Monophosphoryl lipid a attenuates radiation injury through TLR4 activation

Abstract

Ionizing radiation causes severe damage to human body, and normal tissue toxicity in cancer radiotherapy also limits its further application. It is urgently required to develop safe and effective radioprotector. Our previous study has shown that toll like receptor 4 (TLR4) was dispensable for basal radiation resistance. However, severe toxicity of its traditional agonist lipopolysaccharide limits the clinical application. In present study, we demonstrated that monophosphoryl lipid A (MPLA), a potent TLR4 agonist with low toxicity, effectively attenuated radiation injury on in vitro and in vivo. MPLA increased cell survival and inhibited cell apoptosis after irradiation, and cell cycle arrest was also inhibited. Radiosensitive tissues including spleen, intestine, bone marrow and testis were protected from radiation damages in a TLR4 dependent manner. We also found that myeloid differentiation factor 88 (MyD88) accounted more than Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF) for the radioprotective effects of MPLA. In conclusion, our finding suggests TLR4 agonist MPLA as a safe and effective radioprotector for clinical application.

Keywords: MyD88; TRIF; monophosphoryl lipid a (MPLA); radioprotection; toll like receptor 4 (TLR4).

Figure 1. MPLA protected cells against cell death and alleviated DNA damage induced by IR

RAW264.7 cells were treated with MPLA (1ug/mL) before 6Gy irradiation, at 24h after which cell apoptosis was determined with flow cytometry (A) and percentages of Annexin V positive cells were quantified (B). Cell cycle distribution was also analyzed at 12h after irradiation (C). Cell survival of HUVEC (D) and L02 cells (E) were analyzed at 48h post-irradiation by using a CCK-8 method. Comet assay was used to measure DNA damage of irradiated cells at different time points (F). Olive tail moment (G) and tail moment (H) were quantified. ^*P<0.05, ^**P<0.01 Vs IR groups. (n=6)

Figure 2. MPLA induced translocation of NF-kB p65 and activated MAPK signaling pathway

At 0.5h and 2h after 8Gy irradiation, HUVEC cells were stained with NF-kB p65 antibody (A) and p65 fluorescence inside and outside the nuclear were quantified and the ratio was calculated (B). NF-kB luciferase activity was measured in 293T cells after different centration of MPLA treatment (C). Western blot assay of TRIF, MyD88, and MAPK signaling pathway were examined at 0, 0.5 and 8h after irradiation (D). Raw density of TRIF (E), MyD88 (F), p-p38 (G) and pJNK (H), compared to internal control, was analyzed. **P<0.01 Vs IR groups. (n=8)

Figure 3. MPLA alleviated destructions of bone marrow and increased bone marrow nucleated cells following IR

On Day 0, Day 1, Day 3, and Day 7 post 7Gy-irradiation, femurs were isolated and subjected to HE staining (A). On the third day after irradiation, bone marrow nucleated cells (B) and CD34⁺ HSC was analyzed with flow cytometry using CD34-FITC and B220-PC5.5 antibodies (C). The percentages of CD34-FITC⁺ cells were quantified (D). *P<0.05, **P<0.01 Vs IR groups. (n=8).

Figure 4. MPLA alleviated radiation damages on spleen and CD4/CD8 immune imbalance

On Day 0, Day 1, Day 3, and Day 7 post 7Gy-irradiation, spleen from different groups were isolated and subjected to HE staining (A). Splenocytes in single cells suspension was prepared and analyzed for cell apoptosis in on Day 3 post-irradiation (B). The area of white pulps in each group was calculated with Image J software (C). Percentages of CD4⁺ and CD8⁺ cells in splenocytes were analyzed (D) and ratio of CD4+ and CD8+ cells in wild type and TLR4 knockout mice (E). *P<0.05, **P<0.01 Vs IR groups. (n=5)

Figure 5. MPLA suppressed the inflammation cytokines induced by irradiation

On the third day after 7Gy irradiation, blood serum of mice from different groups was isolated. Concentrations of cytokines in blood serum were measured by Elisa assay on the third day after irradiation (A-I). *P<0.05, **P<0.01 Vs IR groups. (n=6)

Figure 6. MPLA protected testis and intestinal damage and increased animal survival in response to IR

On different day after 7Gy total-body irradiation, testis (A) and small intestine (E) were isolated and stained with a HE staining method. Villus height, villus number and the number of crypt in intestine were quantified and analyzed (F). Survival of mice with and without MPLA pretreatment was monitored up to 30 days post 7Gy (B) or 9Gy (C) total body irradiation. We tested survival of mice exposed to 15Gy sub-total irradiation (D). *P<0.05, Vs IR groups. (n=10)

Figure 7. Radioprotective effects of MPLA were abrogated in TLR4-/- mice

TLR4 knockout mice were exposed to 7Gy total-body irradiation, after then femur and spleen were isolated on the third day. Femur (A) and spleen (D) in TLR4-/- mice were subjected to HE staining of sections from on Day 3 post-irradiation. Bone marrow nucleated cells in TLR4-/- mice were also analyzed from different groups (B). Percentages of CD34+ HSC in bone marrow cells from different groups were counted (C). Flow cytometry assay was also used to measure splenocytes apoptosis from TLR4-/- mice (E). NS, non significant Vs IR groups. (n=5)

Figure 8. MPLA showed partial protective effects in TRIF mutant mice

On the third day after 7Gy total-body irradiation, femurs (A) and spleen (D) were isolated from TRIF mutant mice and subjected to HE staining. At 24h after irradiation, cell apoptosis in splenocytes assay was analyzed (B). Ratio of CD4+ and CD8+ splenocytes (C), bone marrow nucleated cells (E) and percentages of CD34+ HSC (F) was analyzed by flow cytometry. *P<0.05, Vs IR groups. (n=10)

Figure 9. Radioprotective effects of MPLA were more dependent on MyD88 than TRIF

At 48h after TRIF siRNA and MyD88 siRNA transfection, cells were treated with MPLA for 12h and subjected to irradiation. Cell apoptosis was detected by Flow cytometry analysis of Annexin V positive cells percentages at 24h after 8Gy irradiation. *P<0.05, Vs IR groups. (n=6).