AhR ligands from LGG metabolites promote piglet intestinal ILC3 activation and IL-22 secretion to inhibit PEDV infection

ABSTRACT

In maintaining organismal homeostasis, gut immunity plays a crucial role. The coordination between the microbiota and the immune system through bidirectional interactions regulates the impact of microorganisms on the host. Our research focused on understanding the relationships between substantial changes in jejunal intestinal flora and metabolites and intestinal immunity during porcine epidemic diarrhea virus (PEDV) infection in piglets. We discovered that Lactobacillus rhamnosus GG (LGG) could effectively prevent PEDV infection in piglets. Further investigation revealed that LGG metabolites interact with type 3 innate lymphoid cells (ILC3s) in the jejunum of piglets through the aryl hydrocarbon receptor (AhR). This interaction promotes the activation of ILC3s and the production of interleukin-22 (IL-22). Subsequently, IL-22 facilitates the proliferation of IPEC-J2 cells and activates the STAT3 signaling pathway, thereby preventing PEDV infection. Moreover, the AhR receptor influences various cell types within organoids, including intestinal stem cells (ISCs), Paneth cells, and enterocytes, to promote their growth and development, suggesting that AhR has a broad impact on intestinal health. In conclusion, our study demonstrated the ability of LGG to modulate intestinal immunity and effectively prevent PEDV infection in piglets. These findings highlight the potential application of LGG as a preventive measure against viral infections in livestock.

IMPORTANCE

We observed high expression of the AhR receptor on pig and human ILC3s, although its expression was negligible in mouse ILC3s. ILC3s are closely related to the gut microbiota, particularly the secretion of IL-22 stimulated by microbial signals, which plays a crucial regulatory role in intestinal immunity. In our study, we found that metabolites produced by beneficial gut bacteria interact with ILC3s through AhR, thereby maintaining intestinal immune homeostasis in pigs. Moreover, LGG feeding can enhance the activation of ILC3s and promote IL-22 secretion in the intestines of piglets, ultimately preventing PEDV infection.

INTRODUCTION

Porcine epidemic diarrhea virus (PEDV), a member of the genus Coronavirus A of the family Coronaviridae, causes acute diarrhea and/or vomiting, dehydration, and high mortality in neonatal piglets (1). Related studies have reported significant changes in the intestinal microbiota of Lactobacillus, Escherichia coli, and Lactococcus in piglets as a result of PEDV infection (2). Specifically, the population of Lactobacillus jejuni and Lactobacillus in the cecum tended to decrease in PEDV-infected piglets, accompanied by significant changes in metabolites (3).

In recent years, intestinal immunity has been shown to play an important role in organismal homeostasis (4). Bidirectional interactions between the microbiota and the immune system coordinate many microbial effects on the host (5). Innate lymphoid cells (ILCs), which continuously interact with the commensal flora in the gut, are the pioneers (6). Moreover, IL-22 production by ILCs is critical for intestinal immunity during the early course of infection (7). Previous studies have shown that the intestinal flora is a key factor in IL-22 production in the gut, but the underlying regulatory mechanisms are unclear (8).

In addition to interacting with a variety of intestinal epithelial cells, ILCs also interact with intestinal stem cells (ISCs) in the crypts to regulate the differentiation and function of ISCs (9). ILC3-derived IL-22 can promote the phosphorylation of signal transducer and activator of transcription 3 (STAT3) on intestinal stem cells through the action of IL-22R, thus inhibiting the apoptosis of intestinal stem cells. Moreover, IL-22 can stimulate the proliferation of intestinal stem cells and alleviate the damage to ISCs caused by chemotherapy (10, 11).

The aryl hydrocarbon receptor (AhR) is essential for the maintenance of ILC3s and the production of IL-22 (12). AhR is another key transcription factor for ILC3s, and the presence of lymphoid tissue in the intestine is associated with intact AhR expression (13). In contrast, the removal of AhR is equivalent to the absence of ILC3s, resulting in the disappearance of intestinal lymphoid tissue (14). AhR is expressed in many mammalian tissues, particularly in the liver, intestine, and kidney. In the intestine, AhR, which is expressed mainly by epithelial cells and innate immune cells, plays an important role in the regulation of innate immunity and the number of lymphocytes within the epithelium. When the energy source is converted from sugar to tryptophan, Lactobacillus lactis amplifies and produces indole-3-aldehyde (I3A), which serves as an AhR ligand that promotes the production of IL-22 by ILC3s (15). IL-22 plays a central role in the protection of host cells against pathogens at the surface of the mucosa through the activation of STAT3 (16). It has been reported that mature porcine IL-22 inhibits PEDV infection and promotes the expression of antimicrobial peptide β-defensins, the cytokine IL-18, and IFN-λ through activation of the STAT3 signaling pathway (17).

An important way in which probiotics combat diarrheal diseases is by modulating the mucosal immune system of the host intestine. Mucosal immunity is an important component of the host immune system, and its main function is to remove pathogenic microorganisms that invade the host through mucosal surfaces. The mucosal immune system has a unique organizational structure and function. It is widely distributed in the mucosal tissues of the respiratory, digestive, and genitourinary tracts and serves as the main site of the local immune response. The mucosal immune system consists of various components, including mucosal epithelial tissue, mucosal-associated lymphoid tissue (MALT), intestinal epithelial cells, immune cells, and the molecules or secretions they produce. Additionally, it includes mucosal microorganisms, such as commensal microorganisms, which normally inhabit the mucosa. Some studies have reported that some Lactic Acid Bacterial (LAB) strains have antiviral activity against PEDV, and oral administration of LAB inhibits PEDV infection (18, 19). It has been reported that the metabolites of L. plantarum reportedly have an inhibitory effect on the replication of PEDV (20). A large number of studies have shown that LGG and its metabolites have a significant effect on improving intestinal immunity and antiviral viruses (21–23). Therefore, LGG can improve the intestinal immunity of piglets to resist PEDV infection.

In this study, a coculture system of porcine jejunal gut tract-like organoids with porcine jejunal lamina propria lymphocytes (LPLs) was established to examine and validate the protective effect of lactobacilli on intestinal epithelial cells. We hypothesize that LGG may secrete substances, such as I3A, which can activate the AhR receptors located on the surface of ILC3s present in the lamina propria of the pig intestinal mucosa. This activation could enhance the secretion of IL-22 by ILC3s. Then, the STAT3 signaling pathway in intestinal epithelial cells was activated, ISC regeneration was induced to repair damage, Paneth cells were induced to secrete Reg3b and Reg3g to inhibit the growth of pathogenic bacteria, and intestinal cells were induced to secrete IFN-λ to inhibit PEDV replication.

RESULTS

PEDV infection affects the intestinal flora and ILC3s in piglets

In our previous work, we found a large number of immune cells in the piglet intestine through flow cytometric sorting and single-cell sequencing (scRNA-seq) (24). In immune cells, in the intestinal lamina propria, there were a large number of ILCs cells in the L/D⁻CD45⁺Lin⁻ population (Fig. 1A), in which ILC3s expressing the signature transcription factor RORC were absolutely dominant (Fig. 1B); these cells also expressed IL7R, AhR, and other surface molecules and secreted corresponding cytokines such as IL-22 and IL-17. The expression of these genes is important for the regulation and protection of intestinal homeostasis (Fig. 1C).

Fig 1.

