Inflammatory Mechanisms Underlying Depressive Behaviors Induced By Chronic Stress

Depression is one of most common psychiatric disorders, which has a complex andmultifactorial aetiology originating from the interaction between environmental andgenetic factors and presents frequent co-morbidity. Stress is well known as one of the mostpathogenic factors of depressive disorder; however, the underlying mechanisms are stillunknown. There is a pressing need for improvements in current depression treatment.Existing antidepressants have many negative effects, and their efficacy is slow and requireschronic treatment. Despite an increasing number of available antidepressants nowadays,there are still many depressed patients do not show improved mood after advanced therapy,or even do not response to any antidepressant therapies. Therefore, to find new potentialtargets and subsequently explore novel antidepressants, it is necessary to clarify themechanisms underlying stress-induced depression.Over the last few decades, researches have made a great progress on the pathogenesis ofdepression. Several theories have been proposed, including the monoamine hypothesis, theneurogenesis hypothesis, the cytokines hypothesis, the hypothalamic-pituitary-adrenal axisdysfunction theory, and so on. The cytokines hypothesis of depression has attractedconsiderable attention ever since it was proposed in1991. It has been and justifieddeveloped by many researchers during the past twenty years. This hypothesis emphases therole of cytokines released by activated immune system in the pathological processunderlying depression.Cytokines are bioactive proteins secreted by activated immune cells. It has beendemonstrated that mice, administrated with LPS acutely or inoculated with BacillusCalmette-Guerin, display depressive behaviors accompanied with the immune systemactivation. Various physiological and psychological stressors can induce the activation ofimmune system and the release of pro-inflammatory cytokines. Exposure to stressedconditions, acute or especially chronic, can induce depressive behaviors in human andanimals. Immune activation and stress exposure have similar ethology effects. Therefore,cytokines-mediated inflammatory reactions may be involved in stress-induced depression.Cytokines functions as part of an integrated network, each of them inducing their ownsynthesis while also inducing the synthesis of other pro-inflammatory cytokines, includingTNFα, IL-1and IL-6, in a “cascade” fashion. In the brain, there is a cytokine networkconsisting of cells, e.g., neurons, microglia and astrocytes, which are able to produce cytokines, express cytokine receptors and amplify cytokine signals. This neural substrateof immune signals under-lies the potent effects of peripheral pro-inflammatory cytokineson pathways involved in the pathophysiology of neuropsychiatric disorders. Peripheralcytokine signals are able to reach the brain through humoral and neural pathways.A number of pathways by which cytokines regulate behaviors have been identified.These pathways include neurotransmitter function, neuroendocrine activity, neuralplasticity and so on. Accompanying with the development of cytokines hypothesis ofdepression, the kynurenine pathway attached much acctention. In mammals, essentialamino acids tryptophan, the synthetic material of neurotransmitter5-HT related todepression, has two metabolic pathways, i.e. kynurenine pathway (KP) and5-HT pathway.The KP is responsible for about90%of the tryptophan metabolism. Indoleamine2,3-dioxygenase (IDO) is a rate-limiting enzyme of KP, breaks down tryptophan intokynurenine. IDO is inflammation inducible, can be stimulated by a number of cytokinesalone or incombination. IDO is activated robustly by IFNγ and TNFα. The activation ofIDO induced by pro-inflammatory cytokines released from activated immune system,enhances the degradation of tryptophan along KP. The breakdown of tryptophan results ina relative decrease in tryptophan bioavailabity, thereby setting the stage for a decrease inserotonin synthesis and serotoninergic neurotransmission. However, it is unclear whetherIDO is involved in stress-induced depression.In this present research, the inflammatory mechanisms underlying chronicstress-induced depressive behaviors were explored based on the cytokines hypothesis ofdepression. Firstly, based on the establishment of the animal model of chronicstress-induced depression, investigated the improvement in stress-induced depressivebehaviors after blocking the inflammation induced by stress exposure withantiinflammatory agents. Secondly, the effects of chronic stress on IDO were evaluated,including its expression and activity. Applying the IDO specific inhibitor1-MT, to estimatethe roles of IDO in stress-induced depression, by investigating the improvement indepressive behaviors induced by stress after the administration of1-MT. Thirdly, found outthe specific cytokines related to depression and the underlying mechanisms regarding toIDO. The details are as follow:1. The establishment and assessment of the animal model of chronic stress-induceddepression. In our present research, the chronic unpredictable mild stress model ofdepression was chosen, combining the physiological and psychological stressors to mimic the complex environments around the human. Applying sucrose preference test, forcedswim test and tail suspension test to evaluate the depressive behaviors induced by chronicstress. Furthermore, the responses of depressed animals to antidepressant fluoxetine wereinvestigated, to further estabilish the animal model. The results showed that chronic stressslows down the increas of weight, and reduces the sucrose preference in a time-dependentmanner. After exposed to stress for one or two weeks, the sucrose preference had nochange compared with control. But the sucrose preference of stress-exposed mice began todecrease at the end of the second week although there was no significance compared withthat of control