The Mechanism Underlying The Depression Induced By Chronic Mild Stress Through Impairing Phagocytosis Of Apoptotic Cells By Microglia

In the nervous system,apoptotic and dying cells can activate neuroimmune cells to produce neuroprotective or neurotoxic effects.Under physiological conditions,apoptotic cells generally promote anti-inflammatory response and immune tolerance at the tissue level.Apoptosis is common during the development of brain.The central nervous system（CNS）requires an effective cleanup system to prevent secondary necrosis of apoptotic cell contents which can trigger the proinflammatory response of surrounding microglia.Defects in the clearance of apoptotic cells can lead to a variety of inflammatory diseases and enhance brain-derived autoimmunity,resulting in damage to surrounding tissue cells.Microglia are resident macrophages of the central nervous system,and their plasticity in function and morphology enables them to actively monitor and respond to changes in their surroundings.Microglia removed apoptotic cells and cell debris by phagocytosis,adjusting the balance between anti-inflammation and pro-inflammation,avoiding excessive inflammation caused by secondary necrosis due to delayed removal of apoptotic cells.Therefore,the normal phagocytic function of microglia is essential to maintain the homeostasis of the central nervous system.Chronic stress is one of the most relevant factors leading to major depression.At present,it is not clear about the changes in the ability of microglia to engulf apoptotic cells under chronic stress and its role in chronic stress-induced depression-like behavior.This study hypothesized that the impaired ability of microglia to phagocytose apoptotic cells promoted the induction of depression-like behavior by chronic stress.We constructed a depression model by applying 4-week chronic stress on mice to explore the changes of depressive-like behavior under chronic stress.Then the activation phenotype of microglia under chronic stress was determined by analyzing the changes of central inflammation level and microglia morphology.The results showed that under chronic stress,the expression of pro-inflammatory factors increased significantly,and the expression of anti-inflammatory cytokines decreased significantly.The branch length of microglia in hippocampal region became shorter,the cell body area of DG and CA1 region increased,and the number of branches decreased.To further explore the changes of microglia phagocytic ability of apoptotic cells under chronic stress,we conducted co-staining of Casapase3 and Iba1 in hippocampal.The results showed that the number of phagocytes and the number of microglia containing one phagocytic cup in the DG region.decreased significantly.The number of apoptotic cells in the three subregions of hippocampus increased significantly.The phagocytic index and the coupling ratio of phagocytosis to apoptosis decreased significantly.It is suggested that chronic stress damages the phagocytic ability of microglia in response to apoptotic cells.The number of neurons decreased significantly by the 4-week chronic stress.To investigate whether blocking phagocytosis of apoptotic cells promotes depression-like behavior induced by 3-week chronic stress,depression-like behavior of mice was assessed by sugar water preference,weight measurement,forced swimming test,tail suspension test,and autonomous activity test.The results showed that the sugar water preference score and body weight of mice were decreased and the total immobile time of forced swimming was prolonged after blocking the phagocytosis of apoptotic cells,suggesting that the blocking the phagocytosis of apoptotic cells can promote the induction of depression-like behavior induced by chronic stress.After blocking the phagocytosis of apoptotic cells,the expression level of inflammatory factors,the number of apoptotic cells in the DG and CA1 regions of the hippocampus increased significantly To further explore the molecular mechanisms underlying the impaired ability of microglia to phagocytose apoptotic cells,we screened multiple receptors associated with phagocytosis of apoptotic cells,including Mer TK,CD33,MFG-E8,P2Y6 R,TREM2,and Vav1.The mRNA levels of P2Y6 R,TREM2 and Vav1 in the tissues were down-regulated by 4-week chronic stress.In this experiment using primary microglia,the level of TREM2 mRNA in primary microglia decreased with increasing LPS-stimulation time.The average fluorescence intensity of immunofluorescence staining decreased.Using the LPS-stimulated BV2 cell line and under 4 weeks of chronic stress,the expression of TREM2 protein levels was down-regulated.These results suggest that inflammation is increased while the ability of microglia to phagocytize apoptotic cells is damaged by four-week chronic stress.Decreased ability of microglia to phagocytose apoptotic cells was accompanied by significant downregulation of hippocampal TREM2.