CD137L Inhibition Ameliorates Hippocampal Neuroinflammation and Behavioral Deficits in a Mouse Model of Sepsis-Associated Encephalopathy

NeuroMolecular Medicine https://doi.org/10.1007/s12017-023-08764-z RESEARCH CD137L Inhibition Ameliorates Hippocampal Neuroinflammation and Behavioral Deficits in a Mouse Model of Sepsis‑Associated Encephalopathy Fang Qiu1,2,3 · Yueming Liu1 · Yang Liu1 · Zhuyun Zhao1 · Lile Zhou1 · Pengfei Chen4 · Yunbo Du5 · Yanmei Wang5 · Huimin Sun6 · Changchun Zeng6 · Xiaokang Wang7 · Yuqiang Liu8 · Haobo Pan2 · Changneng Ke1 Received: 17 May 2023 / Accepted: 20 September 2023 © The Author(s) 2023 Abstract Anxiety manifestations and cognitive dysfunction are common sequelae in patients with sepsis-associated encephalopathy (SAE). Microglia-mediated inflammatory signaling is involved in anxiety, depression, and cognitive dysfunction during acute infection with bacterial lipopolysaccharide (LPS). However, the molecular mechanisms underlying microglia activation and behavioral and cognitive deficits in sepsis have not been in fully elucidated. Based on previous research, we speculated that the CD137 receptor/ligand system modulates microglia function during sepsis to mediate classical neurological SAE symptoms. A murine model of SAE was established by injecting male C57BL/6 mice with LPS, and cultured mouse BV2 microglia were used for in vitro assays. RT-qPCR, immunofluorescence staining, flow cytometry, and ELISA were used to assess microglial activation and the expression of CD137L and inflammation-related cytokines in the mouse hippocampus and in cultured BV2 cells. In addition, behavioral tests were conducted in assess cognitive performance and behavioral distress. Immunofluorescence and RT-qPCR analyses showed that hippocampal expression of CD137L was upregulated in activated microglia following LPS treatment. Pre-treatment with the CD137L neutralizing antibody TKS-1 significantly reduced CD137L levels, attenuated the expression of M1 polarization markers in microglia, and inhibited the production of TNF-α, IL-1β, and IL-6 in both LPS-treated mice and BV2 cells. Conversely, stimulation of CD137L signaling by recombinant CD137-Fc fusion protein activated the synthesis and release of pro-inflammatory cytokines in cultures BV2 microglia. Importantly, open field, elevated plus maze, and Y-maze spontaneous alternation test results indicated that TKS-1 administration alleviated anxiety-like behavior and spatial memory decline in mice with LPS-induced SAE. These findings suggest that CD137L upregulation in activated microglia critically contributes to neuroinflammation, anxiety-like behavior, and cognitive dysfunction in the mouse model of LPS-induced sepsis. Therefore, therapeutic modulation of the CD137L/ CD137 signaling pathway may represent an effective way to minimize brain damage and prevent cognitive and emotional deficits associated with SAE. Keywords CD137L · Microglia · Inflammation · Anxiety · Cognition · TKS-1 Introduction Sepsis, defined as life-threatening multiorgan dysfunction caused by a dysregulated host response infection, is a leading cause of death in intensive care units (Singer et al., 2016). A recent report indicates a pooled incidence of 189 hospital-treated adult sepsis cases per 100,000 personyears with an estimated mortality of 26.7% (FleischmannStruzek et al., 2020). Sepsis-associated encephalopathy Extended author information available on the last page of the article (SAE) is a central brain dysfunction caused by peripheral or systemic inflammatory infection. About 70% of patients with severe sepsis develop SAE, which increases mortality, prolongs hospitalization, and determines excessive consumption of medical resources (Helbing et al., 2018). The symptoms of SAE range from delirium to coma (Gofton & Young, 2012). Therefore, early diagnosis and intervention are very important for the treatment of patients with sepsis. However, the pathological mechanisms of SAE have not been fully elucidated. Microglia, the resident macrophages of the central nervous system (CNS), are closely associated with SAE 13 Vol.:(0123456789) NeuroMolecular Medicine pathogenesis (Li et al., 2020). Uncontrolled neuroinflammation is the main feature of SAE, and the main cause of abnormal brain function and neuronal death associated with this condition (Schwalm et al., 2014). Experiments in both humans and animals have found that microglia activation during SAE is also closely associated with emotional distress, anxiety, depression and cognitive impairment (Lemstra et al., 2007; Szollosi et al., 2018). In animal models of sepsis, microglia are typically activated by lipopolysaccharide (LPS) exposure and play a key role in mediating SAE-related behavioral changes (Osterhout et al., 2022; Cao et al., 2021). Microglia activation states can be classified into two major phenotypes, M1 and M2 (Colton, 2009; Han et al., 2018). M1 microglia produce pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α associated with tissue and nerve injury, whereas M2 microglia produce anti-inflammatory cytokines (IL-4, and IL-10), and wound-healing genes, including arginase-1 (Arg1), which promote tissue and nerve repair (Cherry et al., 2014; Jin & Yamashita, 2016). In sepsis, activated microglia eminently display an M1 phenotype that contributes to disrupting neuronal function and triggers neuronal damage by enhancing the production of inflammatory mediators (Xin et al., 2023). In turn, a recent study showed that cerebroventricular mitochondrial transplantation alleviated brain dysfunction and attenuated behavioral deficits in a mouse model of SAE by inducing microglial M2 polarization (Yan et al., 2020). Therefore, the balance between these contrasting microglial phenotypes is thought to play an essential role in the pathogenesis of SAE (Ren et al., 2020). CD137L (TNFSF9, 4-1BBL) is a transmembrane glycoprotein that belongs to the tumor necrosis factor (TNF) ligand family and plays an important role in regulating the adaptive immune response (Watts, 2005; Croft, 2009). It is expressed on antigen-presenting cells (APCs), and its expression levels increase upon APC activation (Zeng et al., 2019). CD137L cross-links its receptor CD137 (TNFRSF9, 4-1BB), also a transmembrane glycoprotein, expressed on the surface of activated T cells, NK cells, dendritic cells (DC), and vascular endothelial cells. CD137 is a member of the TNF receptor superfamily and a potent co-stimulatory molecule in activated T cells. CD137 signaling in T cells promotes survival, enhances proliferation and effector functions, and confers metabolic sufficiency (Etxeberria et al., 2020; Tu et al., 2012). Signaling through the CD137 receptor/ligand system was shown to activate microglia, contributing to neuroinflammation and neurodegeneration in various CNS pathologies. Blocking CD137L signaling reduces the release of neuroinflammatory mediators, and CD137L knockout mice are largely prevented from developing neurodegenerative disease (Wong & Schwarz, 2020; Mak et al., 2019; Ma et al., 2013). However, the mechanism by which CD137 (...truncated)