Growth differentiation factor 15 aggravates sepsis-induced cognitive and memory impairments by promoting microglial inflammatory responses and phagocytosis

Abstract

Background

Sepsis-associated encephalopathy (SAE) is a severe neurological condition caused by sepsis, and presents with symptoms ranging from delirium and coma to long-term cognitive dysfunction. SAE is acknowledged as a widespread brain impairment characterized by the activation of microglia. However, the specific pathological mechanisms that drive this activation are still not clearly understood. Growth differentiation factor 15 (GDF15) levels have been noted to be considerably increased in patients with sepsis, where they are linked to disease severity and can independently predict short- and long-term mortality risk. Serum levels of GDF15 have also been negatively associated with gray matter volume and predict cognitive impairment in older individuals. However, the impact of GDF15 on sepsis-induced cognitive and memory impairments, as well as the mechanisms behind these effects, are poorly understood.

Methods

To examine the role of GDF15 in SAE, a sepsis model was created in adult C57BL/6J mice using intraperitoneal administration of lipopolysaccharide (LPS). GDF15 levels in plasma and cerebrospinal fluid were measured by ELISA. The anti-GDF15 monoclonal antibody ponsegromab was injected intracerebroventricularly before modeling, and cognitive and memory functions of the septic mice were assessed using fear-conditioning and novel object recognition tests. Microglial activation and phagocytosis were evaluated using immunofluorescence and Golgi staining. Additionally, an in vitro investigation of LPS-stimulated microglia was conducted to evaluate the impacts of GDF15 on inflammatory cytokine productions and microglial phagocytic activity. Mechanisms were explored using RNA sequencing, qPCR, western blotting, flow cytometry, and immunofluorescence assays.

Results

In the cerebrospinal fluid of septic mice, levels of GDF15 were notably elevated after intraperitoneal injection of LPS. Lateral ventricular injection of the anti-GDF15 antibody alleviated both cognitive and memory impairment in the septic mice, together with microglial activation and phagocytosis in the hippocampus, thereby protecting against synaptic loss.

Conclusion

The levels of GDF15 were elevated in the brains of septic mice. Targeting GDF15 with an anti-GDF15 antibody was found to improve sepsis-induced cognitive and memory impairment by reducing the microglial inflammatory response and phagocytosis. These results indicate that GDF15 could serve as an important therapeutic target for treating SAE.

Introduction

Sepsis remains one of the most formidable challenges in contemporary medicine. It is characterized by a life-threatening cascade of organ failure triggered by an abnormal immune response to infection. This challenge is compounded by an alarming rise in annual incidence, poor understanding of its pathophysiology, lack of targeted therapies, and the severe long-term sequelae observed in survivors [1]. Despite extensive research, the mainstay of effective treatment remains the prompt administration of antibiotics and supportive care of failing organ systems [2]. Among the numerous complications associated with sepsis, sepsis-associated encephalopathy (SAE) is particularly concerning. Up to 50% of patients with sepsis develop SAE, characterized by diffuse cerebral dysfunction with pronounced cognitive and memory impairment, despite the absence of direct intracerebral infection [3, 4]. The acute phase of SAE is associated with symptoms such as delirium, agitation, hallucinations, drowsiness, and ultimately coma [5]. These neurological complications may contribute to sepsis-related mortality [1]. The increasing incidence of sepsis and reductions in the overall mortality rate suggests the likelihood of increased prevalence of cognitive sequelae, potentially leading to significant numbers of individuals with disabilities, increasing the burden on healthcare systems globally [1]. However, despite its prevalence, the pathological mechanisms of underlying SAE remain poorly understood. Causes such as neuroinflammation with microglial activation, blood-brain barrier (BBB) dysfunction, ischemic/hemorrhagic lesions, and neurotransmitter dysregulation have been proposed [5, 6]. Notably, the activation of microglia is of particular interest in sepsis-induced cognitive and memory impairment [7, 8].

Microglia serve as the innate immune cells within the central nervous system (CNS) and play a crucial role in various physiological and pathological processes, contributing significantly to brain development and homeostasis [9]. Under normal physiological conditions, microglia sustain both tissue homeostasis and synaptic plasticity [10, 11]. Their surveillance and synaptic pruning activities are crucial for synaptic turnover, the elimination of superfluous synapses, and the establishment of new neuronal circuits, all of which are vital for the refinement of neural circuitry in the adult brain [11]. As the brain’s resident macrophages, microglia are the first responders to brain injury [10]. When microglia become activated, they undergo alterations in shape and gene expression, along with heightened antigen display, engulfment of debris, and the secretion of pro-inflammatory molecules and chemokines [8, 12]. Notably, microglia are observed to cluster around cerebral vessels four hours after injection of lipopolysaccharide (LPS) [13], a potent stimulator of microglial activation. It is possible that dysregulation of microglial activity during SAE may promote and exacerbate damage to brain tissue. Continuous microglial activation is seen in both patients with sepsis and animal models. Postmortem studies have revealed a substantial rise in microglial numbers in the hippocampi of patients with sepsis, coupled with a loss of synaptic proteins in sepsis-affected brains [14, 15]. Moreover, inhibiting microglial activation has been shown to ameliorate cognitive deficits in septic rats [16]. Although these results have been observed, the exact ways in which microglia are involved in cognitive deficits associated with SAE are still not well understood. Therefore, it is essential to conduct more research on how microglial activation and synaptic changes mediated by phagocytosis contribute to the pathology of SAE.

The inflammation-associated hormone, growth differentiation factor 15 (GDF15), also known as nonsteroidal anti-inflammatory drug-activated gene-1 (NAG1), macrophage inhibitory cytokine-1 (MIC-1), and placental bone morphogenetic protein (PLAB), represents an atypical member of the transforming growth factor-β (TGF-β) superfamily [17]. Under physiological conditions, GDF15 expression is generally restricted to the placenta and prostate; however, under disease states like inflammation, tumor, cardiovascular disease, pulmonary embolism, and liver injury, its tissue and blood levels are markedly elevated [17, 18]. GDF15 has garnered attention as a biomarker for various diseases, including certain cancers [19] and heart failure [20]. Several studies have reported that GDF15 is upregulated in sepsis, where it shows a strong association with organ failure [21]. Conversely, Breen et al. demonstrated that subcutaneous administration of GDF15-neutralizing antibody or knockout of GDF15 did not impact the survival of mice with LPS-induced sepsis [22]. These diverse and partially opposing findings have created confusion regarding the role of GDF15 in sepsis, thus limiting the potential for developing GDF15-based therapies. Within the CNS, GDF15 is expressed by the choroid plexus and secreted into the cerebrospinal fluid (CSF) [23]. Elevated GDF15 concentrations in the CSF have been observed in patients with intracranial tumors [24]. Additionally, GDF15 can be upregulated in response to the presence of cortical lesions, with expression and temporal dynamics in established cortical lesion models suggesting an important function in the early response of brain tissue to damage [23]. GDF15 levels are negatively associated with gray matter volumes and are predictive of cognitive decline in older individuals [25, 26]. Moreover, elevated serum GDF15 levels are closely linked with a greater likelihood of developing dementia [27, 28]. Although there are connections noted, the impact of GDF15 on SAE and the mechanisms behind it are still not well understood. Further research is needed to elucidate the function of GDF15 in SAE and to evaluate its potential as a promising target for treatment strategies.

