Abstract

   Changes in the human gastrointestinal microbiome are associated with
   several diseases. To infer causality, experiments in representative
   models are essential, but widely used animal models exhibit
   limitations. Here we present a modular, microfluidics-based model
   (HuMiX, human–microbial crosstalk), which allows co-culture of human
   and microbial cells under conditions representative of the
   gastrointestinal human–microbe interface. We demonstrate the ability of
   HuMiX to recapitulate in vivo transcriptional, metabolic and
   immunological responses in human intestinal epithelial cells following
   their co-culture with the commensal Lactobacillus rhamnosus GG (LGG)
   grown under anaerobic conditions. In addition, we show that the
   co-culture of human epithelial cells with the obligate anaerobe
   Bacteroides caccae and LGG results in a transcriptional response, which
   is distinct from that of a co-culture solely comprising LGG. HuMiX
   facilitates investigations of host–microbe molecular interactions and
   provides insights into a range of fundamental research questions
   linking the gastrointestinal microbiome to human health and disease.
     __________________________________________________________________

   Inline graphic Research on the interactions between the gut microbiota
   and human cells would greatly benefit from improved in vitro models.
   Here, Shah et al. present a modular microfluidics-based model that
   allows co-culture of human and microbial cells followed by 'omic'
   molecular analyses of the two cell contingents.
     __________________________________________________________________

   The human microbiome is emerging as a key player governing human health
   and disease[46]^1,[47]^2. Recent high-resolution molecular analyses
   have linked microbial community disequilibria (dysbiosis), primarily in
   the gastrointestinal tract (GIT), to several idiopathic diseases,
   including diabetes[48]^3, obesity[49]^4, inflammatory bowel
   disease[50]^5, cancer[51]^6 and, most recently, neurodegenerative
   diseases[52]^7. However, a detailed understanding of the fundamental
   molecular mechanisms underlying host–microbe interactions and their
   potential impact on immune regulation, drug metabolism, nutrition and
   infection remain largely elusive[53]^8,[54]^9. More specifically,
   patterns of association between distinct microorganisms, their traits
   and disease states resolved using ‘meta-omics' do not allow direct
   causal inference, and thus experimental validation is essential[55]^10.
   For this, robust experimental models that allow the systematic
   manipulation of variables are required to test the multitude of
   hypotheses that arise from the generated high-dimensional data
   sets[56]^10. Animal models used in human microbiome research are
   physiologically not representative[57]^11. In vitro models that mimic
   microbial processes along the GIT allow the simulation of luminal
   microbial communities[58]^12,[59]^13,[60]^14 and/or mucus-adherent
   microbiota[61]^15,[62]^16, but typically do not include provisions for
   assessing human host responses.

   Host responses to GIT microbiota have traditionally been assessed
   following the exposure of cultured human cells to bacteria-free
   supernatants[63]^17 or through short-term direct-contact co-cultures
   involving, for example, Transwell systems[64]^18, microcarrier
   beads[65]^19 or mouse gut organoid models[66]^20. Recent advances in
   multi-layer microfluidics have led to the development of a
   gut-on-a-chip model that includes a provision for peristalsis[67]^21
   and that has been used to study intestinal inflammation on a
   chip[68]^22. These human–microbial co-culture approaches are, however,
   limited in their scope because they only allow experiments with
   commensal and/or mutualistic microorganisms growing under aerobic
   conditions[69]^21,[70]^22. To overcome these limitations, the recently
   introduced host–microbiota interaction (HMI) module, which interfaces
   with the in vitro simulator of the human intestinal microbial ecosystem
   model, incorporates a semi-permeable membrane between co-cultured human
   enterocytes and bacteria[71]^23. Through inclusion of a partitioning
   membrane between the human and microbial culture chambers, the HMI
   module allows the co-culture of intestinal cells with complex microbial
   communities under microaerophilic conditions[72]^23. This two-chamber
   design requires intermittent perfusion of the human cell culture medium
   to the apical surface of the epithelial cells, which is not
   representative of the continuous supply of nutrients to the basal
   membrane seen in vivo[73]^24,[74]^25,[75]^26. The lack of modularity
   makes it difficult to include additional cell types of relevance to the
   GIT in the HMI module, for example, immune cells. Furthermore, it
   prevents the extraction of biomolecular fractions from the individual
   co-cultured cell contingents following specific experimental regimes
   and thereby renders the HMI module incompatible with downstream
   high-resolution molecular analyses. Although the HMI module currently
   is the most representative in vitro model of gastrointestinal
   host–microbial interactions, there still remains an unmet need for a
   modular, representative in vitro model of the gastrointestinal
   human–microbe interface.

