,
cellular development, and the expansion of metabolic capabilities
(Visconti et al., 2019;Gomaa, 2020). The presence of microbes on
host tissues is an essential part of the host defense repertoire against
pathogens (Kim et al., 2017), and the interaction between microbes and
host cells is known to be important for cellular proliferation and
development (Furusawa et al., 2013). Though many microbe-host
relationships are beneficial to host health and function, pathogenic
microbes present currently unknown short and long-term
consequences to proximal host cells (O’Rourke and Kempf, 2019).
Many stem cells are primed for bacterial interactions and even take cues
from association with commensal bacteria (Nigro and Sansonetti, 2015;
Ferguson et al., 2021), but the unique proliferation and differentiation
properties of stem cells may also make them a target for pathogens
looking to evade the immune system and persist long-term (Granick
et al., 2012;Jain et al., 2020).
MSCsareonehostcelltypewhoseroleinrespondingto
inflammation and infections means interactions with pathogenic
microbes are common occurrence (Brandau et al., 2014;Mezey,
Nemeth, 2015). In light of their known immunomodulatory role in
vivo, MSCs have been investigated as potential therapeutics in instances
of drug-resistant pathogens (Yuan et al., 2014;Johnson et al., 2022).
(Yuan et al., 2014) illustrated the ability of bone marrow-derived MSCs
to increase clearance of methicillin-resistant S. aureus (MRSA) in a rat
model. Further, work by Maiti and colleagues showed that MSC
stimulation with MRSA not only resulted in changes to cell
proliferation but also the induction of inflammatory markers (Maiti
and Jiranek, 2014). MSCs may also act as a potential treatment for the
most drug resistant mycobacterial pathogen, Mycobacterium abscessus
(Kim et al., 2016). The potential application of MSCs in these
pathogenic settings makes it imperative there is a thorough
understanding of the effect of pathogens on MSC cellular activity.
Salmonella, the most common intestinal pathogens, is the
leading causes of foodborne illness (Wilson et al., 2020). The
prevalence of Salmonella as a pathogen, and specifically as an
inflammation-causing intracellular pathogen (Pilar et al., 2013),
makes it an important organism to study in the context of stem
cell activity. In this study, ASCs were vulnerable to microbial
infection in vitro with multiple Salmonella serovars, suggesting
pathogen susceptibility may be a common characteristic,
especially when considered in conjunction with the observations
of Kol et al. (2014). This investigation sought to expand on the
results of Kol et al. (2014) and to provide an insight into the specific
effect S. enterica ssp enterica serotype Typhimurium LT2 invasion
may have on the distinct immunomodulatory behaviors and
differentiation activity of human and goat adipose-derived MSCs.
Important to understanding the results found here is the co-
incubation method used in this study. Other studies have made use
of microbe-associated molecular patterns (MAMPs) like LPS or
long-term, continuous exposure to heat-inactivated pathogens to
study the stem cell response to infectious conditions (Brandau et al.,
2014;Ti et al., 2015;Huang et al., 2020). In this study, the use of
viable bacteria and short exposure time may better mimic the in vivo
physiological conditions in which MSCs interact with infiltrating
microbes. MSCs must migrate to sites of inflammation (Liu H et al.,
2018), where bacteria may be transient, leaving only a narrow
window of opportunity for direct MSC-microbe interaction. The
goat and human MSCs utilized in this study were infected with and
internalized ST within 60 min of co-incubation. Broadly, these ST
challenged MSCs had altered expression of prototypical genes
markers for inflammatory responses, apoptosis, and differentiation.
Responding to sites of inflammation is a key role of MSCs (Liu H
et al., 2018). During the course of infection, Salmonella initiates
epithelial inflammation and the rapid induction of pro-
inflammatory cytokines via calcium-mediated activation of
NFKB1 (Eckmann et al., 1993;Gewirtz et al., 2000). NFKB1
expression was not altered by ST treatment in this study, but
treatment with LPS did result in a significant increase in
expression for gASCs. NFKB1 is a central regulator of innate and
adaptive immune responses and plays a key role in the induction of
inflammation (Liu et al., 2017). NFKB1 proteins are available and
inactive; activity depends on phosphorylation-dependent
degradation of NFKB1 inhibitors, thus the lack of change in
mRNA expression is not unexpected (Oeckinghaus and Ghosh,
2009). It is possible the expression of NFKB1 was transiently
increased in response to ST co-incubation, but then expression
waned as other immunomodulatory genes were activated.
