Swine Enteric Coronaviruses (PEDV, TGEV, and PDCoV) Induce Divergent Interferon-Stimulated Gene Responses and Antigen Presentation in Porcine Intestinal Enteroids

Figures

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  Figure 1. Porcine enteroids are susceptible to PEDV, TGEV, and PDCoV infection. (A) Representative images of the proliferation and differentiation of porcine jejunal enteroids derived from crypt stem cells of jejunum. The isolated intestinal crypts cultured in proliferation medium mixed with Matrigel gradually formed 3D budding organ-like structures on day 7. Bar = 100 μm (day 0, 3) or 200 μm (day 7). (B) Representative images of 2D jejunal enteroid monolayers generated from differentiated 3D enteroids. After culturing for 7 days, 3D jejunal enteroids were dissociated into single cells and seeded onto 96-well plates precoated with Matrigel to differentiate for 3 days. (C) The kinetic curves of SECoVs replication in jejunal enteroids. Jejunal enteroid monolayers were respectively infected with PEDV (MOI = 5), TGEV (MOI = 1), or PDCoV (MOI = 2). The kinetics of SECoVs replication at the indicated time points post infection was determined by RT-qPCR. Each data bar represents the means ± SEMs (n = 3). (D) Examination of SECoVs infection in jejunal enteroids by IFA. Jejunal enteroid monolayers were respectively infected with PEDV (MOI = 5), TGEV (MOI = 1), or PDCoV (MOI = 2) for 24 h, and then…

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  Figure 2. Unique and overlapping transcriptional profiles in porcine enteroids following different SECoVs infection. (A) Confirmation of SECoVs infection in jejunal enteroids by RT-qPCR. Jejunal enteroid monolayers were respectively infected with PEDV (MOI = 5), TGEV (MOI = 1), or PDCoV (MOI = 2) for 24 h, and collected for quantifying the viral genomes of SECoVs. Each data bar represents the means ± SEMs (n = 3). (B) The statistical graph results of differential expressed genes of jejunal enteroids after infection with PEDV, TGEV, or PDCoV. Jejunal enteroid monolayers isolated from three individual SPF 2-day-old piglets were mock-infected or infected with PEDV (MOI = 5), TGEV (MOI = 1), or PDCoV (MOI = 2) for 24 h. Then, the total RNA isolated from enteroids was used for RNA-Seq. (C) Venn diagram of common differentially expressed genes of jejunal enteroids elicited by PEDV, TGEV, or PDCoV. (D) Heat map of differentially expressed genes in jejunal enteroids infected with PEDV, TGEV, or PDCoV. Data are based on log(RPKM) values (n = 3).

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  Figure 3. PEDV and TGEV, not PDCoV, exert a similar effect on the biological function of porcine enteroids. (A, B) Volcano plot of all expressed host genes of PEDV-infected enteroids relative to TGEV-infected enteroids (A) and PDCoV-infected enteroids (B). The y-axis shows the -log10 transformed P-value, and the x-axis shows the log2 fold change in normalized expression; the red, blue, and gray points represent the significantly upregulated, downregulated, and non-significant differentially expressed genes, respectively (P < 0.05). (C, D) The transcriptional expressions of PEDV-elicited top 10 significantly upregulated (C) or downregulated (D) genes in the three SECoVs-infected enteroids were evaluated based on log(RPKM) values (n = 3).

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  Figure 4. GO enrichment analysis of PEDV, TGEV, or PDCoV targets.

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  Figure 5. PEDV infection displays a weaker induction of IFN responses in enteroids than TGEV and PDCoV. (A, D) Heat map of IFNs and IFN receptors expression in enteroids after SECoVs infection. Jejunal enteroid monolayers were mock-infected or infected with PEDV, TGEV, or PDCoV for 24 h. The differential expression profiles of IFNs (A) and IFN receptors (D) were analyzed by RNA-Seq. Data are based on log(RPKM) values (n = 3). (B, C, E). Confirmation of IFNs and IFNAR1 expression in enteroids after SECoVs infection by RT-qPCR. Monolayers of jejunal enteroids were infected with PEDV, TGEV, or PDCoV for 24 h. The mRNA expressions of IFN-β (B), IFN-L1(C), and IFNAR1 (E) were assessed by RT-qPCR. The results are presented as the means ± SEMs (n = 3). The differences between groups were compared using the Student’s t test. Error bars denote the deviations, and the p values are expressed as follows: *p <0.05, and ****p <0.001.

