F4+ ETEC infection and oral immunization with F4 fimbriae elicits an IL-17-dominated immune response

Abstract

Enterotoxigenic Escherichia coli (ETEC) are an important cause of post-weaning diarrhea (PWD) in piglets. Porcine-specific ETEC strains possess different fimbrial subtypes of which F4 fimbriae are the most frequently associated with ETEC-induced diarrhea in piglets. These F4 fimbriae are potent oral immunogens that induce protective F4-specific IgA antibody secreting cells at intestinal tissues. Recently, T-helper 17 (Th17) cells have been implicated in the protection of the host against extracellular pathogens. However, it remains unknown if Th17 effector responses are needed to clear ETEC infections. In the present study, we aimed to elucidate if ETEC elicits a Th17 response in piglets and if F4 fimbriae trigger a similar response. F4(+) ETEC infection upregulated IL-17A, IL-17F, IL-21 and IL-23p19, but not IL-12 and IFN-γ mRNA expression in the systemic and mucosal immune system. Similarly, oral immunization with F4 fimbriae triggered a Th17 signature evidenced by an upregulated mRNA expression of IL-17F, RORγt, IL-23p19 and IL-21 in the peripheral blood mononuclear cells (PBMCs). Intriguingly, IL-17A mRNA levels were unaltered. To further evaluate this difference between systemic and mucosal immune responses, we assayed the cytokine mRNA profile of F4 fimbriae stimulated PBMCs. F4 fimbriae induced IL-17A, IL-17F, IL-22 and IL-23p19, but downregulated IL-17B mRNA expression. Altogether, these data indicate a Th17 dominated response upon oral immunization with F4 fimbriae and F4(+) ETEC infection. Our work also highlights that IL-17B and IL-17F participate in the immune response to protect the host against F4(+) ETEC infection and could aid in the design of future ETEC vaccines.

Figure 1

The mRNA expression profile in PBMCs triggered by F4 ⁺ ETEC infection. Piglets were infected with F4⁺ ETEC on day 0 (D0) and day 1 (D1). PBMCs were isolated from piglets on D0 until D4 after infection. The mRNA expression in the PBMCs of F4⁺ ETEC infected and control piglets was analyzed by qPCR. The mRNA expression level was normalized to the reference genes and then to the control group for every separate day. Then, the data were plotted relative to D0. Data are presented as the mean ± SEM (n = 3 per group). * p < 0.05, ** p < 0.01.

Figure 2

F4 ⁺ ETEC infection induced mRNA expression profile in intestinal tissues. The F4⁺ ETEC infection was performed on day 0 (D0) and day 1 (D1). Intestinal samples were collected on D4. The mRNA expression in intestinal tissues of F4⁺ ETEC infected or control pigs was analyzed by qPCR. The mRNA expression was normalized to the reference genes and then to the control group for all separate intestinal tissues. Data are presented as the mean ± SEM (n = 3 per group). JJ: jejunum without Peyer’s patches, JP: jejunum with Peyer’s patches, IL: ileum without Peyer’s patches, IP: ileum with Peyer’s patches, MLN: mesenteric lymph nodes. * indicates significant differences as compared to the control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Increased presence of IL-17A ⁺ T cells in the ileum of F4 ⁺ ETEC infected piglets. Cryosections were stained with anti-IL-17A (FITC, green) and anti-CD3 mAbs (Texas Red-X, red). The nuclei were counterstained with Hoechst (blue). Images are representative for all piglets in both groups. The arrows indicate colocalization of CD3 and IL-17A.

Figure 4

F4 fimbriae trigger mRNA expression of IL-17 cytokines in PBMCs upon oral immunization. The piglets were immunized with 1 mg F4 fimbriae on day 0 (D0), D1 and D2. PBMCs were isolated from piglets on D0 before immunization and on D4 and D9 after immunization. The mRNA expression in PBMCs isolated from F4-immunized or control piglets was analyzed by qPCR. The mRNA expression level was normalized to the reference genes and then to the control group for every separate day. Data are presented as the mean ± SEM (n = 5 per group). * p < 0.05, ** p < 0.01.

Figure 5

F4 fimbriae elicit an IL-17 dominated cytokine response in naïve PBMCs. PBMCs were isolated from naïve piglets and were stimulated with F4 fimbriae (5 μg/mL) for 24, 48 and 72 h. The mRNA expression profile was analyzed by qPCR. mRNA expression levels were normalized to the reference genes and then to the control group for each time point. Then, the data for every day was plotted relative to day 0. Data are presented as the mean ± SEM (n = 3). NS: non-stimulated. * p < 0.05, ** p < 0.01.

Figure 6

Cytokine secretion by F4 fimbriae stimulated naïve PBMCs. PBMCs were stimulated with endotoxin-free F4 fimbriae (5 μg/mL) or medium for 72 h. The protein level of IL-17A, IL-22, IFN-γ and IL-10 in the supernatant was determined by ELISA. Data are presented as the mean ± SEM (n = 3). NS: non-stimulated, * p < 0.05, ** p < 0.01.

Figure 7

Th17 signature dominates in an antigen recall assay. PBMCs were isolated from F4⁺ ETEC infected animals and stimulated with F4 fimbriae (5 μg/mL) or medium for 48 h. A The mRNA expression profile in PBMCs was analyzed by qPCR. The mRNA expression level was normalized to the reference genes and then to control PBMCs. * indicates significant differences as compared to the control group. B The secretion of IL-17A, IL-22, IFN-γ and IL-10 by PBMCs was determined by ELISA. Data are presented as mean ± SEM (n = 3 per group). NS: non-stimulated, * p < 0.05, ** p < 0.01 and *** p < 0.001.