RESEARCH
pen Acce
Yuan Li ¹² , Bing Zhan ¹ , Xiao Zhuang ¹ , Ming Zhao ¹ , Xiaotong Chen ¹ , Qun Wang ¹ , Qiji Liu ³ and Lining Zhang ¹ ²
Abstract
The dysregulation of pro- and anti-inflammatory processes in the brain has been linked to the pathogenesis of major depressive disorder (MDD), although the precise mechanisms remain unclear. In this study, we discovered that microglial conditional knockout of Pdcd4 conferred protection against LPS-induced hyperactivation of micro- glia and depressive-like behavior in mice. Mechanically, microglial Pdcd4 plays a role in promoting neuroinflamma- tory responses triggered by LPS by inhibiting Daxx-mediated PPARy nucleus translocation, leading to the suppres- sion of anti-inflammatory cytokine IL-10 expression. Finally, the antidepressant effect of microglial Pdcd4 knockout under LPS-challenged conditions was abolished by intracerebroventricular injection of the IL-10 neutralizing antibody IL-10Rα. Our study elucidates the distinct involvement of microglial Pdcd4 in neuroinflammation, suggesting its potential as a therapeutic target for neuroinflammation-related depression.
Introduction
The neuroinflammatory process has been associated with a range of psychiatric disorders, including schizophre- nia, depression, and anxiety [ 1 ]. Accumulating shreds of evidence support the relationship between inflammatory cytokines and affective disorders, including interleukin *Correspondence: Lining Zhang zhanglining@sdu.edu.cn 1 Key Laboratory of Infection and Immunity, Department of Immunology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, 44# Wenhua Xi Road, Jinan 250012, Shandong, China ² Key Laboratory of Endocrine Glucose & Lipids Metabolism and Brain Aging, Department of Endocrinology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China ³ Key Laboratory for Experimental Teratology of the Ministry of Education, Department of Medical Genetics, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) [2]. These results have been confirmed by rodent models, in which mental stress, in addition to leading to inflamma- tory immune activation, produces depressive-like behav- ior [3]. Although studies have focused on the effect of pro-inflammatory factors on depression, the role of anti- inflammatory cytokines and the mechanisms underlying the imbalance between pro- and anti-inflammatory fac- tors need to be explored. Inflammatory stimuli are sensitive to microglia, a resident macrophage in the brain, and it has an immune response function. Under stress conditions, microglia transform into an active state and partici- pate in emotional behaviors through activity-depend- ent regulation of synaptic pruning [ 4 , 5 ]. Peripheral lipopolysaccharide (LPS), an agent for microglial acti- vation, induces microglial pro-inflammatory reactions and depressive-like behavior in mice [ 6 ]. Otherwise, the long-term treatment of minocycline, a microglial The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which s use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the ginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the articles Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. inhibitor, prevented mice from experiencing depres- sive-like behavior due to neuroinflammation [7, 8]. Peroxisome proliferator-activated receptor gamma (PPARy) belongs to the nuclear receptor family, which is a ligand-dependent transcription factor. The dys- function of PPARy is accompanied by mental illness, which is a genetic risk factor for depression [9, 10]. Based on the fact that PPARy play a key role in depres- sion. Such as, neuronal PPARy knockout in the pre- frontal cortex of mice induces depressive-like behavior. Conversely, the PPARy agonist, Rosiglitazone, has an antidepressant-like effect on stressed mice [ 10 – 12 ]. One of the reasons is that PPARy signaling promotes neuroplasticity and neurogenesis by boosting brain- derived neurotrophic factor (BDNF) [ 13 ]. Additionally, PPARy activation results in inhibition of inflammatory responses by increasing anti-inflammatory cytokines expression or suppressing pro-inflammatory molecules, which helps damaged brain repair [ 14 , 15 ]. However, the microglia specific mechanism of PPARy in emo- tional behavior is still unclear. Programmed cell death 4 (Pdcd4) an apoptosis-related molecule, extensively participates in tumorigenesis and inflammatory diseases. Our previous reports have demonstrated Pdcd4 is elevated in brain of patient with depression, and neuronal-expressed Pdcd4 is involved in stress-induced depressive-like behaviors, by block- ing BDNF and up-regulating proinflammatory response [16, 17]. Pdcd4 has a neuroinflammatory effect on emo- tional disorders, however, its biological function in cen- tral nervous system (CNS) neuroinflammation remains unclear. Here, we demonstrated that microglial PPARy nuclear translocation involves inflammatory-related depression. LPS led to the upsurge in Pdcd4 expression in the microglias of depressed mice, and microglial Pdcd4 conditional knockout had antidepressant effects on mice. Functionally, overexpressed Pdcd4 promotes microglia activation by decreasing the levels of IL-10. Mechanically, we discovered that Pdcd4 competed with Daxx to block PPARy nuclear translocation, resulting in interruption of IL-10 transcription. In this study, we emphasized that cellular PPARy distribution regulates neuroinflamma- tion, and microglial Pdcd4 plays a critical role in that.