PEDV infection resulted in abnormal changes in both the intestinal flora and ILCs of piglets. (A) UMAP plots showing the immune landscape of the 12 intestinal immune cell subpopulations. Intestinal immune cells were selected by flow cytometry and identified as L/D⁻CD45⁺Lin⁻ (Lin cells included CD3ε, CD21, γδT, CD11c, CD172a, CD4, CD8, CD163, and CD11b cells). (B) UMAP plots shows the immune landscape of the four clusters of ILCs (ILC regrouping). Cells are color-coded according to the defined subset (ILC3: yellow; ILC1: red; ILC2: green; and NK: blue). (C) Heatmap showing the marker genes with the greatest number of DEGs in each cluster. The abscissas show different clusters, and the ordinates show the names of the DEGs. (D) A total of 7,157 cells from piglets in the CONTROL group and 6782 cells from piglets in the PEDV group. These cells included both immune cells and nonimmune cells. UMAP plots showing the immune landscape of intestinal cells in the jejunum of piglets in both the PEDV-infected and normal states (all intestinal cells, including immune cells and nonimmune cells). The epithelial cells are shown in the yellow dashed box, and the T/ILCs are shown in the black silk dashed box. (E) Green dashed boxes are cells expressing IL23R, mainly ILC3s, and the red dashed boxes are ILC3s expressing the IL-22 gene. The number of ILC3s significantly decreased, whereas the expression of IL-22 significantly increased. (F-I) Proportion analysis of different cell types in the jejunum of piglets in the CON and PEDV groups. (F) Variations in the number of immune cells. (G) Variations in the number of ILC3s. (H) Variations in the number of epithelial cells. (I) Differential changes in the expression of IL-22 by ILC3s. (J) The number of ILC3s in the intestinal lamina propria of PEDV-infected piglets significantly decreased, and the secretion of IL-22 increased (n = 5). (K) Changes in the population of Lactobacillales orders in the intestinal tract of piglets (n = 5). (L) Changes in Lactobacillus genera in the intestinal tract of piglets (n = 5).

Through scRNA-seq analysis, we found that PEDV infection in piglets led to a significant decrease in the number of immune cells in the jejunum, including T cells and ILC3s (Fig. 1D, F, and G). In contrast, nonimmune cells, such as epithelial cells, exhibited abundant proliferation during infection (Fig. 1D and H). Immunofluorescence analysis of the piglet jejunum also confirmed a changed number of CD45⁺ T cells and epithelial cells (Fig. S1B). Notably, despite the significant decrease in the number of ILC3s in the jejunum, the transcription of IL-22 for each ILC3 significantly increased (Fig. 1E and I). Our flow cytometry results were consistent with the single-cell results, and the flow gating strategy is shown in Fig. S1A. These results also confirmed a significant decrease in the number of ILC3s cells in the intestinal lamina propria of PEDV-infected piglets, but despite the decrease in ILC3s, the level of IL-22 secreted per ILC3 has increased significantly (Fig. 1J). Furthermore, our findings revealed significant alterations in the flora of the jejunum in PEDV-infected piglets. PCA revealed significant differences between the CON and PEDV groups (Fig. S1C). At both the order level and the genus level, there was a notable decrease in the population of Lactobacillales and Lactobacillus (Fig. 1K and L). The qPCR analysis revealed a substantial reduction in the intestinal population of Limosilactobacillus reuteri (L. reuteri) and Lacticaseibacillus rhamnosus (L. rhamnosus) in piglets infected with PEDV (Fig. S1D).

To further investigate the underlying cause of the significant changes in ILC3s during PEDV infection, we aimed to discern whether these changes were primarily driven by viral infection or influenced by alterations in the bacterial flora. To explore this issue, we conducted an experiment involving antibiotic treatment to manipulate the structure of the flora. By observing the effects of these changes in the microbial flora on host immunity, we aimed to gain insights into the intricate relationship between the microbial composition and the immune response.

Alterations in the intestinal flora may affect ILC3 development

In our study, we initially established a dysregulation model of the microflora of piglets. We observed significant impacts on the immune development of piglets with dysflora. Specifically, the number of CD4⁺ T cells in the jejunum lamina propria displayed no significant differences (Fig. S2A). However, the development of ILC3s was severely affected, with a significant decrease in cell numbers and an increase in IL-22 secretion (Fig. 2A). Moreover, antibiotic treatment decreased the flora richness and Lactobacillus population in the jejunum of the piglets. α-diversity analysis revealed significantly different taxa between groups, which were significantly enriched from the point of view of order, genus, and species belonging to Lactobacillales, Lactobacillaceae, and Lactobacillus, respectively (Fig. 2B through D). Based on the resulting ASVs, antibiotic treatment significantly altered the population and species of the jejuni flora (Fig. 2E). Boxplot statistics showed that the species did not increase significantly with sample size, and the sample size was sufficient for subsequent data analysis (Fig. 2F). PCA revealed significant differences between the PBS and ABX groups (Fig. S2B). Linear discriminant analysis effect size (LEfSe) analysis revealed an absolute advantage of Lactobacillaceae and Lactobacillales in the PBS group and Pseudomonadaceae and Pseudomonadales in the ABX group (Fig. 2G; Fig. S2C).

Fig 2.

The imbalance of intestinal flora may affect the development of ILC3s. (A) The number of ILC3s in the intestinal lamina propria of piglets with intestinal flora disturbance decreased significantly, and the secretion of IL-22 increased (n = 5). (B–D) Statistical analysis of the relative population (relative) of the species in the samples. (B) Changes in the population of the Lactobacillales order in the intestinal tract of the piglets. (C) Changes in the Lactobacillaceae family in the intestinal tract of piglets. (D) Changes in the Lactobacillus genera in the intestinal tract of piglets. (E) Based on the resulting ASVs, the number of common, unique ASVs between the PBS and ABX groups was plotted as a Venn Graph (α diversity). (F) The horizontal axis represents the amount of sequencing data, and the vertical axis represents the corresponding alpha diversity index. When the curve tends to flatten, the amount of sequencing data is gradually decreased, and a greater amount of data will not have a significant effect on the alpha diversity index (α diversity). (G) LDA effect size (LEfSe) analysis between the PBS and ABX groups (β diversity). Species with LDA scores greater than the set point (default set to 4) were considered biomarkers that were significantly different between groups. The length of the bar chart represents the effect size of the divergent species (LDA score). (H) Changes in differentially abundant metabolites in negative ion mode. (I) Changes in differentially abundant metabolites in the positive ion mode. (J) Different substances were observed between the groups in negative ion mode, with a particular focus on substances that are associated with AhR. (K) Different substances were observed between the groups in negative ion mode, with a particular focus on substances that are associated with AhR. (L) Bubble diagram of the KEGG pathways enriched by differentially abundant metabolites in positive ion mode. (M) UMAP and violin plots show that AhR was prominently expressed in immune cells within pig intestines. (N) UMAP and violin plots show that AhR was prominently expressed in ILC3s within pig intestines. (O) UMAP and violin plots shows that AhR was prominently expressed in ILC3s and ILC1s within the human intestine. (P) UMAP and violin plots showing that AhR was prominently expressed in ILC2s within mouse intestines.

The above results suggest that the number of immune cells, especially ILC3s, was significantly affected under both PEDV infection and antibiotic treatment conditions and that this change persisted when only the flora was changed. The flora may play a very important role in interactions with ILC3s, and if there is a regulatory effect of the flora on ILC3s, by what pathway does the flora regulate ILC3s? The upregulation of indole analogs, including bisindolylmaleimide I and indoxyl sulfate, has been reported in PEDV infection (3). After we fed the piglets with antibiotics, nontargeted metabolomics was further used to analyze the metabolites in jejunum samples. The results showed that the total number of metabolites with significant differences in the negative ion mode was 39, of which 11 were significantly upregulated and 28 were significantly downregulated (Fig. 2H). The total number of metabolites with significant differences in the positive ion mode was 87, of which 36 were significantly upregulated and 51 were significantly downregulated (Fig. 2I). We analyzed the metabolites and found that the levels of some AhR-related regulatory substances significantly changed in the jejunum of the piglets. Negative ion mode contains four substances that can act on AhR receptors (Fig. 2J), and positive ion mode contains 10 substances (Fig. 2K); in particular, many indoles have been repeatedly shown to have a significant activating effect on AhR. We further enriched and analyzed the differentially abundant metabolites and found that the differentially abundant metabolites in the positive ion mode were significantly enriched in important immunomodulatory pathways such as tryptophan metabolism (Fig. 2L). To investigate whether differential microbial communities produce differentially abundant metabolites, we conducted a combined analysis of 16S rRNA and metabolomics. The results revealed a significant decrease in the population of regulatory substances secreted by probiotic bacteria, leading to a reduction in their overall levels. However, unfortunately, we did not directly associate the metabolites with Lactobacillus production (Fig. S2D and E).

