Ionizing radiation can cause acute radiation syndrome, resulting in hematopoietic depletion, gastrointestinal collapse and cerebrovascular injury . The hematopoietic system is highly sensitive to radiation. Even low-dose irradiation can cause significant changes and dysfunction in hematopoiesis . In the pathophysiological process of acute radiation sickness, changes in hematopoietic organs appear earlier, mainly manifested as the reduction and injury of nucleated cells in peripheral blood and bone marrow, hematopoietic dysfunction and even failure, and eventually lead to anemia, hemorrhage, infection, metabolic disorder and death . Hematopoietic radiation injury is obviously time-sensitive and can be used as the basis for classification, diagnosis and prognosis . At present, there is still no research on the radioprotective effects of CRX-527, let alone their effects on the hematopoietic system. In this study, we verified the effect of CRX-527 on the hematopoietic system, determined the phenotypic differentiation of HSCs, and revealed an excitatory effect on the immune system [17, 18]. On this basis, we observed changes in intestinal structure and function in mice after ionizing radiation and revealed the radioprotective mechanism of CRX-527 in TLR4-related pathway activation. This research is a pioneering work.
Irradiation can reduce the survival rate of mice by 30% . In this study, mice were irradiated, the average survival time of mice treated with CRX-527 was significantly prolonged, and the survival rate was significantly improved. Injury to tissues and organs related to the hematopoietic system, including bone marrow, spleen and peripheral blood, was relieved. Mouse HSCs account for only 1% of bone marrow cells enriched in the LSK population . HPCs are mainly enriched in the LK population. IR can cause a decrease in the number and proportion of LSK, while CRX-527 could alleviate it, which could provide necessary guarantees for the self-renewal and peripheral differentiation of hematopoietic stem cells after ionizing radiation. Based on the strong heterogeneity of the LSK cell population, it can be divided into LT-HSCs, ST-HSCs, and MPPs. LT-HSCs are the most primitive HSCs, which can differentiate into ST-HSCs and MPPs and then differentiate into various cells of the whole blood system. CRX-527 can increase the ratio of MPPs and ST-HSCs in mice, while the ratio of LT-HSCs decreased slightly, which indicated that CRX-527 could promote the differentiation of LT-HSCs into MPPs, thus allowing MPPs to differentiate into macrophages .
MPPs can further differentiate into CMPs and CLPs that can continue to differentiate into precursor cells of various lymphocytes and finally differentiate into T cells, B cells, DC cells and NK cells. On the other hand, CMPs can continue to differentiate into GMPs and MEPs. GMPs can further differentiate into monocyte macrophages and granulocytes .
HPCs, similar to MPPs, are the direct source of peripheral blood granulocytes and are sensitive to radiation. At present, the confirmed hematopoietic progenitor cells are as follows: ① Erythrocyte hematopoietic progenitor cells can form red blood cell colonies, also known as red blood cell colony forming units (CFU-Es), under the action of erythropoietin (EPO) . ② Megakaryocyte line hematopoietic progenitor cells can form megakaryocyte colonies, also known as megakaryocyte line colony forming units (CFU-Ms), under the action of thrombopoietin . ③ Neutrophil-macrophage hematopoietic progenitor cells can form neutrophil-macrophage colonies, also known as granulocyte-macrophage colony forming units (CFU-GMs), under the action of granulopoietin (generated by macrophages) . CRX-527 can promote the directional differentiation of HPCs into GMPs, which was consistent with the direct effect on MPP and guaranteed the proliferation of granulocytes and macrophages.
Myeloid-derived suppressor cells (MDSCs) are a group of heterogeneous cells derived from bone marrow . As the precursor of dendritic cells (DCs), macrophages and/or granulocytes, MDSCs have the ability to significantly inhibit the immune cell response and are derived from bone marrow progenitor cells and immature myeloid cells (IMCs) . Under normal circumstances, precursors of DCs, macrophages and granulocytes can quickly differentiate into mature DCs, macrophages and granulocytes and enter corresponding organs and tissues to exhibit normal immune function, in which IMCs account for approximately 0.5% of peripheral blood mononuclear cells. Under pathological conditions such as tumors, infection, inflammation, septicemia, and surgical injury, the maturation of myeloid-derived precursor cells is blocked due to the action of cytokines, thus they remain at various stages of differentiation and become MDSCs with immunosuppressive functions. In previous studies, TLR ligands have been shown to activate many different signaling pathways involved in MDSCs, including STAT6, STAT1 and NF-κB. Notably, CRX-527 can improve the immune response of macrophages and granulocytes by reducing the proportion of MDSCs and the suppression ability of immune cells .
Macrophages and monocytes are both phagocytes that participate in nonspecific defense (innate immunity) and specific defense (adaptive immunity) in vertebrates, in which they phage fragments and pathogens in fixed or dissociated cells and activate lymphocytes or other immune cells to make them respond to pathogens . We found that CRX-527 could stimulate mouse bone marrow to generate a large number of macrophages (CD11b+F4/80+) for immune defense. In addition, the changes in peripheral blood white blood cells (including monocytes, macrophages, neutrophils, DCs and NK cells) are consistent with the clinical stage of radiation sickness in the time phase, so they are often used as one of the criteria for judging the clinical course, condition and prognosis of radiation sickness . CRX-527 alleviated the decrease in leukocyte levels induced by ionizing radiation, mainly by increasing the proportion and number of macrophages in peripheral blood leukocytes. Above all, CRX-527 could maintain the proportion and quantity of LSK, promote the mobilization of HSCs and induce them to differentiate into MPPs-GMPs-macrophages.