Results
Microglial knockout of Pdcd4 ameliorates
neuroinflammation-related depression
To explore intermediate components that modulate neu- roinflammation-related depression, we exposed adult male mice to LPS intraperitoneally for 10 days to mimic depression. (Fig. S1a). Validation of mice with depressive- like phenotypes was achieved by the use of tail suspen- sion test (TST) and forced swimming test (FST) (Fig. S1b, c). We found that Pdcd4 mRNA and protein levels are increased in the prefrontal cortex (PFC) but not in the hippocampus (HIP) of the LPS-induced depressive mice (Fig. 1a–c). Microglias are the response of the cell to LPS triggering neuroinflammation in the central nerv- ous system. To explore whether the expression of Pdcd4 in microglia is elevated after LPS injection, we performed immunofluorescence staining experiments on mouse brain slices using Pdcd4 antibody and Iba1 antibody. The results showed that LPS treatment augmented the level of Pdcd4 in microglias of the PFC and the HIP (Fig. 1d). To investigate the specific effect of microglial Pdcd4 on LPS- induced depressive-like behaviors, we generated Pdcd4 mcKO mice by crossing the floxed Pdcd4 allele (Flx) mice with the Cx3cr1-CreERT2 mice (Fig. 1e). Firstly, we iso- lated the microglial cells in mcKO with tamoxifen and the control without tamoxifen mice by flow cytometry. Western blot showed that Pdcd4 was deleted in micro- glia in the mcKO mice with tamoxifen (Fig. S2a, b). Next, immunofluorescence histochemistry of brain slices showed Pdcd4 expression diminished in the Iba1+ cells of Pdcd4 mcKO mice-treated with tamoxifen (Fig. 1f). We then assessed the depressive-like behaviors in micro- glial Pdcd4 knockout mice exposed to LPS by using FST, TST, and sucrose preference test (SPT). As expected, LPS led to depressive-like behaviors in the mcKO mice without tamoxifen, as indicated by increased immobility and decreased percentage of sucrose preference, which were reversed by the conditional knockout of Pdcd4 in microglia, without affecting the locomotor activ- ity (Fig. 1g–i; Fig. S2c). Interestingly, the LPS-induced increase in anxiety-like behaviors in control mice was not reversed by microglial Pdcd4 knockout (Fig. S2d, e). These results indicate that Pdcd4 deficiency in microglia protects mice from neuroinflammation-related depres- sive-like phenotype.
Microglial Pdcd4 deficiency mitigates LPS-induced
microglial activation via facilitating the PPARy signaling To explore the molecular mechanisms underlying the antidepressant effect observed in the microglial Pdcd4 deletion, we analyzed the mRNA expression profiles in the PFC of LPS-treated microglial Pdcd4 knockout by RNA sequencing (RNA-seq). 586 genes were differen- tially expressed in the PFC of the Pdcd4 microglia con- ditional knockout mice in response to LPS compared to the LPS-treated control (Fig. S3a). KEGG pathway analy- sis showed thermogenesis and oxidative phosphorylation were significantly changed. Gene Set Enrichment Analy- sis (GSEA) indicated the PPAR signaling pathway was enriched in the LPS-treated Pdcd4 microglial knockout group, among which PPARy might be the core gene that was regulated (Fig. S3b–d). To clarify whether PPARy is a core gene in the mice resistant to LPS-induced depres- sive-like behavior, we first measured the mRNA and protein levels of PPARy in the PFC in response to LPS. Compared to the vehicle-treated group, PPARy was markedly decreased in the PFC of the LPS-treated con- trol mice but not the mcKO mice with tamoxifen (Fig. 2a, b). GW9662, a PPARy inhibitor, was able to eliminate the antidepressant effect in microglial Pdcd4 deletion in mice by injecting it into mice simultaneously follow- ing LPS administration (Fig. 2c–f), but it did not exac- erbate depressive-like behavior in WT mice under LPS conditions (Fig. S4a, b). Together, these data suggest that the antidepressant effect of microglial Pdcd4 deficiency in response to LPS might be mediated by the PPARy signaling. Next, we investigated the activation of microglia in the PFC of mice. e number of microglial cells in the PFC of the mcKO mice without tamoxifen was significantly increased after LPS treatment. However, when we treated the mice simultaneously with LPS and GW9662, the number of microglial cells was also obviously up-regu- lated compared to the LPS-treated mcKO with tamoxifen group (Fig. 2g, h). Similarly, the number and length of microglial cell branches were reduced after LPS injection in control mice but not in the Pdcd4 microglia condi- tional knockout mice. Inhibition of the PPARy signaling with GW9662 also decreased the number and length of microglial cell branches in the mcKO with tamoxifen mice in response to LPS (Fig. 2i–k). These results suggest that microglial Pdcd4 deficiency mitigates LPS-induced microglial activation via facilitating the PPARy signaling pathway.
