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. 2026 Oct 1;21(10):e0359086. doi: 10.1371/journal.pone.0359086

Lipopolysaccharide-induced alterations in barrier integrity and inflammatory signaling in porcine small intestinal organoids

Sumin Park 1, Boram Lee 2, Dahye Kim 1, Sheet Sunirmal 1, Min Gook Lee 1, Haesun Lee 1, Sun A Ock 1, Jae Gyu Yoo 3,*
Editor: Prashant Sharma4
PMCID: PMC13630212  PMID: 42821546

Abstract

Inflammation-associated diseases in pigs impair the intestinal tract and impair its function, thereby posing a major threat to their health and productivity. However, current in vitro cell models fail to fully replicate porcine physiological conditions. In this study, we aimed to develop a novel inflammation model using porcine small intestinal organoids derived from adult jejunum stimulated with lipopolysaccharide (LPS). The organoids were exposed to LPS to induce inflammation, and the responses were evaluated by quantifying cytokine mRNA expression, assessing intestinal epithelial barrier integrity, and analyzing changes in the expression and localization of proteins involved in nutrient transport, receptor signaling, and enteroendocrine function using quantitative real-time PCR and immunofluorescence staining. Transcriptome analysis was performed to identify LPS-responsive differentially expressed genes and enriched pathways in porcine small intestinal organoids. LPS stimulation effectively induced inflammation in porcine small intestinal organoids, as evidenced by increased TLR4 and cytokine gene expression, compromised epithelial barrier integrity, and altered expression and localization of proteins involved in nutrient transport, bile acid signaling, and enteroendocrine function. Furthermore, transcriptome profiling revealed LPS-induced broad transcriptional changes in porcine small intestinal organoids, including enrichment of immune- and inflammation-related pathways, as well as functional categories associated with barrier maintenance, membrane function, and adaptation to inflammatory stress. Collectively, these findings demonstrate that porcine small intestinal organoids provide a physiologically relevant in vitro model for studying intestinal inflammation. This model offers a valuable platform for investigating the mechanisms underlying porcine inflammatory bowel diseases and for screening novel therapeutic agents, supporting the 3Rs principle by potentially reducing reliance on animal testing in translational research.

Introduction

The porcine small intestine is highly responsive to infectious agents and environmental stressors [1]. As the primary site of nutrient absorption and a major immune organ, inflammation directly impairs its growth and productivity [2]. Major small intestinal inflammatory diseases include enteritis caused by porcine epidemic diarrhea virus, rotavirus, enterotoxigenic Escherichia coli, and Salmonella spp., which lead to severe diarrhea and dehydration, increasing mortality [3,4]. These conditions involve chronic or recurrent inflammation driven by complex mechanisms, such as dysbiosis, impaired mucosal barrier function, and immune hyperactivation [1,5]. Peyer’s patches in the jejunum and ileum serve as key sites for antigen recognition and immune cell activation in pigs [6]. Accordingly, studying porcine small intestinal inflammatory diseases is essential for improving feeding efficiency and survival rates as well as for elucidating intestinal immune mechanisms and developing models to evaluate novel drugs and vaccines. The need for research on inflammatory diseases using porcine small intestinal organoids (pSIOs) is being increasingly emphasized [7–9].

Lipopolysaccharide (LPS), a major component of the cell walls of Gram-negative bacteria, is a potent pro-inflammatory molecule that exacerbates intestinal inflammatory diseases and impairs barrier function [10,11]. Understanding LPS-induced intestinal inflammation and barrier dysfunction is essential for elucidating the physiological and pathological mechanisms of intestinal diseases. LPS-induced inflammation has been studied using porcine intestinal epithelial cell lines [12–16] or pig models [17–19]; however, both models have limitations. Cell lines are easy to culture and partially reproduce species-specific responses but cannot fully reproduce interactions among multiple intestinal cell types [20–22] and may accumulate genetic and morphological alterations during long-term culture [23–25]. In contrast, in vivo pig models more accurately reflect actual physiological responses but are constrained by substantial ethical and financial burdens, large inter-individual variability, and challenges in monitoring specific signaling pathways and cell-cell interactions [26–29].

To overcome these limitations, organoid models have recently gained significant attention. Organoids are three-dimensional (3D) in vitro models that replicate native organ structure and function and are widely used in disease modeling, drug testing, and nutritional research [7,23,30]. Small intestinal organoids are of particular interest because they accurately recapitulate the structure, cellular composition, and physiological functions of the native small intestine, enabling the study of inflammatory responses in a physiologically relevant context. They enable precise analysis of cellular interactions and immune responses, providing valuable insights into complex physiological phenomena, such as inflammation [31–33]. pSIOs are particularly valuable because of their strong physiological similarity to the human intestine [8,31,34]. Accordingly, organoid models serve as advanced in vitro models that complement the limitations of cell lines and animal models while preserving organ-specific structure and function [20,35,36]. Furthermore, organoids support reproducible, long-term experiments while maintaining species-specific characteristics across various applications [8,23,37,38].

Recent studies have established porcine intestinal organoids as physiologically relevant models for investigating enteric viral infections, host–pathogen interactions, and intestinal epithelial biology [39–41]. Furthermore, two-dimensional organoid-derived monolayer systems have been developed to facilitate barrier function assays and pathogen infection studies [42,43]. However, comprehensive characterization of LPS-induced inflammatory responses in porcine small intestinal organoids, including epithelial barrier disruption, nutrient transporter and receptor regulation, enteroendocrine responses, and global transcriptomic alterations, remains limited. To address this gap, the present study aimed to establish a porcine small intestinal organoid-based inflammatory model that integrates molecular, functional, and transcriptomic analyses to provide a comprehensive platform for investigating intestinal inflammation and evaluating potential therapeutic interventions.

In this study, pSIOs were treated with LPS to establish an intestinal inflammatory model. We analyzed transcriptomic and functional changes, including the expression of inflammation-related genes and the expression and localization of barrier-associated proteins, nutrient transporters, receptors, and enteroendocrine hormones. Collectively, these analyses enabled a comprehensive characterization of LPS-induced inflammatory responses in pSIOs and demonstrated the utility of this model as a physiologically relevant in vitro platform for investigating porcine intestinal inflammation and evaluating potential therapeutic interventions.

Materials and methods

Experimental design and animals

An LPS-induced pSIO model was established using 10-month-old adult pigs. All animal procedures were approved by the Institutional Animal Care and Use Committee of the National Institute of Animal Science (NIAS 2022−0569). Animals were euthanized by intravenous administration of T61 to minimize animal suffering. No anesthesia or analgesia was administered because tissue collection was performed only after euthanasia. Small intestinal tissues were collected immediately after euthanasia. Crypts isolated from porcine small intestines were three-dimensionally cultured in Matrigel® domes to generate organoids (Fig 1A). Mature pSIOs were then exposed to LPS (Sigma-Aldrich, St. Louis, MO, USA) to induce an inflammatory response. Preliminary dose-response experiments were conducted using 5, 10, 20, and 30 μM LPS to identify the appropriate LPS concentration. The 5 and 10 μM concentrations induced minimal inflammatory responses with no apparent morphological changes, whereas 30 μM caused severe structural disruption accompanied by extensive cell death. In contrast, 20 μM reproducibly induced inflammatory responses with only partial morphological alterations and without extensive cell death. Therefore, 20 μM was selected for all subsequent experiments. pSIOs were cultured in IntestiCult™ medium supplemented with 20 μM LPS for 24 h at 37 °C in a 5% CO2 atmosphere. Control pSIOs were maintained under identical conditions without LPS treatment (Fig 1B). Treated pSIOs were analyzed for gene expression, cell viability, and protein expression using quantitative real-time PCR (qRT-PCR), 4′,6-diamidino-2-phenylindole (DAPI)/propidium iodide (PI) staining, immunofluorescence analysis, and transcriptome analysis (Fig 1C).

Fig 1. Experimental design overview.

Fig 1

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(A) Porcine small intestinal crypts were embedded in Matrigel domes and cultured to establish small intestinal organoids. (B) Mature organoids were treated with 20 μM lipopolysaccharide (LPS) for 24 h to induce inflammation. (C) Treated organoids were analyzed using qRT-PCR, DAPI and PI staining, and immunofluorescence to assess gene expression, cell viability, and protein expression.

3D culture of pSIOs

Porcine small intestinal crypts were isolated from 10-month-old pigs as previously described [44]. The isolated crypts were counted using an inverted microscope, and approximately 140–150 crypts were mixed with Matrigel® (Corning Incorporated, Corning, NY, USA) at a 1:1 ratio to form domes. The Matrigel domes containing the mixture were dispensed into the center of preheated 24-well plates and allowed to solidify at 37 °C for 15 min. Subsequently, 1 mL of IntestiCult™ Organoid Growth Medium Human Basal Medium (Stemcell Technologies Inc., Vancouver, BC, Canada) was added to each well, and the cultures were maintained at 37 °C in a 5% CO2 atmosphere. Mature small intestinal organoids were subcultured approximately once per week.

RNA extraction and cDNA synthesis

pSIOs and ear-derived fibroblasts were lysed using the RNeasy Mini Kit (Qiagen, Hilden, NRW, Germany). RNA was extracted according to the manufacturer’s instructions. RNA concentration and purity were measured using an ND-2000 Spectrophotometer (Thermo Fisher Scientific, MA, USA). A total of 500 ng of RNA was reverse transcribed into cDNA using the Omniscript RT Kit (QIAGEN GmbH, Hilden, Germany). The 20 µL reaction mixture contained 2 µL of 10 × RT buffer, 2 µL of dNTP mix (5 mM each), 2 μL of oligo(dT) primer (10 µM), 1 μL of RNase inhibitor (10 units/µL), 1 μL of Omniscript Reverse Transcriptase, RNase-free water, and template RNA. All procedures were performed on ice. Reverse transcription was performed at 37 °C for 60 min. The synthesized cDNA was used immediately for qRT-PCR analysis or stored at −20 °C.

qRT-PCR

Quantitative RT-PCR was performed to compare intestinal epithelial and stem cell marker expression among porcine ear fibroblasts (PEFs) and small intestinal organoids and to analyze inflammatory cytokine expression in LPS-treated organoids.

Each 20 µL reaction contained 10 µL of 2 × SYBR Green Master Mix, 0.5 µL each of forward and reverse primers (10 µM), 1 µL of cDNA template, and 8 µL of RNase-free water. qRT-PCR was performed using SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on a QuantStudio 3 Real-Time PCR Instrument (Thermo Fisher Scientific) with the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Specificity was confirmed by melting curve analysis. For each experiment, organoids were independently cultured, treated, and subjected to RNA extraction, with each independently processed culture considered one biological replicate (n = 3). For each biological replicate, qRT-PCR was performed using five technical replicates. Relative expression levels were calculated using the 2-ΔΔCt method with GAPDH as the endogenous control based on the mean Ct values of the technical replicates. Primer sequences are listed in S1 Table.

Apoptosis analysis (DAPI/PI staining)

Apoptosis was assessed using DAPI/PI staining. LPS-treated pSIOs were washed with phosphate-buffered saline (PBS; Thermo Fisher Scientific) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min. The fixed organoids were washed thrice with PBS and stained with 1 μg/mL PI (Sigma-Aldrich) for 10 min at room temperature in the dark. The stained organoids were washed with PBS, mounted on glass slides using VECTASHIELD® Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA), and nuclear staining was performed simultaneously. Samples were imaged using a confocal laser scanning microscope (AX Series; Nikon Corp., Tokyo, Japan) to assess cell death. DAPI/PI staining was performed using three independent biological replicates (n = 3), and at least five organoids were analyzed for each LPS concentration in each biological replicate.