The intestines have the largest macrophage pool in the body, where macrophages mainly come from intestinal local macrophages and bone marrow HSCs . According to previous research reports, the level of bone marrow-derived macrophages is associated with the complexity of the intestinal flora and mediated by the existing TLR ligands in the serum, which can maintain intestinal mucosal homeostasis, promote intestinal epithelium and crypt regeneration, and regulate positive immune function in the intestine . For example, in the process of senescent cell clearance and tissue remodeling, intestinal macrophages can produce a variety of cytokines, which can stimulate the proliferation of epithelial progenitor cells in the intestinal crypts, regulate the integrity of the epithelial barrier, and maintain intestinal homeostasis . TLR4 was highly expressed on the surface of macrophages, and it was also expressed in small intestinal epithelial cells, but at a much lower level than that in macrophages . We suspected that CRX-527 could not only protect the intestine from radiation damage through the TLR4 pathway but also activate the immune response through macrophages to achieve more effective protection of the intestine. To verify this conjecture, THP-1-HIEC and RAW264.7-MODE-K co-culture systems were used to simulate the therapeutic effect of macrophages on small intestinal epithelial cells , in which CRX-527 behaved well and far exceeded the protective effect of CRX-527 on small intestinal epithelial cells alone, indicating that CRX-527 can activate the TLR4 pathway of macrophages to protect small intestinal epithelial cells from IR-induced damage. In addition, in the co-culture system, CRX-527 also suppressed the generation of ROS, the activation of apoptosis and the release of inflammatory factors in small intestinal epithelial cells.
Studies have shown that ISCs, intestinal epithelial cells, goblet cells, Pan's cells, and some undifferentiated cells have strong sensitivity to irradiation . A certain dose of radiation can cause extensive damage and destruction, eventually leading to the occurrence of intestinal radiation sickness, which is mainly manifested as intestinal mucosal injury and shedding, loss of crypt proliferation ability, villus rupture and peeling, loss of intestinal shielding function, increase of intestinal permeability, and immune deficiency . Patients may show acute symptoms characterized by gastrointestinal symptoms such as vomiting, diarrhea and bloody stool. At present, the recovery rate of intestinal radiation sickness is extremely low clinically, so more research on the molecular mechanism and treatment strategies of intestinal radiation sickness is urgently needed .
Crypt basal ISCs, specially labeled with Lgr5, can generate differentiated intestinal cells, which are necessary to maintain balance in vivo and to regenerate the intestinal crypt-villus structure during irradiation . CRX-527 can preserve the normal crypt-villus structure of mouse small intestinal tissue after ionizing radiation, which was attributed to the constant level of Lgr5+ cells, and reserve the regeneration ability of intestinal cells, including Paneth cells (Lysozyme+), intestinal epithelial cells (Villin+) and transient expansion cells (Ki67+). In addition, CRX527 can also maintain intestinal barrier function and homeostasis. Moreover, CRX-527 protected intestinal tissue by suppressing apoptosis and releasing inflammatory factors .
Hoffmann JA first discovered TLR molecules in Drosophila in 1996 . Subsequently, Burdelya LG reported the effect of TLR5 in radiation prevention in 2008. Since then, the role of TLRs in radiation injury prevention has attracted increasing attention. As a member of the TLR family, TLR4 plays an important role in innate immunity . In previous reports, TLR4 also participated in radiation protection, and the survival rate of irradiated mice was significantly improved after TLR4 was activated by LPS. However, as a proinflammatory agent, LPS itself has inevitable toxicity, so it is crucial that we find a series of radiation protection agents with high efficiency and low toxicity. To date, CRX-527 has been found to be an effective agonist of the TLR4 signaling pathway . After TLR4 dimerization, IRAK1 is phosphorylated by IL-1 receptor-associated kinase (IRAK) via IRAK4 in myD88-dependent pathways . Phosphorylated IRAK1 activates tumor necrosis factor receptor-associated Factor 6 (TRAF6) , leading to the activation of transforming growth factor-β-activated protein kinase 1 (TAK1), which binds TGF-β-activated kinase 1/MAP3K7-binding proteins 1 (TAB1), TAB2, and TAB3. The TAK1/TAB1/TAB2/TAB3 complex then phosphorylates the IKK complex, which has two catalytic subunits, IKKα and IKKβ and IKKγ/NEMO . This is followed by the degradation of the NFκB inhibitor (IκB), which leads to the development of the NF-κB dimer and transfer to the nucleus. For the alternative TRIF pathway, cytoplasmic TRIF-associated adapter molecule (TRAM) induces the recruitment of interferon-β (TRIF) to the TLR4 receptor by an adaptor containing the TIR domain. CRX-527-induced activation of the TLR4 pathway leads to internalization of the TLR4/TRAM/TRIF complex through endosomes. RIP1 signals TRAF6, which results in TNF-R-associated Factor 3 (TRAF3) recruitment to TRIF, thus initiating the activation of IKKε and tank-binding kinase 1 (TBK1) . Interferon regulatory Factor 3 (IRF3) is then transferred to the nucleus, resulting in the generation of inflammatory mediators and type 1 interferon. TLR4/MD-2 complexes expressed on the surface of macrophages, monocytes and dendritic cells sense micromolar concentrations of TLR4 ligand and trigger the generation of various pro-inflammatory mediators, which provides the basis for the TLR4 ligand CRX-527 to reduce intestinal injury after irradiation by activating macrophages.