Pdcd4 regulates the nuclear and cytoplasmic translocation
of PPARy in an LPS-independent manner
To ensure whether or how Pdcd4 regulates the func- tion of PPARy, mouse primary microglias were taken into the experiment. Purified mouse primary microglias were successfully cultured in vitro, as confirmed by Iba1 staining (Fig. S5a). Then the PPARy protein level was determined in the microglias of WT or Pdcd4 knock- out (Pdcd4—/—) with vehicle or LPS treatment. e data showed the protein level of PPARy not changed in microglias of Pdcd4 knockout mice, but Pdcd4 deletion increased PPARy after LPS stimulation for 24 h (Fig. S5b, c). Cycloheximide (CHX) is used to block protein synthe- sis. Under LPS conditions, the expression of PPARy rap- idly decreased along with CHX in WT derived-microglia, while the phenotype disappeared with the loss of Pdcd4 (Fig. S5d, e). Though Pdcd4 didn't alter the total PPARγ at baseline, we separated the proteins of cytoplasm and nucleus in the PFC of the mice, and found that nuclear PPARy was increased in the systemic Pdcd4 knockout mice (Fig. 3a). Meanwhile, the concentrated expression of PPARy in the nucleus was confirmed in the Pdcd4 defi- cient microglia at normal or at LPS treatment (Fig. 3b, c). The further immunofluorescent results showed that loss of Pdcd4 improved nuclear PPARy level (Fig. 3d–f). To examine whether Pdcd4 directly determines PPARy subcellular distribution, HEK293 cells were transfected GFP tagged-PPARy with or without Flag tagged-Pdcd4, GFP scatter is on the behalf of PPARy expression, and the overexpression of Pdcd4 decreased the levels of GFP in the nucleus (Fig. 3g–i). These data suggest that Pdcd4 mediates the subcellular distribution of PPARy.
Pdcd4 inhibits PPARy nuclear expression
through interrupting the interaction between PPARγ
and Daxx
Previous study has demonstrated the interaction between Pdcd4 and Fas death domain-associated pro- tein (Daxx) in restraining p53 activity [ 18 ]. To explore the mechanisms underlying Pdcd4 regulated PPARy nuclear transportation, the immunostaining proved that endogenic PPARy partially co-localized with Daxx in the mouse embryonic fibroblasts (MEF) (Fig. 4a). Next, we co-transfected Myc-tagged Daxx with GFP- tagged PPARγ constructs into HEK293 cells, and the Immunoprecipitation result showed that Daxx could interact with PPARy (Fig. 4b). In order to identify the specific protein domain of Daxx responsible for its interaction with PPARy, we generated mutations in Daxx that lack certain domains (Fig. 4c). By immuno- precipitation with the HA antibody, we found SIM2 domain is necessary for the interaction of Daxx with PPARy, and for SIM2 domain deletion of Daxx dimin- ishing the interaction with PPARγ (Fig. 4d). To test whether Daxx regulates PPARy nuclear transportation, HA tagged-PPARy and Daxx siRNA (siDaxx) were co- transfected into HEK293 cells, and the result showed that knockdown of Daxx decreased the level of PPARy in the nucleus (Fig. 4e, f). Conversely, overexpression of Myc-Daxx up-regulated the nuclear PPARγ expres- sion (Fig. 4i–g). Additionally, GFP-labeled PPARy was co-transfected with siDaxx or Myc-Daxx into HEK293 cells, and immunostaining for GFP was used to assess the localization of PPARy within the cells. Consist- ent with findings from nuclear-cytoplasmic extraction experiments, the expression of Daxx was found to cor- relate with an increase in nuclear distribution of PPARy (Fig. 4g, h, k, l), suggesting Daxx regulates PPARγ nucleus transportation. A previous report has found an association between Daxx and