Immunofluorescence staining

LPS-treated pSIOs were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min. Organoids were blocked with 5% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature. They were incubated overnight at 4 °C with the following primary antibodies (diluted 1:200): Toll-like receptor 4 (TLR4) monoclonal (Thermo Fisher Scientific), epithelial cadherin (E-cadherin) monoclonal (BD Transduction Laboratories™, San Jose, CA, USA), zonula occludens-1 (ZO-1; Tight Junction Protein 1, TJP1) monoclonal (Santa Cruz Biotechnology™, Dallas, TX, USA), sodium-glucose cotransporter 1 (SGLT1) polyclonal (Novus Biologicals, Centennial, CO, USA), peptide transporter 1 (PEPT1) polyclonal (Bioss Antibodies, Woburn, MA, USA), glucose transporter 2 (GLUT2) polyclonal (Novus Biologicals), glucagon-like peptide-1 (GLP-1) polyclonal (MyBioSource, San Diego, CA, USA), and G-protein-coupled bile acid receptor 1 (TGR5) polyclonal (Abcam, Cambridge, MA, USA) antibodies. After washing with PBS, the organoids were incubated for 1 h at room temperature with secondary antibodies (diluted 1:500) as follows: Alexa Fluor® 647-conjugated donkey anti-rat IgG H&L, Alexa Fluor® 647-conjugated goat anti-mouse IgM mu chain, Alexa Fluor® 488-conjugated goat anti-rabbit IgG H&L (all from Abcam, Cambridge, UK), Alexa Fluor™ 488-conjugated goat anti-mouse IgG2b, and Alexa Fluor® 568-conjugated goat anti-rabbit IgG (both from Thermo Fisher Scientific, Waltham, MA, USA). After staining, organoids were mounted on glass slides using VECTASHIELD® Antifade Mounting Medium containing DAPI. Fluorescent signals were imaged using a confocal laser scanning microscope (Nikon Corp.). Immunofluorescence staining was performed using three independent biological replicates (n = 3), and at least five organoids were analyzed for each treatment condition in each biological replicate.

RNA sequencing data processing and quantification

For RNA sequencing, total RNA extracted from three organoid wells derived from the same organoid preparation was pooled to generate a single RNA sample for each experimental group (n = 1 per group). Therefore, RNA sequencing was performed without independent biological replicates. The Illumina NovaSeq 6000 platform was used to generate paired-end reads. Raw BCL files were converted to FASTQ format using bcl2fastq. Read quality assessment and trimming were performed using FastQC (v0.11) and Trimmomatic (v0.39) in paired-end mode by trimming ambiguous N bases and filtering reads with a Phred quality score < 20. Clean reads were aligned to the Sus scrofa reference genome (Sscrofa12.1) using HISAT2 (v2.2.1). Gene-level counts were quantified from aligned BAM files using featureCounts (Subread package v2.0.1), and the resulting count matrix was used for downstream analyses.

Differential gene expression analysis

Differential expression between control and LPS-treated pSIOs was analyzed using the edgeR package in R. Raw gene-level read counts were filtered to remove lowly expressed genes and normalized using the trimmed mean of M-values (TMM) method. Because the experiment included only one biological sample per condition (n = 1), variance estimation from biological replicates was not possible; therefore, differential expression was evaluated under the negative binomial model implemented in edgeR using a fixed dispersion value of 0.1. Raw p-values were adjusted for multiple testing using the Benjamini–Hochberg procedure. Candidate DEGs were defined as those with a log2 fold-change ≥ 1, and genes with a p-value ≤ 0.05 were considered statistically significant. These DEGs were further used for downstream analysis.

Functional and pathway enrichment analyses of DEGs

To interpret the biological relevance of LPS-responsive transcriptional changes in pSIOs, gene ontology (GO) enrichment analysis, including biological process (BP), molecular function (MF), and cellular component (CC), and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed using candidate DEGs identified by edgeR (p-value ≤ 0.05 and log2 fold-change ≥ 1). The DEGs were submitted to DAVID (Database for Annotation, Visualization and Integrated Discovery) for functional annotation. The top 10 significantly enriched GO terms and KEGG pathways were selected for visualization. Pathway plots were generated using the clusterProfiler R package.

Statistical analysis

Statistical analyses were performed using GraphPad Prism (version 5.0; GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± standard deviation (SD) from at least three independent biological replicates, unless otherwise stated. Statistical significance between two groups was determined using an unpaired Student’s t-test. Differences were considered statistically significant at *p < 0.05, **p < 0.01, or ***p < 0.001.

Results

Characterization of porcine intestinal organoids

Gene expression profiles were compared between pEFs and pSIOs using qRT-PCR. Intestinal epithelial markers (villin 1 [VIL1], lysozyme [LYZ], and chromogranin A [CHGA]), intestinal stem cell markers (LGR5, OLFM4, and SOX9), and the proliferation-related marker MKI67 were significantly upregulated in pSIOs compared with pEFs (p < 0.05; Fig 2A–C, D–G).

Fig 2. Characterization of porcine small intestinal organoids (pSIOs).

Fig 2

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Relative mRNA expression in porcine ear fibroblasts (pEFs) and pSIOs was analyzed using qRT-PCR. Intestinal epithelial markers (VIL1, LYZ, and CHGA) (A–C), intestinal stem cell markers (LGR5, OLFM4, and SOX9), and proliferation marker MKI67 (D–G) were significantly upregulated in pSIOs compared with pEFs. Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates. ***p < 0.001.

Apoptosis and barrier protein expression changes in pSIOs following LPS treatment

Phase-contrast microscopy revealed pronounced structural collapse, core darkening, and fragmentation in LPS-treated pSIOs compared with controls (Fig 3A). After LPS treatment (5, 10, 20, and 30 μM), DAPI/PI staining was performed. Red fluorescence intensity increased with LPS concentration, indicating a dose-dependent increase in apoptosis (Fig 3B). To verify epithelial barrier integrity, immunofluorescence staining for E-cadherin was performed in pSIOs (Fig 3C). In the control group, E-cadherin was strongly and continuously expressed along the epithelial cell membrane, whereas the 20 μM LPS-treated group showed disrupted membrane localization and structural disorganization of E-cadherin. Consistently, qRT-PCR analysis showed that CDH1 and TJP1 expression decreased to 0.359-fold (p < 0.001) and 0.483-fold (p < 0.001) compared to the control (0 μM), respectively, in the 20 μM LPS-treated group.

Fig 3. LPS-induced apoptosis and epithelial barrier disruption in pSIOs.

Fig 3

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(A) Phase-contrast images of pSIOs treated with LPS (0, 20, and 30 μM) showing structural disruption, core darkening, and fragmentation at higher concentrations compared with controls. Scale bars: 200 μm. (B) DAPI (blue) and PI (red) staining of pSIOs treated with LPS (5, 10, 20, and 30 μM) showing a dose-dependent increase in apoptosis. Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each LPS concentration in each biological replicate. Scale bars: 200 μm. (C) Representative immunofluorescence images of E-cadherin (green) and DAPI (blue) staining of pSIOs showing reduced and discontinuous E-cadherin expression in LPS-treated organoids compared with controls. Enlarged views of the yellow boxed regions are shown. Low-magnification (200 μm) images and enlarged views (50 μm) are shown in 3C. Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each treatment condition in each biological replicate. (D) Relative gene expression levels of CDH1 and TJP1 in pSIOs treated with LPS (20 μM) compared with controls (0 μM), as determined by qRT-PCR. LPS treatment significantly decreased the expression of CDH1 and TJP1, indicating disruption of epithelial barrier integrity. Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates. ***p < 0.001.

LPS-induced TLR4 and inflammatory cytokine gene expression in pSIOs

To evaluate the inflammatory responsiveness of pSIOs, organoids were treated with LPS (20 μM), and the TLR4 protein and cytokine gene expression were analyzed. Immunofluorescence staining showed TLR4 expression in both the control and LPS-treated groups, with significantly higher fluorescence intensity in the LPS-treated group (Fig 4A). To further characterize the inflammatory response, the mRNA expression levels of pro- and anti-inflammatory cytokines were analyzed by qRT-PCR (Fig 4B). The pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-8 (p < 0.01) and anti-inflammatory cytokines, IL-4 and IL-10 (p < 0.05) were significantly upregulated in the LPS-treated organoids, showing 2.61-fold, 3.61-fold, 3.65-fold, 12.15-fold, and 3.40-fold increases, respectively. In contrast, transforming growth factor beta (TGF-β) expression increased 1.41-fold but did not reach statistical significance.

Fig 4. Cytokine gene expression changes in pSIOs following LPS treatment.

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(A) Immunofluorescence staining of TLR4 in pSIOs after LPS (20 μM) treatment. Nuclei were stained with DAPI (blue), and TLR4 expression is shown in green. Representative images suggest higher TLR4 immunofluorescence in the LPS-treated group than in the control group. Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each treatment condition in each biological replicate. Scale bar = 100 μm. (B) qRT-PCR analysis of pro- and anti-inflammatory cytokines in pSIOs after LPS (20 μM) exposure. Pro-inflammatory cytokines TNF-α, IL-1β, and IL-8 were significantly upregulated in the LPS-treated group compared with the control group. Anti-inflammatory cytokines IL-4 and IL-10 also showed significantly increased expression, whereas TGF-β expression was increased but did not reach statistical significance following LPS stimulation. Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates. *p < 0.05, **p < 0.01.

Changes in metabolic, endocrine, and receptor-related expression in pSIOs following LPS treatment

The pSIOs used in this study represent a basal-out model, in which the basolateral surface of intestinal epithelial cells is exposed to the culture medium. Following 20 μM LPS treatment, immunofluorescence analysis revealed alterations in proteins related to nutrient transport, endocrine function, and receptor signaling (Fig 5A). Consistently, qPCR demonstrated significant changes in the mRNA expression levels of SLC5A1 (SGLT1), SLC15A1 (PEPT1), SLC2A2 (GLUT2), GPBAR1 (TGR5), and GCG (GLP-1) (Fig 5B).

Fig 5. LPS-induced alterations in intestinal transport and enteroendocrine marker expression in pSIOs.

Fig 5

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(A) Immunofluorescence of pSIOs stained with DAPI (blue) to visualize nuclei and immunolabeled for SGLT1 (green), PEPT1 (green), GLUT2 (green), TGR5 (red), and GLP-1 (green). Representative untreated (upper rows) and LPS-treated (lower rows) organoids are shown. Untreated organoids exhibited distinct localization of SGLT1, PEPT1, GLUT2, and TGR5 along the epithelial membrane with strong expression and cytoplasmic GLP-1 expression in scattered enteroendocrine-like cells within the epithelial layer. LPS-treated organoids exhibited reduced expression intensity, irregular protein distribution, and partial loss of membrane localization. Enlarged views of the yellow boxed regions further highlight differences in expression patterns and subcellular localization between untreated and LPS-treated organoids. Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each treatment condition in each biological replicate. Scale bars: 50 μm. (B) qRT-PCR analysis showing significant alterations in nutrient transporter and enteroendocrine-related gene expression following LPS treatment. Decreased SLC5A1 (SGLT1) and SLC15A1 (PEPT1) expression and increased SLC2A2 expression were observed after LPS exposure. Among enteroendocrine-related genes, GPBAR1 expression was markedly decreased, whereas GCG (the precursor gene of GLP-1) expression was significantly increased after LPS exposure.Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates. **p < 0.01, ***p < 0.001.

In the untreated control group, the glucose uptake transporter SGLT1, the peptide transporter PEPT1, and the glucose efflux transporter GLUT2 exhibited continuous and uniform immunofluorescence signals primarily along the epithelial cell membrane. In contrast, in the LPS-treated group, these transporters exhibited an overall reduction in immunofluorescence signal intensity and uneven and discontinuous distribution. Consistent with the immunofluorescence findings, qPCR analysis revealed that the mRNA expression levels of SLC5A1 (SGLT1) and SLC15A1 (PEPT1) decreased 0.826-fold and 0.527-fold, respectively, following 20 μM LPS treatment. In contrast, the mRNA expression of SLC2A2 (GLUT2) increased 1.299-fold compared with the control group.

The bile acid receptor, TGR5, exhibited normal expression patterns, with relatively uniform distribution throughout the cell membrane and cytoplasm in the untreated control group. However, after LPS treatment, its immunofluorescence signal intensity decreased, and an irregular distribution pattern was observed with weakened localization at the cell membrane. Consistently, qPCR analysis demonstrated that the mRNA expression level of GPBAR1 (TGR5) decreased 0.345-fold in the 20 μM LPS-treated group compared with the control group.