Out of the seven immunomodulatory markers surveyed in this
study, IL8 was the most significantly altered in both human and goat
ASCs as a response to ST co-incubation, confirming previous
observations (Gewirtz et al., 2000). IL8 can be rapidly expressed
and secreted by multiple cell types and is utilized as a clinical
biomarker of inflammation (Bernhard et al., 2021). Previous
studies showed a dose-dependent increase in IL8 by human bone
marrow-MSCs in response to LPS treatment (Rougier et al., 1998)
and by human intestinal epithelial stem cells in response to dietary
compound forskolin (Wang et al., 2018). IL8 is an important
cytokine for the recruitment of neutrophils to sites of
inflammation, but elevated levels of circulating IL8 in cancer
patients are associated with poorer health outcomes (Matsushima
and YangOppenheim, 2022). In conjunction with this finding,
IL8 has also been shown to have stimulatory effects on stem
cells, encouraging proliferation and differentiation related to
malignant tumor growth (Matsushima and YangOppenheim,
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2022). In the context of these previous findings, the increased
production of IL8 as a response to ST treatment in the stem cells
of this study is a potentially concerning result. The association of
increased IL8 with more negative host health outcomes indicates
Salmonella infection may drive host stem cells towards more
harmful outcomes, though these findings are preliminary and
require follow-up work to confirm in an in vivo setting.
Another pleiotropic cytokine involved in innate tissue response to
injury and maintenance of undifferentiated MSC status is IL6 (Tie et al.,
2019). In this study, a significant increase in IL6 expression was
observed in LPS-treated gASCs. A similar trend was noted in ST-
treatedgASCsandhASCs,althoughthis change was not statistically
significant. IL6 expression is known to decrease during osteogenic
differentiation (Pricola et al., 2009). While mature osteoblasts display
enhanced osteogenic differentiation, primitive MSCs experience a
decrease in proliferation following IL6 treatment (Cho et al., 2007).
This implies that the influence of IL6 on osteogenesis is complex and
dependent on the differentiation status of targeted MSCs (Cho et al.,
2007). The divergent responses of MSCs to IL6 by cellular status
suggests stem cell response to cytokine-promoting infections, like
ST, changes the secretion of small molecules that are capable of
crosstalk between inflammatory and differentiation pathways.
Exposure to MAMPs alone, without an associated viable pathogen,
is sufficient to influence ASC signaling. The MSC response to these
microbial components is mediated by Toll-like receptors (TLRs); the
hASCs used in this study are known to express TLR1-6 and TLR9 (Hwa
Cho et al., 2006). LPS, a key component of the Salmonella cell wall
(Hoshino et al., 1999), is a TLR4 agonist. LPS has been shown to
influence osteogenesis in hASCs and BM-MSCs by increasing
mineralization, ALP activity, and expression of osteogenic markers
(Hwa Cho et al., 2006;Cho et al., 2010;Raicevic et al., 2012;Fiedler et al.,
2013). These LPS-induced changes in differentiation may be in part
mediated by TLRs expressed on MSCs and dependent on the
aforementioned NFKB1 activation (Rougier et al., 1998;Bernhard
et al., 2021). The overall pattern of immunomodulatory gene
expression differed between the human and goat cells, indicating a
potential difference in activity between these two groups. Goat cells may
be a more easily accessible sample type, but the heightened expression of
immunomodulatory markers in response to LPS in comparison to the
humanMSCsisanareathatrequiresfurtherexplorationifgoatMSCs
are to act as model for human regenerative medicine.
While the conditions of this study did not lead to an observed
induction of TLR gene expression in MSCs, previous reports
highlight the role of TLR activation in MSC physiology. In non-
induced mouse BM-MSCs, TLR2 activation inhibited spontaneous
adipogenic differentiation and increased osteogenesis, but inhibited
trilineage differentiation in induced cultures (Pevsner-Fischer et al.,
2007). Osteogenic markers in non-induced ST-treated ASCs were
upregulated in our study, supporting these previous observations in
BM-MSCs. Furthermore, TLR-activated MSCs recruit immune cells;
TLR-activated macrophages secrete oncostatin M, a cytokine that
induces osteogenesis and inhibits adipogenesis in BM-MSCs
(Guihard et al., 2012). Providing further evidence for a link
between microbe induced TLR signaling and osteochondrogenic
pathway induction are the diminished capabilities of myeloid
differentiation primary response 88 (MYD88)deficient MCSs.
