Preliminary Study On The Mechanism Of LPS-induced Autophagy Regulating The Proliferation In Haemocytes Of Crassostrea Gigas

Haematopoiesis plays an important role in maintaining immune homeostasis for organism.Haemocytes are the main immune cells of invertebrates,and their proliferation and differentiation are the basis of immune defense.In the immune response of vertebrates,autophagy can enhance innate immunity by regulating the proliferation and differentiation of immune cells,realize antigen presentation,pathogen clearance and dampening inflammation.However,there are few reports on the regulation of autophagy in the proliferation of invertebrate haemocytes.In this study,the effects of ROS levels on haemocytes proliferation were investigated and the mechanism of ROS-induced autophagy of haemocytes was explored in C.gigas was revealed by real-time PCR,Western blot,flow cytometry and immunofluorescence assay,which preliminarily revealed the potential regulatory effect of autophagy on haemocytes proliferation in antimicrobial immunity of C.gigas.The results are as follows:1.ROS increased after LPS stimulation,which promoted the proliferation of haemocytes in C.gigas.The seawater（SW）,lipopolysaccharides（LPS）and N-acetylcysteine（NAC）,a ROS scavenger,were employed to treat oysters,respectively.The ROS levels in haemocytes of C.gigas were detected by flow cytometry,an increase of ROS was observed in oyster haemocytes after LPS injection,and a decrease of ROS was detected in the haemocytes after the injection of NAC.Meanwhile,the percentage of Ed U positive cells（Ed U⁺cells）in haemocytes is significantly different.Among them,the percentage of Ed U⁺haemocytes significantly increased at 12 h and 24 h after LPS injection,which was 2.98-fold（p<0.001）and 2.04-fold（P<0.001）of that in SW group,respectively.On the contrary,the percentage of Ed U⁺haemocytes significantly decreased at 12 h and 24 h after NAC injection,which was 0.27-fold（p<0.001）and 0.63-fold（p<0.05）of SW group,respectively.The percentage of Ed U⁺agranulocytos,semi-granulocytes and granulocytes significantly increased at 12 h after LPS injection,which was 2.22-fold（p<0.05）,1.53-fold（p<0.01）and 4.13-fold（p<0.001）of that in SW group,respectively,and the percentage of Ed U⁺agranulocytos,semi-granulocytes and granulocytes significantly increased at 24 h after LPS injection,which was 1.88-fold（p<0.01）,1.56-fold（p<0.01）and 2.36-fold（p<0.001）of that in SW group,respectively.The percentages of Ed U⁺semi-granulocytes and granulocytes significantly decreased at 12 h after NAC injection,which were0.24-fold（p<0.001）and 0.26-fold（p<0.001）higher than that in SW group,and the percentages of Ed U⁺agranulocytos,semi-granulocytes and granulocytes significantly decreased at 24 h after NAC injection,which were 0.31-fold（p<0.01）,0.66-fold（p<0.05）and 0.62-fold（p<0.05）higher than that in the SW group,respectively.These results indicated that LPS can induce ROS production and the increase of Ed U⁺cells.2.ROS increased after LPS stimulation,which induced autophagy of haemocytes in C.gigas.The m RNA and protein expression levels of Cg LC3 in haemocytes were detected after treatment with SW,LPS or NAC.The m RNA expression level of Cg LC3 was significantly up-regulated at 12 h after LPS stimulation,which was 1.25-fold（p<0.01）of that in the SW group.While it was significantly down-regulated at 12 h after NAC stimulation,which was 0.49-fold（p<0.001）of that in the SW group.Western Blotting analysis was employed to examine the conversion of LC3.LPS stimulation induced a significant increase of the transformation of LC3-I to LC3-II,and NAC stimulation induced a significant decrease of the transformation of LC3-I to LC3-II.The formation of autophagosomes in haemocytes of C.gigas after stimulation by SW,LPS and NAC was investigated by flow cytometry and confocal microscopy.Results show that the percentage of autophagosome CYTO-ID^?-labeled haemocytes after LPS stimulation for 12 h and 24 h was significantly higher than that in the SW group,which was 1.17-fold（p<0.01）and 1.19-fold（P<0.05）that in the SW group,respectively.In contrast,the percentage of CYTO-ID^?-labeled haemocytes after NAC stimulated for12 h and 24 h was significantly lower in SW group,which was 0.41-fold（p<0.001）and 0.28-fold（p<0.001）than that in SW group,respectively.The green CYTO-ID^?labeling of autophagosomes was observed under a confocal microscope.Meanwhile,compared with the weak green fluorescent signals of haemocytes in SW treated oysters,the green fluorescence intensity of autophagosome puncta in LPS-treated haemocytes was significantly enhanced,while there was almost no green fluorescence signal observed in NAC-treated haemocytes.The above research shown that the increased ROS level after LPS stimulation induced autophagy in haemocytes of C.gigas.3.LPS-induced autophagy regulates the proliferation of granulocytes in C.gigas.To explore the potential role of autophagy in ROS-induced haemocytes proliferation,the ammonium chloride（NH₄Cl）was used to inhibit the degradation of autophagosomes.Results showed that in LPS+NH₄Cl group,the percentage of autophagy CYTO-ID^?labeled haemocytes was significantly higher than that in LPS group,which was 1.27-fold（p<0.01）than that in LPS group.Flow cytometry quantitatively detected the percentage of Ed U⁺cells in haemocytes.The results showed that the percentage of Ed U⁺cells in LPS+NH₄Cl group was significantly higher than that in LPS group,which was 1.22-fold（p<0.01）of that in LPS group.Meanwhile,the percentage of Ed U⁺agranulocytos and semi-granulocytes decreased significantly after LPS+NH₄Cl stimulated for 12 h,which were 0.51-fold（p<0.05）and 0.55-fold（p<0.001）of that in LPS group,while the percentage of Ed U⁺granulocytes increased significantly,which was 1.70-fold（p<0.01）of that in LPS group.The above research indicated that NH₄Cl stimulation inhibited the autophagosomes degradation of haemocytes and promoted the proliferation of granulocytes.It is suggested that LPS induces autophagy to regulate the proliferation of granulocytes in C.gigas.LPS can increase the ROS level in haemocytes,and ROS level mediated the autophagy to regulate the proliferation of granulocytes in C.gigas.In a word,this study reveals the regulation effect of autophagy on the proliferation of oyster haemocytes in antimicrobial immunity,provided an important reference for further study on the haematopoietic development of C.gigas and the mechanism of haematopoietic regulation in invertebrates.