Finally, GLP-1 was expressed locally in the cytoplasm of a subset of cells within the epithelial layer in the untreated control group. In the LPS-treated group, a reduction in fluorescence intensity was observed, accompanied by structural disruption and fragmentation of GLP-1-positive regions. qRT-PCR analysis revealed that the mRNA expression level of GCG increased 1.978-fold in the 20 μM LPS-treated group compared with the control group.

Transcriptome analysis and DEG identification

To identify transcriptional changes induced by LPS in pSIOs, differential expression analysis was performed using a cut-off criterion of p ≤ 0.05 and |log2 fold-change| ≥ 1. Significant upregulated and downregulated DEGs were identified in the LPS-treated group compared with the control group (Fig 6A). Analysis of the top 10 DEGs based on fold change identified the gene group with the most pronounced transcriptional responses to LPS (Fig 6B). The statistical significance and expression differences of DEGs were comprehensively analyzed using a volcano plot, and the top five genes showing the most pronounced expression differences were identified as DUOX1, AQN-1, CCL26, BST2, and SFTPA1 (Fig 6C). Heatmap analysis and hierarchical clustering of the top 100 DEGs showed that the two groups are separated by treatment condition and that LPS treatment induces a distinct expression pattern compared to the control group (Fig 6D).

Fig 6. Identification of DEGs in pSIOs after LPS treatment.

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(A) Total number of upregulated and downregulated DEGs (p ≤ 0.05 and |log2 fold-change| ≥ 1). (B) Top 10 upregulated and downregulated DEGs ranked by fold change. (C) Volcano plot of DEGs with top five genes highlighted. (D) Heatmap and hierarchical clustering of the top 100 DEGs. RNA sequencing was performed using one pooled RNA sample generated from three organoid wells for each experimental group, resulting in one biological replicate (n = 1) per group. DEG, differentially expressed gene.

GO and KEGG pathway enrichment in LPS-treated pSIOs

To elucidate the regulatory mechanisms of transcriptome changes induced by LPS stimulation in pSIO, we performed GO function enrichment analysis (BP, MF, and CC) and analysis of the top 10 KEGG pathways. In the BP category, the immune and host defense programs were the most prominent, including defensive responses to external stimuli, responses to other organisms and external biological stimuli, and immune activation regulatory processes (Fig 7A). In the MF category, the top 10 significantly enriched terms (p < 0.05) included chemokine activity, receptor-ligand activity, cytokine activity, and chemokine receptor binding (Fig 7B). In the CC category, the top 10 enriched terms (p < 0.05) included membrane and epithelial interface structures, including the apical plasma membrane, the plasma membrane region, and the apical part of the cell (Fig 7C). This suggests that LPS-responsive genes are preferentially localized to pathways operating at the cell surface and the apical epithelial compartment.

Fig 7. Gene Ontology (GO) enrichment analysis of DEGs in LPS-treated pSIOs.

Fig 7

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Top 10 significantly enriched (p < 0.05) GO terms in (A) biological process (BP), (B) molecular function (MF), and (C) cellular component (CC).

KEGG pathway enrichment analysis in LPS-treated pSIOs

KEGG pathway enrichment analysis of DEGs in LPS-treated pSIOs identified immune and inflammatory signaling pathways as the dominant transcriptional responses (p < 0.05). Gene-pathway network analysis showed that a significant number of DEGs were involved in multiple pathways, forming close interactions between inflammatory signaling networks (Fig 8A). Analysis of the top 10 enriched pathways (Fig 8B) identified cytokine–cytokine receptor communications as the most prominent, followed by complement and coagulation cascades, viral protein interaction with cytokines and cytokine receptors, and the IL-17 signaling pathway. Other significantly enriched pathways were NF-κB signaling, TNF signaling, and other infection-related pathways.

Fig 8. KEGG pathway enrichment analysis of DEGs in LPS-treated pSIOs.

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Top 10 significantly enriched pathways (p < 0.05) are shown. (A) Cnetplot showing specific genes in multiple enriched pathways. (B) Dot plot visualization of the top 10 enriched pathways.

Discussion

Organoids are next-generation in vitro models that replicate the 3D architecture and cellular diversity of an organ. pSIOs are a robust model for studying intestinal inflammation because of their physiological similarity to the human intestine and experimental reproducibility. In this study, basal-out pSIOs derived from adult jejunum were used to evaluate epithelial inflammatory responses to LPS. LPS treatment induced organoid structural disruption and increased pro-inflammatory cytokine expression, accompanied by significant alterations in nutrient transport, receptor signaling, and endocrine function markers. These findings support pSIOs as a physiologically relevant in vitro model for investigating innate immune responses and inflammation-associated epithelial alterations.

In the present study, immunofluorescence analyses were performed using three-dimensional (3D) organoids to preserve the native epithelial architecture, cell–cell interactions, and spatial organization that closely resemble those of the in vivo intestine. Although organoid-derived two-dimensional (2D) monolayer systems provide improved antibody accessibility and facilitate more precise visualization of protein localization, they do not fully retain the three-dimensional structural complexity of the intestinal epithelium [30,35,43]. Therefore, we selected the 3D organoid model to evaluate protein expression within an intact epithelial context. Nevertheless, we acknowledge that the 3D configuration may limit antibody penetration and make precise subcellular localization more challenging than in 2D monolayer systems. This should be considered when interpreting the immunofluorescence findings.

Compared with the previous applications of porcine intestinal organoids, which have mainly focused on enteric infections, host–pathogen interactions, and intestinal epithelial biology, the present study expands their application by providing an integrated characterization of LPS-induced inflammatory responses. While earlier studies have often relied on evaluating a limited set of inflammatory markers or specific barrier proteins, we simultaneously evaluated epithelial barrier integrity, inflammatory cytokine expression, nutrient transporter regulation, enteroendocrine responses, and global transcriptomic alterations within a single experimental platform. This integrated approach provides a more comprehensive understanding of LPS-induced intestinal inflammation and supports the use of porcine small intestinal organoids as a physiologically relevant in vitro model for mechanistic studies and the evaluation of potential therapeutic or nutritional interventions. The physiological relevance of this model was further supported by the observation that pSIOs exhibited characteristics representative of diverse intestinal epithelial cell types. Elevated epithelial marker (VIL1, LYZ, and CHGA) expression suggests the presence of absorptive enterocytes, Paneth cells, and enterochromaffin cells, consistent with previous studies reporting that small intestinal organoids recapitulate multiple epithelial lineages observed in vivo, highlighting species-specific characteristics between human and porcine organoids [8,20,23,43,45]. Expression of stem cell markers (LGR5, OLFM4, and SOX9) confirms the maintenance of a stem cell population within pSIOs [46], supporting intrinsic self-renewal capacity [47–49]. Increased MKI67 expression further indicates active proliferation, reflecting the rapid turnover typical of the intestinal epithelium [50]. Collectively, these results demonstrate that the pSIOs in this study possess both epithelial cell characteristics and stem cell-driven regenerative capacity.

Microscopic observation revealed that LPS treatment induced concentration-dependent morphological changes in pSIOs. LPS-untreated organoids maintained a typical budding structure and stable morphology, whereas structural stability gradually decreased with increasing LPS concentration. The findings indicate that increasing concentrations progressively compromise organoid structural integrity. Furthermore, PI staining revealed a concentration-dependent increase in cell death, with localized cell death at lower doses and widespread PI-positive signals at higher concentrations. These findings are consistent with those of previous reports that LPS simultaneously impairs morphological stability and cell survival of intestinal organoids. Similar fragmentation and increased apoptosis have been observed in a porcine small intestinal epithelial cell line (IPI-2I) [51] and human intestinal organoids following LPS treatment [52]. In vivo animal models further show that LPS induces mucosal barrier disruption, epithelial shedding, and cell-cell junction damage in porcine and rodent models [53]. Moreover, LPS downregulate E-cadherin, weakens cell–cell adhesion and compromises barrier integrity [54–56]. Immunofluorescence analysis showed continuous and uniform E-cadherin localization along lateral membranes in control organoids, whereas LPS-treated organoids exhibited markedly reduced E-cadherin intensity and discontinuous localization along cell-cell borders. Therefore, LPS stimulation disrupts the structural integrity of adherens junctions. qRT-PCR further revealed significant downregulation of CDH1 (E-cadherin) and TJP1 (ZO-1), the latter indicating disruption of tight junctions, leading to increased epithelial permeability [57–59]. E-cadherin mediates cell–cell adhesion, whereas ZO-1 stabilizes tight junction complexes [60–62]. Their concurrent downregulation indicates that epithelial barrier disruption involves coordinated impairment of multiple intercellular junctional complexes. This observed disruption further supports the applicability of pSIOs as an in vitro model that recapitulates intestinal barrier dysfunction. These findings are consistent with those of previous studies demonstrating that LPS-induced barrier dysfunction involves TLR4/NF-κB signaling, leading to decreased tight junction protein expression and increased permeability [55,63]. Collectively, these findings demonstrate the utility of the pSIO model as a reliable screening platform for identifying pathological mechanisms of LPS-induced barrier damage and for screening novel barrier-protecting compounds.

In this study, TLR4 expression increased in LPS-treated pSIOs compared with the control group. TLR4 recognizes LPS from gram-negative bacteria and initiates downstream inflammatory signaling [64–66]. The increased TLR4 fluorescence intensity following LPS stimulation shows that pSIOs partially possess functional innate immune sensing mechanisms that can recognize and respond to bacterial endotoxins. LPS also triggered an inflammatory response in pSIOs, as demonstrated by pro-inflammatory cytokine upregulation. Concurrent anti-inflammatory cytokine upregulation indicates activation of a compensatory anti-inflammatory response to maintain immune homeostasis [67]. In contrast, TGF-β expression exhibited a non-significant increase, reflecting that its expression and function can vary depending on the stimulation conditions, cell state, and tissue environment [68]. These results are consistent with those of previous studies demonstrating a dynamic balance between pro- and anti-inflammatory cytokines following LPS stimulation [52,69]. The multifaceted cytokine expression patterns identified in the pSIO model demonstrate its utility as a valuable in vitro platform for studying porcine intestinal inflammation and epithelial immune responses.

Transcriptome analysis further supported the LPS-induced inflammatory phenotype in pSIOs. Differential expression profiling identified a distinct set of LPS-responsive genes, with more upregulated than downregulated DEGs, indicating a predominantly activated transcriptional response. Notably, highly upregulated genes, including DUOX1, DUOXA1, and BST2, are associated with epithelial defense and inflammatory signaling, suggesting that LPS robustly activated a defense system for the outer layer of the organoids. Functional enrichment analysis indicated that LPS exposure in pSIOs triggers coordinated epithelial responses that extend beyond inflammatory signaling, including processes related to barrier maintenance, membrane-associated functions, and cellular adaptations to inflammatory stress [70]. Consistently, KEGG pathway analysis showed significant enrichment of immune- and inflammation-related pathways, including cytokine-cytokine receptor interaction, complement and coagulation cascades, viral protein interaction with cytokine and cytokine receptor, IL-17, NF-κB, and TNF signaling [71], and other infection-associated pathways, reflecting overlap between LPS-responsive genes and conserved host-defense pathways [72].

The effects of 20 μM of LPS on gene and protein expression and intracellular localization of key transporters, receptors, and hormones involved in nutrient absorption, metabolic regulation, and signaling were further analyzed. SGLT1 and PEPT1, responsible for nutrient absorption in the intestinal epithelium, are crucial in maintaining metabolic homeostasis [73,74]. The decreased signal intensity of these transporters and membrane fragmentation observed after LPS treatment in this study suggest that inflammatory stimuli inhibit nutrient absorption. This is consistent with the observed reduction in SLC5A1 and SLC15A1 gene expression and with previous studies showing that inflammation inhibits intracellular glucose and peptide uptake at the transcriptional level [75,76]. The decreased TGR5 signal intensity and loss of membrane localization observed after LPS treatment indicate disruption of bile acid receptor signaling within small intestinal organoids [77,78]. TGR5, a bile acid-sensing receptor, is involved in energy metabolism and immune regulation, and the transcriptional downregulation of GPBAR1 (TGR5) identified in this study supports the disruption of this signaling axis under inflammatory conditions. Given the central role of bile acids in host-microbiota metabolic communication, TGR5 disruption can lead to dysregulation of the microbe-bile acid-host interaction network, ultimately leading to impaired metabolic homeostasis of the intestinal epithelium [79–83].

