关键词: 旋毛形线虫, 白细胞介素-4, 肠道期, 感染, 抵抗作用

Abstract:

Objective To investigate the protective effects of interleukin 4 (IL-4) to Trichinella spiralis infection in the intestinal phase in mice. Methods The muscle samples of T. spiralis reservation mice were digested for collection of muscle larvae, which were sonicated and centrifuged to collect supernatant for preparing Trichinella antigen. Ten wild-type BALB/c mice were randomly divided into the non-infection group and wild-type infection group, with 5 mice each group, while additional 4 IL-4 knockout mice (IL-4KO) were assigned to IL-4KO infection group. The mice of wild-type infection group and IL-4KO infection group were given with 400 muscle larvae each by gavage respectively, while the non-infection group received the same volume of PBS. Eight days after infection, orbital blood samples were obtained to collect sera by centrifugation, and the content of monocyte chemotactic protein-1 (MCP-1) was detected by ELISA. The mice of all groups were dissected to collect the duodenum and proximal jejunum for preparation of paraffin sections. The sections were stained with Hematoxylin and eosin (HE). Measure the length of villi and crypts as well as the number and size of goblet cells useing Aperio ImageScope 12.4.3, and calculate the villus length/crypt length (V/C) and the ratio of the number of goblet cell/villus length (GC/V). Lymphocytes isolated from the mesenteric lymph nodes were cultured with Trichinella antigen (10 μg/ml) for 72 hours, and the supernatant was collected. A Luminex assay was performed to measure the levels of cytokines including IL-1β, IL-12p70, IL-2, IL-5, IL-6, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). The data was analyzed using SPSS 26.0 software with independent sample t-tests for pairwise comparisons and one-way ANOVA for multiple sample comparisons. Results ELISA results showed that serum MCP-1 levels were (344.90 ± 21.80), (350.50 ± 38.30) and (467.94 ± 190.01) pg/ml in the non-infection, wild-type infection and IL-4KO infection group, respectively, the IL-4KO infection group was higher than the wild-type infection group (t = 0.681, P < 0.05). HE staining revealed intact mucosa and normal villous architecture with no signs of inflammation in the non-infection group, whereas both the wild-type infection and IL-4KO infection groups showed vacuolated goblet cells, shortened villi and marked inflammation in the duodenum and jejunum, with more severe inflammation in the IL-4KO infection group. The IL-4KO infection group had V/C of 2.62 ± 0.12 and 2.78 ± 0.25 in the duodenum and jejunum, which were both lower than those in the wild-type infection group (3.46 ± 0.05, 3.65 ± 0.12) (F = 24.09, 20.46, P < 0.01, 0.05). The non-infection group had higher V/C of 4.69 ± 0.16 in the jejunum (F = 25.43, P < 0.01). The IL-4KO infection group had GC/V of 9.66 ± 0.88 and 7.33 ± 0.88 in the duodenum and jejunum, which were both higher than those in the wild-type infection group (5.33 ± 1.20 and 4.33 ± 0.33) (F = 17.12, 16.78, both P < 0.05). The goblet cell size in the duodenum and jejunum of the IL-4KO infection group were (12.39 ± 1.17) and (11.05 ± 0.60) μm, both larger than the wild-type infection group, which had (8.33 ± 0.44) and (8.44 ± 0.58) μm (F = 18.47, 16.22, both P < 0.05). The Luminex results showed that the levels of IL-1β, IL-12p70, IL-2, IL-5, IL-6, IFN-γ and TNF-α in the lymphocyte culture supernatants of the IL-4KO infection group were (0.80 ± 0.37), (2.70 ± 0.94), (49.76 ± 16.40), (25.25 ± 3.26), (12.51 ± 4.86), (51.20 ± 8.93), (15.86 ± 2.74) pg/ml, whereas the wild-type infection group had levels of (0.45 ± 0.03), (1.03 ± 0.04), (1.00 ± 0.38), (0.64 ± 0.16), (0.62 ± 0.24), (0.57 ± 0.09), (0.94 ± 0.31) pg/ml, respectively. The IL-4KO infection group were higher than the wild-type infection group (F = 5.52, 24.73, 48.72, 5.00, 123.10, 50.55, P < 0.05 or 0.01) except IL-1β (F = 0.87, P > 0.05). Conclusion IL-4 plays a protective immunological role during the intestinal phase of T. spiralis infection in mice. It could reduce the serum MCP-1, mitigate inflammation response in the duodenum and jejunum and suppress the secretion of inflammatory cytokines.

Key words: Trichinella spiralis, IL-4, Intestinal phase, Infection, Protective effect