In this research, we report that GDF15 levels in the brains of septic mice were significantly elevated following intraperitoneal injection of LPS. Intracerebroventricular injection of an anti-GDF15 monoclonal antibody (mAb) mitigated sepsis-induced cognitive and memory deficits in the mice. Silencing of GDF15 effectively suppressed microglial activation, resulting in reduced microglial phagocytosis and the generation of pro-inflammatory molecules. The NF-κB signaling pathway was identified as the mediator of the effects exerted by GDF15. Additionally, it was observed that neutralization of GDF15 in the CNS alleviated microglial activation-mediated synapse engulfment and synaptic loss, ameliorating sepsis-induced cognitive and memory decline. Collectively, the findings reveal a previously unrecognized role of GDF15, secreted by activated microglia, in the pathological mechanism underlying microglial activation and microglia-mediated synaptic damage during sepsis-induced cognitive and memory impairment.

Materials and methods

Animals

Adult male C57BL/6J mice, aged 8 to 9 weeks and weighing between 20 and 25 g, were obtained from Slac Laboratory Animal Co., Ltd. in Shanghai, China. These mice were kept in a controlled environment featuring a 12-hour light/dark schedule and a temperature range of 20 to 24 °C, with free access to food and water at all times.

To establish the sepsis models, the mice were administered intraperitoneal (i.p.) injections of LPS from Sigma-Aldrich (St. Louis, MO, USA) at a concentration of 10 mg/kg, prepared in a solution of phosphate-buffered saline (PBS). For sample collection, the mice were deeply anesthetized with avertin (Sigma-Aldrich) via i.p. injection. Blood specimens were obtained from the inner canthal orbital vein, allowed to coagulate at ambient temperature for 2 h, and then subjected to centrifugation at 2000 x g for 20 min. The sera were then transferred to new Eppendorf (EP) tubes. The CSF was collected by cisterna magna puncture. Lastly, the whole brain was carefully dissected. The collected samples were stored immediately in a precisely controlled environment at − 80 °C until further use. The levels of GDF15 in both serum and CSF were measured utilizing ELISA. GDF15 expression of the whole brain was measured by western blotting (WB).

ELISA

GDF15 concentrations in serum and CSF from septic mice were quantified using a mouse GDF15 ELISA kit from R&D Systems (Minneapolis, MN, USA), following the manufacturer’s guidelines. In a comparable approach, the levels of IL-6 and TNF-α in the BV2 cell culture media were quantified using specific ELISA kits designed for mouse IL-6 and TNF-α, sourced from BioTNT (China). Briefly, the ELISA kits were allowed to reach room temperature for 30 min before being utilized. The samples were diluted with the diluent provided in the kits before being loaded onto the microtiter plate. The plate was prepared with wells designated for blanks, standards, samples, and duplicates. Samples were then added to the wells in accordance with the protocol. Absorbances were measured employing a microplate spectrophotometer (BioTek, Winooski, VT, USA) as specified in the protocol. A standard curve was generated by plotting the standard concentrations against their respective absorbance readings. The sample concentrations were calculated based on reference curve and modified according to the dilution ratio to derive the accurate protein levels.

WB analysis

Total protein extraction from brain tissue and cells was conducted using RIPA lysis buffer (Share-Bio, China), which was supplemented with a cocktail of protease and phosphatase inhibitors (Beyotime, China). Protein concentrations in the resulting lysates were measured using the BCA Protein Assay Kit (EpiZyme, China). Following quantification, equal protein amounts were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel (EpiZyme, China), and then transferred to polyvinylidene fluoride membranes (Millipore, USA) following standard procedures. The membranes were then blocked with 5% non-fat milk (EpiZyme, China) at room temperature for 2 h to prevent non-specific binding. After this step, they were incubated with primary antibodies specific to the target proteins at 4 °C overnight. The bound primary antibodies were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies, either anti-mouse IgG (H + L) or goat anti-rabbit IgG (H + L) (Beyotime, China), depending on the primary antibody used. The primary antibodies employed in the WB included: rabbit anti-GDF15 (Proteintech, USA), rabbit anti-NRF2 (Affinity Biosciences, China), rabbit anti-p-NF-κB p65 (Ser536) (Cell Signaling Technology, USA), rabbit anti-NF-κB p65 (ZEN-BIOSCIENCE, China), rabbit anti-p-p38 (Thr180/Tyr182) (ZEN-BIOSCIENCE, China), rabbit anti-p38 MAPK (Abclonal, China), rabbit anti-p-p44/42 MAPK (Erk1/2) (Cell Signaling Technology, USA), rabbit anti-ERK1/ERK2 (Abclonal, China), rabbit anti-p-JNK (Tyr185) recombinant antibody (Proteintech, USA), rabbit anti-JNK1/2 (Abclonal, China), rabbit anti-CD68 (Proteintech, USA), rabbit anti-Synapsin I (Abcam, UK), rabbit anti-PSD95 (Proteintech, USA), mouse anti-Iba1 (Oasisbiofarm, China), mouse anti-α-Tubulin (Proteintech, USA), mouse anti-GAPDH (Proteintech, USA), and mouse anti-β-actin (Proteintech, USA). Signals were detected using Super ECL Detection Reagent (Yeasen, China), as per the manufacturer’s protocols, visualized with the Tanon 5200 Imager, and analyzed using Image-J software.

Surgeries for intracerebroventricular injection

Mice were sedated using 1% isoflurane and placed on a warming pad to ensure their body temperature remained stable. Following anesthesia, a stereotactic apparatus was used to stabilize the mice. A central incision was created to reveal the scalp, and the periosteum covering the skull was meticulously excised. After drilling a small hole, a fine glass electrode containing the antibody was inserted into one side of the lateral ventricle. The measurements for the lateral ventricle were determined to be anteroposterior (AP) -0.22 mm, mediolateral (ML) ± 1.05 mm, and dorsoventral (DV) -2.5 mm, based on the guidance from the stereotactic apparatus. The anti-GDF15 monoclonal antibody ponsegromab (30 µg per mouse) (CSNpharm, USA) or negative control antibody IgG (CSNpharm) was then injected into the lateral ventricle. The glass probe was maintained in place for an additional 10 min to enhance the antibody diffusion process. The incision was then carefully sutured with single stitches, and the mice were placed under a warming lamp and monitored until they had recovered fully. Four hours following the administration of the antibody, the mice received an injection of LPS (10 mg/kg, intraperitoneally) to induce the sepsis model.

Behavioral tests

Open field tests (OFTs) were performed to evaluate the natural movement and behavior of the mice following intracerebroventricular injection and sepsis induction. Cognitive and memory functions were assessed using fear conditioning (FC), novel object recognition (NOR), and novel location recognition (NLR) tests. All behavioral assessments were performed between 13:00 and 21:00. Prior to each experiment, the mice were accommodated in the testing environment for one hour to facilitate habituation, and the assessments were carried out in well-lit conditions. The devices used for behavior testing were cleaned meticulously with 75% ethanol to eliminate odor cues from previous subjects. The apparatus, such as the instruments, video monitoring systems, and analytical software utilized for the behavioral assessments, was obtained from AniLab Scientific Instruments based in Ningbo, China.