   Here we present a modular microfluidics-based human–microbial
   co-culture model, HuMiX, which overcomes the majority of the
   limitations of existing in vitro models and allows the partitioned yet
   proximal co-culture of representative human and microbial cells
   followed by downstream molecular analyses of the individual cell
   contingents. More specifically, we demonstrate the viable co-culture of
   differentiated human epithelial cells (Caco-2) with either a
   facultative anaerobe, Lactobacillus rhamnosus GG (LGG), grown solely
   under aerobic or anaerobic conditions, or grown in combination with an
   obligate anaerobe, Bacteroides caccae, under anaerobic conditions.
   Co-culture experiments were followed by detailed molecular analyses of
   the effects of the induced co-cultures on the physiology of human and
   bacterial cells. Comparison of our results with published in vitro and
   in vivo data sets demonstrates the ability of HuMiX to representatively
   mimic the gastrointestinal human–microbe interface.

Results and Discussion

Design and characterisation of the HuMiX model

   To overcome the limitations of existing in vitro models[76]^10,[77]^23,
   we developed a modular microfluidics-based device, which allows the
   establishment of a model of the gastrointestinal human–microbe
   interface, named HuMiX (human-microbial crosstalk) ([78]Fig. 1a–c). The
   device consists of three co-laminar microchannels: a medium perfusion
   microchamber (henceforth referred to as the ‘perfusion microchamber'),
   a human epithelial cell culture microchamber (henceforth referred to as
   the ‘human microchamber') and a microbial culture microchamber
   (henceforth referred to as the ‘microbial microchamber'; [79]Fig. 1a,b;
   [80]Supplementary Fig. 1a,b). Each microchamber has a dedicated inlet
   and outlet for the inoculation of cells as well as for the precise
   control of physicochemical parameters through the perfusion of laminar
   streams of dedicated culture media ([81]Fig. 1d,e). Dedicated outlets
   provide means to collect eluates from the individual chambers for
   downstream characterisation ([82]Fig. 1d; [83]Supplementary Fig. 1a,b).
   By juxtaposing the human and microbial cell contingents at a distance
   of 0.5-1 mm across a separatory nanoporous membrane, the HuMiX model is
   representative of a healthy intact epithelial barrier[84]^10
   ([85]Supplementary Note 1). Furthermore, the model integrates oxygen
   sensors (optodes) for the real-time monitoring of the dissolved oxygen
   concentrations within the device ([86]Fig. 1a,b,d; [87]Supplementary
   Fig. 1c). Given the challenges associated with measuring
   transepithelial electrical resistance (TEER) on a chip[88]^27, a
   specially designed version of HuMiX, which allows the insertion of a
   commercial chopstick style electrode (STX2; Millipore), was fabricated
   to monitor TEER for the characterisation of cell growth and
   differentiation within the device ([89]Fig. 1d; [90]Supplementary Fig.
   1d).

Figure 1. The HuMiX model.