Notably, GLUT2, which is localized to the cell membrane and mediates glucose export, also exhibited downregulation and loss of membrane localization following LPS treatment, indicating that LPS-induced inflammation disrupts not only glucose uptake by the intestinal epithelium but also overall metabolic homeostasis through glucose transport systems and the epithelial barrier [84]. This result is consistent with those of previous studies reporting that LPS-induced inflammatory responses lead to metabolic imbalance in intestinal epithelial cells by disrupting glucose transport [85,86]. However, the mRNA expression of SLC2A2, which codes for GLUT2, was significantly upregulated after LPS treatment. This reflects a transcriptional compensatory response in which cells attempt to maintain metabolic function in response to LPS-induced physical barrier disruption and protein degradation, or metabolic reprogramming in response to inflammation [87]. The ‘asymmetric regulation’ pattern, characterized by the simultaneous upregulation of SLC2A2 (GLUT2) and downregulation of SLC5A1 (SGLT1), suggests that glucose influx and efflux pathways in the intestinal epithelium are disrupted by different mechanisms under inflammatory conditions.

The overall decrease in GLP-1 expression in LPS-treated pSIOs suggests a decline in secretory capacity because of dysfunction of L-cells, which are a specialized intestinal epithelial cell subtype distributed within the epithelial layer of villus-like structures in organoids [88,89]. Considering that GLP-1 is crucial in promoting insulin secretion and maintaining intestinal barrier integrity, this downregulation implies simultaneous impairment of metabolic regulation and intestinal defense mechanisms under inflammatory conditions [90–94]. The upregulation of GCG, the precursor gene encoding GLP-1, following LPS treatment may represent an adaptive or compensatory endocrine response to inflammatory stimuli, suggesting reprogramming of intestinal endocrine function under inflammatory conditions [95–98]. Collectively, these findings suggest that LPS-induced inflammatory conditions in intestinal epithelial organoids affect not only immune-related responses but also broader metabolic and functional networks, including nutrient transporters, bile acid receptor signaling, and intestinal endocrine regulation. Thus, inflammatory stimuli may lead to coordinated alterations in metabolic stability and signaling regulation within the intestinal epithelium.

Nevertheless, this study has some limitations. First, the analyses were performed at a single time point using only LPS as the inflammatory stimulus, which limits the generalizability of the findings to other inflammatory conditions. In addition, further functional validation, including time-course analyses and TEER measurements, will be necessary to strengthen the utility of this model.

Conclusions

This study demonstrates that pSIOs derived from adult pigs effectively mimic LPS-induced epithelial structural damage and increased inflammatory cytokine expression. Disruption of nutrient transporters, bile acid receptors, and endocrine factors was observed at both transcriptional and protein levels, accompanied by structural disruption of intestinal barrier proteins. These results demonstrate the value of pSIOs as a species-specific in vitro platform that has the potential to reduce reliance on animal models while retaining key physiological characteristics. Although this study is limited to a single time point and a specific stimulus, future studies incorporating time-course analyses and additional functional assessments, such as TEER measurements, will further validate the utility of this model.

Supporting information

S1 Table. Primers used for gene expression analysis using quantitative RT-PCR.

(DOCX)

pone.0359086.s001.docx (18.6KB, docx)