OFT

The OFT was performed in a square box (40 × 40 × 40 cm) situated in a quiet and dimly lit room. The mice were positioned in the middle of the enclosure and allowed 5 min for unrestricted exploration. The overall distance covered, rate of movement, frequency of central grid crossings, and duration spent within the central grid were measured automatically and analyzed.

NOR and NLR

The NOR test was employed to evaluate short-term working memory, and consisted of three distinct phases, namely, habituation (day 1), familiarization (day 2), and testing (day 3), with a 24-h interval between each. The OFT conducted on day 1 served as the habituation phase. On the second day, the mice were introduced into the arena containing two identical items and allowed 5 min for unrestricted exploration. On day 3, mice were placed in the arena alongside one item from the previous day and a novel object, with 5 min allotted for unrestricted exploration. The duration of exploration for both the familiar and new objects was automatically logged. The object recognition metric was subsequently calculated using the equation: dicrimination index = (duration spent with the new item − duration spent with the familiar item) ÷ overall exploration duration. After a 120-min rest, the NLR test was conducted. The mice were reintroduced to the arena where one object had been moved to a new location while another remained in its original position, and were given 5 min to explore freely. Their exploration times for both locations were recorded, and the place discrimination index was computed using the formula: discrimination index = (duration spent in the unfamiliar area − duration spent in the original area) ÷ overall exploration duration.

FC

The FC test, a reliable method for assessing the learning and memory capabilities of rodents through the association of a conditioned stimulus with an aversive unconditioned stimulus, was carried out over three sessions, each separated by 24-h intervals, specifically: habituation (day 1), training (day 2), and re-exposure (day 3). On the first day, the mice were placed in a fear-conditioning environment featuring white walls and a metal grid floor, where they were permitted to roam freely for a duration of 5 min. On the second day, the mice were situated in the identical fear-conditioning chamber and allowed to investigate for a period of 180 s. This was followed by five trials of conditioned stimuli (a tone at 70 dB, lasting for 30 s) paired with unconditioned stimuli (foot shock of 0.45 mA for 2 s), with a 30-second interval between each trial. On day 3, the contextual FC test was performed by re-exposing the mice to the same chamber for 5 min in the absence of the tone or electric shock to assess context association. After a 120-min rest, the cued FC test was conducted in a different context (a triangular space with two black tops and a solid white floor). Following a 180-second exploration period, the mice were subjected to the same sound stimulus (tone of 70 dB for 30 s) as used in the training period but without the electric foot shock, for four trials. The movement speed of the mice was recorded by an infrared camera. Freezing time and the percentage of freezing time during the contextual and cued FC tests were analyzed to evaluate memory function in the mice after induction of sepsis.

Primary cell sorting

Primary cell sorting was carried out utilizing the Adult Brain Dissociation Kit (Miltenyi Biotec, Germany), adhering to the protocols provided by the manufacturer. In summary, the mice were euthanized via cervical dislocation. Subsequently, the entire brain tissues were excised and rinsed in chilled D-PBS solution. The brains were then diced into smaller fragments using a scalpel and placed into a gentleMACS C tube (Miltenyi Biotec, Germany) which contained enzyme mixture 1. Following this, enzyme mix 2 was introduced into the C tube. The tube was then inverted and attached to the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec, Germany) for a one-hour dissociation process. Upon completion of the dissociation, the resulting cell suspension was filtered through a 70-µm nylon mesh (Miltenyi Biotec, Germany) and the cells were centrifugated. Ultimately, the cells were resuspended to eliminate debris and red blood cells. The isolation of microglia (CD11b (Microglia) MicroBeads, human and mouse, Miltenyi Biotec, Germany), neurons (Neuron Isolation Kit, mouse, Miltenyi Biotec, Germany), and astrocytes (Anti-ACSA-2 MicroBead Kit, mouse, Miltenyi Biotec, Germany) was accomplished through the MACS technology.

Cell cultivation and treatment

Five mouse cell lines, including BV2 microglial cells, C8-D1A astrocytes, HT22 hippocampal neuronal cells, N2a neuroblastoma cells, and RAW 264.7 cells, were grown in DMEM (BasalMedia, China) enriched with 10% fetal bovine serum (FBS) (Thermo Fisher, USA) and preserved in a moisture-controlled incubator with 5% CO₂ at 37 °C. Mouse bEnd.3 brain microvascular endothelial cells were grown in DMEM supplemented with 20% FBS. All cells were treated with LPS for the specified durations and then harvested for quantitative PCR assessment. Additionally, BV2 microglial cells were collected for WB assays, and proteins in their culture supernatants were measured by ELISA. In certain experiments, BV2 cells were pretreated with various inhibitors, such as the ERK inhibitor FR180204 (Beyotime), the JNK inhibitor SP600125 (Beyotime), p38 inhibitor SB203580 (Beyotime), NF-κB inhibitor BAY11-7082 (TargetMoi, USA), the NRF2 inhibitor ML385 (Selleck, USA), or recombinant GDF15 protein (200 ng/ml, R&D) before being exposed to LPS (1 µg/ml). HT22 cells underwent pretreatment with recombinant GDF15 protein (10, 100, or 1000 ng/ml) before exposure to TNF-α (10 ng/ml) (PeproTech, USA).

For transient transfection, BV2 cells were cultured in 60 mm dishes until they reached 80–90% confluence. The cells were subsequently moved to 12-well plates, with a density of 8 × 10⁴ cells per well, and permitted to incubate overnight. On the subsequent day, the cells were transfected with siRNA duplexes at a concentration of 50 nM for a duration of 48 h, utilizing the jetPRIME transfection reagent (Polyplus, France) in accordance with the guidelines provided by the manufacturer. The sequences of the siRNA primers were listed below:

siGdf15-1 forward, 5′-GCAGGCAACUCUUGAAGACUU-3′, reverse, 5′-AAGUCUUCAAGAGUUGCCUGC-3′;

siGdf15-2 forward, 5′-GUGUCACUGCAGACUUAUGAUTT-3′, reverse, 5′-AUCAUAAGUCUGCAGUGACACTT-3′ (purchased from BioTNT);

siNrf2 forward, 5′-CAGAAAUGGACAGCAAUUATT-3′, reverse, 5′-UAAUUGCUGUCCAUUUCUGTT-3′.