MSCs deficient in MYD88, crucial for TLR signaling (Kawai
et al., 1999), lack both osteogenic and chondrogenic potential
(Pevsner-Fischer et al., 2007), supporting the interconnectedness
of MSC immunomodulatory and differentiation activity.
Broader examination of the transcriptome of ST treated hASCs
in this study suggests a physiological shift in favor of cell survival and
proliferation. Previous studies have confirmed Salmonella is able to
exist intracellularly long term, evading the host defense through
shelter in immune-privileged cells (Ross et al., 2014;Goldberg et al.,
2018). Caco2 cells in this study displayed a marked upregulation in
genes regulating the apoptotic pathway when exposed to ST, while
the infected hASCs showed no difference in the expression of these
same genes. Under oxidative stress, like that surrounding an ST
infection, MSCs display a reduced ability to repair tissue and an
increased propensity towards senescence (Zhu et al., 2006;Kim et al.,
2010). These stress conditions decrease the MSC capacity for
osteogenesis in favor of adipogenic commitment (Kim et al.,
2010). Through upregulation of redox mediators, hASCs respond
to and mitigate oxidative stress, helping to ensure cell viability,
multipotency, and promote immune suppression (Hu et al., 2018).
As versatile immune privileged cells, MSCs presented with a
microbial challenge may function as a safeguard by generating an
anti-inflammatory environment, creating an atmosphere conducive
for infection clearance by resident phagocytes (Evans et al., 2021).
Results of this study indicate ST infection may induce the expression
of immunomodulatory genes in MSCs, but more interestingly
indicate that ST association decreases apoptotic responses,
ultimately driving MSC towards proliferation, differentiation, or
senescence rather than cell death. However immuno-privileged stem
cells may be, it is unlikely that the hASC response to infection is
without physiological consequences. The influence of inflammatory
mediators, which come about in response to such an infection, on
lineage commitment appears to prime hASCs towards a pro-
osteogenic phenotype.
ST treatment of hASCs had a significant effect on osteogenic
differentiation. Consistent with findings by Fiedler et al. (2013),
there was an observed increase in ALPL expression for
osteogenically-induced and ST-treated hASCs. Increased ALPL
expression is also consistent with previous work showing
increased expression in LPS-treated hASCs at 10 days post-
osteogenic induction (Hwa Cho et al., 2006). gASCs in this study
did not have any detectable ALPL expression. Cells were harvested
21 days post-induction, when the mineralization phase was likely
occurring (Owen et al., 1990), and ALPL expression at that point
may have already decreased (Owen et al., 1990;Choi et al., 1996).
ALPL expression is a marker of early osteogenesis and MSCs lacking
this crucial gene experience alterations in their cellular fate (Liu W
et al., 2018). While differentiation stage is a known effector of ALPL
expression, cell density is also factor (Mao et al., 2016). (Mao et al.,
2016) illustrated ALPL expression was increased in higher density
stem cell environments, indicating cell-to-cell signaling is also an
important regulator of differentiation status. ALPL is a key marker of
osteogenesis, but consistent trends were not seen across the human
and goat cells or across treatments in this work. The lack of this early
osteogenic marker may be attributable more to the time of collection
rather than an actual deficiency of ALPL expression overall.
Coinciding with the lack of early osteogenic marker ALPL,
upregulation of SPP1 (a late marker of osteogenesis) (Owen
et al., 1990;Cowles et al., 1998), was observed in ST-treated
gASCs. SPP1 was not significantly upregulated in induced and
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ST-treated cells; it is possible that the osteoinductive effect of ST-
treatment in induced ASCs was masked, as the medium contained
additives that already strongly induce osteogenesis. In addition to
osteogenic commitment, the 6-glycosylated phosphoprotein
SPP1 has a significant role in cellular stress and immunity.