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by the Rural Development Administration (RDA), Republic of Korea, under the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01671901). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Tang X, Xiong K, Fang R, Li M. Weaning stress and intestinal health of piglets: A review. Front Immunol. 2022;13:1042778. doi: 10.3389/fimmu.2022.1042778 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Lee IK, Kye YC, Kim G, Kim HW, Gu MJ, Umboh J, et al. Stress, Nutrition, and Intestinal Immune Responses in Pigs - A Review. Asian-Australas J Anim Sci. 2016;29(8):1075–82. doi: 10.5713/ajas.16.0118 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.López-Figueroa C, Cano E, Navarro N, Pérez-Maíllo M, Pujols J, Núñez JI, et al. Clinical, Pathological and Virological Outcomes of Tissue-Homogenate-Derived and Cell-Adapted Strains of Porcine Epidemic Diarrhea Virus (PEDV) in a Neonatal Pig Model. Viruses. 2023;16(1):44. doi: 10.3390/v16010044 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kim K, Song M, Liu Y, Ji P. Enterotoxigenic Escherichia coli infection of weaned pigs: Intestinal challenges and nutritional intervention to enhance disease resistance. Front Immunol. 2022;13:885253. doi: 10.3389/fimmu.2022.885253 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Upadhaya SD, Kim IH. Maintenance of gut microbiome stability for optimum intestinal health in pigs - a review. J Anim Sci Biotechnol. 2022;13(1):140. doi: 10.1186/s40104-022-00790-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Furukawa M, Ito S, Suzuki S, Fuchimoto D, Onishi A, Niimi K, et al. Organogenesis of Ileal Peyer’s Patches Is Initiated Prenatally and Accelerated Postnatally With Comprehensive Proliferation of B Cells in Pigs. Front Immunol. 2020;11:604674. doi: 10.3389/fimmu.2020.604674 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Vermeire B, Gonzalez LM, Jansens RJJ, Cox E, Devriendt B. Porcine small intestinal organoids as a model to explore ETEC-host interactions in the gut. Vet Res. 2021;52(1):94. doi: 10.1186/s13567-021-00961-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Zhang M, Lv L, Cai H, Li Y, Gao F, Yu L, et al. Long-Term Expansion of Porcine Intestinal Organoids Serves as an in vitro Model for Swine Enteric Coronavirus Infection. Front Microbiol. 2022;13:865336. doi: 10.3389/fmicb.2022.865336 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Yen L, Nelli RK, Twu N-C, Mora-Díaz JC, Castillo G, Sitthicharoenchai P, et al. Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus. Front Immunol. 2024;15:1451154. doi: 10.3389/fimmu.2024.1451154 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Cao S, Zhang Q, Wang C, Wu H, Jiao L, Hong Q, et al. LPS challenge increased intestinal permeability, disrupted mitochondrial function and triggered mitophagy of piglets. Innate Immun. 2018;24(4):221–30. doi: 10.1177/1753425918769372 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Mazgaeen L, Gurung P. Recent Advances in Lipopolysaccharide Recognition Systems. Int J Mol Sci. 2020;21(2):379. doi: 10.3390/ijms21020379 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Zhao X, Dong B, Friesen M, Liu S, Zhu C, Yang C. Capsaicin Attenuates Lipopolysaccharide-Induced Inflammation and Barrier Dysfunction in Intestinal Porcine Epithelial Cell Line-J2. Front Physiol. 2021;12:715469. doi: 10.3389/fphys.2021.715469 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Omonijo FA, Liu S, Hui Q, Zhang H, Lahaye L, Bodin J-C, et al. Thymol Improves Barrier Function and Attenuates Inflammatory Responses in Porcine Intestinal Epithelial Cells during Lipopolysaccharide (LPS)-Induced Inflammation. J Agric Food Chem. 2019;67(2):615–24. doi: 10.1021/acs.jafc.8b05480 [DOI] [PubMed] [Google Scholar]
  • 14.Dong N, Xu X, Xue C, Wang C, Li X, Bi C, et al. Ethyl pyruvate inhibits LPS induced IPEC-J2 inflammation and apoptosis through p38 and ERK1/2 pathways. Cell Cycle. 2019;18(20):2614–28. doi: 10.1080/15384101.2019.1653106 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Hu P, Zhao F, Wang J, Zhu W. Lactoferrin attenuates lipopolysaccharide-stimulated inflammatory responses and barrier impairment through the modulation of NF-κB/MAPK/Nrf2 pathways in IPEC-J2 cells. Food Funct. 2020;11(10):8516–26. doi: 10.1039/d0fo01570a [DOI] [PubMed] [Google Scholar]
  • 16.Li H, Han R, Yong F, Fan Y, Zhao B, Hu X, et al. The protective effect of Eleutheroside E against the mechanical barrier dysfunction triggered by lipopolysaccharide in IPEC-J2 cells. Res Vet Sci. 2023;154:1–7. doi: 10.1016/j.rvsc.2022.11.002 [DOI] [PubMed] [Google Scholar]
  • 17.Sun X, Cui Y, Su Y, Gao Z, Diao X, Li J, et al. Dietary Fiber Ameliorates Lipopolysaccharide-Induced Intestinal Barrier Function Damage in Piglets by Modulation of Intestinal Microbiome. mSystems. 2021;6(2):e01374-20. doi: 10.1128/mSystems.01374-20 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Fu R, Liang C, Chen D, Tian G, Zheng P, He J, et al. Yeast hydrolysate attenuates lipopolysaccharide-induced inflammatory responses and intestinal barrier damage in weaned piglets. J Anim Sci Biotechnol. 2023;14(1):44. doi: 10.1186/s40104-023-00835-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Wen X, Zhong R, Dang G, Xia B, Wu W, Tang S, et al. Pectin supplementation ameliorates intestinal epithelial barrier function damage by modulating intestinal microbiota in lipopolysaccharide-challenged piglets. J Nutr Biochem. 2022;109:109107. doi: 10.1016/j.jnutbio.2022.109107 [DOI] [PubMed] [Google Scholar]
  • 20.Beaumont M, Blanc F, Cherbuy C, Egidy G, Giuffra E, Lacroix-Lamandé S, et al. Intestinal organoids in farm animals. Vet Res. 2021;52(1):33. doi: 10.1186/s13567-021-00909-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Matsumoto K, Namai F, Miyazaki A, Imamura Y, Fukuyama K, Ikeda-Ohtsubo W, et al. Development of an intestinal epithelial cell line and organoids derived from the same swine and characterization of their antiviral responses. Biosci Microbiota Food Health. 2024;43(4):342–51. doi: 10.12938/bmfh.2024-0046 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Wen G, Eder K, Ringseis R. Comparative evaluation of the modulatory role of 1,25-dihydroxy-vitamin D3 on endoplasmic reticulum stress-induced effects in 2D and 3D cultures of the intestinal porcine epithelial cell line IPEC-J2. J Anim Sci Biotechnol. 2024;15(1):153. doi: 10.1186/s40104-024-01112-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Ma P, Fang P, Ren T, Fang L, Xiao S. Porcine Intestinal Organoids: Overview of the State of the Art. Viruses. 2022;14(5):1110. doi: 10.3390/v14051110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.He Z, Wilson A, Rich F, Kenwright D, Stevens A, Low YS, et al. Chromosomal instability and its effect on cell lines. Cancer Rep (Hoboken). 2023;6(6):e1822. doi: 10.1002/cnr2.1822 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.de Vos J, Crooijmans RPMA, Derks MFL, Kloet SL, Dibbits B, Groenen MAM, et al. Detailed molecular and epigenetic characterization of the pig IPEC-J2 and chicken SL-29 cell lines. iScience. 2023;26(3):106252. doi: 10.1016/j.isci.2023.106252 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Ly Pham L, Watford S, Pradeep P, Martin MT, Thomas R, Judson R, et al. Variability in in vivo studies: Defining the upper limit of performance for predictions of systemic effect levels. Comput Toxicol. 2020;15(August 2020):1–100126. doi: 10.1016/j.comtox.2020.100126 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Shi D, Mi G, Wang M, Webster TJ. In vitro and ex vivo systems at the forefront of infection modeling and drug discovery. Biomaterials. 2019;198:228–49. doi: 10.1016/j.biomaterials.2018.10.030 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Marshall LJ, Bailey J, Cassotta M, Herrmann K, Pistollato F. Poor Translatability of Biomedical Research Using Animals - A Narrative Review. Altern Lab Anim. 2023;51(2):102–35. doi: 10.1177/02611929231157756 [DOI] [PubMed] [Google Scholar]
  • 29.Soufizadeh P, Mansouri V, Ahmadbeigi N. A review of animal models utilized in preclinical studies of approved gene therapy products: trends and insights. Lab Anim Res. 2024;40(1):17. doi: 10.1186/s42826-024-00195-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Hoffmann P, Schnepel N, Langeheine M, Künnemann K, Grassl GA, Brehm R, et al. Intestinal organoid-based 2D monolayers mimic physiological and pathophysiological properties of the pig intestine. PLoS One. 2021;16(8):e0256143. doi: 10.1371/journal.pone.0256143 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Li X-G, Zhu M, Chen M-X, Fan H-B, Fu H-L, Zhou J-Y, et al. Acute exposure to deoxynivalenol inhibits porcine enteroid activity via suppression of the Wnt/β-catenin pathway. Toxicol Lett. 2019;305:19–31. doi: 10.1016/j.toxlet.2019.01.008 [DOI] [PubMed] [Google Scholar]
  • 32.Huang Y, Huang Z, Tang Z, Chen Y, Huang M, Liu H, et al. Research Progress, Challenges, and Breakthroughs of Organoids as Disease Models. Front Cell Dev Biol. 2021;9:740574. doi: 10.3389/fcell.2021.740574 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Kar SK, Wells JM, Ellen ED, Te Pas MFW, Madsen O, Groenen MAM, et al. Organoids: a promising new in vitro platform in livestock and veterinary research. Vet Res. 2021;52(1):43. doi: 10.1186/s13567-021-00904-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Lee S-A, Lee HJ, Gu N-Y, Park Y-R, Kim E-J, Kang S-J, et al. Evaluation of porcine intestinal organoids as an in vitro model for mammalian orthoreovirus 3 infection. J Vet Sci. 2023;24(4):e53. doi: 10.4142/jvs.23017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Aguilar C, Alves da Silva M, Saraiva M, Neyazi M, Olsson IAS, Bartfeld S. Organoids as host models for infection biology - a review of methods. Exp Mol Med. 2021;53(10):1471–82. doi: 10.1038/s12276-021-00629-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Liu X, Zhou Z, Zhang Y, Zhong H, Cai X, Guan R. Recent progress on the organoids: Techniques, advantages and applications. Biomed Pharmacother. 2025;185:117942. doi: 10.1016/j.biopha.2025.117942 [DOI] [PubMed] [Google Scholar]
  • 37.Zhang Y, Yang N, Li Q, Tang Y, Bai B, Liu G. Intestinal organoids: A novel and ideal in vitro platform for swine enteric coronavirus investigations. Virology. 2025;610:110595. doi: 10.1016/j.virol.2025.110595 [DOI] [PubMed] [Google Scholar]
  • 38.Yang S, Hu H, Kung H, Zou R, Dai Y, Hu Y, et al. Organoids: The current status and biomedical applications. MedComm (2020). 2023;4(3):e274. doi: 10.1002/mco2.274 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Li Y, Yang N, Chen J, Huang X, Zhang N, Yang S, et al. Next-Generation Porcine Intestinal Organoids: an Apical-Out Organoid Model for Swine Enteric Virus Infection and Immune Response Investigations. J Virol. 2020;94(21):e01006-20. doi: 10.1128/JVI.01006-20 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Lee J, Lee S, Jeon H, Kim G, Kim S, Kim J. Intestinal organoid as a research platform for the virus-host interaction. J Bacteriol Virol. 2023;53:51–60. doi: 10.4167/jbv.2023.53.1.051 [DOI] [Google Scholar]
  • 41.Nakamura T. Recent progress in organoid culture to model intestinal epithelial barrier functions. Int Immunol. 2019;31(1):13–21. doi: 10.1093/intimm/dxy065 [DOI] [PubMed] [Google Scholar]
  • 42.Roodsant T, Navis M, Aknouch I, Renes IB, van Elburg RM, Pajkrt D, et al. A Human 2D Primary Organoid-Derived Epithelial Monolayer Model to Study Host-Pathogen Interaction in the Small Intestine. Front Cell Infect Microbiol. 2020;10:272. doi: 10.3389/fcimb.2020.00272 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.van der Hee B, Loonen LMP, Taverne N, Taverne-Thiele JJ, Smidt H, Wells JM. Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids. Stem Cell Res. 2018;28:165–71. doi: 10.1016/j.scr.2018.02.013 [DOI] [PubMed] [Google Scholar]
  • 44.Lee BR, Ock SA, Park MR, Lee MG, Byun SJ. Establishing porcine jejunum-derived intestinal organoids to study the function of intestinal epithelium as an alternative for animal testing. J Anim Reprod Biotechnol. 2024;39:2–11. doi: 10.12750/JARB.39.1.2 [DOI] [Google Scholar]
  • 45.Joo SS, Gu B-H, Lee E, Oh E, Kim M, Jung H, et al. Functional characteristics of a pig 2D intestinal organoid model as an in vitro platform for nutritional studies. J Anim Sci Technol. 2026;68(1):236–57. doi: 10.5187/jast.2024.e116 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Schaaf CR, Polkoff KM, Carter A, Stewart AS, Sheahan B, Freund J, et al. A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease. FASEB J. 2023;37(6):e22975. doi: 10.1096/fj.202300223R [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Yin L, Yang Q, Zhang Y, Wan D, Yin Y, Wang Q, et al. Dietary Copper Improves Intestinal Morphology via Modulating Intestinal Stem Cell Activity in Pigs. Animals (Basel). 2021;11(9):2513. doi: 10.3390/ani11092513 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Zhou J-Y, Zan G-X, Zhu Q-J, Gao C-Q, Yan H-C, Wang X-Q. Recombinant Porcine R-Spondin 1 Facilitates Intestinal Stem Cell Expansion along the Crypt-Villus Axis through Potentiating Wnt/β-Catenin Signaling in Homeostasis and Deoxynivalenol Injury. J Agric Food Chem. 2022;70(34):10644–53. doi: 10.1021/acs.jafc.2c02013 [DOI] [PubMed] [Google Scholar]
  • 49.Duchesne C, Randuineau G, Normand LL, Romé V, Laraqui S, Arnaud AP, et al. Initial pig developmental stage influences intestinal organoid growth but not phenotype. bioRxiv. 2024;2024.01:26.577507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Li L, Fu F, Guo S, Wang H, He X, Xue M, et al. Porcine Intestinal Enteroids: a New Model for Studying Enteric Coronavirus Porcine Epidemic Diarrhea Virus Infection and the Host Innate Response. J Virol. 2019;93(5):e01682-18. doi: 10.1128/JVI.01682-18 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Dong L, Yuan Q, Qiu G, Zhang Y, Wang H, Yu L. Protective Effects of Tea Tree Oil on Inflammatory Injury of Porcine Intestinal Epithelial Cells Induced by Lipopolysaccharide In Vitro. Animals (Basel). 2024;14(17):2577. doi: 10.3390/ani14172577 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Huang S, Zhang S, Chen L, Pan X, Wen Z, Chen Y, et al. Lipopolysaccharide induced intestinal epithelial injury: a novel organoids-based model for sepsis in vitro. Chin Med J (Engl). 2022;135(18):2232–9. doi: 10.1097/CM9.0000000000002348 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Williams JM, Duckworth CA, Watson AJM, Frey MR, Miguel JC, Burkitt MD, et al. A mouse model of pathological small intestinal epithelial cell apoptosis and shedding induced by systemic administration of lipopolysaccharide. Dis Model Mech. 2013;6(6):1388–99. doi: 10.1242/dmm.013284 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Chen Y, Zhang L, Zhang Y, Bai T, Song J, Qian W, et al. EphrinA1/EphA2 Promotes Epithelial Hyperpermeability Involving in Lipopolysaccharide-induced Intestinal Barrier Dysfunction. J Neurogastroenterol Motil. 2020;26(3):397–409. doi: 10.5056/jnm19095 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Li Q, von Ehrlich-Treuenstätt V, Schardey J, Wirth U, Zimmermann P, Andrassy J, et al. Gut Barrier Dysfunction and Bacterial Lipopolysaccharides in Colorectal Cancer. J Gastrointest Surg. 2023;27(7):1466–72. doi: 10.1007/s11605-023-05654-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Li C, Wang J, Yang H, Luo S, Lu Q. Oxyberberine alleviates lipopolysaccharide-induced intestinal barrier disruption and inflammation in human colonic Caco-2 cells in vitro. Front Pharmacol. 2025;15:1496874. doi: 10.3389/fphar.2024.1496874 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Ning W, Gao G, Zhou Y, Li W-Q, Yang H-H, Duan X-B, et al. Calcitonin gene-related peptide ameliorates sepsis-induced intestinal injury by suppressing NLRP3 inflammasome activation. Int Immunopharmacol. 2023;116:109747. doi: 10.1016/j.intimp.2023.109747 [DOI] [PubMed] [Google Scholar]
  • 58.He Y, Li Z, Xu T, Luo D, Chi Q, Zhang Y, et al. Polystyrene nanoplastics deteriorate LPS-modulated duodenal permeability and inflammation in mice via ROS drived-NF-κB/NLRP3 pathway. Chemosphere. 2022;307(Pt 1):135662. doi: 10.1016/j.chemosphere.2022.135662 [DOI] [PubMed] [Google Scholar]
  • 59.Chen X, Luo D, Jia G, Zhao H, Liu G, Huang Z. L-theanine attenuates porcine intestinal tight junction damage induced by LPS via p38 MAPK/NLRP3 signaling in IPEC-J2 cells. Food Chem Toxicol. 2023;178:113870. doi: 10.1016/j.fct.2023.113870 [DOI] [PubMed] [Google Scholar]
  • 60.Jia X, Zhao Y, Li H, Fan S, Hu H. Downregulation of MUC6 improves esophageal epithelial barrier dysfunction and inhibits epithelial-mesenchymal transition in reflux esophagitis. BMC Gastroenterol. 2026;26(1):53. doi: 10.1186/s12876-025-04336-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Rubtsova SN, Zhitnyak IY, Gloushankova NA. Dual role of E-cadherin in cancer cells. Tissue Barriers. 2022;10(4):2005420. doi: 10.1080/21688370.2021.2005420 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Kuo W-T, Odenwald MA, Turner JR, Zuo L. Tight junction proteins occludin and ZO-1 as regulators of epithelial proliferation and survival. Ann N Y Acad Sci. 2022;1514(1):21–33. doi: 10.1111/nyas.14798 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Nighot M, Al-Sadi R, Guo S, Rawat M, Nighot P, Watterson MD, et al. Lipopolysaccharide-Induced Increase in Intestinal Epithelial Tight Permeability Is Mediated by Toll-Like Receptor 4/Myeloid Differentiation Primary Response 88 (MyD88) Activation of Myosin Light Chain Kinase Expression. Am J Pathol. 2017;187(12):2698–710. doi: 10.1016/j.ajpath.2017.08.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Ciesielska A, Matyjek M, Kwiatkowska K. TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cell Mol Life Sci. 2021;78(4):1233–61. doi: 10.1007/s00018-020-03656-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Park BS, Lee J-O. Recognition of lipopolysaccharide pattern by TLR4 complexes. Exp Mol Med. 2013;45(12):e66. doi: 10.1038/emm.2013.97 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Chen J, Wu C, Yang R, Chen Z, Yang X, Xu Y, et al. LPS-Binding Hydrogel for TLR4-Mediated Microbiota-Immune Modulation. Adv Mater. 2026;38(9):e14484. doi: 10.1002/adma.202514484 [DOI] [PubMed] [Google Scholar]
  • 67.Kulkarni OP, Lichtnekert J, Anders H-J, Mulay SR. The immune system in tissue environments regaining homeostasis after injury: is “inflammation” always inflammation? Mediat Inflamm. 2016;2016:2856213. doi: 10.1155/2016/2856213 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Abnaof K, Mallela N, Walenda G, Meurer SK, Seré K, Lin Q, et al. TGF-β stimulation in human and murine cells reveals commonly affected biological processes and pathways at transcription level. BMC Syst Biol. 2014;8:55. doi: 10.1186/1752-0509-8-55 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Hyun J, Romero L, Riveron R, Flores C, Kanagavelu S, Chung KD, et al. Human intestinal epithelial cells express interleukin-10 through Toll-like receptor 4-mediated epithelial-macrophage crosstalk. J Innate Immun. 2015;7(1):87–101. doi: 10.1159/000365417 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Luissint A-C, Parkos CA, Nusrat A. Inflammation and the Intestinal Barrier: Leukocyte-Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair. Gastroenterology. 2016;151(4):616–32. doi: 10.1053/j.gastro.2016.07.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Sahoo DK, Borcherding DC, Chandra L, Jergens AE, Atherly T, Bourgois-Mochel A, et al. Differential Transcriptomic Profiles Following Stimulation with Lipopolysaccharide in Intestinal Organoids from Dogs with Inflammatory Bowel Disease and Intestinal Mast Cell Tumor. Cancers (Basel). 2022;14(14):3525. doi: 10.3390/cancers14143525 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Kinnebrew MA, Pamer EG. Innate immune signaling in defense against intestinal microbes. Immunol Rev. 2012;245(1):113–31. doi: 10.1111/j.1600-065X.2011.01081.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Gorboulev V, Schürmann A, Vallon V, Kipp H, Jaschke A, Klessen D, et al. Na(+)-D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion. Diabetes. 2012;61:187–96. doi: 10.2337/db11-1029 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.Spanier B. Transcriptional and functional regulation of the intestinal peptide transporter PEPT1. J Physiol. 2014;592(5):871–9. doi: 10.1113/jphysiol.2013.258889 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75.Wuensch T, Ullrich S, Schulz S, Chamaillard M, Schaltenberg N, Rath E, et al. Colonic expression of the peptide transporter PEPT1 is downregulated during intestinal inflammation and is not required for NOD2-dependent immune activation. Inflamm Bowel Dis. 2014;20(4):671–84. doi: 10.1097/01.MIB.0000443336.71488.08 [DOI] [PubMed] [Google Scholar]
  • 76.Barrenetxe J, Sánchez O, Barber A, Gascón S, Rodríguez-Yoldi MJ, Lostao MP. TNFα regulates sugar transporters in the human intestinal epithelial cell line Caco-2. Cytokine. 2013;64(1):181–7. doi: 10.1016/j.cyto.2013.07.004 [DOI] [PubMed] [Google Scholar]
  • 77.Xiao X, Cheng Y, Fu J, Lu Z, Wang F, Jin M, et al. Gut Immunity and Microbiota Dysbiosis Are Associated with Altered Bile Acid Metabolism in LPS-Challenged Piglets. Oxid Med Cell Longev. 2021;2021:6634821. doi: 10.1155/2021/6634821 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 78.Tian S, Wang J, Gao R, Zhao F, Wang J, Zhu W. Galacto-Oligosaccharides Alleviate LPS-Induced Immune Imbalance in Small Intestine through Regulating Gut Microbe Composition and Bile Acid Pool. J Agric Food Chem. 2023;71(46):17615–26. doi: 10.1021/acs.jafc.3c00419 [DOI] [PubMed] [Google Scholar]
  • 79.He Y, Shaoyong W, Chen Y, Li M, Gan Y, Sun L, et al. The functions of gut microbiota-mediated bile acid metabolism in intestinal immunity. J Adv Res. 2026;80:351–70. doi: 10.1016/j.jare.2025.05.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 80.Song G, Xie Y, Yi L, Cheng W, Jia H, Shi W, et al. Bile acids affect intestinal barrier function through FXR and TGR5. Front Med (Lausanne). 2025;12:1607899. doi: 10.3389/fmed.2025.1607899 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 81.Zhai Z, Niu K-M, Liu Y, Lin C, Wu X. The Gut Microbiota-Bile Acids-TGR5 Axis Mediates Eucommia ulmoides Leaf Extract Alleviation of Injury to Colonic Epithelium Integrity. Front Microbiol. 2021;12:727681. doi: 10.3389/fmicb.2021.727681 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 82.Liu Z, Qin X, Zhang B, Nong K, Chen W, Yang Z, et al. Proline rich-39 (PR-39) antimicrobial protein alleviated lipopolysaccharide-induced intestinal barrier dysfunction in piglets by altering intestinal flora associated bile acid metabolism and in turn regulating TGR-5/NF-κB/MLCK/MLC pathway. Int J Biol Macromol. 2025;307(Pt 1):141930. doi: 10.1016/j.ijbiomac.2025.141930 [DOI] [PubMed] [Google Scholar]
  • 83.Sorrentino G, Perino A, Yildiz E, El Alam G, Bou Sleiman M, Gioiello A, et al. Bile Acids Signal via TGR5 to Activate Intestinal Stem Cells and Epithelial Regeneration. Gastroenterology. 2020;159(3):956–68.e8. doi: 10.1053/j.gastro.2020.05.067 [DOI] [PubMed] [Google Scholar]
  • 84.Tang X, Xiong K. Epidermal growth factor activates EGFR/AMPK signalling to up-regulate the expression of SGLT1 and GLUT2 to promote intestinal glucose absorption in lipopolysaccharide challenged IPEC-J2 cells and piglets. Ital J Anim Sci. 2022;21:943–54. doi: 10.1080/1828051X.2022.2073832 [DOI] [Google Scholar]
  • 85.Koepsell H. Glucose transporters in the small intestine in health and disease. Pflugers Arch. 2020;472(9):1207–48. doi: 10.1007/s00424-020-02439-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 86.Schmitt CC, Aranias T, Viel T, Chateau D, Le Gall M, Waligora-Dupriet A-J, et al. Intestinal invalidation of the glucose transporter GLUT2 delays tissue distribution of glucose and reveals an unexpected role in gut homeostasis. Mol Metab. 2016;6(1):61–72. doi: 10.1016/j.molmet.2016.10.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 87.Wang B, Jiang L, Wu Z, Dai Z. L-Tryptophan Differentially Regulated Glucose and Amino Acid Transporters in the Small Intestine of Rat Challenged with Lipopolysaccharide. Animals (Basel). 2022;12(21):3045. doi: 10.3390/ani12213045 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 88.Petersen N, Reimann F, Bartfeld S, Farin HF, Ringnalda FC, Vries RGJ, et al. Generation of L cells in mouse and human small intestine organoids. Diabetes. 2014;63(2):410–20. doi: 10.2337/db13-0991 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 89.Sanchez JG, Enriquez JR, Wells JM. Enteroendocrine cell differentiation and function in the intestine. Curr Opin Endocrinol Diabetes Obes. 2022;29(2):169–76. doi: 10.1097/MED.0000000000000709 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 90.Abdalqadir N, Adeli K. GLP-1 and GLP-2 Orchestrate Intestine Integrity, Gut Microbiota, and Immune System Crosstalk. Microorganisms. 2022;10(10):2061. doi: 10.3390/microorganisms10102061 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 91.Wang W, Zhang C, Zhang H, Li L, Fan T, Jin Z. The alleviating effect and mechanism of GLP-1 on ulcerative colitis. Aging (Albany NY). 2023;15(16):8044–60. doi: 10.18632/aging.204953 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 92.Mehdi SF, Pusapati S, Anwar MS, Lohana D, Kumar P, Nandula SA, et al. Glucagon-like peptide-1: a multi-faceted anti-inflammatory agent. Front Immunol. 2023;14:1148209. doi: 10.3389/fimmu.2023.1148209 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 93.Lee Y-S, Jun H-S. Anti-Inflammatory Effects of GLP-1-Based Therapies beyond Glucose Control. Mediators Inflamm. 2016;2016:3094642. doi: 10.1155/2016/3094642 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 94.Migliorisi G, Gabbiadini R, Dal Buono A, Ferraris M, Privitera G, Petronio L, et al. GLP-1 receptor agonists in IBD: exploring the crossroads of metabolism and inflammation. Front Immunol. 2025;16:1610368. doi: 10.3389/fimmu.2025.1610368 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 95.Panaro BL, Yusta B, Matthews D, Koehler JA, Song Y, Sandoval DA, et al. Intestine-selective reduction of Gcg expression reveals the importance of the distal gut for GLP-1 secretion. Mol Metab. 2020;37:100990. doi: 10.1016/j.molmet.2020.100990 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 96.Lebrun LJ, Lenaerts K, Kiers D, Pais de Barros J-P, Le Guern N, Plesnik J, et al. Enteroendocrine L Cells Sense LPS after Gut Barrier Injury to Enhance GLP-1 Secretion. Cell Rep. 2017;21(5):1160–8. doi: 10.1016/j.celrep.2017.10.008 [DOI] [PubMed] [Google Scholar]
  • 97.Colwill M, Povlsen S, Pollok R, Patel K, Goodhand J, Ahmad T, et al. Glucagon-like peptide-1 (GLP-1) receptor agonists in inflammatory bowel disease: mechanisms, clinical implications, and therapeutic potential. J Crohns Colitis. 2025;19(9):jjaf167. doi: 10.1093/ecco-jcc/jjaf167 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 98.Zietek T, Rath E. Inflammation Meets Metabolic Disease: Gut Feeling Mediated by GLP-1. Front Immunol. 2016;7:154. doi: 10.3389/fimmu.2016.00154 [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Prashant Sharma

