Protective Role of Rabbit Nucleotide-Binding Oligomerization Domain-2 (NOD2)-Mediated Signaling Pathway in Resistance to Enterohemorrhagic Escherichia coli Infection

Abstract

Nucleotide-binding oligomerization domain 2 (NOD2), a member of the NOD-like receptors (NLRs) family that is well-known to play a key role in innate immune responses and is involved in innate antibacterial responses. In this study, rabbit NOD2 (rNOD2) was cloned from rabbit kidney (RK) cells. It was distributed in various tissues, and the highest level of rNod2 was detected in spleen. Moreover, the expression of rNod2 was significantly upregulated in the heart, liver, and spleen induced by enterohemorrhagic Escherichia coli (EHEC). Overexpression of rNOD2 induced the expression of pro-inflammatory cytokine, including Il1β, Il6, Ifn-γ, and Tnf, as well as defensins, including Defb124, Defb125, and Defb128 through the nuclear factor (NF)-κB signaling pathway. Furthermore, overexpression of rNOD2 inhibited the growth of EHEC, and knockdown of rNOD2 or inhibition of the NF-κB pathway promoted its replication. In addition, our results suggest that rNOD2 can significantly activate NF-κB signaling and trigger antibacterial defenses to increase the expression of pro-inflammatory cytokine and defensins after stimulation by EHEC. These findings are useful to further understanding the innate immune system of rabbits and providing a new perspective for the prevention of bacterial diseases in rabbits.

Keywords: EHEC; NF-κB; NOD2; antibacterial ability; rabbits.

Figure 1

A phylogenic tree based on rNOD2 between Oryctolagus curiculus and other species. A neighbor-joining tree was performed with MEGA 5.0. The tree was bootstrapped 1,000 times and the percentage of the bootstrapped values are shown. The sequences used are: Ovis aries NOD2, XP_014955920.1; Orcinus orca NOD2, XP_004264937.1; Sus scrofa NOD2, BAH24204.1; Canis lupus familiaris NOD2, NP_001273968.1; Mustela putorius furo NOD 2, XP_004744216.1; Pteropus Alecto NOD2, XP_006908812.1; Pteropus vampyrus NOD2, XP_011358488.1; O. princeps NOD2, XP_004584173.1; M. nemestrina NOD2, XP_011757602.1; P. troglodytes NOD2, NP_001098710.2; H. sapiens NOD2, NP_071445.1; Cavia porcellus NOD2, XP_003477732.2; Mus musculus NOD2, AAN84594.1; Danio rerio NOD2, NP_001314973.1; Ctenopharyngodon idella NOD2, ACX71753.1; Clupea harengus NOD2, XP_012682426.1; Larimichthys crocea NOD2, AJF23836.1; Takifugu rubripes NOD2, NP_001035913.1; Salmo salar NOD2, XP_014031576.1; Oncorhynchus mykiss NOD2, NP_001188484.1.

Figure 2

Expression analysis of rNOD2 in rabbits. (A) The tissue distribution of rNOD2 transcripts in healthy rabbits. The relative rNOD2 mRNA level in the liver (B), spleen (C), and kidneys (D) in rabbits in the early stages of an EHEC (10⁸ CFU) infection. Bars represent the means ± standard deviations (n = 5). A significant difference is indicated with an asterisk (*). ND, not detected in skin using qRT-PCR.

Figure 3

rNOD2 recombinant expression at protein level. The expression of rNOD2 protein in RK-13 cells was detected with immunofluorescence labeling. rNOD2 appears in green. Magnification was 100 × (A) and 400 × (B). (C) Immunoblotting of rNOD2 after transfected with pC-rNOD2. pCDNA3.1 (+) vector was used as control.

Figure 4

Effect of NF-κB and IFN-β by rNOD2. pC-rNOD2, pCDNA3.1 (+) vector, NF-κB (A), the IFN-β (B) reporter plasmid, and pRL-TK plasmid were transfected into cells for 24 h. The cells were harvested for dual-luciferase assays. Bars represent the means ± standard deviations (n = 3). A significant difference is indicated with an asterisk (*).

Figure 5

Expression of immune-related genes induced by rNOD2 overexpression in RK-13 cells. Two micrograms of pC-rNOD2 or pCDNA3.1 (+) vector were transfected for 24 h. The expression of immune-related genes induced by overexpression of rNOD2 were determined using RT-PCR. Bars represent the means ± standard deviations (n = 3). A significant difference is indicated with an asterisk (*).

Figure 6

rNOD2 induces EHEC-stimulated NF-κB and IFN-β. (A) Silencing efficiency of siRNA targeting rNOD2. RK-13 cells were transfected with the indicated siRNA1, 2, and 3 for 24 h. qRT-PCR was used to analyze the expression of rNOD2. pC-rNOD2, pCDNA3.1 (+) vector, Si-rNOD2, and NC siRNA with NF-κB (B) and IFN-β (C) reporter plasmid, and pRL-TK plasmid were transfected into RK-13 cells. After 24 h, the cells were infected with EHEC. The cells were then harvested for dual-luciferase assays. Bars represent the means ± standard deviations (n = 3). A significant difference is indicated with an asterisk (*).

Figure 7

Effect of overexpression or knockdown of rNOD2 on expression of immune-related genes induced by EHEC in RK-13 cells. Expression of Il1β (A), Il6 (B), Il8 (C), Tnf (D), Ifn-γ (E), Defb124 (F), Defb125 (G), Defb128 (H), and Np5 (I) in EHEC-infected RK-13 cells transfected with pC-rNOD2 or si-rNOD2. Bars represent the means ± standard deviations (n = 3). A significant difference is indicated with an asterisk (*).

Figure 8

Effect of MDP on expression of immune-related genes induced by EHEC in RK-13 cells. RK-13 cells were treated with 500 ng of MDP for 12 h and then were infected with EHEC. Bars represent the means ± standard deviations (n = 3). A significant difference is indicated with an asterisk (*).

Figure 9

Effect of overexpression of rNOD2 on expression of immune-related genes induced by EHEC after inhibition of NF-κB in RK-13 cells. pC-rNOD2 or pCDNA3.1-empty were transfected into cells for 24 h. RK-13 cells were pretreated with 10 μM of BAY11-7082 for 12 h to inhibit NF-κB pathways and then infected with EHEC. Bars represent the means ± standard deviations (n = 3). A significant difference is indicated with an asterisk (*).

Figure 10

The antimicrobial activity of rNOD2. (A) pC-rNOD2 and pCDNA3.1 (+) vector were transfected into RK-13 cells for 24 h. (B) Si-rNOD2 and NC siRNA were transfected into RK-13 cells for 24 h. (C) After transfection of pC-rNOD2, cells were treated with 10 μM of BAY11⁻7082 for 12 h. The cells were infected with EHEC. The cells were then lysed in 1% Triton X-100, and intracellular bacteria were calculated. Bars represent the means ± standard deviations (n = 3). A significant difference is indicated with an asterisk (*).