To date, the expression of the AhR receptor has been studied in immune cells in humans and mice, but its expression has not been reported in pigs. Furthermore, the expression of AhR in immune cells in the intestine is unknown. scRNA-seq analysis revealed that immune cells in the porcine intestine highly expressed AhR receptors (Fig. 2M), and further analysis revealed that ILC3s in the intestine expressed high levels of AhR (Fig. 2N). In human intestinal ILCs, both ILC3s and ILC1s highly expressed AhR receptors (Fig. 2O), whereas only ILC2s expressed AhR in mouse intestinal ILCs (Fig. 2P). These results suggest that porcine and human intestinal ILC3s exhibit high levels of AhR activation, indicating that ILC3s in the intestine may respond significantly to stimulation by AhR ligand-related substances.

Feeding piglets LGG can enhance their immunity against PEDV infection

To explore whether Lactobacillus can prevent PEDV infection in the gut, we selected four types of Lactobacillus, including Lactiplantibacillus plantarum (L. plantarum), L. reuteri, Lacticaseibacillus casei (L. casei), and L. rhamnosus. The results of the TCID50 and qPCR experiments found that L. rhamnosus could help IPEC-J2 cells to alleviate PEDV infection (Fig. S3A and B). Therefore, we used the standard L. rhamnosus strain LGG to supplement piglets with an intestinal flora imbalance and then carried out a PEDV challenge experiment. Our objective was to determine whether LGG feeding could enhance intestinal immunity, specifically by activating ILC3s, thereby promoting intestinal homeostasis and conferring resistance against PEDV infection.

Our results revealed that LGG supplementation significantly reduced the pathological changes caused by PEDV infection in piglets, and the intestinal tract was healthy in the CON group. The villi of the jejunum of piglets in the ABX + LGG + PEDV group were shortened and atrophied, and the epithelial cells were mildly diseased. The villi of the jejunum in piglets from the ABX + PEDV group exhibited shortened, fragmented, and broken structures. Additionally, the intestinal epithelial cells underwent typical histopathological changes such as vacuolization, fusion lesions, necrosis, and detachment following PEDV infection (Fig. 3A). The VH/CD ratio of the jejunal villi of piglets in each infection group was significantly lower in the ABX + PEDV group than in the ABX + LGG + PEDV group (Fig. 3B). The diarrhea scoring results proved that feeding piglets LGG alleviated diarrhea (Fig. 3C). Next, we found significant differences in viral loads in the duodenum, jejunum, and ileum between the ABX + LGG + PEDV group and the ABX + PEDV group, especially in the jejunum and ileum, where the viral copy number was significantly decreased after LGG feeding (Fig. 3E).

Fig 3.

LGG can help piglets resist PEDV infection. (A) LGG feeding can significantly alleviate the pathological changes in the jejunum of piglets (n = 5). (B) The ratio of villus height to crypt depth (VH/CD) of the intestinal villi of the piglets. (C) Piglet diarrhea score. 0 = normal stool, 1 = soft but formed stool, 2 = semiliquid stool, and 3 = watery diarrhea, with a score of 2 or more indicating diarrhea. (D) Flow cytometry showing the changes in ILC3 quantity and IL-22 secretion in the intestinal tract of piglets. (E) qPCR results show the viral load in the intestines of the piglets. (F) Immunofluorescence analysis of the expression of EpCAM and VILLIN in the gut via Image J software. (G) Bar chart analysis of the genus-level microbiota in the jejunum of piglets from different groups. (H) Alpha diversity refers to the analysis of differences between groups, and box plots can visually represent the median, dispersion, maximum value, minimum value, and outliers of species diversity within each group. The horizontal axis of the box chart represents the grouping, whereas the vertical axis represents the corresponding alpha diversity index (Chao1).

The flow cytometry results also demonstrated a significant increase in the number of ILC3s in the intestines of the ABX + LGG + PEDV group, which was close to that of the CON group and significantly greater than that of the ABX + PEDV group. The secretion of IL-22 was also significantly lower than that in the ABX + PEDV group (Fig. 3D). LGG induced a dramatic improvement in the abnormal activation state of the intestinal tract through stimulation to promote the proliferation of epithelial cells and the production of IL-22 after viral infection, including the expression of proteins such as EpCAM and VILLIN in the intestinal tract (Fig. 3F). There was also a significant reduction in the expression of the cell proliferation-related proteins LGR5 and Ki-67 (Fig. S3A).

LGG feeding also significantly increased the population of intestinal flora in piglets, particularly the number of Lactobacillus genera, and elevated the content of other probiotics, such as Streptococcus spp (Fig. 3G). Moreover, the alpha diversity index of the microbial community significantly increased (Fig. 3H). The results of heatmap analysis of microbial communities at the genus level also showed that LGG feeding can promote the recovery of the intestinal flora (Fig. S3B). PCA revealed that the intestinal microbiota varied significantly among the different groups (Fig. S3C). The results of the β diversity analysis also showed that feeding LGG promoted the restoration of intestinal flora diversity, and the coefficient of difference between the CON group and the ABX + LGG + PEDV group was significantly smaller than that between the CON group and the ABX + PEDV group (Fig. S3D).

Our results at the mRNA level were consistent with the results at the protein level, revealing that the expression of inflammation-related genes such as IL-22 and IL-1β, the epithelial gene EpCAM, the villi gene VILLIN, the proliferation- and differentiation-related gene MKI-67, the ISC-encoding genes LGR5, Ascl2, Olfm4, and the Paneth cell activation genes Lyz1 and REG3b were all significantly reduced (Fig. S3E).

Extracellular vesicles secreted by LGG (L-EVs) can promote ILC3 activation and the secretion of IL-22

We first successfully constructed an in vitro culture model of ILC3s (Fig. 4A). In previous experiments, Lin⁻ (Lin includes CD3ε, CD21, γδT, CD11c, CD172a, CD4, CD8, CD163, and CD11b) cell collection species were shown to be dominated by ILC3s (accounting for more than 80%); hence, the ILCs were collected by magnetic bead sorting (Fig. S4A). Then, a flow cytometry assay was performed under different stimulant-inducing conditions, and a gating strategy was developed (Fig. S4B). We added heat-killed LGG and LGG metabolites (obtained by ultracentrifugation) to ILCs. We found that L-EVs significantly promoted ILC3 proliferation and the secretion of IL-22 (Fig. 4B).

Fig 4.

L-EVs promote ILC3 activation. (A) Figure depicting an in vitro coculture model of ILC3s (through the immune magnetic bead separation technique). No irritants were added to the culture wells of the CON group, 50 µg of HK LGG was added to each well in the HK LGG group, and 50 µg of L-EVs was added to each well in the L-EVs group. Flow cytometry was performed after 12 h of culture. (B) Flow cytometry showed that L-EVs promoted the number of ILC3s and the secretion of IL-22 under coculture conditions (n = 5). (C) Results of nanoflow cytometry detection of L-EVs. (D) Observation of L-EVs under an electron microscope. (E) Detection of L-EVs and classification of LGG metabolites in positive and negative ion modes. The pie chart reflects the classification of detected metabolites, and the number contained in each classification. (F) KEGG enrichment analysis was conducted for LGG metabolites. The abscissa represents the number of metabolites, and the ordinate represents the annotated KEGG pathway. This figure shows the number of metabolites annotated in each secondary classification under the pathway’s primary classification.