Pdcd4 [ 19 ], we sought to explore whether Pdcd4 affects the PPARγ-Daxx complex formation. Firstly, the protein level of Daxx was detected by Western blot on the PFC of Pdcd4 microglial knockout mice, and found there have no changes in the groups of LPS admin- istration (Fig. S6a, b). Meanwhile, the endogenous inter- action between Daxx and PPARy was increased in the Pdcd4 knockout peritoneal macrophages compared to the WT control (Fig. 5a). Moreover, knockdown of Pdcd4 could strengthen the interaction between Daxx and PPARy in cultured HEK293 cells (Fig. 5b). Conversely, the overexpression of Pdcd4 resulted in a decrease in the interaction between PPARy and Daxx in HEK293 cells (Fig. 5c). Moreover, we performed the immunostaining to observe the subcellular distribution of PPARy and Daxx in peritoneal macrophage from WT and Pdcd4 deficient mice, and found that Pdcd4 knockout enhanced the colo- calization of PPARy with Daxx and the PPARy nucleus distribution (Fig. 5d–f). Above all, these results suggest that Pdcd4 interrupts the interaction between PPARγ and Daxx. Furthermore, to explore whether Pdcd4 affects Daxx-mediated PPARy nuclear transportation, we co- transfected siPdcd4 and siDaxx into HEK293 cells. e findings indicated that the inhibition of Daxx prevented the effect of siPdcd4 on enhancing the nuclear PPARy protein level (Fig. 5g, h). Similarly, siDaxx also decreased the nucleus PPARy expression in the MEF from Pdcd4 knockout mice (Fig. 5i, j). Taken together, Pdcd4 hin- dered Daxx-mediated PPARγ nuclear translocation by disrupting the formation of the complex. Pdcd4 regulates PPARy-related IL-10 transcription We next explored the mechanism underlying microglial Pdcd4 deficiency-induced antidepressant effect in neuro- inflammation-associated depression. Multiple cytokines have been regulated by PPARγ, such as Tnf-α, Inos, Il-1β, (Fig. 6a; Fig S7a). To prove whether Pdcd4-regulated IL-10 expression was mediate by PPARy, the cytokines expression in the PFC of the microglial Pdcd4 knockout mice was detected. e data showed that Tnf-α, Inos, Il-1β, Ccl2 and Il-10 were increased or decreased in the LPS-treated control mice but not in the Pdcd4 condi- tional deletion mice. Moreover, GW9662 treatment sig- nificantly reduced the mRNA and protein expression of IL-10 in the microglial Pdcd4 knockout compared to the saline-treated group under LPS injection conditions, but it did not affect the mRNA expression of Tnf-α, Inos, Il-1β, and Ccl2 (Fig. 6a, b; Fig. S7b-e). These data sug- gested the involvement of Pdcd4 in the PPARγ/IL-10 axis. Although IL-10 as a neuroinflammatory factor is involved in depression, the transcriptional regulatory mechanism of IL-10 in the microglia has not been eluci- dated. As previously reported, analysis of the promoter in the IL-10 gene revealed multiple PPARy response ele- ments (PPREs) (Fig. 6c). Next, we performed the lucif- erase reporter assay in HEK293 cells to detect the effect of Pdcd4 on IL-10 expression. e activation of PPARγ by the agonist rosiglitazone (Rosi) markedly enhanced the transcription of IL-10; conversely, the overexpres- sion of Pdcd4 reversed this effect (Fig. 6d). While rosigli- tazone has been shown to enhance IL-10 transcription, the knockdown of Pdcd4 results in a heightened exacer- bation of this effect. However, the stimulation of IL-10 transcription by rosiglitazone was found to be signifi- cantly reduced in the absence of PPREs within the IL-10 promoter (Fig. 6e). Collectively, these data indicated an important role for Pdcd4/PPARy axis in IL-10 expression.