   [91]Figure 1
   [92]Open in a new tab

   (a) Conceptual diagram of the HuMiX model for the representative
   co-culture of human epithelial cells with gastrointestinal microbiota.
   (b) Annotated exploded view of the HuMiX device. The device is composed
   of a modular stacked assembly of elastomeric gaskets (thickness:
   700 μm) sandwiched between two polycarbonate (PC) enclosures, and each
   gasket defines a distinct spiral-shaped microchannel with the following
   characteristics: length of 200 mm, width of 4 mm and height of 0.5 mm,
   amounting to a total volume of 400 μl per channel. Semi-permeable
   membranes affixed to the elastomeric gaskets demarcate the channels.
   The pore sizes of the membranes were chosen for their intended
   functionality. A microporous membrane (pore diameter of 1 μm), which
   allows diffusion-dominant perfusion to the human cells, is used to
   partition the perfusion and human microchambers. A nanoporous membrane
   (pore diameter of 50 nm) partitions the human and microbial
   microchambers to prevent the infiltration of microorganisms, including
   viruses, into the human microchamber. (c) Photograph of the assembled
   HuMiX device (scale bar, 1 cm). (d) Diagram of the experimental set-up
   of the HuMiX model with provisions for the perfusion of dedicated oxic
   and anoxic culture media as well as the monitoring of the oxygen
   concentration and transepithelial electrical resistance. The oxygen
   concentration in the anoxic medium is maintained at 0.1% by
   continuously bubbling the medium with dinitrogen gas. (e) Diagrammatic
   overview of the HuMiX co-culture protocol.

   Following the conceptualisation and engineering of the HuMiX model
   ([93]Supplementary Note 1), we developed an optimised protocol for the
   co-culture of human epithelial cells with gastrointestinal microbes
   ([94]Fig. 1e). The human cell line and bacterial isolates used for the
   co-culture experiments were originally obtained from the human large
   intestine and, together with the physical characteristics of the model
   ([95]Supplementary Note 1), allowed the assembly of a model
   representing the human–microbe interface of the human colon.
   Nonetheless, given the modularity of the device and the flexibility of
   its set-up, other sections of the human GIT may also be modelled
   following appropriate modifications to the presented model
   ([96]Supplementary Note 1). The protocol includes an extensive
   sterilisation and handling procedure that enables the culture of human
   epithelial cells (Caco-2) in antibiotic-free DMEM medium to allow their
   subsequent co-culture with bacteria in HuMiX. The Caco-2 cell line was
   chosen because it represents the most widely used model for the human
   gastrointestinal epithelial barrier, as it exhibits essential
   functional and physiological traits of the intestinal
   epithelium[97]^25,[98]^28. The differentiation of the epithelial cells
   was evaluated by measuring TEER of the Caco-2 cell monolayer ([99]Fig.
   2a) and through microscopic observation of the expression of the tight
   junction protein occludin ([100]Fig. 2b).

Figure 2. In vitro co-culture of human and microbial cells inside the HuMiX
device.

   [101]Figure 2
   [102]Open in a new tab

   (a) Characterisation of epithelial cell monolayer formation in HuMiX in
   comparison with the standard Transwell system. In both cases, the
   transepithelial electrical resistance (TEER) was determined on
   7-day-old Caco-2 cell layers using standard chopstick electrodes. The
   error bars indicate the s.e.m. (n=3). * Indicates a statistically
   significant difference (paired Student's t-test, P<0.05). (b)
   Immunofluorescent microscopic observation of the tight junction protein
   occludin (green) in Caco-2 cells following 24 h of co-culture with LGG
   grown under anaerobic conditions. The cell nuclei are stained with
   4,6-diamidino-2-phenylindole and appear in blue. (c,d) Viability
   assessment of Caco-2 cells and LGG at 24 h post co-culture,
   respectively. The cells were stained using a live–dead stain and
   observed using a fluorescence microscope. The live cells appear in
   green, whereas the dead cells appear in red. The collagen-coated
   microporous membrane does support the attachment and proliferation of
   the Caco-2 cells, whereas the mucin-coated nanoporous membrane provides
   a surface for the attachment and subsequent proliferation of the
   bacteria. (e) Representative electropherogram of an RNA fraction
   obtained from the Caco-2 cells co-cultured in HuMiX. The RNA Integrity
   Number (RIN) is provided. (f) Sampled eluates from the HuMiX device
   following a 24 h co-culture with LGG. (g) Oxygen concentration profiles
   within the perfusion and microbial microchambers upon initiation of the
   co-culture with LGG. ⋄indicates the pre-inoculation oxygen
   concentration of 2.6% in the microbial microchamber. (h) The relative
   abundances (in %) of Lactobacillus spp. and Bacteroides spp. following
   24 h of co-culture with Caco-2 cells determined by 16S rRNA gene
   amplicon sequencing (n=4). Scale bars, 10 μm (b–d).