Primary microglial culture

Primary microglial cultures were established from cerebral cortices and hippocampi of 1 to 3-day-old neonatal C57BL/6J mice. For each batch of primary microglial culture, the brains of 4 to 6 neonatal pups from the same litter were surgically excised and combined. The procedure involved decapitation followed by rapid brain extraction and immersion in ice-cold PBS. Following the removal of other brain regions, meninges, and blood vessels, the cortices and hippocampi were isolated. After a process of mechanical and enzymatic dissociation with 0.25% trypsin (Gibco, USA) for a 3 min at 37 °C, the digestion was terminated. The resulting suspension was filtered through a 70-µm cell strainer, followed by centrifugation and collection. Then, the cells were seeded and cultured in DMEM/F12 (Gibco, USA) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, and preserved in a moisture-controlled incubator with 5% CO₂ at 37 °C. The culture medium was initially replaced 24 h after seeding and subsequently every 3 days. Upon reaching confluence on day 13, the medium was exchanged, and the culture flask was then placed on a 37 °C constant temperature shaker (200 r/min) and shook for 2 hours. The resultant suspension, containing the isolated primary microglial cells, was then collected and directly seeded into culture flasks or multi-well plates at a density of 4 × 10⁴-10 × 10⁴ cells/cm². Experiments commenced 24 h after cell isolation.

RNA sequencing (RNA‑seq)

BV2 cells were harvested and sent to Magigen Biotechnology Co., Ltd (Guangzhou, China) for the preparation and sequencing of the RNA-seq library. The sequencing data underwent quality control and filtering using FastQ (v0.23.2) (https://github.com/OpenGene/fastp). Gene expression levels for each group were determined using RSEM (v1.3.3). Differentially expressed genes (DEGs) were identified and grouped based on their expression trends using DESeq2 (v1.34.0). Functional analysis of the DEGs was carried out using GO and KEGG pathway enrichment, using ClusterProfiler (v4.2.2) (http://www.bioconductor.org/packages/release/bioc/html/clusterProfiler.html).

RNA isolation and qPCR

Total RNA was isolated utilizing TRIzol reagent (Thermo Fisher) in accordance with the guidelines provided by the manufacturer. The integrity and concentration of the RNA were evaluated utilizing a NanoDrop spectrophotometer (Thermo Fisher), calibrated with 1 µl of the identical nuclease-free water used for RNA extraction. RNA concentrations were determined from the absorbance measurements. The RNA was subsequently reverse-transcribed into cDNA with HiScript II Q RT SuperMix (5 x) (Vazyme, China). The resulting cDNA was diluted 10-fold for subsequent experiments. qPCR assays were arranged in 96-well or 384-well reaction plates (Axygen, USA). Gapdh served as an internal control, and gene expression changes were determined with the 2^−ΔΔCt approach. The primer sequences were: Gdf15 forward, 5′-AATGCCTGAACAGCGACCCTC-3′, reverse, 5′-CCTGGAAGCGACCCCGTAG-3′ (purchased from BioTNT);

Nrf2 forward, 5′-CTGAACTCCTGGACGGGACTA-3′, reverse, 5′-CGGTGGGTCTCCGTAAATGG-3′;

Il-6 forward, 5′-ACCAAGACCATCCAATTCATC-3′, reverse, 5′-CTGACCACAGTGAGGAATGTC-3′ (purchased from BioTNT);

Il-1β forward, 5′-GCTTCAGGCAGGCAGTATC-3′, reverse, 5′-AGGATGGGCTCTTCTTCAAAG-3′;

Tnf-α forward, 5′-CCTGTAGCCCACGTCGTAG-3′, reverse, 5′-GGGAGTAGACAAGGTACAACCC-3′;

Ccl-2 forward, 5′-TAAAAACCTGGATCGGAACCAAA-3′, reverse, 5′- GCATTAGCTTCAGATTTACGGGT-3′.

Analysis of scRNA-seq data

Single-cell RNA-seq (scRNA-seq) data for cells obtained from the motor cortex of mice treated with either PBS or LPS (40 mg/kg) were retrieved from the GEO database. The dataset can be accessed under the accession number GSE211099 [29]. The Seurat v3 anchor method [30] was used for integration of the data from the LPS and PBS treatments. The outcomes of the integration were utilized to perform t-distributed stochastic neighbor embedding (t-SNE) for dimensionality reduction. Cell clustering was conducted employing the Louvain algorithm, alongside the application of canonical markers to identify various cell types [29]. The expression profiles of the selected genes were visualized in the un-integrated space using the FeaturePlot function from Seurat.

Cell viability assay

HT22 cells were plated in 96-well plates at a density of 5,000 cells per well and allowed to incubate overnight. Subsequently, the cells were treated with different concentrations of TNF-α (0, 10, 100, or 1000 ng/ml) for a duration of 48 h. Certain cells received a pre-treatment with various concentrations of recombinant GDF15 protein (10, 100, or 1000 ng/ml) before exposure to TNF-α (10 ng/ml). Cell viability was subsequently evaluated with CCK-8 assays (NCM Biotech, China).

Microglial uptake of latex beads

The phagocytosis assay was conducted as previously described [31]. BV2 cells and primary microglia were plated in 12-well plates at a density of 80,000 cells per well and allowed to incubate overnight. The cells were then transfected with Gdf15 siRNA and incubated for 48 h, after which they were treated with LPS (1 µg/ml) for an additional 24 h. Each well received 1 µl/ml of latex beads (from Sigma-Aldrich), which were incubated at 37 °C for a duration of 2 h. The cells were subsequently rinsed thrice with PBS to remove any unphagocytosed beads and then fixed in 4% paraformaldehyde (PFA) for 10 min. Immunofluorescence assays were conducted as detailed below, and cells were observed with a fluorescence device (Zeiss, Germany). For flow cytometry analysis, the samples were washed and collected before assessment of phagocytosis by flow cytometry (Beckman Coulter, Brea, CA, USA) after resuspension in PBS. The engulfing function of the cells was assessed utilizing FlowJo software.

Immunofluorescence assays

To minimize confounding factors, sample collections were performed between 08:00 and 12:00. Mice were deeply anesthetized with avertin (i.p.) and underwent transcardiac perfusion with cold PBS and 4% PFA using a perfusion pump. After perfusion, the brains were harvested and then fixed in 4% PFA overnight. Subsequently, the fixed brains were subjected to dehydration by immersing them in 20% sucrose for 24 h, followed by 30% sucrose for an additional 48 h. After embedding in OCT compound, the brains were sectioned into 10 μm slices using a cryostat microtome (Leica, Germany). The resulting slices were then incubated for one hour in a blocking solution of 5% bovine serum albumin (BSA) in 0.3% Triton X-100 PBS. After this blocking step, the sections were left to incubate with primary antibodies overnight at 4 °C. The next day, after three PBS washes, the sections were treated with secondary antibodies labeled with Alexa Fluor-488 or Alexa Fluor-594 for 2 h at room temperature. To stain the nuclei, an antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) was used. Primary antibodies used here included Iba1 mouse mAb (Oasisbiofarm, China), GDF15 rabbit polyclonal antibody (pAb) (Proteintech, USA), C1q rabbit mAb (Abcam, UK) and CD68 rabbit pAb (Proteintech, USA). Images were acquired using a confocal microscope (Zeiss, Germany).