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  Figure 6. PEDV infection elicits a poorer ISG response in enteroids than TGEV and PDCoV. (A) Heat map of robustly upregulated ISGs expression in enteroids following SECoVs infection. Jejunal enteroid monolayers infected with PEDV, TGEV, or PDCoV, and collected at 24 hpi for the analysis of ISGs expression profiles by RNA-Seq. Data are based on log(RPKM) values (n = 3). (B–G) Verification of ISGs expression in enteroids after SECoVs infection by RT-qPCR. The mRNA levels of OAS1 (B), OASL (C), RSAD2 (D), IFIT2 (E), IFIM3 (F), and ISG15 (G) in jejunal enteroids following PEDV, TGEV, and PDCoV were measured by RT-qPCR. The results are presented as the means ± SEMs (n = 3). The differences between groups were compared using the Student’s t test. Error bars denote the deviations, and the p values are expressed as follows: *p <0.05, **p <0.01, and ****p <0.001.

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  Figure 7. The differential ability of SECoVs to manipulate PRRs and NLRs expression in enteroids. (A) Heat map of PRRs and NLRs expression in jejunal enteroids infected SECoVs. Total RNA was extracted from enteroids after infection with or without PEDV, TGEV, or PDCoV for 24 h, and the transcriptional profiles of PRRs and NLRs were determined by RNA-Seq. Data are based on log (RPKM) values (n = 3). (B-C). Verification of RIG-I and NLRC5 expression in jejunal enteroids by RT-qPCR. Monolayers of jejunal enteroids were infected with PEDV (MOI = 5), TGEV (MOI = 1), or PDCoV (MOI = 2) for 24 h, and the mRNA levels of RIG-I (B) and NLRC5 (C) were detected by RT-qPCR. Data represent the means ± SEMs (n = 3). The differences between groups were compared using the Student’s t test. Error bars denote the deviations, and the p values are expressed as follows: *p <0.05, and **p <0.01. (D) The expression kinetics of NLRC5 in jejunal enteroids after SECoVs infection. Following PEDV, TGEV, or PDCoV infection, jejunal enteroid monolayers were collected at the indicated time points for the quantification of NLRC5 expression by RT-qPCR. The scale data of panel D were taken as log2 (fold change) values.

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  Figure 8. TGEV and PDCoV, not PEDV, robustly upregulate the expression of SLA-I and its associated antigen-presentation genes expression in enteroids. (A) Heat map of SLA molecules and genes associated with antigen-presentation in enteroids following infection with SECoVs. The transcriptional profiles of SLA molecules (SLA-I, SLA-II, and SLA-III) and genes associated with antigen-presentation in enteroids following infection with PEDV, TGEV, and PDCoV were determined by RNA-Seq. Data are based on log(RPKM) values (n = 3). (B–E) Verification of the expression of SLA-I and it’s associated with antigen-presentation by RT-qPCR. Jejunal enteroid monolayers were mock-infected or infected with PEDV, TGEV, or PDCoV for 24 h. The mRNA expressions of β2M (B), PSMB9 (C), TAP1 (D), and SLA-1 (E) were measured by RT-qPCR. (F, G) The expression kinetics of SLA-1 in jejunal enteroids induced by TGEV (F) and PEDV (G) were assessed by RT-qPCR. The differences between groups were compared using the Student’s t test. Error bars denote the deviations, and the p values are expressed as follows: *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. ns, not significant.

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  Figure 9. TGEV and PDCoV, not PEDV, strongly promote the expression of chemokines related to T cell immunity in enteroids. (A) Heat map of significant induction of chemokines related with T cell immunity in enteroids infected with SECoVs. Monolayers of jejunal enteroids infected with PEDV, TGEV, and PDCoV were harvested at 24 hpi. The transcriptional expression of chemokines in enteroids was confirmed by RNA-Seq. Data are based on log(RPKM) values (n = 3). (B, C) Jejunal enteroids monolayers were infected with PEDV, TGEV, and PDCoV, and collected at 24 hpi to analyze the expression of CXCL10 (B), and CXCL11 (C) by RT-qPCR. (D–F). Following infection with PEDV, TGEV, and PDCoV, jejunal enteroid were collected at the indicated time points. The expression kinetics of CXCL10 (D), CXCL11 (E), and CD86 (F) were quantified by RT-qPCR. Data are presented as the means ± SEMs (n = 3). The scale data of panel D-F were taken as log2 (fold change) values. The differences between groups were compared using the Student’s t test. Error bars denote the deviations, and the p values are expressed as follows: *p < 0.05.

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  Figure 10. IFNs treatment increases the expression of SLA-I and its associated antigen-presentation genes. (A–D) IPEC-J2 cells were respectively pretreated with IFN-β, IFN-γ, and IFN-L at the indicated concentrations for 24 h. The expression levels of NLRC5 (A), SLA-1 (B), SLA-2 (C), and SLA-3 (D) were examined by RT-qPCR. Data are presented as the means ± SEMs (n = 3). The scale data of panel (A) were taken as log2 (fold change) values. The differences between groups were compared using the Student’s t test. Error bars denote the deviations, and the p values are expressed as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.