   Following the establishment of differentiated Caco-2 cell monolayers,
   we initiated co-cultures of these cells with LGG grown in anoxic DMEM
   medium ([103]Supplementary Fig. 2a). LGG of the phylum Firmicutes was
   chosen, as it represents a commensal facultative anaerobic bacterium
   originally isolated from the human GIT[104]^29,[105]^30,[106]^31.
   Importantly, extensive data exist on its physiological impacts on
   mammalian mucosal tissues in vivo[107]^32,[108]^33,[109]^34. The
   developed co-culture protocol ([110]Fig. 1e) first results in the
   establishment and maintenance of an epithelial cell monolayer. The
   Caco-2 cells adhere to the collagen-coated microporous membrane
   ([111]Fig. 1a,e; [112]Supplementary Note 1), proliferate and
   differentiate into confluent cell monolayers that form tight junctions
   between adjacent cells ([113]Fig. 2a,b). The diffusion-based perfusion
   of the cell culture medium to the basal side of the Caco-2 cells
   through the microporous membrane mimics the intestinal blood supply and
   provides shear-free conditions accelerating the growth of the human
   cells[114]^35.

   Co-culture with LGG was initiated after 7 days of epithelial cell
   culture (day 9 of the HuMiX co-culture protocol; [115]Figs 1e and
   [116]2a,b). This first involved the introduction of anaerobically grown
   LGG cell suspensions into the microbial microchamber through the port
   on a three-way connector ([117]Fig. 1d).

   Following the co-culture, the modular device architecture allows access
   to individual cell contingents on disassembly, whereby one half of each
   of the cell contingents can be used for microscopic evaluation and the
   other half can be used for the extraction of intracellular biomolecules
   (DNA, RNA, proteins and metabolites) for subsequent high-resolution
   molecular analyses[118]^36. The viability of the co-cultured
   contingents was determined via live–dead staining and subsequent
   fluorescence microscopy, demonstrating that no apparent cytotoxic
   effects were induced in either cell contingent following their
   co-culture ([119]Fig. 2c,d). RNA electropherograms confirmed that
   high-quality biomolecular fractions were obtained from the individual
   co-cultured contingents ([120]Fig. 2e).

   Due to the laminar flow profiles within the microchambers, eluate
   samples ([121]Fig. 2f) can be recovered from each microchamber, thereby
   providing a means to continually monitor the effects of the co-culture
   on the individual co-cultured cell contingents through various
   analyses, such as the use of cytokine assays and metabolomic profiling.
   Visible differences in the eluates from the three proximal
   microchambers support the notion of distinct microenvironments in each
   of the microchambers ([122]Fig. 2f).