Microglial Pdcd4 knockout resolves LPS-induced
depressive behavior by rescuing IL-10
To investigate the potential role of IL-10 in Pdcd4- associated depressive-like behavior, we administered a neutralizing IL-10 antibody, IL-10Rα, via intracer- ebroventricular injection to Pdcd4 mcKO mice prior to conducting behavioral assessments (Fig. 7a). We found that IL-10Rα treatment significantly blocked the decreased immobility in TST and FST trials and the improved preference to sucrose in SPT in the LPS treated mcKO mice (Fig. 7b–d). Meanwhile, we confirmed that IL-10Rα treatment did not worsen depressive-like behav- ior in WT mice injected with LPS (Fig. S4a, b). Finally, e expression of Tnf-α, Il-1β, Ccl2, Inos, and B2m was dramatically elevated in the PFC of the IL-10Rα- injected Pdcd4 microglial knockout mice in response to LPS (Fig. 7g–k). In summary, the data indicates that the increased expression of IL-10 plays a role in the antide- pressant properties observed in microglial Pdcd4 knock- out mice following exposure to LPS.
Discussion
Neuroinflammation is thought to drive changes in neuro- transmitters and neurocircuits that lead to major depres- sive disorder (MDD). In this article, we provide direct evidence that microglial Pdcd4 mediates neuroinflam- mation-associated microglia activation and consequent depressive-like behaviors in mice by interrupting the PPARy mediated IL-10 transcription. Firstly, we confirmed Pdcd4 is involved in inflamma- tory-related depression via regulating microglia activa- tion. Regardless of the devasting role of neuronal Pdcd4 in chronic restraint stress (CRS)-induced depression [ 16 , 17], we found microglia Pdcd4 knockout protect mice from LPS-induced depressive-like behaviors. LPS chal- lenge, mimicking an acute inflammatory response, stim- ulated peripheral immunity, and subsequently evoked neuroinflammation and depressive-like behaviors [ 20 ]. Mechanically, LPS stress caused more microglia activa- tion and stronger cytokines expression than CRS, result- ing in a significant difference between the LPS and CRS models in aspects of intensity and duration of stress. Though the prefrontal cortex, hippocampus, striatum, and hypothalamus are well-known brain areas that have a relationship with depression, multiple studies have referred to significant cytokines' expression and micro- glia activation in the PFC rather than in the HIP [21]. Our study suggests that the expression profile of Pdcd4 may play a role in neuroinflammatory responses across various brain regions under LPS conditions. Western blot and RT-PCR techniques are limited in their abil- ity to detect changes in Pdcd4 at the single-cell level. Interestingly, pathological staining revealed an upregula- tion of Pdcd4 in both microglia and non-microglia cells in the prefrontal cortex. These findings may be attrib- uted to variations in cell types present in different brain regions [ 22 ]. Understanding the upstream regulatory mechanism of Pdcd4 expression can reveal how different brain regions respond to stress. Recently report found LPS required the NF-κB signaling activation to boost Pdcd4 expression in microglia, perhaps discovering the NF-kB signaling response pattern during the LPS chal- lenge will help us to answer that question [ 23 ]. In sum- mary, we discovered that microglial Pdcd4 plays a role in LPS-induced depressive behavior, highlighting the vari- ous functions of Pdcd4 in nerve cells and brain regions, particularly its immunoregulatory role in the PFC during neuroinflammation-related depression. Secondly, we discovered the involvement of the Pdcd4/ PPARy axis in depressive-like behavior. A previous study has referred that lncRNA-H19 facilitated Pdcd4 expres- sion in microglia via sponging miR-21, which initiated the Ischemia–reperfusion (I/R)-induced inflammation [24]. However, the specific mechanism underlying Pdcd4-reg- ulated neuroinflammation in depression is still unknown. RNA-sequencing analysis elucidates the regulatory role and potential mechanism of Pdcd4 in microglia, with enrichment of the KEGG pathway in thermogenesis and oxidative phosphorylation. Mitochondrial dysfunction is recognized as a contributing factor to emotional dis- orders, while recent research indicates impaired energy metabolism in individuals with depression, specifically affecting mitochondrial oxidative phosphorylation in glu- tamatergic neurons [ 25 , 26 ]. Therefore, abnormal energy regulation of synapses is a potential mechanism for emotional disorders [ 27 ]. Our prior studies have shown that Pdcd4 plays a detrimental role in metabolic disor- ders by inhibiting the transition of white adipose tissue to beige adipose tissue and facilitating the formation of stress