Page 31 - Read Online

Huang et al. Microbiome Res Rep 2024;3:30 https://dx.doi.org/10.20517/mrr.2024.14 Page 9 of 13

in GuMI-FP vs. GuMI-NB [Figure 4A, Supplementary Tables 3 and 4]. In contrast, only 27 TF genes were
changed considerably in GuMI-NB vs. Static culture, despite a similar number of TF genes (1,306) being
detected [Figure 4B, Supplementary Tables 3 and 4]. Among all changed TF genes, only 10 TFs are specific
in GuMI-NB. These results suggest that despite the apical compartment being switched from Static and
oxygenated to a fluidic and anerobic environment, it induces relatively mild effects on the transcriptome of
colonic epithelial cells. In contrast, F. prausnitzii specifically altered the expression of 243 TFs in the human
colonic epithelial cells in GuMI [Figure 4C]. Many of these TFs belong to the families involved in cell
metabolism (bHLH) and cell differentiation (Homeobox). For instance, the disheveled binding antagonist
of beta-catenin 3 (DACT3) and FOSL1 are genes linked to canonical and non-canonical Wnt signaling
pathways essential for cell differentiation. Here, we found that DACT3 and FOSL1, transcription factor
genes JUNB and JUND in colonic epithelial cells were significantly increased by F. prausnitzii in GuMI-FP.

Interestingly, we also found that interferon transcription factors IRF1, IRF6, and IRF7 were significantly
increased in GuMI-FP. IRF7 is a master transcription factor of Type I interferon-dependent immune
responses , making it a potential target for infection control . Notably, early growth response 1 (EGR1)
[30]
[29]
transcription is significantly reduced by F. prausnitzii in GuMI [Supplementary Tables 3 and 4].
DISCUSSION
We demonstrated that the GuMI microphysiological system is feasible for multiple donors from healthy
individuals and patients of IBD. Using metabolomics, we found that F. prausnitzii dramatically modulates
the chemicals derived from the host and medium. Coculture of F. prausnitzii with colonic epithelium
induces specific transcriptional changes of transcription factor genes in the colonic epithelium [Figure 5].
These results suggest that metabolomics, in combination with the GuMI microphysiological system, enables
us to discover novel metabolic activities of F. prausnitzii that may contribute to the host-microbiome
crosstalk in the human intestine.

Similar to our observations, Lenoir et al. found that F. prausnitzii supernatant induces the expression of
DACT3 in vitro in HT-29 cells and in vivo in mice, and identified butyrate as the effector metabolite .
[11]
[15]
Indeed, butyrate was produced and maintained at mM level in GuMI-FP . Contradictory to these findings,
Lukovac et al. found that F. prausnitzii supernatant has a minimal effect on the gene expression of mouse
ileal organoids despite a high level of butyrate (8.03 mM) in the supernatant . Multiple variants could
[31]
explain this discrepancy in the experiments with GuMI vs. organoids, including cell sources (human colon
vs. mouse ileum), exposure time (48 h vs. 3 h), exposure routes (continuous exposure on the apical side vs.
static exposure on the basolateral side), and microenvironment (anoxic-oxic gradient vs. oxic). Future
research needs to clarify this discrepancy by taking these factors into account.

[32]
Gut microbiota are known to mediate resistance to infections through multiple mechanisms . Here, we
observed that F. prausnitzii significantly changed the expression of transcription factors in colonic epithelial
cells, including IRFs and EGR1 related to viral and bacterial infection. Pathogenic bacteria such as
Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella strains, and E. coli can upregulate EGR1 in
[33]
different host epithelial cells , independent of bacterial adherence to epithelial cells. The downregulation of
EGR1 and increase of IRFs by F. prausnitzii might serve as new mechanisms contributing to the pathogen
resistance of the gut in a homeostatic scenario.

While it is not fully understood how F. prausnitzii precisely modifies the expression of TF genes in colonic
epithelium, several mechanisms may contribute to the observed effects. For instance, short-chain fatty acids
and amino acid metabolites can directly modify histones such as methylation, acetylation, and