We enriched and collected the L-EVs through ultrafast centrifugation and found the diameter of the L-EVs was approximately 70 nm by nanoflow cytometry (Fig. 4C). The signature horseshoe-shaped structure of the L-EVs, which contained a large amount of LGG metabolites, was visualized under an electron microscope (Fig. 4D). Subsequently, we conducted untargeted metabolome detection. In total, 326 substances were identified in the positive mode (NEG), whereas 414 substances were detected in the negative mode (POS). We observed a significant presence of lipids and lipid-like molecules, as well as organic acids and derivatives, in both the POS and NEG modes (Fig. 4E). We found a wide range of substances that can interact with AhR, including indole compounds such as L-5-hydroxytryptophan and indole-3-acrylic acid, as well as several ketone compounds and some ketones. Research has indicated that in addition to indoles, ketones can also interact with AhR (25). We conducted KEGG enrichment analysis on the total metabolites and found significant enrichment in the metabolism pathway. Additionally, enrichment was observed in pathways such as Cellular Processes, Environmental Information Processing, and Genetic Information Processing (Fig. 4).

L-EVs promote the ability of ILC3s to resist PEDV through the AhR receptor

To explore whether L-EVs can promote the activation of ILC3s through AhR, we added an AhR receptor agonist (indole-3-carbinol, I3C), an L-EVs, and an AhR antagonist (CH-223191, CH) to the ILC3 culture medium. We found that both L-EVs and I3C could significantly promote ILC3 proliferation and the secretion of IL-22 through the activation of AhR. The effect disappeared when CH was added (Fig. 5A).

Fig 5.

L-EVs protect IPEC cells from PEDV infection by activating the AhR receptor of ILC3s. (A) Flow cytometry showed that I3C and L-EVs promoted the number of ILC3s and the secretion of IL-22 through AhR under coculture conditions, and these effects increased after the addition of CH (n = 5). (B) Transcriptional levels of ILC3-related cytokines (IL-22 and IL-17) stimulated by LGG metabolites were measured by qPCR (ILC3-related cytokines were assayed after coculture with L-EVs for 12 h). (C) ILC3-related chemokines (CXCL2 and CXCL8) stimulated by LGG metabolites. (D) Schematic diagram of an in vitro coculture model of ILC3s with IPEC-J2 cells. (E) Statistics of the number of IPEC-J2 cells. The transcription level of the STAT3 gene in IPEC-J2 cells was detected by qPCR. (F) Flow cytometry was used to calculate the percentage of cells that died. The Annexin V⁻/PI⁺ cells in the upper left quadrant may represent cell fragments lacking cell membranes or dead cells resulting from other causes. The Annexin V⁻/PI⁻ cells in the lower left quadrant were considered normal and alive. The Annexin V⁺/PI⁺ cells in the upper right quadrant are indicative of late apoptotic cells. Finally, the Annexin V⁺/PI⁻ cells in the lower right quadrant also corresponded to early apoptotic cells.

Previous correlational studies have indicated the possibility of ILC transformation within an organism, particularly when induced by specific conditions (26). To investigate whether the change in the number of ILC3s is due to the transformation of other immune cells, we conducted magnetic bead sorting on ILCs and subsequently examined them using SFSE staining. We discovered that ILC3s are the primary cell population within ILCs where proliferation occurs. The proliferation of ILCs dominated by ILC3s was significantly greater than that of other non-ILC cells (Fig. S5A). The expression of ILC3-related cytokines, including IL-22, IL-17A, IL-17B, CXCL2, and CXCL8, was significantly upregulated (Fig. 5B and C).

Our study investigated whether the activation of ILC3s and the secretion of IL-22 promoted by L-EVs are effective in enhancing the resistance of porcine epithelial cells to PEDV infection. We established a coculture model of ILC3s and IPEC-J2 cells (Fig. 5D) and observed that IL-22 secreted by L-EVs-promoted ILC3s had a positive impact on IPEC-J2 cell proliferation and the activation of STAT3 (Fig. 5E). Furthermore, the addition of L-EVs and ILC3s to cocultured IPEC-J2 cells after PEDV infection, it significantly influenced the outcome of PEDV infection by preventing IPEC-J2 cell apoptosis (Fig. 5F); the gating strategy is shown in Fig. S5B.

These results show that L-EVs can stimulate ILC3 proliferation and the secretion of IL-22 through AhR. This process promotes epithelial cell proliferation and activation and enhances the expression of the STAT3 gene. Therefore, it alleviates IPEC-J2 cell apoptosis caused by PEDV infection.

L-EVs can activate ILC3s and secrete IL-22 to promote the development of porcine intestinal organoids

We successfully constructed an in vitro model of organoids from the jejunum of piglets. The size of the isolated intestinal crypts was approximately 15 µm, and the crypts were cultured with Matrigel. The first generation of organoids grew slowly, developed into mature bodies with a diameter of approximately 200 µm by the eighth day, and continued to be cultured. Then, they died and fragmented, displayed a large number of buds in 5–8 days, and could be propagated through passaging culture (Fig. 6A). Subsequently, the cultured organoids grew rapidly after passaging, reaching a size of 100 µm within 3 days and maturing into bodies with a diameter of 200 µm within 5 days (Fig. S6A). Similar to human and murine intestinal organoids, porcine intestinal organoids also consist of intestinal Iumen, Villi, Crypt, Paneth cells, and other parts (Fig. S6B).

Fig 6.

L-EVs can promote the development of porcine intestinal organoids by activating ILC3s to secrete IL-22. (A) Crypts isolated from piglet intestines were cultured in vitro, and the images depict the changes in organoid size over 8 days. (B) HK LGG, L-EVs, IL22, and anti-IL-22 were added to the intestinal organoid culture wells. (C) The coculture system consisted of intestinal organoids, along with associated stimulatory molecules and ILC3s. (D) Effects of HK LGG and L-EVs on the growth of intestinal organoids. The size of the organoids treated with/without HK LGG and L-EVs, the total number of organoids, and the percentage of budding organoids among the total number of organoids per well (day 3) (n = 6 wells per group) were measured. (E) The effects of adding ILC3s, ILC3s + L EVs, ILC3s + L-EVs + anti-IL-22, IL-22, and IL-22 + anti-IL-22 to organoid medium on organoid growth were statistically analyzed (day 3) (n = 6 wells per group). (F) Organoids were stained with EdU (red). Nuclei are stained blue. EdU-positive cells were found in transit-amplifying regions, and there were obvious differences in the percentages of EdU-positive cells among the samples (n = 5 organoids per group). (G) Changes in the transcription of the EpCAM, VILLIN, MKI-67, LGR5, Ascl2, Olfm4, Lyz1, and REG3b genes were detected by qPCR (n = 3 wells per group).

Previous experiments have shown that L-EVs can stimulate the secretion of IL-22 from ILC3s. Therefore, it is worth investigating whether IL-22, secreted by ILC3s, can promote the growth of organoids. In our study, we initially supplemented the intestinal organoid medium with HK LGG and L-EVs (Fig. 6B). We then monitored the number of organoids, organoid size, and germination rate throughout the organoid growth process. Interestingly, we observed that the addition of HK LGG or L-EVs alone did not have any noticeable effect on the development of organoids (Fig. 6D).