   Integrated oxygen sensors (optodes) allow continuous monitoring of the
   dissolved oxygen concentrations in the perfusion and microbial
   microchambers ([123]Fig. 1e; [124]Supplementary Fig. 1c). The
   simultaneous perfusion of oxic (21% dissolved O[2]) and anoxic (0.1%
   O[2]) media through the perfusion microchamber and the microbial
   microchamber, respectively, allowed the establishment and maintenance
   of an oxygen gradient representative of the in vivo situation
   ([125]Fig. 2g). The measured dissolved oxygen concentrations in the
   perfusion microchamber stabilised to 5.43±0.137% for the final 12 h of
   co-culture between the Caco-2 cells and LGG, which is comparable to the
   actual recorded concentrations in human intestinal tissues, that is,
   4.6% (ref. [126]^37; [127]Fig. 2g). The oxygen profiles in the
   microbial microchamber were characterised by a rapid decrease in the
   oxygen concentration (from 2.6 to ≤0.8% of dissolved oxygen), following
   an intermittent spike due to the introduction of small amounts of
   oxygen into the microbial microchamber during the inoculation process
   of LGG ([128]Fig. 2g). The established anoxic conditions are analogous
   to those observed in vivo between the mucus layer and the luminal
   anaerobic zone (∼0.88%; ref. [129]38) and such oxygen concentrations
   have been reported to be favourable for the growth of diverse
   microbiota, including obligate anaerobes[130]^39. The gradient of
   oxygen in the HuMiX model was maintained through the continuous
   perfusion of anoxic media (0.1%) into the microbial microchamber and
   further shaped by the consumption of oxygen by Caco-2 cells and the
   facultative anaerobe LGG ([131]Fig. 2g).

   Through the consumption of oxygen, anaerobic niches are established in
   the microbial microchamber, which subsequently allow colonisation of
   the microbial microchamber by obligate anaerobes[132]^40. To showcase
   the ability of HuMiX to sustain culture of an obligate anaerobe, we
   initiated co-cultures using a simple microbial consortium comprising
   LGG in combination with B. caccae ([133]Supplementary Figs 2b and 3).
   B. caccae was chosen as it represents an obligate anaerobic commensal
   that belongs to the phylum Bacteroidetes, the other dominant phylum
   apart from the Firmicutes (LGG) constituting the human GIT
   microbiome[134]^41. Both organisms were inoculated in equal starting
   proportions (optical density (OD) ∼1) and co-cultured with Caco-2 cells
   for 24 h ([135]Supplementary Fig. 2b). The consortium was sustained via
   continuous perfusion of anoxic DMEM medium. The consortium structure
   was determined using 16S rRNA gene amplicon sequencing after 24 h of
   co-culture, and the relative abundances of Bacteroides spp. and
   Lactobacillus spp. were found to be 69 and 31%, respectively ([136]Fig.
   2h). These results confirm the ability of the HuMiX model to support
   the growth of an obligate anaerobic microbial strain. Human cells still
   exhibited tight junctions ([137]Supplementary Fig. 3a) and both
   contingents were viable ([138]Supplementary Fig. 3b,c). It follows from
   these experiments that the inclusion of more complex communities into
   the HuMiX model is possible but goes beyond the scope of the reported
   proof-of-concept experiments.

   Furthermore, to demonstrate the ability to incorporate other cell types
   within HuMiX, we cultured non-cancerous colonic cells, i.e., CCD-18Co,
   in the human microchamber ([139]Supplementary Fig. 4a,b). In addition,
   to demonstrate that HuMiX can be used in a three-layered set-up for
   addressing specific research questions, we cultured primary CD4+ T
   cells in the perfusion microchamber of HuMiX ([140]Supplementary Fig.
   4c,d). The primary CD4+ T cells were cultured in the absence
   ([141]Supplementary Fig. 4c,d) or presence of LGG ([142]Supplementary
   Fig. 4e,f) over 48 h and did not exhibit any significant differences in
   terms of cell viability. These experiments highlight the potential of
   HuMiX to be used for investigating the cellular mechanisms involved in
   the interplay between GIT bacteria and different human cell types.