25 Jun 2026

Dear Dr. Yoo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The revised manuscript should particularly address the following points:

  • Better position the study within the existing literature on porcine intestinal organoids and clearly highlight the novelty of the work.

  • Provide a stronger justification for the use of three-dimensional organoids for immunofluorescence analyses and discuss the limitations regarding protein localization.

  • Clarify the rationale for selecting the 20 μM LPS concentration.

  • Report the number of biological and technical replicates for all experiments.

  • Include quantitative or semi-quantitative analyses of immunofluorescence data where appropriate.

  • Replace all figures with high-resolution images and correct inconsistencies between Figure 4 and its legend.

  • Review the manuscript for consistency in terminology, nomenclature, figure labeling, and overall presentation.

Please submit your revised manuscript by Aug 24 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Prashant Sharma, Ph.D.

Academic Editor

PLOS One

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Please state what role the funders took in the study.  If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.""

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5. Thank you for stating the following in the Acknowledgments Section of your manuscript: [This work was supported by a grant from the Rural Development Administration (PJ01671901). The authors gratefully acknowledge the financial support provided by the Korean government. We also extend our sincere gratitude to all individuals who contributed directly or indirectly to the completion of this research.]

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: [This work was supported by the Rural Development Administration (RDA), Republic of Korea, under the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01671901).]

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Two major comments have to be considered in order to improve the quality of the present version of this paper:

1. The authors have described known effects of LPS in connection with inflammatory processes and epithelial integrity in detail, however, they have not considered adequately to what extent such processes have already been described from other studies based on porcine organoids. This should be added either in the Introduction or in the Disccusion of their results.

2. A further major comment deals with the question why the authors have used three-dimensional rather than two-dimensional tissues for their immunofluorescence approaches. From their pictures it is hardly possible to identify the localization of the different protein. The potential use of two-dimensional tissues obtained from the original organoids has been described in a number of studies.

Reviewer #2: This study successfully established a lipopolysaccharide (LPS)-induced inflammatory model using porcine small intestinal organoids (pSIOs) derived from adult porcine jejunum. The authors comprehensively investigated LPS-mediated changes in intestinal structure, cell apoptosis, epithelial barrier function, inflammatory response, nutrient transporters, enteroendocrine factors, and global transcriptomic profiles via multiple techniques including qRT-PCR, immunofluorescence, DAPI/PI staining and RNA-seq. The overall experimental design is rigorous, data are sufficient, logical framework is clear, and discussion is objective. However. multiple defects exist in experimental description, data presentation, figure quality and nomenclature standardization. Extensive revisions are required to meet the journal’s publication criteria. Therefore, this manuscript is recommended for Major Revision.

1. The term 'lipopolysaccharide' is written in lowercase multiple times in the abstract. It is recommended that on first occurrence in abstract, it be uniformly standardized as the abbreviation LPS (with the full name preceding the abbreviation).

2. Lines 98–100 specify that “Organoids were cultured in IntestiCult™ medium supplemented with 20 μM LPS for 24 h at 37 °C in a 5% CO₂ atmosphere”. Please clarify the rationale for selecting 20 μM LPS as the working concentration. State whether this dosage was determined based on your preliminary experiments or cited from previous published studies.

3. All figures presented in the manuscript suffer from poor resolution and blurry display. Please replace all figures with high-definition, clear versions to meet the journal's image requirements.