In the next phase of our study, we successfully established a coculture system involving intestinal organoids, stimulatory molecules, and ILC3s (Fig. 6C). Initially, we attempted to culture ILC3s by adding them directly to Matrigel. However, this method proved unsuitable for the survival of ILC3s and resulted in severe cell death and fragmentation within 24 h. To overcome this issue, we performed cocultures using a transwell system with a pore size of 0.4 µm. We renewed the upper layer of ILC3s every 24 h. Notably, we observed that the growth of the organoids was significantly enhanced in the coculture system when L-EVs, ILC3s, and IL-22 were added. Within the first 2 days, the organoids experienced rapid growth, reaching a size of 70 µm. Subsequently, they started to exhibit significant budding and divided into multiple crypts on the third day. The organoids continued to grow rapidly, whereas the promotional effect of L-EVs and IL-22 on organoid growth disappeared upon the addition of an antibody specific to IL-22. Importantly, the number of organoids was not affected (Fig. 6E). By comparing the experimental group (L-EVs + ILC3, IL-22) with the control group (including ILC3, ILC3 + L-EVs + anti-IL-22, IL-22 + anti-IL-22), we found that the experimental group significantly promoted the growth of the organoids. Notably, a significant difference was observed on the third day of germination, and by the sixth day, the size of the organoids in the experimental group was significantly different from that in the control group (Fig. S6C).

Next, immunofluorescence revealed significant increases in the expression of proliferation-related proteins, including 5-ethynyl-2′-deoxyuridine (EdU) (Fig. 6F), the ISC activation-related protein LGR5, and the cell proliferation-related protein Ki-67, in organoids after the addition of L-EVs + ILC3 s and IL-22; moreover, a significant increase in the expression of the epithelial protein EpCAM and the chorionic protein VILLIN was also found (Fig. S6D). qPCR also revealed that the epithelial gene EpCAM, the chorionic gene VILLIN, the proliferation and differentiation-associated gene MKI-67, and the ISC marker genes LGR5, Ascl2, and Olfm4 were expressed in the organoids following the incorporation of cocultures of L-EVs + ILC3 s and IL-22, and the expression of the Paneth cell activation genes Lyz1 and REG3b was significantly increased (Fig. 6G). These results demonstrated that the EVs of LGG could significantly promote the growth and development of organoids and activate ISCs, Paneth cells, epithelial cells, and other cells in organoids.

Our study revealed that the metabolites of LGG are rich in AhR ligands, such as indole compounds. This study provides insights into the interaction between ILC3s in the porcine small intestine and Lactobacillus, aiming to understand better how ILC3s interact. We demonstrated that oral LGG administration is a potential approach for preventing PEDV infection in pigs. It can alleviate intestinal inflammation, intestinal damage, and clinical diarrhea symptoms caused by PEDV infection. We found that L-EVs can activate ILC3s and promote IL-22 secretion, thereby influencing intestinal stem cell regeneration and epithelial protection. Ultimately, this helps purify the gut environment and resist PEDV infection. In summary, our research demonstrated that oral LGG may be a potential method for preventing PEDV infection in pigs. Furthermore, this study opens up new avenues for preventing PEDV infection in piglets.

DISCUSSION

Recently, we discovered the presence of ILCs in the jejunum of piglets. These cells are predominantly ILC3s and play a crucial role in maintaining intestinal immune homeostasis. In our study, we further elucidated the interaction between ILC3s and the gut microbiota, particularly Lactobacillus, in the jejunum of piglets to maintain intestinal homeostasis. Based on our findings and the literature, we constructed a pathway diagram illustrating how L-EVs facilitate the secretion of IL-22 by ILC3s. IL-22, in turn, acts on downstream targets such as epithelial cells, Paneth cells, and ISCs to regulate intestinal immune function, thereby conferring protection against PEDV infection (Fig. 7).

Fig 7.

The pathway by which L-EVs maintain intestinal immune homeostasis in piglets.

The gut microbiota plays a crucial role in the development and maintenance of the host immune system, and its complexity is undeniable. It stabilizes mucosal function by maintaining the integrity of the gut barrier and balances the inflammatory response with immune tolerance through the induction of T lymphocyte differentiation. Additionally, the gut microbiota promotes the development of early B lymphocytes in the lamina propria of the mouse small intestine (27, 28). At the same time, the immune system also regulates the microbial community in a certain way to maintain relative balance. However, changes in this balance are often associated with the occurrence or progression of disease. Studies have reported that the gut flora changes significantly in multiple types of viral infections, in addition to viruses that infect the gut (29, 30). These results also suggest that during the process of viral infection, the host and intestinal flora may interact in a variety of ways, which is worthy of further study. Probiotics have a positive effect on the gut microbiota, especially on the innate and adaptive immune systems. Preclinical studies and clinical practice have shown that the use of probiotics can limit the overgrowth of pathogenic bacteria and control the host’s pathological processes (31). Furthermore, the interaction between bacteria and host cells is a complex process that has yet to be fully understood and deserves further exploration. Our research revealed significant changes in the gut microbiota during PEDV infection, including a decrease in the population of beneficial bacteria such as Lactobacillus. However, oral administration of LGG can greatly alleviate dysbiosis and increase the diversity of the gut microbiota.

AhR is a widely expressed transcription factor in immune system cells, which plays a crucial role in intestinal immune cells. AhR activation specifically alters innate and adaptive immune responses and participates in the regulation of cell differentiation and inflammation-related gene expression. Furthermore, inducible AhR activation may serve as a mechanism by which the gut microbiota promotes mucosal homeostasis. In contrast to its role in intestinal immune cells, the function of AhR in intestinal epithelial cells (IECs) has not been extensively studied. Previous research has revealed a close association between AhR in IECs and the maintenance of epithelial barrier function (32). One of the endogenous ligands for AhR is a metabolite of tryptophan. The gut microbiota can convert tryptophan into various molecules, including ligands for AhR, such as indole and its derivatives (e.g., IAld, IAA, IPA, IAAld, and indole-3-acrylic acid). The AhR signaling pathway is an essential component for maintaining barrier immune responses, promoting epithelial cell recovery, preserving barrier integrity, and regulating certain immune cell functions to maintain gut homeostasis. The gut microbiota and tryptophan and its metabolites interact closely in various aspects, including gut barrier function, gut immunity and endocrine activity, and intestinal motility (33). Current research has described a few commensal bacteria, such as Lactobacillus, capable of producing AhR ligands (34). However, the significance of these complex phenomena in the intestinal system still requires further investigation. One study revealed that adaptive lactobacilli can expand and produce the AhR ligand, I3A, which contributes to the AhR-dependent transcription of IL-22. Therefore, the microbiota-AhR axis may serve as an important strategy for modulating host mucosal immune responses during symbiotic evolution (15).

Additionally, lactobacilli can regulate immune cells through AhR by producing substances such as tryptophan and expressing tryptophanase, thus promoting the production of indole-3-propionic acid, which is associated with human health (35). Our research revealed that in a healthy piglet gut microbiota, there was a greater proportion of Lactobacillus. Additionally, we observed a significant presence of substances in the gut that interact with AhR. Moreover, AhR notably upregulated porcine immune cells, particularly ILC3s, indicating a potentially stronger regulatory relationship between ILC3s and the gut microbiota via AhR in the pig gut. In states of PEDV infection or dysbiosis, the levels of substances that interact with AhR significantly increase in the gut. The activation of ILC3s and the secretion of IL-22 are subsequently promoted through AhR, thereby exacerbating inflammation and accelerating the stimulation and activation of downstream receptor cells by IL-22. However, oral administration of LGG to piglets leads to significant restoration of the gut microbiota balance and the regulatory relationship between metabolites and AhR.

IL-22 is a member of the IL-10 family that can be produced by ILCs and CD4⁺ T cells. The IL-22-IL-22R signaling axis plays a crucial role in integrating immune responses with mucosal surface barrier function (36, 37). Increasing evidence suggests that IL-22 plays an important role in inflammatory bowel disease (IBD) (38). IL-22 produced by ILC3s is essential for maintaining intestinal homeostasis and provides early protection to epithelial barrier function during inflammation and injury (39). The interaction between the microbiota and IL-22 is central to regulating the barrier sites in intestinal homeostasis.

