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. 2022 Nov 30;11(12):1747.
doi: 10.3390/biology11121747.

Porcine Interleukin-17 and 22 Co-Expressed by Yarrowia lipolytica Enhance Immunity and Increase Protection against Bacterial Challenge in Mice and Piglets

Junjie Peng  1 , Fang Yang  2 , Jianlin Chen  3 , Shaohua Guo  1 , Linhan Zhang  1 , Dinghao Deng  1 , Jiangling Li  4 , Xuebin Lv  4 , Rong Gao  1
Affiliations

Porcine Interleukin-17 and 22 Co-Expressed by Yarrowia lipolytica Enhance Immunity and Increase Protection against Bacterial Challenge in Mice and Piglets

Junjie Peng et al. Biology (Basel). .

Abstract

Drug resistance in economic animals to pathogens is a matter of widespread concern due to abuse of antibiotics. In order to develop a safe and economical immunopotentiator to raise the immunity and antibacterial response as a replacement for antibiotics, a recombinant yeast co-expressing pig interleukin-17 (IL-17) and IL-22 was constructed and designated as Po1h-pINA1297-IL-17/22. To evaluate the immunoregulator activities of Po1h-pINA1297-IL-17/22, two experiment groups (oral inoculation with Po1h-pINA1297 or Po1h-pINA1297-IL-17/22) and a negative control group (PBS) were set up using 4-week-old female BALB/c mice (10/group). The level of cytokines, including IL-2, IL-4, IL-10, and IFN-γ, were detected by ELISA, and the circulating CD4+ and CD8+ lymphocytes were quantified by flow cytometry. The IgG and secretory IgA (SIgA) levels in both small intestine and fecal matter were also measured by ELISA. The results indicated that the IgG antibody titer and SIgA concentration increased significantly in the Po1h-pINA1297-IL17/22 group in comparison with the controls (p < 0.05) and so did the cytokine levels in the serum (IL-2, IL-4, IL-10, and IFN-γ). In addition, CD4+ and CD8+ T cells were also obviously elevated in the Po1h-pINA1297-IL17/22 group on 35th day (p < 0.05). After challenge with pathogenic Salmonella typhimurium, the Po1h-pINA1297-IL17/22 group showed a relatively higher survival rate without obvious infectious symptoms. On the contrary, the mortality of control group reached 80% due to bacterial infection. As for the piglet experiment, 30 healthy 7-day piglets were similarly attributed into three groups. The oral inoculation of piglets with Po1h-pINA1297-IL17/22 also markedly improved the growth performance and systemic immunity (up-regulations of IL-4, IL-6, IL-15, IL-17, IL-22, and IL-23). Overall, the results indicated that Po1h-pINA1297-IL17/22 effectively promoted the humoral and cellular immunity against bacterial infection. These proved the promising potential of Po1h-pINA1297-IL-17/22 to be a potent immunopotentiator for the prevention of microbial pathogen infections.

Keywords: Yarrowia lipolytica; bacterial infection; immunity; immunopotentiator; interleukin-22; pig interleukin-17.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The construction of the Po1h-pINA1297-IL-17/22 strain. Analysis of the rIL-17/22 gene in Po1h by PCR. Lane M: DL 5000 DNA marker; Lane 1: Po1h-pINA1297-IL-17/22.
Figure 2
Figure 2
The expression analysis of Po1h-pINA1297-IL-17/22. Expression analysis of rIL-17 and rIL-22 in Po1h by ELISA (a,b) and by Western blot (c,d).
Figure 3
Figure 3
Cell viability analysis via CCK8. The data with different lowercase letter are significantly different, p < 0.05, and the following are the same as here.
Figure 4
Figure 4
Changes in IgG levels in the serum of the experimental mice. The data with different lowercase letter are significantly different, p < 0.05).
Figure 5
Figure 5
Concentrations of IL-2 (a), IL-4 (b), IL-10 (c), and IFN-γ (d) in the serum of the mice post inoculation. The data with different lowercase letter are significantly different, p < 0.05.
Figure 5
Figure 5
Concentrations of IL-2 (a), IL-4 (b), IL-10 (c), and IFN-γ (d) in the serum of the mice post inoculation. The data with different lowercase letter are significantly different, p < 0.05.
Figure 6
Figure 6
Changes in CD4+ and CD8+ T cells in the peripheral blood of mice on the 35th day post inoculation. (a) Changes in the CD4+ and CD8+ T cells. (b) The counts of CD4+ T cells. (c) The counts of CD8+ T cells. The data with different lowercase letters are significantly different, p < 0.05.
Figure 7
Figure 7
Total SIgA concentrations (a,b) in the feces and intestines of the mice. The data with different lowercase letters are significantly different, p < 0.05.
Figure 8
Figure 8
The survival percentage of mice after challenge.
Figure 9
Figure 9
Hematoxylin and eosin staining of small intestine tissues. Histologic examination showed the fluff height, crypt depth, and intestinal wall thickness in PBS (a), Po1h-pINA1297 (b) and Po1h-pINA1297-IL-17/22 groups (c) and their statistical comparison (d) respectively. Scale bar = 100 µm. The data with different letter are significantly different, p < 0.05.
Figure 9
Figure 9
Hematoxylin and eosin staining of small intestine tissues. Histologic examination showed the fluff height, crypt depth, and intestinal wall thickness in PBS (a), Po1h-pINA1297 (b) and Po1h-pINA1297-IL-17/22 groups (c) and their statistical comparison (d) respectively. Scale bar = 100 µm. The data with different letter are significantly different, p < 0.05.
Figure 10
Figure 10
Changes in piglet weights during 8 weeks of observation (n = 10/group). The data with different lowercase letter are significantly different, p < 0.05.
Figure 11
Figure 11
Change in IL-17 (a), IL-22 (b), IL-4 (c) and IL-6 (d) levels in the plasma of the piglets. The data with different lowercase letters are significantly different, p < 0.05.
Figure 11
Figure 11
Change in IL-17 (a), IL-22 (b), IL-4 (c) and IL-6 (d) levels in the plasma of the piglets. The data with different lowercase letters are significantly different, p < 0.05.
Figure 12
Figure 12
Changes in the IL-15 (a) and IL-23 (b) levels in the plasma of the piglets.
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