   In summary, HuMiX exhibits the following essential characteristics: (1)
   modular microfluidic device architecture consisting of three
   microchambers engineered to facilitate the proximal co-culture of human
   and microbial cells; (2) ability to perfuse the device with dedicated
   culture media to allow the establishment of aerobic conditions for
   human cell culture and anaerobic conditions for GIT bacteria; (3)
   real-time monitoring of oxygen concentrations; (4) easy access to the
   individual cell contingents following specific experimental regimes;
   and (5) compatibility with end point microscopic assays as well as
   high-resolution multi-omic analyses.

HuMiX recapitulates in vivo responses

   Given the demonstrated ability to establish conditions representative
   of the human GIT in HuMiX, we conducted further validation experiments
   to assess the human cellular responses with respect to different
   co-culture conditions in HuMiX. LGG has been widely used in several
   human clinical trials aimed at understanding the efficacy of probiotic
   treatments in humans[143]^32,[144]^33. More specifically, gene
   expression differences have been documented in human intestinal mucosal
   biopsy samples after the administration of LGG to either healthy
   subjects[145]^32 or as a therapeutic supplement for male individuals
   suffering from esophagitis[146]^33. Therefore, to validate our in vitro
   co-culture approach, we performed detailed experiments involving the
   co-culture of Caco-2 cells maintained under aerobic conditions with LGG
   cultured under anaerobic conditions ([147]Supplementary Fig. 2a) and
   compared the resulting Caco-2 gene expression data with reference data
   from clinical studies[148]^32,[149]^33. For this, total RNA was first
   extracted from Caco-2 cells following their co-culture with LGG grown
   under anaerobic conditions as well as their corresponding LGG-free
   controls (anoxic medium was perfused through the microbial
   microchamber, but no bacteria were inoculated, [150]Supplementary Fig.
   2a). The RNA was then subjected to DNA microarray-based messenger RNA
   and microRNA (miRNA) profiling.

   Overall, we identified 208 genes that were differentially expressed
   following co-culture with LGG grown under anaerobic conditions (fold
   change (FC)>1.5 and equivalently with swapped conditions for decreased
   expression, P<0.01, empirical Bayes moderated t-statistic (BtS);
   [151]Fig. 3a; [152]Supplementary Fig. 5a; [153]Supplementary Table 1).
   Given the lack of detail regarding the identities of the majority of
   genes found to be differentially expressed in vivo, we limited our
   subsequent analyses and discussions to genes that were explicitly
   highlighted in the in vivo clinical studies and that showed
   statistically significant differences in our study ([154]Table 1).
   Among the top differentially expressed genes, we validated the gene
   expression of four genes—ccl2, pi3, egr1 and mt2a—using quantitative
   PCR with reverse transcription (RT–qPCR) analyses. The RT–qPCR results
   showed differential expression patterns analogous to those observed in
   the microarray data ([155]Supplementary Fig. 5b).

Figure 3. Validation of the HuMiX model by transcriptomic, metabolomics and
immunological analyses.

   [156]Figure 3
   [157]Open in a new tab

   (a) Heat map highlighting the top 30 differentially expressed genes and
   miRNAs in Caco-2 cells co-cultured with LGG growing under anaerobic
   conditions compared with their corresponding LGG-free controls (n=3).
   The threshold parameters used were FC>2 and P<0.01, as determined using
   the empirical Bayes moderated t-statistic[158]^65. Ranking was based on
   the π-values calculated using the log-fold changes and P values (BtS).
   An average linkage hierarchical clustering with the Euclidean distance
   metric was performed to determine the ordering of the genes. (b)
   Extracellular CCL20/MIP3A and IL-8 cytokine levels before and 24 h
   after the initiation of co-culture with LGG. Eluate samples were
   obtained from the perfusion microchamber (n=3). (c) Heat map of
   intracellular metabolites from Caco-2 cells co-cultured with LGG
   growing under anaerobic conditions compared with their corresponding
   LGG-free controls (n=3). The threshold parameter used was P<0.1 (StT).
   An average linkage hierarchical clustering with the Euclidean distance
   metric was performed to determine the ordering of the metabolites.