4. The manuscript only states that experiments were performed with at least three independent experiments in the statistical section, but the exact number of biological replicates is not clearly specified for each individual experiment, including qRT-PCR, DAPI/PI staining, immunofluorescence and RNA sequencing. Please supplement the number of biological replicates for all in vitro assays in the Materials and Methods section as well as all figure legends. Meanwhile, clearly distinguish biological replicates from technical replicates.

5. There are multiple errors and mismatches regarding Figure 4 and its legend. The main text clearly states that Figure 4 consists of two parts: Panel A shows TLR4 immunofluorescence results, and Panel B presents qRT-PCR data of pro- and anti-inflammatory cytokines. However, the current figure legend incorrectly describes non-existent subpanels: “Pro-inflammatory cytokines TNF-α, IL-1β, and IL-8 (A–C) and anti-inflammatory cytokines IL-4, IL-10, andTGF-β (D–F) (Lines 286-287), and completely omits the description of TLR4 immunofluorescence. Please revise the figure legend to accurately match the actual content of Figure 4.

6. Only representative immunofluorescence images are presented in Figure 3, Figure 4, and Figure 5. Please add semi-quantitative analysis of fluorescence intensity and corresponding statistical results to improve the objectivity and persuasiveness of the findings.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2026 Oct 1;21(10):e0359086. doi: 10.1371/journal.pone.0359086.r002

Author response to Decision Letter 1

18 Aug 2026

Response to Reviewers: Manuscript ID: PONE-D-26-24948

Title: Lipopolysaccharide-induced alterations in barrier integrity and inflammatory signaling in porcine small intestinal organoids

General Response: We sincerely thank the Editor and the Reviewers for their careful evaluation of our manuscript and for their valuable comments and suggestions. We have revised the manuscript accordingly. All changes have been highlighted using Track Changes.

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

(Response) We have carefully reviewed the manuscript and all associated files to ensure full compliance with the PLOS ONE style requirements, including the formatting and file naming guidelines. The manuscript and all submission files have been revised accordingly.

Changes in manuscript: The title page and all submission files have been revised to comply with the PLOS ONE style and file naming requirements.

2. To comply with PLOS One submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

(Response) We have revised the Materials and Methods section to include the requested information regarding the animal experiments. Specifically, we have added details on the method of euthanasia, including intravenous administration of T61 to minimize animal suffering, clarified that no anesthesia or analgesia was administered because tissue collection was performed only after euthanasia, and stated that small intestinal tissues were collected immediately after euthanasia. The requested information has been added to the Materials and Methods section (Page 5, Lines 174–179) as below.

“An LPS-induced pSIO model was established using 10-month-old adult pigs. All animal procedures were approved by the Institutional Animal Care and Use Committee of the National Institute of Animal Science (NIAS 2022-0569). Animals were euthanized by intravenous administration of T61 to minimize animal suffering. No anesthesia or analgesia was administered because tissue collection was performed only after euthanasia. Small intestinal tissues were collected immediately after euthanasia.”

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

(Response) We have carefully reviewed the funding information and confirmed that the correct grant number for this study is PJ01671901. The Funding Statement has been revised accordingly to ensure consistency between the Funding Information and Financial Disclosure sections. Changes in manuscript: (Page 25, Lines 835-863).

“This work was supported by the Rural Development Administration (RDA), Republic of Korea, under the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01671901). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

4. Thank you for stating the following financial disclosure: [This work was supported by the Rural Development Administration (RDA), Republic of Korea, under the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01671901)]. Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

(Response) We thank the editor for this helpful comment. In accordance with the journal's guidelines, we have revised the Funding Statement to explicitly include the role of the funders. We have also included the amended Role of Funder statement in the cover letter, as requested. Changes in manuscript: (Page 25, Lines 835-863) as below.

“This work was supported by the Rural Development Administration (RDA), Republic of Korea, under the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01671901). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

5. Thank you for stating the following in the Acknowledgments Section of your manuscript: [This work was supported by a grant from the Rural Development Administration (PJ01671901). The authors gratefully acknowledge the financial support provided by the Korean government. We also extend our sincere gratitude to all individuals who contributed directly or indirectly to the completion of this research.] We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: [This work was supported by the Rural Development Administration (RDA), Republic of Korea, under the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01671901).] Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

(Response) In accordance with the journal's guidelines, we have removed all funding-related statements from the Acknowledgments section. In addition, we have revised the Funding Statement to provide the complete funding information and the role of the funders. The Acknowledgments have been revised to include only a general expression of gratitude.

Changes in manuscript: (Page 25, Lines 826-828).

“The authors sincerely thank all individuals who contributed directly or indirectly to this study.”

6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

(Response) We have added the captions for all Supporting Information files at the end of the manuscript and updated the corresponding in-text citations accordingly. In addition, the primer sequence table has been moved to the Supporting Information and is now presented as S1 Table.

Changes in manuscript: The in-text citation to S1 Table has been added to the Materials and Methods section (Page 8, Line 278), and the captions for all Supporting Information files have been added at the end of the manuscript (Page 46). “Primer sequences are listed in S1 Table.”

7. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

(Response) We carefully reviewed all reviewer comments and confirmed that no specific references were explicitly recommended for citation. Nevertheless, in response to Reviewer 1's suggestion to better position our study within the existing literature, we added several relevant references on porcine intestinal organoids where appropriate. Relevant references on porcine intestinal organoid research have been added to the Introduction and Discussion (Refs. 31, 36, and 40–44).

Reviewer 1

(Comment 1) The authors have described known effects of LPS in connection with inflammatory processes and epithelial integrity in detail, however, they have not considered adequately to what extent such processes have already been described from other studies based on porcine organoids. This should be added either in the Introduction or in the Discussion of their results.

(Response 1) We have revised both the Introduction and the Discussion to better discuss the current state of research on porcine intestinal organoids and to more clearly position our study within the existing literature.

In the Introduction, we have added a summary of previous applications of porcine intestinal organoids, including studies on enteric infections, host–pathogen interactions, intestinal epithelial biology, and organoid-derived two-dimensional monolayer systems. We also clarified that comprehensive characterization of LPS-induced inflammatory responses in porcine small intestinal organoids remains limited, thereby emphasizing the rationale and significance of the present study.

In the Discussion, we further compared our findings with previous reports and highlighted that, unlike previous studies focusing primarily on infectious disease models, our study provides an integrated characterization of LPS-induced inflammatory responses by simultaneously evaluating epithelial barrier integrity, inflammatory cytokine expression, nutrient transporter regulation, enteroendocrine responses, and global transcriptomic alterations. These revisions better clarify how our study differs from previous porcine intestinal organoid studies and highlight the novelty and contribution of the present work.

Changes in manuscript: The relevant text has been added to the Introduction (Page 4, Lines 131–169) and the Discussion (Page 19, Lines 672–685.

(Introduction)

“Recent studies have established porcine intestinal organoids as physiologically relevant models for investigating enteric viral infections, host–pathogen interactions, and intestinal epithelial biology (40-42). Furthermore, two-dimensional organoid-derived monolayer systems have been developed to facilitate barrier function assays and pathogen infection studies (43, 44). However, comprehensive characterization of LPS-induced inflammatory responses in porcine small intestinal organoids, including epithelial barrier disruption, nutrient transporter and receptor regulation, enteroendocrine responses, and global transcriptomic alterations, remains limited. To address this gap, the present study aimed to establish a porcine small intestinal organoid-based inflammatory model that integrates molecular, functional, and transcriptomic analyses to provide a comprehensive platform for investigating intestinal inflammation and evaluating potential therapeutic interventions.

In this study, pSIOs were treated with LPS to establish an intestinal inflammatory model. We analyzed transcriptomic and functional changes, including the expression of inflammation-related genes and the expression and localization of barrier-associated proteins, nutrient transporters, receptors, and enteroendocrine hormones. Collectively, these analyses enabled a comprehensive characterization of LPS-induced inflammatory responses in pSIOs and demonstrated the utility of this model as a physiologically relevant in vitro platform for investigating porcine intestinal inflammation and evaluating potential therapeutic interventions.”

(Discussion)

“Compared with the previous applications of porcine intestinal organoids, which have mainly focused on enteric infections, host–pathogen interactions, and intestinal epithelial biology, the present study expands their application by providing an integrated characterization of LPS-induced inflammatory responses. While earlier studies have often relied on evaluating a limited set of inflammatory markers or specific barrier proteins, we simultaneously evaluated epithelial barrier integrity, inflammatory cytokine expression, nutrient transporter regulation, enteroendocrine responses, and global transcriptomic alterations within a single experimental platform. This integrated approach provides a more comprehensive understanding of LPS-induced intestinal inflammation and supports the use of porcine small intestinal organoids as a physiologically relevant in vitro model for mechanistic studies and the evaluation of potential therapeutic or nutritional interventions.”

(Comment 2) A further major comment deals with the question why the authors have used three-dimensional rather than two-dimensional tissues for their immunofluorescence approaches. From their pictures it is hardly possible to identify the localization of the different protein. The potential use of two-dimensional tissues obtained from the original organoids has been described in a number of studies.

(Response 2) We appreciate the reviewer’s critical observation regarding the clarity of our immunofluorescence images. We agree that 2D monolayer systems offer distinct advantages for high-resolution imaging. We have revised the Discussion to clarify the rationale for using three-dimensional (3D) organoids for immunofluorescence analyses.

Specifically, we explained that 3D organoids were selected because they preserve the native epithelial architecture, cell–cell interactions, and spatial organization of the intestinal epithelium, thereby providing a more physiologically relevant context for evaluating protein expression. We also acknowledged that, although organoid-derived two-dimensional (2D) monolayer systems facilitate antibody accessibility and more precise visualization of protein localization, they do not fully retain the three-dimensional structural complexity of the intestinal epithelium.

Furthermore, we have discussed the limitation that the 3D configuration may restrict antibody penetration and make precise subcellular localization more challenging than in 2D monolayer systems. These revisions clarify the rationale for our methodological approach and acknowledge the limitations associated with 3D immunofluorescence analyses.

Changes in manuscript: The relevant text has been added to the Discussion (Page 19, Lines 662-671)

“In the present study, immunofluorescence analyses were performed using three-dimensional (3D) organoids to preserve the native epithelial architecture, cell–cell interactions, and spatial organization that closely resemble those of the in vivo intestine. Although organoid-derived two-dimensional (2D) monolayer systems provide improved antibody accessibility and facilitate more precise visualization of protein localization, they do not fully retain the three-dimensional structural complexity of the intestinal epithelium (31, 36, 44). Therefore, we selected the 3D organoid model to evaluate protein expression within an intact epithelial context. Nevertheless, we acknowledge that the 3D configuration may limit antibody penetration and make precise subcellular localization more challenging than in 2D monolayer systems. This should be considered when interpreting the immunofluorescence findings.”

Reviewer 2

(Comment 1) The term 'lipopolysaccharide' is written in lowercase multiple times in the abstract. It is recommended that on first occurrence in abstract, it be uniformly standardized as the abbreviation LPS (with the full name preceding the abbreviation).

(Response 1) The Abstract has been revised accordingly. The first occurrence has been standardized as “lipopolysaccharide (LPS)”, and the abbreviation “LPS” has been used consistently throughout the remainder of the Abstract.

Changes in manuscript: This has been revised in the Abstract (Page 2, Lines 28-38).

(Comment 2) Lines 98–100 specify that “Organoids were cultured in IntestiCult™ medium supplemented with 20 μM LPS for 24 h at 37 °C in a 5% CO₂ atmosphere”. Please clarify the rationale for selecting 20 μM LPS as the working concentration. State whether this dosage was determined based on your preliminary experiments or cited from previous published studies.

(Response 2) The rationale for selecting the 20 μM LPS concentration has now been clarified in the Materials and Methods section. The working concentration was determined based on our preliminary dose-response experiments using 5, 10, 20, and 30 μM LPS. Phase-contrast microscopy and DAPI/PI staining demonstrated a dose-dependent increase in structural damage and apoptosis following LPS treatment. While lower concentrations induced relatively mild changes, 20 μM consistently produced pronounced inflammatory and epithelial alterations while maintaining sufficient organoid integrity for subsequent molecular and functional analyses. In contrast, 30 μM caused extensive structural disruption and cell death, making it less suitable for downstream experiments. Therefore, 20 μM was selected as the optimal concentration for establishing the LPS-induced inflammatory model.