granules [ 28 , 29 ]. Further exploration is needed to determine whether Pdcd4 plays a role in mitochondria function, which is implicated in cell metabolism and may contribute to the development of depression. PPARy is one of the three isoforms of PPARs, and is activated by thiazolidinediones such as pioglitazone and is applied for insulin resistance treatment. Previous reports have demonstrated that neuronal PPARγ directly mediated stress-induced emotional disorders and PPARy agonists have an antidepressant effect, suggesting the essential role of PPARy in depression [ 11 ]. Based on the enrich- ment of PPARy expression in microglia, PPARy also has anti-inflammatory properties [ 30 ]. In this study, we observed that the deletion of microglial Pdcd4 resulted in the inhibition of LPS-induced microglial activation by up-regulating PPARy and enhancing nuclear transporta- tion. Previous studies have established a causal relation- ship between PPARy protein stability and its subcellular localization within the nucleus, as cytosolic PPARγ is rapidly degraded through ubiquitin–proteasome-medi- ated mechanisms [ 31 ]. While several E3 ubiquitin ligases, such as NEDD4-1, TRIM25, FBXO9, MKRN1, CUL4B, and FBXO4, have been implicated in the regulation of PPARy protein levels, the mechanism of LPS-mediated protein degradation of PPARy remains poorly under- stood [ 32 – 37 ]. In conclusion, microglial PPARy is neces- sary for neuroinflammatory response. Thirdly, we illustrated Daxx as an adaptor, which is critical for PPARy cytosol-nuclear shuttle. Evidence sug- gests that PPARy is exported from the nucleus in a MEK- dependent manner through serine 112 phosphorylation [38, 39]. However, the specific process by which PPARy nucleus translocation in various cell types, particularly microglia, has yet to be fully elucidated. Herein, we found Daxx is recruited by PPAR γ in a dynamic manner, lead- ing to the translocation of PPARy into the nuclear com- partment. e interaction between Pdcd4 and Daxx results in a reduction in the half-life of Daxx, ultimately disrupting the formation of the PPARγ/Daxx complex [19]. Therefore, Pdcd4 is a switch for Daxx-mediated PPARy nuclear translocation, and Pdcd4 deletion sta- bilizes PPARγ in the nuclei. As previously mentioned, Daxx functions as a co-repressor in macrophages, inhib- iting the expression of NF-κB-targeted pro-inflammatory genes through its interaction with histone deacetylases flammatory gene transcription. Finally, our data demonstrated the antidepressant role of IL-10. Research on IL-10 knockout mice found an increase in depressive-like behaviors that were reversed with IL-10 injection [ 41 ]. In addition, the drugs for depression treatment, such as Fluoxetine and Sertraline, have displayed anti-inflammatory properties, includ- ing IL-10 up-regulation [3]. However, further research is needed to elucidate the mechanisms underlying the
Behavioral tests
Open field test
Elevated plus maze
Tail suspension test
Methods
Animals
Forced swimming test
Sucrose preference test
LPS-induced depressive behavior
Primary microglias isolation

observed changes in relation to IL-10. Our study indi- cates that the inhibition of PPARy leads to a suppres- sion of IL-10 expression in microglia during depression. By demonstrating the ability of pioglitazone to improve depression-like behaviors through the promotion of a neuroprotective microglial phenotype, we have provided direct evidence supporting the anti-inflammatory func- tion of PPARy in enhancing mRNA transcription via binding to the IL-10 gene promoter. Overall, we empha- sized the therapeutic role of IL-10 for depression. In conclusion, to the limitation of traditional therapy of depression, we are urged to find the new pathological mechanism of the disease. Our findings show that neu- roinflammatory reaction, including microglia activation and cytokines secretion is integrated into depressive-like behaviors via activation of Pdcd4 mediated PPARγ/IL-10 axis. Mouse is placed in the area (40×40×35 cm, L×W×H) with 60 lux lighting for 10 min. A SMART tracking sys- tem (Panlab, DC, USA) is used to record the movement of mice and analyze the traveled distance. e apparatus sets to a height of 50 cm beyond the ground and has black stainless steel with two closed and open arms (30×5×10 cm walls or no wall). Use LED lights to control the brightness of the light. To simulate the sense of security of animals in natural environments, the closed arm brightness is set 100 lux. Mouse is placed into the center platform facing an open arm and a video tracking system scores the time spent in open arms. Each mouse is hung to a grid bar of 30 cm which is the height from the ground by the tail with tape. Then