Golgi-Cox staining

Golgi staining was performed utilizing a Golgi staining kit (Servicebio, China) following the instructions provided by the manufacturer. The harvested brain samples were first fixed in a Golgi solution and then stained with the Golgi-Cox staining solution for 14 days at room temperature in a cool ventilated and light-protected environment. After staining, the samples were transferred to the provided tissue treatment solution for a duration of 1 h, after which the solution was substituted with a new one and incubated for 3 days at 4 °C away from light. Coronal slices of the brains (60 μm in the thickness) were sectioned with a cryostat microtome (Leica). The tissue treatment solution was applied to the surface of the brain tissue slices. The slices were subsequently rinsed with ultra-pure water and immersed in Golgi developer solution for 30 min. The samples were analyzed using a wide-format MIDI scanner (3DHISTECH, Hungary). Pyramidal neurons in the CA1 area were examined, and three randomly chosen basal dendrite segments, each at least 30 μm in length, were analyzed for spine density utilizing ImageJ software.

Statistical analysis

Statistical evaluations were performed using GraphPad Prism 9 software (GraphPad Software, USA). Results are presented as mean ± standard error of the mean (SEM). The statistical relevance for differences between two groups was evaluated using unpaired t-tests, whereas the analysis of multiple groups was conducted using one-way ANOVA. P-values less than 0.05 were considered statistically meaningful.

Results

GDF15 levels are elevated in the brains of septic mice

To investigate the potential function of GDF15 in the central nervous system during sepsis, GDF15 levels in the peripheral blood, CSF, and brain tissue were first measured. Consistent with the findings of prior studies [22], GDF15 levels in the sera of the septic mice, induced by i.p. administration of LPS at 10 mg/kg, increased rapidly, peaking at 2 h post-injection (Fig. 1A). Notably, GDF15 levels were also significantly elevated in both the CSF and brain tissue of septic mice compared to controls (Fig. 1B and C). These results suggest that GDF15 is elevated in the brain during sepsis, suggesting a potential role in sepsis-induced cerebral injury.

Fig. 1

GDF15 levels are elevated in the brains of septic mice. (A) Enzyme-linked immunosorbent assay (ELISA) results of plasma GDF15 levels in septic mice following intraperitoneal injection of LPS (10 mg/kg) for the indicated times. (B) ELISA measurements of GDF15 levels in the CSF of septic mice. (C) Western blots showing GDF15 expression levels in the brain tissues of septic mice. n = 3 mice per group, and bars indicate the means ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001

Anti-GDF15 mAb alleviates cognitive and memory impairment in septic mice

To clarify the role of GDF15 within the brain of septic mice, a specific GDF15 neutralizing antibody, ponsegromab, was administered by intraventricular injection to block GDF15 effect in the brain. Behavioral tests, including FC, NOR, and NLR, were used to assess the impact of central GDF15 blockade on neurocognitive deficits in the mice (Fig. 2A). It was found that mice with sepsis showed significant learning and memory dysfunction, measured in the FC, NOR, and NLR tests (Fig. 2D - I, Fig. S1A-S1C), while their exploratory and locomotory functions remained unaffected (Fig. 2B and C). Notably, pre-treatment with ponsegromab before sepsis induction resulted in reduced learning and memory deficits compared to control animals (Fig. 2D - I), indicating the importance of GDF15 in sepsis-induced cognitive and memory dysfunction. Interestingly, ponsegromab treatment also alleviated anxiety-like behaviors in septic mice (Fig. S2A-S2C).

Fig. 2

Administration of an anti-GDF15 monoclonal antibody alleviates cognitive and memory impairment in septic mice. (A) Schematic diagram of the experimental procedure. Mice received intracerebroventricular injection of the anti-GDF15 monoclonal antibody ponsegromab or IgG control and intraperitoneal injection of LPS (10 mg/kg) on day 1. The mice were then subjected to an open field test (OFT) on day 5 and a fear-conditioning test (FC), novel object recognition test (NOR), and novel location recognition test (NLR) on days 6 and 7 after intraperitoneal injection of LPS (10 mg/kg) or the same volume of PBS. (B-C) The total distance (B) and average locomotory speed (C) during the OFT were measured on day 5 after intracerebroventricular injection of the anti-GDF15 monoclonal antibody ponsegromab or IgG control and intraperitoneal injection of LPS (10 mg/kg). (D-E) The contextual memory test (D) and cue memory test (E) were performed on day 7 following injection of ponsegromab or IgG control and intraperitoneal injection of LPS (10 mg/kg). (F-G) Example of movement traces (F) and object discrimination indices (G) in the NOR test. (H-I) Example of movement traces (H) and location discrimination indices (I) in the NLR test. Each symbol represents one mouse (n = 10–12). Bars indicate means ± SEM. ns, not significant; *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001

Microglia are the main source of GDF15 in the cerebral tissues of mice experiencing sepsis induced by LPS

To investigate the origin of GDF15 within the cerebral structures of mice affected by sepsis triggered by LPS, an scRNA-seq dataset from motor cortex cells of adult wild-type (WT) mice administered a fatal amount of LPS (40 mg/kg) or PBS was first analyzed. The data showed that Gdf15 was expressed mainly in the microglia after LPS challenge (Fig. S3A-S3D). Next, we isolated primary microglia, astrocytes, and neurons from the cortices and hippocampi of septic mice, cerebral regions that have been well demonstrated to be responsible for cognition and memory, and found that Gdf15 was preferentially increased in the sorted microglia (Fig. 3A and B). Consistent with this result, immunofluorescence staining showed that GDF15 was co-localized with microglia in the hippocampi of septic mice (Fig. 3C and D). To further verify this conclusion, mouse cell lines, including BV2 microglial cells, C8-D1A astrocytes, bEnd.3 brain microvascular endothelial cells, HT22 hippocampal neuronal cells, N2a neuroblastoma cells, and RAW 264.7 macrophage-like cells, were exposed to LPS, and the mRNA levels of Gdf15 were assessed by qPCR. Notably, significant upregulation of Gdf15 mRNA was observed only in BV2 cells after LPS stimulation (Fig. 3E), which was corroborated by RNA-seq and WB analyses (Fig. 3F and G). Furthermore, the levels of GDF15, a secreted protein, in the culture supernatants of BV2 cells were also increased following LPS stimulation (Fig. 3H). The upregulation of Gdf15 in BV2 cells demonstrated a dose-response relationship with the LPS dose (Fig. 3I). These findings suggest that the elevated levels of GDF15 observed in the brains of septic mice were predominantly derived from activated microglia.