Table 1. Differentially expressed genes in Caco-2 cells following their
HuMiX-based co-culture with LGG in comparison with in vivo data.

   Gene In vitro HuMiX-based co-cultures
     __________________________________________________________________

   In vivo data
     __________________________________________________________________

   Function
     LGG culture conditions Expression logFC P value Subject Expression
   Reference
   EGR1 Only differentially expressed when LGG growing under anaerobic
   conditions Down −2.56 0.0012 Human Down [159]33 Transcription
   regulation, transcription factor activity for the regulation of cell
   proliferation and apoptosis, anti-cancer effect and IL-8 suppression
   CCL2   Up +1.51 0.0005 Human Up [160]32 Chemotactic factor that
   attracts monocytes and basophils and binds to the chemokine receptors
   CCR2 and CCR4
   SLC9A1   Down −0.29 0.0168 Human Down [161]32 Signal transduction,
   regulation of pH homeostasis, cell migration, cell volume and
   anti-inflammatory effect
   UBD   Up +0.61 0.0261 Human Up [162]33 Proteasomal degradation,
   cytokine response, antimicrobial response and apoptosis

   ELF3 Differentially expressed when LGG growing under both anaerobic and
   aerobic conditions Up +0.55 0.0022 Human & GF Piglet Down
   [163]33,[164]34 ets family member, epithelial-specific function,
   transcriptional mediator of angiogenesis during inflammation and
   epithelial cell differentiation
   CXCR4   Up +0.94 0.0019 Human Up [165]33 Chemotaxis, cell arrest,
   angiogenesis, cell survival, maintenance of the epithelial barrier
   function and HIV-1 co-receptor
   MYBL2   Down −0.26 0.0446 Human Down [166]33 Anti-apoptopic function,
   regulation of cell cycle and transcription, and epithelial cell
   differentiation
   PIM1   Up +1.13 0.0039 Human Up [167]32 Cell survival, cell
   proliferation, cell growth and signal transduction

   CYP1A1   Up +0.82 0.0090 Human Up [168]33 Drug metabolism and
   xenobiotic transformation
   GADD45B   Up +0.60 0.0134 Human Down [169]33 Cell growth and apoptosis
   PILRB   Down −0.45 0.0374 Human Up [170]33 Receptors involved in the
   regulation of the immune system and cellular signalling
   CDK9   Down −0.22 0.0467 Human Up [171]33 Cell proliferation,
   regulation of the cell cycle, and transcription elongation factor

   SOX4 Only differentially expressed when LGG growing under anaerobic
   conditions Down −0.31 0.0295 GF piglet Unspecified [172]34
   Transcription factor, regulation of cell fate and apoptosis pathway,
   and prognostic marker in colon and gastric cancer
   CEBPA   Up +0.55 0.0172 GF piglet Unspecified [173]34 Transcription
   factor, cell cycle regulation and regulation of metallothioneins
   PTGS2   Up +1.13 0.0145 GF piglet Down [174]34 Prostaglandin
   biosynthesis and metabolism, inflammation, and mitogenesis
   IGFBP2   Up +0.5 0.015 GF piglet Up [175]34 Regulation of IGF-mediated
   growth and developmental rates
   GSTA1   Down −0.5 0.005 GF piglet Down [176]34 Detoxification of
   carcinogens, drugs, environmental toxins and products of oxidative
   stress
   CTNNB1   Up +0.54 0.003 GF piglet Up [177]34 Regulation of cell growth
   in addition to the creation and maintenance of epithelial layers
   TPD52   Up +0.42 0.008 GF piglet Up [178]34 Molecular marker in human
   cancer and target for immunotherapy
   [179]Open in a new tab

   FC, fold change; GF, germ free; LGG, Lactobacillus rhamnosus GG; IL-8,
   interleukin-8; IGF, insulin-like growth factor

   References indicating the functions of the highlighted genes are