[Construction of a Toll-like receptor 5-intestine-specific overexpression mouse model and its protective effects against Salmonella infection].

Objective

To construct a intestine-specific Toll-like receptor 5 (TLR5) overexpression mouse model and investigate its protective role against Salmonella infection.

Methods

In-Fusion cloning technology was used to construct a conditional TLR5 overexpression homologous recombination vector. This vector was integrated into the genome of C57BL/6 mice via gene editing. The F0-generation mice were bred to obtain stable F1-generation progeny, which were further crossed with intestine-specific Cre mice to generate TLR5-overexpressing mice (TLR5 group). The littermates lacking the transgene served as wild-type controls (WT group). Both groups consisted of 4-6 weeks old mice weighing 22-25 g. Eight mice in TLR5 group and WT group were selected 3 times each for the experiment: phosphate buffered saline (PBS) buffer was intraperitoneally injected in the first time (blood was taken at 0 hour after injection), flagellin was injected intraperitoneally in the second time (blood was taken at 1 hour after injection) and the third time (blood was taken at 4 hour after injection) to activate TLR5. The expression levels of inflammatory factors, such as serum levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured in both groups. Another 8 mice in TLR5 group and WT group were selected to establish Salmonella enteric infection model by oral feeding, and fecal bacterial loads were monitored on the 1st, 3rd, and 5th day. Kaplan Meier method was used to draw survival curves to compare the survival time of mice infected with Salmonella. Eight mice in TLR5 group were divided into TLR5 infected group and TLR5 uninfected group, and 8 mice in WT group were divided into WT infected group and WT uninfected group, with 4 mice in each group. TLR5 infected group and WT infected group were fed salmonella orally, and TLR5 uninfected group and WT uninfected group were fed with PBS buffer orally. All mice were euthanized on day 5 to compare splenic and colonic tissue damage.

Results

Gel electrophoresis confirmed the successful construction of the homologous recombination vector, as evidenced by a distinct band at 16 943 bp, consistent with the theoretical size. After the reproduction of F0 generation mice, some offspring mice produced by the hybridization of F1-generation mice and gut-specific Cre mice were identified by PCR with 3.7 kb fragment amplified by 5' arm and 4.0 kb fragment amplified by 3' arm. Subsequent gene sequencing further confirmed that TLR5 was gut-specific overexpressed mice. Following flagellin injection, serum IL-6 level in the TLR5 group was higher than that in the WT group at 1 hour [M (Q1, Q3), 1 079 (516, 1 498) vs 71 (54, 236) ng/L, P<0.05]. Serum IL-6 level [1 512 (1 247, 1 522) vs 75 (17, 964) ng/L] and serum TNF-α level [248 (188, 464) vs 21 (6, 119) ng/L] in the TLR5 group were higher than those in the WT group at 4 hours (all P<0.05). The survival time of TLR5 group was longer than that of WT group [9.0 (8.0, 10.5) vs 8.0 (7.5, 8.5) d, P<0.05]. Fecal salmonella test of mice showed that on the 5th day after salmonella infection, the bacteria amount of Salmonella in TLR5 group was lower than that in WT group [650 (148, 2 175) vs 44 (1, 128)×104 colony forming units (CFU)/g, P<0.05]. The experiment of dissecting mice on the 5th day after infection showed that the colonic length of TLR5 infected mice was longer than that of WT infected mice [(9.1±1.1) vs (7.0±0.7) cm, P<0.05].

Conclusions

In this study, an intestine-specific TLR5 overexpression mouse model is successfully constructed. The intestine-specific TLR5 overexpression plays a protective role against Salmonella infection in mice.