Novel chimeric TLR2/NOD2 agonist CL429 exhibited significant radioprotective effects in mice

Abstract

Severe ionizing radiation causes the acute lethal damage of haematopoietic system and gastrointestinal tract. Here, we found CL429, the novel chimeric TLR2/NOD2 agonist, exhibited significant radioprotective effects in mice. CL429 increased mice survival, protected mice against the lethal damage of haematopoietic system and gastrointestinal tract. CL429 was more effective than equivalent amounts of monospecific (TLR2 or NOD2) and combination (TLR2 + NOD2) of molecules in preventing radiation-induced death. The radioprotection of CL429 was mainly mediated by activating TLR2 and partially activating NOD2. CL429-induced radioprotection was largely dependent on the activation of TLR2-MyD88-NF-κB signalling pathway. In conclusion, the data suggested that the co-activation of TLR2 and NOD2 could induce significant synergistic radioprotective effects and CL429 might be a potential high-efficiency selective agent.

Keywords: Cl429; NOD2; TLR2; radiation; radioprotection.

FIGURE 1

CL429 exhibited significant radioprotective effects in vivo. (A/C) C57BL/6 mice (n = 10) were pre‐treated with PBS, CL429 (5 mg/kg), LPS (2.5 mg/kg) and WR2721 (150 mg/kg) via peritoneal injection 24 and 2 h before 7.5 Gy or 9.0 Gy TBI. Then, the survival was recorded. (B/D) C57BL/6 mice were administered with PBS, CL429 (5 mg/kg), LPS (2.5 mg/kg) and WR2721 (150 mg/kg) via peritoneal injection immediately after 7.5 Gy or 9.0 Gy TBI. The survival was recorded

FIGURE 2

CL429 protected mice against lethal radiation‐induced haematopoietic system injury. C57BL/6 mice were treated with PBS or CL429 before 7.5 Gy TBI. (A) Representative images of HE‐stained bone marrow sections with the indicated treatment at 1, 5 and 10 d after 7.5 Gy TBI. (B) The number of BMCs was determined at 1, 5 and 10 d after 7.5 Gy TBI. (C) BMCs were isolated from mice 24 h after radiation; then, the apoptosis of BMCs was analysed by flow cytometry. (D) The data of apoptosis were presented as mean ± SD. (E) The number of LKS+ and LKS‐ cells were analysed by flow cytometry 24 h after 7.5 Gy radiation. (F) The data of LSK cells were presented as mean ± SD. *P < .05 and **P < .01 for control vs CL429 treatment

FIGURE 3

CL429 prevented lethal radiation‐induced intestinal injury. C57BL/6 mice were pre‐treated with PBS or CL429 before 9.0 Gy TBI. (A) Representative images of HE‐stained intestinal sections with the indicated treatment at 1.0, 3.5 and 5.0 d after TBI. Scale bar: 100 μm. (B) The relative length of small intestine villus at 96 h after radiation. (C) The representative images of Ki67‐stained intestinal sections. Scale bar: 100 μm. (D) The quantification of Ki67 positive area per section. (E) The representative images of TUNEL‐stained intestinal sections. Scale bar: 100 μm. (F) The quantification of TUNEL⁺ cells per crypt. *P < .05 and **P < .01 for control vs CL429 treatment

FIGURE 4

CL429 induced synergistic radioprotective effects compared with the combination of separate ligands. (A) C57BL/6 mice were pre‐treated with CL429, Pam3CSK (TLR2 ligand), MDP (NOD2 ligand), and Pam3CSK + MDP before TBI, and then, the survival was monitored. (B) C57BL/6 mice were treated with CL429, Pam3CSK (TLR2 ligand), MDP (NOD2 ligand), and Pam3CSK + MDP immediately after TBI, and then, the survival was monitored. (C) The representative pathological images of haematopoietic system and gastrointestinal tract at 3.5 d after radiation

FIGURE 5

The mechanism for the radioprotective effect of CL429. (A) C57BL/6 mice and TLR2 KO mice were treated with CL429 before TBI, and then, the survival was monitored (WT n = 11, TLR2 KO n = 7). (B) The representative pathological images of haematopoietic system and gastrointestinal tract at 3.5 d after radiation. (C) The protein expression of TLR2 and NOD2 after siRNA knock‐down. Cell viability was determined using CCK‐8 at 24 h after radiation. *P < .05 vs IR groups. (D) The effects of CL429 on TLR2, NOD2, MyD88 and p‐IKK at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h were measured by Western blot assay. (E) IL‐6, IL‐11, IL‐12 and TNF‐α were detected by ELISA 24 h after radiation. *P < .05, **P < .01 vs IR + PBS groups

FIGURE 6

Possible mechanism of CL429 radioprotection