the test is video-captured for 6 min. The immobile time is counted, and immobility is defined as the absence of escape-orientated movement. The littermate wild type (WT) and Pdcd4-deficient male mice in this study have been previously described. Also, LoxP-flanked Pdcd4 mice were generated by Biocyto- gen Co., Ltd (Beijing, China) using CRISPR/Cas 9 which has been referred to before [ 16 ]. Briefly, by crossing with Cx3cr1-CreERT2 (Jax stock no. 021160), we get micro- glial conditional Pdcd4 knockout mice, and Pdcd4fl/fl mice are used for littermate control. For inducing gene knockout, tamoxifen (50 mg/kg, ig) diluted into corn oil is treated to conditional knockout or control mice at 56 days after birth. Male mice are utilized in behavioral and biochemical experiments. All animal experiments and protocols are approved by the Animal Care and Utili- zation Committee of Shandong University. e mouse is individually placed in a glass cylinder (25×18 cm diameter) which contains water (15 cm height, 22±0.5 °C). e experiments are videotaped by a camera for tracking mice's reactions and scored the latency to immobility. e time of immobility is determined by the absence of motion except for slight actions to maintain the head above the water. Mice are habituated to drink sucrose solution (1%) over 2 days. After deprivation of water for 22 h, mice are given free access to two bottles separately containing water or 1% sucrose solution for 2 h. At the next day’s test, the site of the two bottles is exchanged to avoid a side bias. Finally, the fluid consumption is indicated by the weight loss of the bottles. Sucrose preference is calculated as follows: Sucrose preference (%) = sucrose intake/total fluid consumption × 100%. Male C57BL6/J mice (2 months) are intraperitoneally injected with LPS dissolved in sterile 0.9% saline at 0.5 mg/kg for 10 days. Saline or LPS injection is admin- istered between 09:00 and 09:30 a.m. daily for 10 days. Regents used in animal experiments were list in table below. Coat T-75 culture flask with 10 μg/mL PDL for 2 h, and wash the flask bottom with sterile water 3 times before use. Postnatal 1–3 days WT or Pdcd4–/– mouse were anesthetized, and brains were rapidly dissected and placed into ice-cold PBS. Brain material was cut off small pieces, and placed into 15 mL tube with 0.25% Trypsin–EDTA for 20 min at 37 °C. Aspirate the super- natant and resuspend the pellet with 5 mL warmed DMEM/F12 containing 10%FBS, 100 IU/mL penicil- lin and 100 μg/mL streptomycin, after that centrifuge the 15 mL tubes at 400xg for 5 min. Plate the cells into coated T-75 flask and change the culture medium the next day to remove cell debris. In 5–7 days, microglia and some oligodendrocytes grow on the top of astro- cytic layer. Tapping the flasks vigorously on the bench top, the floating cells were collected in culture media. The microglial cells are ready to use the next day.
Cell culture
HEK293 is purchased from the National Collections of Authenticated Cell Cultures (Shanghai, China). Mouse embryonic fibroblasts (MEFs) of Pdcd4 conventional knockout or littermate mice were prepared from E13.5 embryos. e 6–8 weeks-old mice are injected (i.p.) with 6% broth starch solution, and 3 days later perito- neal cells are collected. All media are Dulbecco's modi- fied eagle medium (DMEM) supplemented with 10% FBS (Cat. 10099-141, Gibco, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Cat. 03-031-1B, Biological Industries, Israel). Cells were all cultured in a humidified cell incubator with an atmosphere of 5% CO 2 at 37 °C.
Immunohistochemistry
A mouse heart is instilled in vivo with 4% polyformal- dehyde after anesthesia by 5% chloral hydrate (7.5 mL/ kg, i.p). e brain is harvested and embedded into OCT, after then, it is sectioned 40 μm thick. For the immuno- fluorescence, slides are incubated in 0.4% TritonX-100 diluted donkey serum to 10% for 1 h. Anti-Iba1 or anti- Pdcd4 antibody is used to slide incubation overnight at 4 °C, after that, those are washed with PBS three times. The secondary antibody incubated the slides at room temperature for 1 h. Washing in PBS three times, slides are mounted with cover glass. All the images are captured with an Olympus VS120 microscope (Tokyo, Japan). Pic- tures are analyzed by NIH Image J.
Real-time PCR
Mice are sacrificed immediately after behavioral tests. The hippocampus, prefrontal cortex, and hypothalamus are collected using a mouse brain slicer. Total RNA is extracted by Trizol reagent (Tiangen, Beijing, China) fol- lowing the manufacturer's instructions. Then, the puri- fied total RNA (1000 ng) is reversed to cDNA by using the cDNA Synthesis Kit (TAKARA, Tokoyo, Japan). Real- time PCR primers were listed in table below.