Fig. 3

Microglia are the main source of GDF15 in the brains of septic mice. (A-B) Quantitative polymerase chain reaction (qPCR) analysis of Gdf15 mRNA expression in the indicated primary cells sorted from the cortices (A) and hippocampi (B) of LPS-induced septic mice (n = 3). (C) Representative images of immunofluorescence staining of Iba1 and GDF15 in the hippocampi of septic mice. Red, Iba1; green, GDF15; blue, DAPI; scale bar, 50 μm and 5 μm. (D) Quantification of the ratio of GDF15⁺Iba1⁺/Iba1⁺ cells in (C). (E) qPCR analysis of Gdf15 mRNA expression in the indicated cells treated with LPS (1 µg/ml) for the specified times. (F) Volcano plots of BV2 cells treated with LPS (1 µg/ml) for 6 h. Red, upregulated genes; blue, downregulated genes. (G) Western blotting showing GDF15 protein expression in BV2 cells (upper panel) and primary microglia (lower panel) treated with LPS (1 µg/ml) for the specified times. (H) ELISA measurements of GDF15 concentrations in the culture supernatants of BV2 cells treated with LPS (1 µg/ml) for the specified times. (I) qPCR analysis of Gdf15 mRNA expression in BV2 cells treated with the indicated LPS concentrations for 3 h. Gapdh was used as an endogenous reference for qPCR. Bars indicate means ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001

GDF15 is upregulated by NRF2 signaling in LPS-stimulated microglia

To explain the process through which LPS upregulates GDF15 in microglia, BV2 cells were treated with inhibitors targeting the downstream pathways of Toll-like receptor 4 (TLR4), including the ERK blocker FR180204, JNK blocker SP600125, p38 blocker SB203580, and NF-κB blocker BAY11-7082. Following LPS stimulation, Gdf15 mRNA levels were assessed by qPCR. The findings indicated that the LPS-triggered upregulation of GDF15 in BV2 cells remained unaltered after treatment with downstream TLR4 pathway inhibitors (Fig. 4A), suggesting that other mechanisms are involved in this process. A previous study observed that NRF2 can regulate GDF15 expression [32]. Here, increased Nrf2 mRNA expression was observed in BV2 cells after LPS stimulation (Fig. 4B). Pre-exposure of BV2 cells using the NRF2 inhibitor ML385 was observed to suppress the LPS-induced upregulation of GDF15 significantly (Fig. 4C). To further verify that GDF15 upregulation in microglia induced by LPS was mediated by the NRF2 signaling pathway, BV2 cells were transfected with Nrf2-specific siRNA. The efficiency of Nrf2 silencing was assessed using qPCR and WB analyses (Fig. 4D and E). Subsequent qPCR analysis revealed that LPS-induced upregulation of GDF15 was inhibited in BV2 cells after Nrf2 knockdown (Fig. 4F). These findings indicate that LPS upregulates GDF15 expression in microglia through the NRF2 signaling pathway.

Fig. 4

GDF15 is upregulated by NRF2 signaling in LPS-stimulated microglia. (A) qPCR analysis of Gdf15 mRNA expression in BV2 cells pretreated for 30 min with inhibitors of downstream TLR4 pathway components, including the ERK inhibitor FR180204, JNK inhibitor SP600125, p38 inhibitor SB203580, and NF-κB inhibitor BAY11-7082, before treatment with LPS (1 µg/ml) for 3 h. (B) qPCR analysis of Nrf2 mRNA expression in BV2 cells treated with LPS (1 µg/ml) for the specified times. (C) qPCR analysis of Nrf2 mRNA expression in BV2 cells pretreated with the NRF2 inhibitor ML385 (1 µM) for 24 h before treatment with LPS (1 µg/ml) for 3 h. (D) qPCR analysis of Nrf2 mRNA in BV2 cells after transfection with Nrf2 siRNA for 48 h. (E) Western blotting showing NRF2 protein expression in BV2 cells after transfection with Nrf2 siRNA for 48 h. (F) qPCR analysis of Gdf15 mRNA expression in BV2 cells after transfection with Nrf2 siRNA for 48 h before treatment with LPS (1 µg/ml) for the indicated times. Gapdh was used as an endogenous reference for qPCR. Bars indicate means ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001

GDF15 silencing reduces inflammatory cytokine production in LPS-stimulated microglia through regulation of NF-κB signaling

The results described above established that GDF15 was elevated in the brains of septic mice and LPS-stimulated microglia, and that inhibiting GDF15 in the brain ameliorates cognitive and memory impairment in the mice. For further investigation, GDF15 was silenced in BV2 cells and primary microglia using Gdf15-specific siRNAs, resulting in a notable decrease in both mRNA and protein expression of GDF15 (Fig. S4A-S4D). To assess the function of GDF15 in the inflammatory response, GDF15-silenced BV2 cells were treated with LPS, followed by RNA sequencing. Discrepancies in the expression of genes linked to inflammatory responses triggered by LPS in BV2 cells were investigated between the siNC and siGdf15 groups. RNA sequencing revealed a significant decrease in the expression of numerous proinflammatory cytokine genes, such as Tnf, Il-1, and Il-6, in GDF15-silenced BV2 cells (Fig. 5A). Subsequently, qPCR and ELISA were used to confirm the effect of GDF15 silencing on cytokine production at both the messenger RNA and protein levels. The qPCR measurements indicated marked reductions in Il-6, Il-1β, Tnf-α, and Ccl-2 mRNA levels in GDF15-silenced BV2 cells and GDF15-silenced primary microglia compared to controls (Fig. 5B - E). Additionally, the ELISA results showed significant reductions in the concentrations of IL-6 and TNF-α in the culture media of GDF15-silenced BV2 cells compared to siNC controls (Fig. 5F and G). Furthermore, the proinflammatory Il-6 gene was considerably upregulated in LPS-triggered BV2 cells pretreated with recombinant GDF15 (Fig. 5H). These data confirm that GDF15 promotes proinflammatory cytokine production in microglia in response to pathogenic stimuli.

Fig. 5

GDF15 silencing reduces inflammatory factor production in LPS-stimulated microglia through regulation of NF-κB signaling. (A) Heatmap of mRNA expression of inflammatory factors in BV2 cells after transfection with Gdf15 siRNA for 48 h and subsequent treatment with LPS (1 µg/ml) for 6 h. (B-E) qPCR analysis of Il-6 (B), Il-1β (C), Tnf-α (D), and Ccl-2 (E) mRNA expression in BV2 cells (left panel) and primary microglia (right panel) after transfection with Gdf15 siRNA for 48 h and subsequent treatment with LPS (1 µg/ml) for the indicated times. (F-G) ELISA measurements of IL-6 and TNF-α production in the culture supernatants of BV2 cells after transfection with Gdf15 siRNA for 48 h and subsequent treatment with LPS (1 µg/ml) for 12 h. (H) qPCR analysis of Il-6 mRNA expression in BV2 cells pretreated with recombinant GDF15 (200 ng/ml) for 30 min and then treated with LPS (1 µg/ml) for 3 h. (I) Western blotting showing levels of phosphorylated p65, p38, ERK, and JNK, or total proteins, in BV2 cell lysates after transfection with Gdf15 siRNA for 48 h and subsequent treatment with LPS for the indicated times. Gapdh was used as an endogenous reference for qPCR. Bars indicate means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

The observed reduction in inflammatory cytokine production in LPS-stimulated microglia following GDF15 silencing led to further exploration into the possible process through which GDF15 regulates the inflammatory reaction in microglia. Interactions between TLRs and their ligands is known to lead to the triggering of JNK, ERK1/2, p38, and NF-κB signaling cascades, resulting in the transcription of inflammatory cytokine genes [33]. WB confirmed that GDF15 silencing resulted in reduced phosphorylation of the NF-κB p65 subunit, rather than p38, ERK, or JNK, in BV2 microglial cells following TLR4 activation, suggesting that GDF15 modulates the inflammatory response in microglia primarily through NF-κB signaling (Fig. 5I, S4E).

