After the phagocytosis or internalization, pathogens are transferred to phagosomes, which subsequently fuse with intracellular granules to form phagolysosomes, within these structures, pathogens are killed by a combination of nonoxidative and oxidative mechanisms (Plüddemann et al., 2011; Stuart and Ezekowitz, 2005). A bacterial clearance assay was conducted to further investigate the effect of CiCXCL12a-mediated phagocytosis. The results showed that the numbers of A. hydrophila are significantly decreased after the addition of CiCXCL12a, and the numbers of A. hydrophila are markedly increased after Cyto D blockage (Figure 6A). Microbial cargo undergoes degradation through progressive fusions of the phagosome with early endosomes, late endosomes, and ultimately with lysosomes (Egami et al., 2011; Verma and Datta, 2017). The small GTPases, Rab5 and Rab7, are the key regulators of the transport of newly endocytosed vesicles to early and late endosomes (Jordens et al., 2005). To explore the potential involvement of CiCXCL12a in the expression of early endosomes markers (Rab5) and late endosomes markers (Rab7) proteins, we initially analyzed the protein expressions of Rab5 and Rab7. Our findings revealed that Rab5 and Rab7 are upregulated in monocytes/macrophages challenged with CiCXCL12a (Figure 6B, B’). Subsequent analysis of Rab5 and Rab7 expression patterns following bacterial challenge demonstrated that two small GTPases (Rab5 and Rab7) increase significantly when compared with that of the control and CiCXCL12a treatment alone (Figure 6C, C’). Furthermore, CiCXCL12a and A. hydrophila synergistically increase the expressions of Rab5 and Rab7 (Figure 6C, C’). These results suggested that Rab5 and Rab7 play crucial roles in resistance against bacterial pathogens and Rab5 and Rab7 are participated in CiCXCL12a-mediated phagocytosis of monocyte/macrophage. We further detected that Rab5 and Rab7 are colocalized with A.hydrophila in grass carp monocytes/macrophages in a time dependent manner (Figure 6D and E). Also, FITC-labeled A.hydrophila was found to be colocalized with lysosomes of monocyte/macrophage stained with LysoBrite Red (Figure 6F). To confirm the involvement of lysosomes in A. hydrophila degradation, the lysosomal inhibitor chloroquine (CLQ) was utilized to inhibit lysosomal activity (Chen et al., 2021), and the elimination of A. hydrophila was assessed. Although, the numbers of surviving A. hydrophila are obviously decreased after addition of CiCXCL12a, monocytes/macrophages appear to have lost their A. hydrophila degradation role after pretreating CLQ (Figure 6G). Taken together, the results suggested that Rab5 and Rab7 are involved in CiCXCL12a-mediated phagocytosis and lysosomes are involved in the elimination of A. hydropila.
CiCXCL12 suppresses cell apoptosis by activating the PI3K-AKT signal pathway
Apart from its conserved antibacterial activity and phagocytosis, CiCXCL12a treatments increase the phosphorylation of AKT, mTOR, STAT5 and ERK in a dose-dependent manner (Figure 7A-D). However, the degradation of phosphorylation indued by CiCXCL12 are rapid within 30 min (Figure 7E and F). These results are consistent with human CXCL12 (HsCXCL12), which induces the activations of the PI3K-AKT/mTOR, JAK-STAT5 and MAPK-ERK signaling pathways (Delgado-Martín et al., 2011; Mowafi et al., 2008). Previous study showed that HsCXCL12 suppresses cell apoptosis by activating the PI3K-AKT pathway (Fu et al., 2018; Ho et al., 2010). To validate whether CiCXCL12a also inhibits cell apoptosis. We successfully constructed an LPS-induced apoptosis model in monocytes/macrophages and the precent of monocyte/macrophage apoptosis is suppressed after the addition of CiCXCL12a (Figure 7G), suggesting CiCXCL12a, similar to HsCXCL12, also has a conserved ability to attenuate cell apoptosis. Then, we used specific phosphorylation inhibitors for PI3K, AKT, STAT5 and ERK to inhibit the activation of these pathways (Figure 7H). Monocytes/macrophages were respectively pretreated with four specific phosphorylation inhibitors for 3 h, then incubation with 0.2 μM CiCXCL12a for 3 h, and finally LPS was added each well for 16 h. STAT5 and ERK specific phosphorylation inhibitors have no effect for CiCXCL12a-suppressed monocyte/macrophage apoptosis induced by LPS (Figure 7I, I’). However, significant inhibition of monocyte/macrophage apoptosis induced by CiCXCL12a is alleviated after pretreating with both PI3K and AKT specific phosphorylation inhibitors (Figure 7I, I’). These results indicated that CXCL12 has a conserved role in inhibiting cell apoptosis by activating the PI3K-AKT pathway. 帮我润色英文语法