Changes in manuscript: The relevant explanation has been added to the Materials and Methods section (Page 6, Lines 197–203).

“Preliminary dose-response experiments were conducted using 5, 10, 20, and 30 μM LPS to identify the appropriate LPS concentration. The 5 and 10 μM concentrations induced minimal inflammatory responses with no apparent morphological changes, whereas 30 μM caused severe structural disruption accompanied by extensive cell death. In contrast, 20 μM reproducibly induced inflammatory responses with only partial morphological alterations and without extensive cell death. Therefore, 20 μM was selected for all subsequent experiments.”

(Comment 3) All figures presented in the manuscript suffer from poor resolution and blurry display. Please replace all figures with high-definition, clear versions to meet the journal's image requirements.

(Response 3) We thank the reviewer for this valuable comment. In response, we have replaced all figures in the revised manuscript with high-resolution versions to improve image clarity and meet the journal's image quality requirements. In addition, the font sizes of figure labels, axis titles, and annotations have been increased to improve readability and ensure that all text is clearly visible. All figures were regenerated from the original source files and exported at high resolution to provide clear visualization of images, graphs, and text.

Changes in manuscript:

All figures have been replaced with high-resolution versions, and the font sizes of figure labels and annotations have been increased to improve readability throughout the manuscript.

(Comment 4) The manuscript only states that experiments were performed with at least three independent experiments in the statistical section, but the exact number of biological replicates is not clearly specified for each individual experiment, including qRT-PCR, DAPI/PI staining, immunofluorescence and RNA sequencing. Please supplement the number of biological replicates for all in vitro assays in the Materials and Methods section as well as all figure legends. Meanwhile, clearly distinguish biological replicates from technical replicates.

(Response 4) We have revised the Materials and Methods section to clearly specify the number of biological and technical replicates for each in vitro assay. Specifically, for qRT-PCR, each independently processed organoid culture was considered one biological replicate (n = 3), and each biological replicate was analyzed using five technical replicates. For DAPI/PI staining and immunofluorescence analyses, experiments were performed using three independent biological replicates (n = 3), and at least five organoids were analyzed for each treatment condition within each biological replicate.

For RNA sequencing, RNA extracted from three organoid wells was pooled to generate a single RNA sample for each experimental group, resulting in one biological replicate (n = 1) per experimental group. While we acknowledge the limitation of lacking biological replicates for the RNA-seq analysis, this pooling strategy was carefully implemented to minimize intra-well variability and establish a stable, representative global transcriptomic profile. Importantly, the RNA-seq data was utilized primarily as an exploratory screening tool to map broad pathway alterations. To ensure the reliability of our results, all key transcriptomic findings—including the significant shifts in inflammatory cytokines, tight junction proteins, nutrient transporters, and enteroendocrine markers—were successfully cross-validated and confirmed using statistically powered, independent biological replicates (n = 3) via qRT-PCR and immunofluorescence assays. We believe this comprehensive validation robustly supports the biological reliability of our transcriptomic data despite the n = 1 design.

In addition, the corresponding figure legends have been revised to clearly indicate the number of biological replicates for each experiment and to distinguish biological replicates from technical replicates where applicable.

Changes in manuscript: The relevant explanations have been added to the Materials and Methods section (Pages 8–17, Lines 273–611), including the qRT-PCR, DAPI/PI staining, immunofluorescence staining, and RNA sequencing subsections. The corresponding figure legends (Figures 2, 3, 4, 5, and 6) have also been revised accordingly.

(qRT-PCR)

“For each experiment, organoids were independently cultured, treated, and subjected to RNA extraction, with each independently processed culture considered one biological replicate (n = 3). For each biological replicate, qRT-PCR was performed using five technical replicates. Relative expression levels were calculated using the 2-ΔΔCt method with GAPDH as the endogenous control based on the mean Ct values of the technical replicates.”

(Apoptosis analysis (DAPI/PI staining)

“DAPI/PI staining was performed using three independent biological replicates (n = 3), and at least five organoids were analyzed for each LPS concentration in each biological replicate.”

(Immunofluorescence staining)

“Immunofluorescence staining was performed using three independent biological replicates (n = 3), and at least five organoids were analyzed for each treatment condition in each biological replicate.”

(RNA sequencing data processing and quantification)

“For RNA sequencing, total RNA extracted from three organoid wells derived from the same organoid preparation was pooled to generate a single RNA sample for each experimental group (n = 1 per group). Therefore, RNA sequencing was performed without independent biological replicates.”

(Differential gene expression analysis)

“Because the experiment included only one biological sample per condition (n = 1), variance estimation from biological replicates was not possible”

(Statistical analysis)

“Statistical analyses were performed using GraphPad Prism (version 5.0; GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± standard deviation (SD) from at least three independent biological replicates, unless otherwise stated. Statistical significance between two groups was determined using an unpaired Student's t-test.”

(Figure 2.)

“Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates.”

(Figure 3B.)

“Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each LPS concentration in each biological replicate.”

(Figure 3C.)

“Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each treatment condition in each biological replicate.”

(Figure 3D.)

“Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates.”

(Figure 4A.)

“Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each treatment condition in each biological replicate.”

(Figure 4B.)

“Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates.”

(Figure 5A.)

“Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each treatment condition in each biological replicate.”

(Figure 5B.)

“Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates.”

(Figure 6.)

“RNA sequencing was performed using one pooled RNA sample generated from three organoid wells for each experimental group, resulting in one biological replicate (n = 1) per group.”

(Comment 5) There are multiple errors and mismatches regarding Figure 4 and its legend. The main text clearly states that Figure 4 consists of two parts: Panel A shows TLR4 immunofluorescence results, and Panel B presents qRT-PCR data of pro- and anti-inflammatory cytokines. However, the current figure legend incorrectly describes non-existent subpanels: “Pro-inflammatory cytokines TNF-α, IL-1β, and IL-8 (A–C) and anti-inflammatory cytokines IL-4, IL-10, and TGF-β (D–F) (Lines 286-287), and completely omits the description of TLR4 immunofluorescence. Please revise the figure legend to accurately match the actual content of Figure 4.

(Response 5) We thank the reviewer for this valuable comment. We have carefully revised Figure 4 to ensure complete consistency between the figure, its legend, and the corresponding description in the Results section. Specifically, the Figure 4 legend has been corrected to accurately describe Panel A as the TLR4 immunofluorescence analysis and Panel B as the qRT-PCR analysis of pro- and anti-inflammatory cytokines. The incorrect descriptions of the non-existent subpanels (A–F) have been removed, and a detailed description of the TLR4 immunofluorescence results has been added. In addition, the corresponding text in the Results section has been revised to maintain consistency with the updated figure and legend.

Changes in manuscript: The Figure 4 legend has been revised to accurately describe Panel A as the TLR4 immunofluorescence analysis and Panel B as the qRT-PCR analysis of pro- and anti-inflammatory cytokines. The corresponding description in the Results section (Page 13–14, Lines 468–493) has also been revised to ensure consistency between the figure, legend, and text.

“To evaluate the inflammatory responsiveness of pSIOs, organoids were treated with LPS (20 μM), and the TLR4 protein and cytokine gene expression were analyzed. Immunofluorescence staining showed TLR4 expression in both the control and LPS-treated groups, with significantly higher fluorescence intensity in the LPS-treated group (Figure 4A). To further characterize the inflammatory response, the mRNA expression levels of pro- and anti-inflammatory cytokines were analyzed by qRT-PCR (Figure 4B). The pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-8 (p < 0.01) and anti-inflammatory cytokines, IL-4 and IL-10 (p < 0.05) were significantly upregulated in the LPS-treated organoids, showing 2.61-fold, 3.61-fold, 3.65-fold, 12.15-fold, and 3.40-fold increases, respectively. In contrast, transforming growth factor beta (TGF-β) expression increased 1.41-fold but did not reach statistical significance.

“Figure 4. Cytokine gene expression changes in pSIOs following LPS treatment.

(A) Immunofluorescence staining of TLR4 in pSIOs after LPS (20 μM) treatment. Nuclei were stained with DAPI (blue), and TLR4 expression is shown in green. Representative images suggest higher TLR4 immunofluorescence in the LPS-treated group than in the control group. Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each treatment condition in each biological replicate. Scale bar = 100 μm. (B) qRT-PCR analysis of pro- and anti-inflammatory cytokines in pSIOs after LPS (20 μM) exposure. Pro-inflammatory cytokines TNF-α, IL-1β, and IL-8 were significantly upregulated in the LPS-treated group compared with the control group. Anti-inflammatory cytokines IL-4 and IL-10 also showed significantly increased expression, whereas TGF-β expression was increased but did not reach statistical significance following LPS stimulation. Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates. *p < 0.05, **p < 0.01.”

(Comment 6) Only representative immunofluorescence images are presented in Figure 3, Figure 4, and Figure 5. Please add semi-quantitative analysis of fluorescence intensity and corresponding statistical results to improve the objectivity and persuasiveness of the findings.

(Response 6) We sincerely thank the reviewer for this valuable and constructive suggestion and fully agree that quantitative analysis of immunofluorescence images can provide additional information under appropriate experimental conditions.

However, in the present study, immunofluorescence was not intended to quantify protein abundance or fluorescence intensity. Instead, it was performed to visualize the spatial localization of proteins associated with epithelial barrier integrity (E-cadherin), inflammatory responses (TLR4), nutrient transport (SGLT1, PEPT1, and GLUT2), bile acid signaling (TGR5), and enteroendocrine function (GLP-1), as well as to evaluate LPS-induced structural alterations within intact three-dimensional organoids.

Because individual three-dimensional organoids inherently differ in size, morphology, orientation, and imaging plane, direct comparison of fluorescence intensity among organoids is technically challenging and may introduce substantial bias. Therefore, our conclusions were not based solely on immunofluorescence observations but were supported by complementary quantitative analyses, including qRT-PCR and transcriptomic analyses, which consistently demonstrated LPS-induced inflammatory responses and epithelial alterations.

Similar approaches have also been reported in previous organoid studies. For example, Sarvestani et al. (Nature Communications, 2021) used immunofluorescence to assess the subcellular localization and co-localization of E-cadherin and β-catenin in human ulcerative colitis-derived organoids, while transcriptomic analyses were performed as part of the comprehensive characterization of the organoid model. Likewise, Zietek et al. (Scientific Reports, 2015) used immunofluorescence to determine the expression and spatial localization of intestinal nutrient transporters (SGLT1, PEPT1, and GLUT2), the bile acid receptor TGR5, and the enteroendocrine marker GLP-1 in small intestinal organoids. Notably, the immunofluorescence findings for GLP-1 were complemented by Gcg (encoding GLP-1) mRNA expression analysis, which showed the same expression pattern and demonstrated that the physiological characteristics of the organoids were preserved in vitro.

Based on these considerations, we respectfully believe that the representative immunofluorescence images presented in this study provide appropriate support for demonstrating the spatial localization of biologically relevant proteins and LPS-induced structural alterations in intact three-dimensional organoids. Accordingly, we have clarified this rationale in the revised manuscript.

Sarvestani et al. (Nature Communications, 2021)

“To confirm the tight association of E-cadherin/β-catenin proteins, as well as significant differences in their sub-cellular co-localization, we performed dual IF staining in iHNOs and iHUCOs (Fig. 2g).”

Zietek, Tamara, et al.(Scientific reports, 2015)

“We first determined the expression and localization of some known solute carriers as well as of the bile acid receptor TGR5 in polarized SI organoids by immunofluorescent staining (Fig. 1a and Supplementary Fig. S1).”

“Immunofluorescent staining confirmed the presence of GLP-1- and GIP-containing EEC in SI organoids (Fig. 2a,e). It is established that the number of GLP-1-containing cells increases from proximal SI/duodenum to distal SI/ileum and vice versa for GIP-containing cells. When preparing organoids from different parts of the SI, we obtained the same expression pattern for the Gcg (encoding GLP-1) and Gip mRNA showing that physiology is preserved in vitro (Fig. 2b,f).”

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<p>Lipopolysaccharide-induced alterations in barrier integrity and inflammatory signaling in porcine small intestinal organoids

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    S1 Table. Primers used for gene expression analysis using quantitative RT-PCR.

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