To further investigate whether GDF15 affects neurons directly, HT22 cells were exposed to different concentrations of TNF-α as an in vitro model of neuronal injury mediated by inflammatory factors. To identify appropriate TNF-α concentrations, morphological examinations and CCK-8 assays were performed, showing a dose-response relationship between HT22 cell viability and TNF-α concentration, with a TNF-α concentration of 10 ng/ml inducing significant neuronal damage (Fig. S5A, S5B). However, pretreatment with recombinant GDF15 protein did not affect the viability of cells exposed to 10 ng/ml of TNF-α (Fig. S5C). The results indicate that GDF15 does not have a direct effect on inflammatory cytokine-mediated neuronal damage.

GDF15 silencing inhibits microglial phagocytosis in vitro

To gain a deeper understanding of the function of GDF15 in microglia, transcriptomic analysis was performed on GDF15-silenced BV2 cells activated by LPS. This identified 1292 DEGs, of which 866 were up-regulated and 426 were down-regulated, using the criteria of log₂FC > 1 and < − 1. The identification of DEGs revealed that silencing of GDF15 not only reduced the expression of the microglial marker Iba1 editing gene Aif1, but also decreased the transcript levels of the C1q complement components C1qb and C1qc (Fig. 6A). GO functional enrichment of the DEGs indicated significant enrichment in GO Biological Process (GO-BP) terms such as “pseudopodium organization” and “pseudopodium assembly”, indicating altered microglial migration following GDF15 silencing (Fig. S6A). Additionally, the GO Cell Component (GO-CC) terms including “endosome,” “lysosome,” “endocytic vesicle,” and “phagocytic vesicle” also showed significant enrichment (Fig. S6C). These processes have been frequently associated with microglial phagocytic activity. KEGG pathway analysis also revealed enrichment in pathways related to “complement and coagulation cascades,” “phagosome,” and “lysosome”, indicating the participation of the complement system and phagocytosis (Fig. 6B).

Fig. 6

GDF15 silencing inhibits microglial phagocytosis in vitro. (A) Heatmap of top 10 differentially expressed genes (DEGs) in BV2 cells transfected with Gdf15 siRNA for 48 h and then treated with LPS (1 µg/ml) for 6 h. Values represent the log2 fold change of Gdf15-knockdown BV2 cells over control BV2 cells in the respective groups. (B) KEGG pathway analysis of the DEGs shown in (A). (C) Representative flow cytometry dot plots of red latex beads engulfed by BV2 cells transfected with Gdf15 siRNA for 48 h and then treated with LPS (1 µg/ml) for 24 h. (D) Quantitative analysis of microglial phagocytosis in (C). (E) Representative images of immunofluorescence analysis of red latex beads engulfed by BV2 cells (upper panel) and primary microglia (lower panel) transfected with Gdf15 siRNA for 48 h and then treated with LPS (1 µg/ml) for 24 h. Red, latex beads; blue, DAPI. Scale bar, 100 μm. **P < 0.01, ***P < 0.001, ****P < 0.0001

To examine the impact of GDF15 on microglial phagocytosis, a phagocytosis assay was performed using fluorescent latex beads. The findings demonstrated that LPS exposure significantly enhanced the uptake of latex beads by BV2 cells and primary microglia. However, GDF15 silencing significantly reduced the phagocytic activity (Fig. 6C - E). Collectively, these results demonstrate that silencing of GDF15 in microglial cells impaired LPS-induced microglial activation and phagocytosis, indicating the critical role of GDF15 in regulating these functions.

Anti-GDF15 mAb reduces microglial phagocytosis-mediated synaptic loss in the hippocampi of septic mice

Earlier research has indicated that microglial cells in the hippocampi of both patients and mice with sepsis are activated during the acute phase of SAE, and C1q-labeled synapses are phagocytosed by activated microglia, resulting in severe neuronal damage [15, 34]. Notably, the present study observed diminished phagocytic activity in GDF15-silenced microglial cells. To address this further, the GDF15-neutralizing antibody ponsegromab was injected into the lateral ventricles of mice to determine whether this could mitigate microglial phagocytosis and counteract the sepsis-induced reduction in dendritic spines. Colocalization analysis of Iba1 and CD68 showed an increase of microglial activation in the hippocampi during experimental sepsis, which was reduced after ponsegromab administration (Fig. S7A, S7B). Furthermore, septic mice showed increased numbers of C1q-positive synapses engulfed by microglia, which was significantly reversed by ponsegromab treatment (Fig. 7A and B). To further assess the impact of ponsegromab on synaptic phagocytosis, the levels of the synaptic markers PSD95 and Synapsin1 were measured in microglia sorted from the hippocampi of septic mice using western blotting. As shown in Fig. 7C, the levels of PSD95 and Synapsin1 were increased in these sorted microglia, which were reduced following ponsegromab treatment. Additionally, the synaptic spine density and number of dendritic branches at a specified distance from the soma in the hippocampal CA1 region were reduced in septic mice. However, these changes were prevented by ponsegromab treatment (Fig. 7D - F). Taken together, the results suggest that anti-GDF15 mAb treatment effectively reduced microglial activation and phagocytosis in the hippocampi of septic mice and ameliorated the sepsis-induced loss of dendritic spines. These findings underscore the therapeutic potential of targeting GDF15 in mitigating neuronal damage associated with sepsis.

Fig. 7

Anti-GDF15 monoclonal antibody treatment reduces microglial phagocytosis-mediated synaptic elimination in the hippocampi of septic mice. (A) Representative images of immunofluorescence staining of Iba1 and C1q in the hippocampi of mice after intracerebroventricular injection of the anti-GDF15 monoclonal antibody ponsegromab or IgG control and intraperitoneal injection of LPS (10 mg/kg) for 7 days. Green, Iba1; Red, C1q; blue, DAPI; scale bar, 50 μm and 5 μm. (B) Quantification of the ratio of C1q⁺Iba1⁺/Iba1⁺ cells in (A). (C) Western blots showing PSD95 and Synapsin1 expression levels in primary microglia sorted from the hippocampi of mice after intracerebroventricular injection of the anti-GDF15 monoclonal antibody ponsegromab or IgG control and intraperitoneal injection of LPS (10 mg/kg) for 7 days. (D) Representative images of Golgi-Cox staining of hippocampi in mice after intracerebroventricular injection of the anti-GDF15 monoclonal antibody ponsegromab or IgG control and intraperitoneal injection of LPS (10 mg/kg) for 7 days. (E) Representative images of hippocampal pyramidal neuronal dendritic spines in mice after intracerebroventricular injection of ponsegromab or IgG control and intraperitoneal injection of LPS (10 mg/kg) for 7 days. Scale bar, 10 μm. (F) Quantification of dendritic spine densities in the CA1 region of mice after intracerebroventricular injection of ponsegromab or IgG control and intraperitoneal injection of LPS (10 mg/kg) for 7 days. n = 3 mice per group, and in each mouse, 3 fields from the hippocampal CA1 region were analyzed. **P < 0.01. ***P < 0.001

Discussion

The findings of this study demonstrate a unique role for GDF15 during the early phase of SAE (Fig. 8). During sepsis, GDF15 was found to be produced mainly by activated microglia. This was associated with synaptic loss and induced phagocytosis-mediated synaptic pruning in an autocrine manner resulting from microglial activation. Treatment with a GDF15-neutralizing antibody to block the action of GDF15 in the brains of septic mice was observed to result in significant improvements in sepsis-induced neuronal and cognitive dysfunction. These results suggest that GDF15 plays an indispensable function in the development of SAE and underscore its promise as a potential therapeutic focus for the management of sepsis-related cognitive impairment.