SPP1 is an inflammatory mediator and is reported to have anti-
inflammatory effects in acute colitis (Wang and Denhardt, 2008)
and SPP1-deficient mice have an impaired ability to clear Listeria
monocytogenes (Ashkar et al., 2000). Conversely, in chronic disease
states increased SPP1 expression is thought to have the opposite
effect, as it decreases survival time for patients with lung cancer and
is associated with increased Irritable Bowel Disease symptoms (Miao
et al., 2021;Nguyen et al., 2022). The observation in this study of
increased SPP1 expression in ST-treated but uninduced gASCs
implies a complex microbial-dependent response in MSCs. Taken
along with previous findings and the other results of this study, the
upregulation of SPP1 in response to microbe-induced inflammation
may drive affected MSCs towards osteogenic commitment.
Differential markers of expression following ST treatment were
observed in non-induced ASCs. We observed a concomitant
decrease in chondrogenic differentiation in response to ST
treatment, as illustrated by a decrease in SOX9 expression in
ASCs. SOX9 is required for commitment to chondrogenic lineage
(Akiyama, 2008). To our knowledge, this is the first report on the
direct effect of bacterial association on MSC chondrogenesis.
Osteogenic markers were observed to increase, important to note
as osteogenesis and chondrogenesis are tightly coupled processes
(Joyce et al., 1990), both are regulated by proteins in the TGFβ
superfamily (Wan and Cao, 2005). Though, an inverse relationship
between osteogenic and chondrogenic differentiation has been
demonstrated, where microRNAs targeting genes important for
osteogenesis were upregulated during chondrogenesis and vice
versa (Suomi et al., 2008). The demonstrated differential
regulation of trilineage differentiation markers in non-induced
but pathogen-treated cells highlights the potential direct influence
of microbes on ASC lineage commitment.
ST association in this study was shown to impact key modulators
of both apoptosis and trilineage differentiation in adipose-derived
MSCs. Treatment with the intracellular pathogen ST resulted in
increased expression of pro-proliferative MSC pathways and of
mineralization related SPP1. Pathways dictating the response of
MSCs to injury, microbial products, and inflammation intersect
with those regulating cellular differentiation, providing a route for
pathogen influence on lineage commitment. However, the extent to
which pathogens can influence MSC differentiation and the
mechanisms responsible for this potential control have yet to be
fully described, though it is clear that microbial reprogramming of
host cells is a possible consequence of association. With MSCs
poised as a potential therapeutic in regenerative medicine, where
pathogen association is a likely factor, the consequences of
pathogenic interactions on MSC activities must be further
investigated for safe application and use.
Data availability statement
The data are available on NCBI in the 100K Pathogen Genome
BioProject assession number of PRJNA239251.
Author contributions
NM-F:Conceptionanddesign,collectionand/orassemblyofdata,
data analysis and interpretation, manuscript writing; CS: Assembly of
data, data analysis and interpretation, manuscript writing; SF: Conception
and design, collection and/or assembly of data, data analysis and
interpretation, manuscript writing; DS: Informatics, data analysis; PD:
Collection and/or assembly of data; JD: Collection and/or assembly of
data; NK: Administrative support, collection and/or assembly of data; AK:
Collection and/or assembly of data, data analysis and interpretation; DB:
Conception and design, provision of study material; MH: Data analysis
and interpretation, manuscript writing; JM: Conception and design,
financial support, administrative support, provision of study material,
data analysis and interpretation, manuscript writing, final approval of
manuscript.; BW: Conception and design, financial support,
administrative support, provision of study material, data analysis and
interpretation, manuscript writing, final approval of manuscript.
Funding
Jastro Shields Research Fellowship—funded lead author USDA-
CREES W2171 Regional Research Project—funded resources 100K
Pathogen Genome Project—funded resources.
Acknowledgments
We would like to thank Azarene Foutouhi for her helpful
discussions and assistance with reagents during the progression
of this project. We also thank Majlis Amanah Rakyat (Malaysia) and
the Jastro Shields Research Fellowship, and the USDA-CREES
W2171 Regional Research Project for funding a portion of the
work described in this paper (NM-F).
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fcell.2023.1077350/
full#supplementary-material
Frontiers in Cell and Developmental Biology frontiersin.org17
Mohamad-Fauzi et al. 10.3389/fcell.2023.1077350

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