On one hand, IL-22 promotes intestinal barrier function by inducing antimicrobial peptides, mucins, and other beneficial factors from epithelial cells, thereby modulating the composition of the gut microbiota. On the other hand, the gut microbiota also regulates the production of intestinal IL-22, although the underlying mechanisms are not fully understood. Research has reported that many functions of the gut microbiota in regulating health and disease are mediated through its metabolites (40). In our in vitro experiments using IPEC-J2 cells and piglet intestinal organoids, we found that L-EVs can promote the activation of ILC3s and the production of IL-22. The generated IL-22 can act on IPEC-J2 cells, promoting their proliferation, activating the STAT3 signaling pathway, and conferring resistance against PEDV infection. Moreover, the produced IL-22 can also act on Paneth cells, ISCs, and epithelial cells within the organoids to promote their growth and development.

Investigators have shown that IFN-γ and IL-22 act synergistically to induce the expression of interferon-stimulated genes and control rotavirus infection. Therefore, this pathway may not be specific to PEDV infection and could play a role in other diseases as well (41). There are a variety of probiotics in the gut that can improve intestinal immunity by regulating host immunity. Although we observed significant changes in the secretion of ILC3s and IL-22 in the jejunum of piglets following exposure to Lactobacillus, it is still unknown whether other bacteria can significantly stimulate ILC3s to secrete IL-22. We speculate that other lactic acid bacteria may also have regulatory effects that promote intestinal immune homeostasis. First, the expression of AhR in the intestines is crucial for regulating intestinal immune function (42, 43). Second, studies have reported that Lactobacillus reuteri tryptophan metabolism promotes host susceptibility to CNS autoimmunity through AhR (44). Research has also shown that Lacticaseibacillus paracasei GM-080 ameliorates allergic airway inflammation in children with allergic rhinitis through AhR (45). In summary, if other lactic acid bacteria can produce AhR ligands, they can also regulate intestinal immunity in piglets by regulating the AhR receptors on ILC3s. Could it be possible that the balance between lactobacilli and ILC3s is disrupted, leading to dysregulation of the existing regulatory relationship and a significant increase in the number of harmful bacteria, thereby further stimulating ILC3 activation? These questions remain unanswered, and we hope to address them in future research.

Currently, there is increasing interest in studying the interactions between microorganisms and the immune system. On one hand, the immune system can regulate and shape the microbial community, and on the other hand, the established microbial community can promote the development of the host immune system and provide signals for subsequent immune responses. However, our understanding of the interactions between microorganisms and the immune system is still limited, and unraveling these complexities requires interdisciplinary collaboration and innovation. Numerous intrinsic factors influence balance within animals. Although our research focused on the interaction between intestinal lactobacilli and ILC3s through AhR, it is important to note that there are countless regulatory pathways in animals. Moreover, research on pigs is relatively scarce, and much of our knowledge comes from studies conducted on humans and mice. We discovered that pigs have some differences between humans and mice, for example, Fig. 1 shows the clustering of cells with similar gene expression after single-cell sequencing. In T cells, the clustering locations of CD3⁺ T cells and CD4⁺ T cells are close and thus represented by CD3⁺ T cells, whereas the CD8⁺ T-cell subset has the highest expression of CD8 (the CD8⁺ T-cell subset includes CD3⁺CD4⁻CD8⁺ T cells and CD3⁻CD4⁻CD8⁺ T cells); hence, only CD3⁺ and CD8⁺ T cells are shown in Fig. 1A. In Fig. S2A, they were not subjected to flow-sorting enrichment; the number of CD3⁻CD4⁻CD8⁺ T cells was very low, and it was difficult to show significant flow without cell enrichment. The results of our previous study revealed that pigs exhibit differences between humans and mice (46). This study of iNKT cells in the thymus revealed that pigs exhibit differences between humans and mice (47).

In summary, we cannot fully comprehend the complete role of ILCs in the intestinal immunity of pigs. Furthermore, considering that ILC3s are an emerging cell type in pigs, our work is only beginning. In the future, we will explore the changes in this cell population during intestinal immunity and disease states, laying the foundation for studying intestinal immunity in pigs.

MATERIALS AND METHODS

Animals and sample collection

We used 3-day-old SPF piglets, which were purchased from the Harbin Veterinary Research Institute. All the piglets were transported in enclosed carts designed for SPF conditions. We collected samples from the jejunum and other tissues of the piglets. Then, we analyzed the immune cells in the jejunal tissues using flow cytometry, processed and analyzed the samples using qPCR and other experimental methods, and sectioned and stained the tissue samples. All animal experiments complied with the requirements of the Animal Management and Ethics Committee of Jilin Agricultural University.

10× ScRNA-seq analysis

The methods used for the analysis of the ScRNA-seq data in the manuscript are described in detail in my previously published manuscript (20). The source of the jejunal scRNA-seq data for piglet jejunum ILCs in PRJNA907920 (the data are presented in Fig. 1A through C, 2L, and M). The source of the jejunal scRNA-seq data after PEDV infection in piglets was GSE175411 (the data are presented in Fig. 1D and E). We compared human intestinal ILCs (GSA-human: HRA000919) and mouse intestinal ILCs (GSE166266) (the data are presented in Fig. S2D and E).

Cell separation

Cell samples obtained from the jejunum of piglets were subjected to subsequent flow cytometry, in vitro cell culture, and qPCR. First, after the piglets were euthanized, approximately 5 cm of jejunum was dissected longitudinally, rinsed with PBS, and divided into 1 cm intestinal fragments, which were then transferred to separation solution [15 mL of RPMI-1640, 1% penicillin, and streptomycin, 1% HEPES, 5 mM EDTA, 2 mM DTT, and 2% heat-inactivated fetal bovine serum (FBS)] and incubated for 28 min in a shaking incubator at 37°C and 200 rpm. After incubation for 28 min in a shaking incubator at 37°C and 250 rpm, the intestinal fragments were rinsed and incubated in enzyme digestion solution (8 mL of RPMI-1640 medium, 1% penicillin and streptomycin, 1% HEPES, 50 mg of collagenase IV, 1 mg of DNase I, and 2% FBS), incubated for 28 min in a shaking incubator at 37°C and 250 rpm before being removed, and then filtered through a 70 µm cell strainer to obtain the LPL cells in the jejunum of the piglets. The immune magnetic bead separation technique was performed using a STEMCELL EasySep magnet (19F99020).

Preparation of the IL-22 protein and IL-22 monoclonal antibody

The porcine IL-22 (Gene ID: 595104) gene was synthesized by consulting NCBI, the structural domains were examined to determine the protein expression scheme, the prokaryotic and eukaryotic expression vectors were prepared, the IL-22 protein was obtained after the prokaryotic expression was purified, the IL-22 protein was immunized to the mice and then taken from the B cells for the fusion of hybridoma cells, and large quantities of monoclonal antibodies were prepared by means of ascites preparation. A large number of monoclonal antibodies were prepared from ascites, and the antibodies were labeled with an antibody labeling kit (ab201795) after purification. This part of the work was performed with the help of Shanghai Company. We preserved IL-22 and anti-IL-22 for use in subsequent in vitro experiments and flow cytometry experiments.

Strains of Lactobacillus, LGG, and L-EVs acquisition

Four strains of Lactobacillus were purchased from ATCC. Included L. plantarum (ATCC BAA-793), L. reuteri (ATCC BAA-2837), L. casei (ATCC 393), L. rhamnosus (ATCC BAA-3227 ).

After culturing overnight, LGG (ATCC 53103) was inoculated at a 1:100 ratio in fresh MRS broth and grown under anaerobic conditions until it reached the mid-log phase. Then, the concentration of LGG was adjusted to 1 × 10⁹ colony-forming units (CFU)/mL.