Fig. 8

Model of GDF15 regulation of microglial-mediated neuroinflammation and phagocytosis in septic mice

Previous research has documented elevated GDF15 levels in the plasma of patients with sepsis [21, 35], and the plasma GDF15 content has been proposed to possess predictive significance, with elevated levels linked to increased mortality [21]. Despite these findings, the role of GDF15 in sepsis remains ambiguous. In vivo studies have suggested that GDF15 has immunomodulatory actions. For example, research on LPS-induced sepsis has suggested that GDF15 may have anti-inflammatory functions, as GDF15 deficiency exacerbates the inflammatory response and worsens LPS-induced renal and cardiac injury, whereas its overexpression can protect against LPS-induced organ dysfunction [36]. Conversely, bone marrow chimeras with GDF15 deficiency in the bone marrow compartment showed protection against atherosclerosis and reduced macrophage levels in atherosclerotic plaques, implying that GDF15 might play a pro-inflammatory role in this context [37]. However, Luan et al. observed no significant changes in pathogen control or inflammatory cytokine levels after GDF15 blockade, indicating that the effects of GDF15 may be associated with increased tissue tolerance rather than enhanced immunity [38]. Further, Breen et al. demonstrated that inhibition of GDF15, whether acute inhibition with neutralizing antibodies or chronic genetic ablation, did not improve survival in LPS-induced sepsis [22]. These findings cast doubt on the idea that GDF15 plays a role in regulating sepsis tolerance. These diverse and partially opposing functions proposed for GDF15 offer no clarity on its role in sepsis, and additional studies are required to thoroughly clarify the role of GDF15 in sepsis and the underlying mechanisms. The findings of this study align with earlier reports indicating the swift increase of GDF15 during sepsis. Specifically, this study is the first to show that CSF GDF15 levels are elevated in LPS-induced septic mice and promote local inflammation in the brain. The study findings provide clear and convincing evidence that GDF15, secreted by activated microglia, is responsible for the exacerbation of microglial activation and phagocytosis during SAE, as microglial activation and microglial phagocytosis-mediated synaptic elimination could be alleviated by neutralization of GDF15 using a specific blocking antibody. Moreover, silencing of GDF15 in microglial cells effectively suppressed microglial activation, reducing both the inflammatory reaction and microglial phagocytosis.

Recent investigations have indicated the involvement of microglial activation in SAE pathogenesis, with several potential therapeutic targets suggested by recent studies. Notably, the development of the NLR family pyrin domain containing 3 (NLRP3) inflammasome [39], increased cytokine expression [40], and activation of the complement system [41] have been identified as key areas of interest. Neuroinflammation is increasingly recognized as a critical driver of SAE pathogenesis, leading to profound brain dysfunction. In sepsis, inflammatory substances and signals travel to various areas of the brain through various pathways, including physiological fluids and nerves, resulting in brain inflammation and significant activation of microglial cells [42]. Overactivation of microglia and other immune cells can harm both neurons and endothelial cells, as well as damaging the BBB and exacerbating brain dysfunction [43]. Recent research has shown that the prevention of microglial activation may partially alleviate the cognitive impairment associated with SAE [16, 34]. Consistent with these findings, the present research demonstrated that inhibition of GDF15, both experimental studies conducted in living organisms and those performed in a controlled environment, resulted in decreased microglial activation and alleviated sepsis-induced cognitive decline.

Apart from preventing neuroinflammation, reducing microglia-dependent phagocytosis may contribute to the therapeutic benefits of inhibiting cerebral GDF15 in SAE. Pathway enrichment analysis of the DEGs showed significant enrichment in GO-BP terms such as “pseudopodium organization,” and “pseudopodium assembly,” supporting altered microglial migration following silencing of GDF15 in BV2 cells (Fig. S6A). GO-CC terms such as “endosome,” “lysosome,” “endocytic vesicle,” and “phagocytic vesicle” were also enriched, suggesting altered microglial phagocytosis (Fig. S6C). KEGG pathway analysis further confirmed this with significant enrichment in the “phagosome” and “lysosome” categories, thus corroborating the findings of reduced microglial phagocytosis in GDF15-silenced BV2 cells. Furthermore, reduced microglial phagocytosis was observed in GDF15-silenced primary microglia as well as decreased microglia-mediated synaptic engulfment in anti-GDF15 mAb-treated mice, resulting in improvements in neurocognitive function.

Microglia are integral to both neuroinflammation and synaptic pruning, and they are also a critical source of complement factors [44]. Evidence indicates that microglial activation and engagement of the complement system are pivotal in mediating synaptic pruning following endotoxemia induced by LPS [40]. Recent research has also indicated the initial participation of complement factor C1q in synaptic tagging after experimental sepsis, with microglia being key in the clearance of C1q-tagged synapses during the development of SAE [34]. Consistent with these findings, the present study demonstrated that silencing GDF15 in BV2 microglia cells resulted in a downregulation of genes linked to microglial activation and migration, such as Aif1. Moreover, the expression of the complement factor C1q components, C1qb and C1qc, was also decreased, underlining the relationship between GDF15, microglia, and complement-mediated synaptic damage.

GDF15 has garnered attention as a biomarker for a range of pathological conditions, including chronic inflammation and cancer, as well as cardiovascular, liver, kidney, and neurological diseases [17, 45]. Elevated GDF15 levels have been consistently linked to increased mortality and disease progression in various conditions. Specifically, studies on aging and age-related disorders have proposed GDF15 as a potential biomarker for cognitive aging and dementia [25]. In the context of SAE, several potential biomarkers have been investigated, including neuron-specific enolase (NSE), Tau, glial fibrillary acidic protein (GFAP), S-100β, and ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) [46]. However, the diagnostic accuracy of these biomarkers remains low. In addition, studies using inflammatory-associated biomarkers to assess delirium in critically ill patients have shown conflicting results. To date, the diagnosis of SAE relies primarily on clinical assessments, and there are no reliable biomarkers for identifying and evaluating the severity of SAE. The present study demonstrates that peripheral administration of LPS results in rapid induction of elevated GDF15 levels in the CSF and circulation of septic mice, indicating that GDF15 could potentially act as a marker for SAE. Nonetheless, additional experimental and clinical investigations are required to clarify the exact function of GDF15 in SAE diagnosis and evaluation of severity.

Conclusions

In conclusion, the results of this research indicated that the injection of an anti-GDF15 monoclonal antibody into the lateral ventricle significantly improved cognitive and memory impairments, decreased microglial activation, and enhanced synaptic activity in SAE. These therapeutic benefits seem to be influenced, at least in part, by the regulation of the NF-κB pathway. These results indicate that targeting GDF15 in the CNS may offer a promising therapeutic strategy for the early intervention and management of SAE.