group. After exposed to stress for three or four weeks, the sucrosepreference decreased remarkablely. Antidepressant fluoxetine blocked the decrease ofsucrose preference induced by chronic stress. The immobility time of mice increasedsignificantly in the forced swim test and tail suspension test, after exposed to stress for fourweeks. These results indicated the establishment of the animal model of stress-induceddepression.2. The roles of inflammation in chronic stress-induced depressive behaviors. Based onthe relations between stress and immune activation, besides the relations between immuneactivation and depression, we proposed that inflammation initiated by immune activationmay be involved in the process of induction of depressive behaviors by chronic stress.Administrated mice with minocycline when exposed them to various stressors, theimprovement in depressive behaviors were observed by blocking inflammatory reactionswith minocycline. The results showed that chronic stress and minocycline had no effect ondepressive behaviors after exposed to stress for two weeks, including sucrose preferenceand immobility time in the forced swim test. After exposed to stress for three weeks, thesucrose preference of stress-exposed mice decreased significantly compared with that ofcontrol. Stress exposure also increased the immobility time remarkablely in the forcedswim test. Minocycline abrogated the depressive behaviors induced by stress exposurecompared with that of the stress group, including surose preference and immobility time.After exposed to stress for four weeks, the results were similar as those of three weeks.Exposure to stress for four weeks induced depressive behaviors obviously, including thedecrease of sucrose preference and increase of immobility time. These above depressivebehaviors were abrogated by minocycline compared with that of the stess group. There wasalmost no chang behviors of the mice treated with minocycline alone. Moreover, to furtherdetermine the roles of inflammation in stress-induced depression, the depressive behaviors of mice treated with minocycline and stress were investigated after the removal ofminocycline. The results showed that after removal of minocycline for two weeks,depressive behaviors were induced by chronic stess compared with that of the controlgroup, including the decrease of sucrose preference and increase of immobility time in theforced swim test and tail suspension test. The effects of chronic stress on peripheral andcentral inflammatory mediators were also determined. As shown, the levels of circulatingproinflammatory cytokines were significantly increased by stress exposure, includingTNFα、IL-1β and IL-18. The protein expression of TNFα of stress-exposed mice increasedremarkablely in the cortex, rather than the hippocampus or midbrain. Stress had no effecton the expression of proinflammatory cytokines in the cortex, hippocampus and midbrainof stress-exposed mice, including IL-1β、IL-18、IL-6、IFNγ. Microglias were activated inthe cortex of stress-exposed mice, indicating by the increased expression of CD11b.Minocycline abrogated completely the increase of proinflammatory cytokines and theactivation of microglia induced by chronic stress. The results of Nissl staining showed thatneurons in the cortex of stress-exposed mice were damaged obviously, indicating by thereduction and dissolution of nissl bodies. Minocycline attenuated the reducdion of nisslbodies compared with that of the stress group, protected neuron from damage induced bystress.3. The roles of IDO in chronic stress-induced depressive behaviors. Our present researchshowed that the peripheral and central immune systems were activated by stress exposure,accompanying by increased expression of proinflammatory cytokines. IDO is aninflammation inducible enzyme. So it was proposed that the activation of IDO, induced byinflammation intiated from stress exposure, account for the dysbolism of neutransmittersand subsequent depressive behaviors. Therefore, the effects of chronic stress on theexpression and activity of IDO were detected. The results showed that both of the mRNAexpression and activity of IDO are enhanced by stress exposure compare with that of thecontrol group. Furthermore, administrated mice with the IDO specific inhibitor1-MTwhen they were subjected to various stressors, and the improvement in depressivebehaviors were detected. As shown,1-MT blocked the enhancement of IDO transcript andactivity, and accompanying depressive behaviors induced by stress, including thedecreased sucrose preference and increased immobility time. These results indicated thatIDO is involved in the pathogenesis of chronic stress-induced depression.4. The roles of TNFα in chronic stress-induced depressive behaviors. The above data demonstrated that TNFα was significantly upregulated in periphery and cortex after stressexposure for four weeks. TNFα can activate IDO robustly. Therefore, we hypothesized thatTNFα might be one of the key biomarkers in this chronic stress-induced depression model.To confirm this hypothesis, we measured the expression of TNFα and depressive behaviorin the presence or absence of TNFα monoclonal antibody infliximab. As shown, the levelof TNFα in stressed mice was back to the control level with infliximab pretreatment.Along with the downregulation of TNFα, infliximab pretreatment abrogated the decreaseof sucrose preference and the increase of immobility time in FST induced by stress. Therewas no significant change in behaviors of the mice treated with infliximab alone. So,blockage of TNFα with infliximab abrogated the depressive behavior induced by stress.The cortex was selected for morphological assessment to investigate the effects ofinfliximab pretreatment on cortex neurons. As shown, normal neurons were observed incontrol group and stress plus infliximab group, while damaged neurons obviouslyincreased in stress group. Furthermore, infliximab pretreatment abrogated this decrease ofundamaged neurons.