Nuclear and cytoplasmic extraction
The experiment follows the manufacturer's instruc- tion for NE-PER Nuclear and Cytoplasmic Extraction kit (Cat. 78833, Thermo Fisher, USA). Briefly, cultured cells are harvested with trypsin–EDTA and then centri- fuge at 500xg for 5 min. After washing cells with PBS, we add ice-cold CER I to the cell pellet and incubate on ice for 10 min, after that, add ice-cold CER II to the tube and centrifuge that for 5 min at maximum speed. Next, suspend the insoluble fraction in ice-cold NER for 40 min, and centrifuge the tube at maximum speed for 10 min. Prepare the supernatants for Western blot.
Western blot and ELISA
Mice brain tissue or cultured cell is homogenized in lysis buffer with protease inhibitors and ready for western blot and ELISA. The IL-10 level of the mouse brain is determined by IL-10 ELISA Kit according to the instructions (Cat. 431414, Biolegend, USA). All the primary and secondary antibodies have been listed in Table.

Flow cytometry
A mouse heart is instilled in vivo with PBS after anes- thesia by 5% chloral hydrate (7.5 mL/kg, i.p). Collect the brain and cut it into 1–2 mm³ pieces with small scissors. Transfer the sample to a 50 mL conical using a 10 mL digestion cocktail per brain at room temperature for 15 min. Resuspend the pellet in 37% percoll, and then on top of that layer slowly pipette 4 mL of 30% percoll, fol- lowed by 2 mL of HBSS. Centrifuge gradient 40 min at 200×g. Collect the 70–37% interphase into a clean 50-mL conical tube, and wash it with HBSS 3 times. Obtained single-cell suspension is labeled by using antibodies, and data are obtained on Cytoflex S (Backman, USA).
Immunocytochemistry
Cultured cells are fixed with 4% paraformaldehyde. For the immunofluorescence, slides are incubated in 0.4% TritonX-100 diluted donkey serum to 10% for 1 h. Pri- mary antibodies are used for samples incubation over- night at 4 °C, after that, those are washed with PBS three times. Secondary antibodies incubate the slides at room temperature for 1 h. Then, the samples are washed three times with PBS and mounted on slides in Prolong Gold medium (Invitrogen, USA). All the images were captured with a Zeiss LSM880 confocal microscope fitted with a 63× oil-immersion objective lens.
Immunoprecipitation
Cell proteins are extracted with TNE buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol, and protease inhibitors). The cell lysates are precipitated with antibody or Protein A/G beads overnight at 4 °C. The beads are rinsed 4 times with the TNE buffer.
Luciferase assay
Luciferase activity is measured with a dual luciferase assay system (Cat. E1910, Promega, USA) in HEK293 cells with siRNA or overexpression plasmids, and the readout is determined by using a microplate luminom- eter (Centro LB 960, Germany).
RNA-Seq
A total of 3 μg RNA was isolated using TRIzol, and sequencing libraries are generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA). RNA sequencing (RNA-seq) was performed on an Illumina HiSeq 4000 platform accord- ing to the manufacturer’s instructions and using a paired- end approach with 150-bp reads. Raw sequence read files are quality checked by FastQC software.
Statistical analysis
Data are displayed as the mean ± SEM and analyzed by using GraphPad Prism 8.0 (GraphPad Software, CA, USA). Normal distributions are compared by using the Kolmogorov–Smirnov test. One-way ANOVA followed by Tukey’s post hoc test and unpaired Student’s t-tests are performed. Statistical tests were two-sided and *p< 0.05, °°p < 0.01 was regarded as statistically significant. All data points provided are biological replicates and represent n.
Supplementary Information
The online version contains supplementary material available at https://doi. org/10.1186/s12974-024-03142-3.
Acknowledgements We thank the Translational Medicine Core Facility of Shandong University and the Key Laboratory of Endocrine Glucose & Lipids Metabolism and Brain Aging (Shandong First Medical University) for consultation and instrument availabil- ity that supported this work. Author contributions YL and LZ designed the experiments; YL, XZ, and BZ performed experi- ments, interpreted data, and wrote the manuscript; MZ and XC provided input on data analysis and computational approaches; QL undertook project management. Funding The National Natural Science Foundation of China (No. 82202015, 82173105, 81971471, 81771775) and The Natural Science Foundation of Shandong Province (No. ZR2023MH322) supported this study.
Availability of data and materials
Data supporting the present study are available from the corresponding author upon reasonable request.
Declarations
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