First, the precipitate of overnight cultured LGG was discarded, and the supernatant was collected in a centrifuge tube. Then, the samples were centrifuged for 1 h at 150,000 × g and 4°C. Finally, the precipitate was collected, and the sample was tested for nontarget metabolism. Each sample was tested using 100 mg of L-EVs. L-EVs can be stored at −80°C for later use. In the culture stimulation experiment of ILCs, 50 µg of L-EVs were added to each culture well (a 24-well plate was used as an example).

The size distribution and morphology of the L-EVs were analyzed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), respectively (HT7800, HITACHI).

PEDV information

The PEDV strain (PEDV LJX01/2014, PEDV N gene GenBank number: MK252703) was provided by Professor Guangliang Liu, Lanzhou Veterinary Research Institute, China. The propagation and titration of the PEDV LJX strain were described previously (48). Piglets were infected by oral feeding using a virus with a copy number of 1.35 × 10⁴ RNA copies/g, and each 7-day-old piglet was infected with 3 mL of virus solution. A total of 10 mL of virus solution was added to a 17-day-old piglet. The virus infects piglets orally and only needs to be fed once.

Experimental animal model construction

1. PEDV-infected piglets: We divided the 7-day-old SPF piglets into two groups of six animals each and then orally administered PEDV to the infected piglets. The piglets were euthanized 72 h later.

2. Gut microbiota imbalance in piglets: We divided the 3-day-old SPF piglets into two groups of six animals each. Then, every piglet was treated with oral antibiotics, namely, 3 mg gentamicin, 20 mg metronidazole, and 100 mg ampicillin per kg of piglet feed once a day for 7 days.

3. ABX + LGG/PBS + PEDV: We divided the 3-day-old SPF piglets into three groups of six animals each, and the CON group was fed PBS for 14 days as a control group. The ABX + LGG + PEDV group continued to be fed LGG (each pig was fed 2 × 10⁹ CFU LGG per day) for 7 days after antibiotic feeding and then infected with PEDV. The ABX + PEDV group continued to be fed PBS for 7 days after antibiotic feeding and then were infected with PEDV. Seventeen-day-old piglets were orally administered the PEDV virus, and the piglets were subsequently euthanized 72 h later.

Flow cytometry detection of ILCs

Flow cytometry detection of ILCs

After the piglets were euthanized, a single-cell suspension of intestinal innate immune cells was prepared, and antibodies were added to tubes containing 1–5 × 10⁶ cells, mixed thoroughly, and stained for 30 min at 4°C in the dark. One milliliter of PBS was added, and the mixture was centrifuged at 2,000 rpm at 4°C for 5 min, after which the supernatant was discarded. The cells can then optionally be fixed and permeabilized, and after permeabilization, the antibody can be used to continue the staining. The staining was completed and detected using a flow cytometric analyzer (BD). It is worth noting that the ILCs used for detection are not the same as the ILC staining methods used for culture. Direct detection of IL-22 secreted by ILC3s requires the cells to be fixed and permeabilized (the staining and gating method diagram is shown in Fig. S1). In vitro, the sorting of ILCs does not require fixation, and the magnetic bead sorting method is used for sorting (the diagram of the staining and gating methods is shown in Fig. 4A; Fig. S4A). ILCs detected after in vitro culture were fixed and permeabilized (the gating method diagram is shown in Fig. S4B).

In vitro culture method for ILCs

The in vitro culture medium for ILCs was RPMI-1640 (1% penicillin and streptomycin, 1% HEPES, and 10% heat-inactivated FBS). In Fig. 1 to 3, ILCs extracted from the intestines of piglets need to be stimulated in vitro for 8 h. PMA and ionomycin need to be added to the medium, and BFA (Brefeldin A) needs to be added at the same time. In Fig. 4 to 6, there is no need to add PMA and ionomycin to the medium of ILCs; only specific stimulants are added, but BFA still needs to be added.

Antibody and reagent information

We obtained the following from Invitrogen: CD2 Monoclonal Antibody (14–0029-82), CD3e Monoclonal Antibody (MA5-28774), CD3e Monoclonal Antibody (Biotin) (MA5-28771), CD11b Monoclonal Antibody (MA5-16604), CD11c Monoclonal Antibody (APC) (17–0116-42), CD21 Monoclonal Antibody (A1-19243), CD21 Monoclonal Antibody (PE) (MA1-19754), CD45 Monoclonal Antibody (FITC) (MA5-28383), CD117 (c-Kit) Monoclonal Antibody (APC) (17–1171-82), CD163 Monoclonal Antibody (PE) (MA5-16476), CD172a Monoclonal Antibody (MA5-28299), CD335 (NKp46/NCR1) Monoclonal Antibody (PE) (MA5-28352), SLA Class II DR Monoclonal Antibody (MA5-28503), Gata-3 Monoclonal Antibody (PE-Cyanine7) (25–9966-42), T-bet Monoclonal Antibody (PE) (12–5825-82), and Goat anti-Mouse IgG H&L (PE- Cyanine5) (M35018).

We obtained the following from BD Pharmingen: Fixable Viability Stain 780 (565388), Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) (553142), Rat Anti-Pig γδ T Lymphocytes (PE) (551543), Rat Anti-Pig γδ T Lymphocytes (561486), and Mouse anti-GATA3 (558686).

We obtained the following from Abcam: Streptavidin protein (Alexa Fluor 594) (ab272189), Streptavidin protein (APC) (ab243099), Goat Anti-Mouse IgG H&L (FITC) (ab6785), Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (ab150077), Goat Anti-Mouse IgG H&L (Alexa Fluor 488) (ab150113), Goat Anti-Rat IgG H&L (Alexa Fluor 488) (ab150165), Goat F(ab')2 Anti-Mouse IgG H&L (PE) (ab7002), Goat F(ab')2 Anti-Rat IgG H&L (PE-Cyanine5) (ab130803), Goat Anti-Mouse IgG H &L (Alexa Fluor 647) (ab150115), Rabbit Anti-Goat IgG H&L (Alexa Fluor 488) (ab150141), Rat Anti-Mouse IgG1 H&L (PE) (ab99605), Anti-Villin antibody (ab244292), Anti-EpCAM antibody(ab71916), Anti-EpCAM antibody (ab71916), Anti-Ki-67 antibody (ab15580), and Anti-LGR5 antibody (ab273092).

We obtained the following from Sigma: Penicillin And Streptomycin (V900929), Collagenase IV (V900893-1G), Ionomycin (56092–81-0), DNase I (10104159001), DTT (3483–12-3), HEPES (H3375), EDTA (E8008), FBS (F8318), and BFA (87022601).

PE Goat anti-Mouse (SPP101), FITC Rabbit anti-Goat (SPP101-100), and PE anti-Mouse (SHP501) were purchased from 4A Biotech., Ltd. CFSE (S1076), PMA (P6741), D-PBS (D1040), and PBS (P1010) were purchased from Solarbio. Percoll (17089101) was from GE Healthcare. APC anti-pig IL-4 (1644057) was from R&D Systems. EasySep PE Positive Selection Kit II (17684) was from STEMCELL. Indole-3-carbinol (HY-N0170) and CH-223191 (HY-12684) were from MCE.

16S rRNA-seq experiment

16S rRNA amplicon sequencing data for PRJNA526581, PRJNA1048561, and PRJNA1048648. Data on changes in the jejuni flora after PEDV infection in piglets are available at PRJNA526581 (the data are presented in Fig. 1K and L). Data on changes in the jejuni flora after antibiotic treatment of piglets are available at PRJNA1048561 (the data are presented in Fig. 2B through G). The piglets were first treated with antibiotics, and then, during the PEDV virus infection experiments, the changes in the C. jejuni flora in the piglets were recorded in the PRJNA1048648 (the data are presented in Fig. 3G and H; Fig. S3B and C).

