Leptin Receptor Gene Knockout Induced Microglial Cell Activation In Rats And CCL18 Induced The Phagocytosis Of HMC3 Through Activating The CCR8/NF-κB/Src Pathway

Background:Leptin receptor(LEPR)was the functional receptor of leptin.Leptin can bindto LEPR and play roles in inhibiting appetite,promoting energy consumption,reducingfat and regulating body weight.LEPR was expressed in microglia and astrocytes,it might interact with glia cells,andbe involved in neurodegenerative diseases.However,we know little about the effect of LEPR on microglia by now,and it is still not clear that whether LEPR stimulatsor inhibits the activation of microglia.Objective:LEPR knockout rats(LEPR-/-)were used to investigate the morphology and cytokine expression of microglia in the tissues from wild type(LEPR+/+)and LEPR-/-rats and in the primary culture rat microglia.The function of LEPR on microglia was illsutarted comparatively in vitro and vivo.Methods:(1)Primary microglia,astrocytes and neurons were isolated from LEPR+/+or LEPR-/-rats.RT-PCR was used to detect the expression of LEPR mRNA in primary glia and neurons and the expression of cytokines.(2)The rats was treated with LPS at two months old and survival rates were recorded.The sensitivity to LPS-induced inflammationin was then evaluated in the LEPR+/+and LEPR-/-rats.(3)Immunofluorescent was used to detectedphagocytosis of Dextran and Aβ in microglia from LEPR+/+and LEPR-/-rats.(4)WB was used to detect efficiency of the LEPR knockoutin primary microglia and the phosphorylation of the key proteins in PI3K-AKT signaling pathway in the tissues from LEPR+/+and LEPR-/-rats.Results:(1)LEPR was expressed in primary microglia,astrocytes and neurons.LEPR protein was completely knockout in primary microglia of LEPR-/-rats.(2)LEPRdeletion increased sensitivity to inflammation induced by LPS.After injecting LPS,survival ratewithin 48 hours of LEPR+/+and LEPR-/-rats were respectively 75%(n=10,*P<0.05)and 0%(n=10,**P<0.01)compared with that of non-treatment group.Immunohistology showed that LEPR deletion was resulted in the activation of microglia in brain tissues of rats.The activated microglia(hypertrophicandamoeboid)was increased by 170%in the brain tissues of LEPR-/-rats compared with that of LEPR+/+ rats(n=6,**P<0.01).In the presence of LPS,the activated microglia was increased by 30%in the brain tissues ofLEPR-/-rats compared with that of LEPR+/+rats(n=6,**P<0.01).(3)The mRNA expression of cytokines such as IL1-β,iNOS,IL-6 and TNF-α were increased in primary microglia from LEPR-/-rats and the phagocytosis of Dextran and Aβ was also enhanced as well.(4)The phosphorylationofPI3K and AKTprotein were significantly enhanced in the LEPR-/-rats.Conclusion:LEPR knockout induced the microglia differenciated onto pro-inflammatory and pro-phagocytic phenotype.LEPR knockout elevated mRNA expression of pro-inflammatory cytokines and enhanced phagocytosis ability of fluorescent markers.LEPRdeletion increased sensitivity to inflammation induced by LPSand increased activated microglia.LEPRdeletion was resulted in loss of negative control of PI3K/AKT signaling,which could be one of the mechanisms to restrict the hyper-activation of microglia.Our findings suggest that the Leptin/LEPR axis probably played roles in microglial activation and neuroinflammation.Background:CCL18 is a CC-type chemokine,which is involed in multi-diseases such as tumors and chronic inflammation.The most reports on CCL18 were focued on cancer and human chronic inflammatory diseases,such as atopic dermatitis and atopic asthma.Limited literatures show that CCL18 is involed in the neuroinflammation and its expresiosn levels were positively correlated with the pathological process of,but the mechanisms is unclear yet.Objective:Microglia were the main inflammatory cells in central nervous system.CCL18 might have regulatory effects on microglia.In this curent study,the role and mechanism of CCL18 were investigated using HMC3 cells,a human microglia cell line.Methods:(1)The phagocytosis of HMC3 on fluorescent labeled Aβ1-42 and Dextran was observed by laser scanning confocal microscopy,to analyze the regulation of CCL18 on the phagocytosis of HMC3 cells.(2)The expression of cytokine mRNA in HMC3 cells stimulated by CCL18 was detected by RT-PCR,and the regulatory function of CCL18 on expression of inflammatory factors was analyzed.(3)Western blot was used to detect the phosphorylation of signaling molecules downstream of receptor CCR8 and PITPNM3 of CCL18.(4)CCR8 and PITPNM3 were knockdown by shRNA respectively.Results:(1)Immunofluorescence showed that the Aβ1-42 and Dextran phagocytosis of HMC3 cells was increased by 2.3 folds and 2.5folds(n=3,*P<0.05)compared with the control in the presence of 1 Ong/mL CCL18.(2)RT-PCR results showed that CCL18 did not significantly change the expression of IL-1β,and IL-6,but increased the expression of MRC-1 and ARG-1.(3)With CCL 18 treatment,the receptor CCR8 on the surface of HMC3 cells was aggregated and internalized in the cells,but the receptor PITPNM3 was aggregated and internalized in the cells.CCL 18 treament induced the phosphorylation of NF-κB,PyK2 and Src.(4)When CCR8 was knockdown by shRNA agaist CCR8,CCL 18 induced phosphorylation of NF-κB an Src was removed,subsequntly,the CCL18 induced phagocytosis of Aβ 1-42 were significantly decreased in HMC3 cells.In contrast,PITPNM3 knockdown with shRNA agaist CCR8 PITPNM3 was slightly decrease in phosphorylation of Pyk2 and was not changed the phosphorylation of Src and did not inhibit the phagocytosis responding the CCL18 treatment.Conclusions:CCL18 was not alter the expression of cytokines in HMC3 cells,but enhanced phagocytosis.CCR8 was the main functional receptor of CCL18 on HMC3 cells.CCL18/CCR8/NF-κB/Src signaling pathway promoted the phagocytosis of microglia,rather than CCL18/PITPNM3.These findings suggest that CCL18 was not sitimulted the pro-imflammation,but it enhanced the phagocytosis of microglia.