1. Paired-end Reads Assembly and Quality Control: The tags were compared with the reference database using UCHIME. An algorithm (http://www.drive5.com/usearch/manual/uchime_algo.html) was used to detect chimeric sequences, after which the chimeric sequences were removed. Then, the effective tags were finally obtained.

2. ASV Denoise and Species Annotation: The absolute population of ASVs was normalized using a standard of sequence number corresponding to the sample with the fewest sequences. Subsequent analyses of alpha diversity and beta diversity were all performed based on the output normalized data.

3. Alpha Diversity and Beta Diversity.

4. Association analysis: Correlations between metabolites and flora.

Metabolomic data analysis

These metabolites were annotated using the KEGG database (https://www.genome.jp/kegg/pathway.html), HMDB (https://hmdb.ca/metabolites), and the LIPIDMaps database (http://www.lipidmaps.org/). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed with metaX (a flexible and comprehensive software for processing metabolomics data). We applied univariate analysis (t test) to calculate the statistical significance (P value). The metabolites with VIP >1 and P value < 0.05 and fold change ≥2 or FC ≤ 0.5 were considered to be differentially abundant metabolites. Volcano plots were generated to filter metabolites of interest based on the log2 (fold change) and −log10 (P value) values of the metabolites via ggplot2 in R language.

For clustering heatmaps, the data were normalized using z-scores of the intensity areas of differentially abundant metabolites and were plotted by the Pheatmap package in R language. The correlations between differential metabolites were analyzed by cor () in R (method = pearson). Statistically significant correlations between differentially abundant metabolites were calculated by cor.mtest() in R language. A P value < 0.05 was considered to indicate statistical significance, and correlation plots were generated with the corrplot package in R. The functions of these metabolites and metabolic pathways were studied using the KEGG database. The metabolic pathway enrichment of differentially abundant metabolites was performed when the ratio was satisfied by x/n > y/N, metabolic pathways were considered enriched, and when P value of the metabolic pathway was <0.05, metabolic pathways were considered significantly enriched.

qPCR experiment

Total RNA was extracted according to the manufacturer’s instructions, and 1 mg of RNA was reverse transcribed into cDNA by reverse transcriptase (Promega). In the real-time quantitative PCR system of the Applied Biosystems 7500, quantitative PCR was performed using a SYBR Green mixture (TakBR Green). In the real-time quantitative PCR system of the Applied Biosystems 7500, quantitative PCR was performed using a SYBR Green mixture (Takara Bio). The average mRNA fold changes were calculated by the 2^-ΔΔCT method, and all primer sequences are shown in Supplementary Data 1.

Cell experiments

We stained the total LPLs extracted from the lamina propria with antibodies against Live/Dead, CD45, Lin1 (CD3ε, CD21, γδ T, CD11c, and CD172a), and Lin2 (CD4, CD8, CD163, and CD11b) and then selected L/D⁻CD⁺Lin1⁻Lin2⁻ cells by flow sorting or magnetic bead sorting (STEMCELL EasySep PE Positive Selection Kit II # 17684). After collection, the cells were cultured in vitro and then subjected to a flow assay.

Coculture model of ILC3s and IPEC-J2 cells

A schematic diagram of the cocultivation process is shown in Fig. 5D. Culture medium 1 was RPMI-1640 (1% penicillin and streptomycin, 1% HEPES, and 10% heat-inactivated FBS). Culture medium 2 was DMEM (1% penicillin and streptomycin, 1% HEPES, and 10% heat-inactivated FBS). Transwell plates (0.4 µM, Corning-CLS3413) were used.

Organoid experiment

1. Piglets were euthanized by removing 8 cm of jejunum, removing the mesentery, dissecting the intestinal segments longitudinally, washing them with cold PBS until they were cleaned, cutting the segments into 0.5 cm pieces, washing them with cold PBS by blowing gently, and adding 15 mL of PBS (2 mM EDTA). The mixture was allowed to stand at room temperature for 40 min.

2. The supernatant was discarded, 10 mL of DPBS was added, and the mixture was blown two times. The supernatant was collected in a 50 mL centrifuge tube through a 70 µm cell sieve, labeled #1, and this process was repeated four times. Filtrates 3 and 4 were centrifuged at 300 × g for 5 min, after which the supernatant was discarded. The supernatant was resuspended in 1 mL of DME/F12 (1% penicillin and streptomycin), transferred to a 1.5 mL centrifuge tube, and centrifuged at 200 × g for 3 min, after which the supernatant was discarded.

3. Then, 250 µL of complete medium and 250 µL of Matrigel (operating on ice) were added to the precipitate, which was subsequently mixed. Then, 50 µL of solution was added to the center of a 24-well plate and placed in the incubator for 30 min. Then, 500 µL of medium (STEMCELL #6000) was added to each well, and 500 µL of PBS was added to the remaining wells.

4. When the organoids start to germinate, they are passaged. First, discard the old medium was discarded, 2 mL of DME/F12 was added, the mixture was blown up and down, and the mixture was recycled into a centrifuge tube. After centrifugation, the supernatant was discarded, and the solution was reintroduced into the complete medium and Matrigel.

The morphology of the organoids was observed, the number of organoids in each culture hole (organoid number), the average diameter of the organoids (organoid size), and the ratio of budding organoids to all the organoids (building organoids) were statistically analyzed, and 20 organoids from each well were randomly selected for statistical analysis.

Coculture model of intestinal organs

A schematic diagram of the in vitro stimulation and culture model of the organoids is shown in Fig. 6B. HK LGG, L-EVs, IL22, anti-IL-22, and other substances were added to observe their effects on the growth and development of organoids. In the coculture model of ILC3s and intestinal organs shown in Fig. 6C, culture medium 1 was RPMI-1640 (1% penicillin and streptomycin, 1% HEPES, and 10% heat-inactivated FBS). Culture medium 2 was Organoid media (STEMCELL #6000). L-EVs and anti-IL-22 can be added to culture medium 1.

Statistics

Statistical analyses were performed using the statistical computer package, GraphPad Prism version 6 (GraphPad Software Inc., San Diego, CA). The results are expressed as the means ± SEMs. Statistical comparisons were made using a two-way analysis of variance with Tukey’s post hoc test, or statistical comparisons were made using a two-way analysis of variance with Tukey’s post hoc test or Student’s t-test, where appropriate. Differences were considered to be significant at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 indicate significant differences among the groups.

ETHICS APPROVAL

The animal management procedures and all laboratory procedures abided by the regulations of the Animal Care and Ethics Committees of Jilin Agriculture University, China.

DATA AVAILABILITY

The raw data for this article were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database, Gene Expression Omnibus (GEO) database, and EBI database. The source of the jejunal scRNA-seq data after PEDV infection in piglets was GSE175411. scRNA-seq data of piglet jejunum ILCs may be accessed at PRJNA907920. We compared human intestinal ILCs (GSA-human HRA000919) and mouse intestinal ILCs (GSE166266). 16S rRNA amplicon sequencing data may be accessed at PRJNA526581, PRJNA1048561, and PRJNA1048648. This paper does not report the original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jvi.01039-24.

Fig. S1. jvi.01039-24-s0001.tif.

Gating of ILC subsets.

Fig. S2. jvi.01039-24-s0002.tif.

Statistical analysis of flow cytometry results.

Fig. S3. jvi.01039-24-s0003.tif.

TCID₅₀ detection of 4 Lactobacillus.

Fig. S4. jvi.01039-24-s0004.tif.

Magnetic bead sorting.

Fig. S5. jvi.01039-24-s0005.tif.

Proliferation assay of ILCs.

Fig. S6. jvi.01039-24-s0006.tif.

Growth dynamics of porcine intestinal organoids.

Supplemental data. jvi.01039-24-s0007.docx.

PEDV correlation detection plasmid and primer synthesis; q-PCR primer.

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