Extracellular Nucleosomes Accelerate Microglial Inflammation via C-Type Lectin Receptor 2D and Toll-Like Receptor 9 in mPFC of Mice With Chronic Stress

Abstract

Damage-associated molecular patterns (DAMPs) are the primary promoter of progressive neuroinflammation and are associated with chronic stress-related emotional disorders. The present study investigated the role and mechanism of extracellular nucleosomes and histones, the newly defined DAMPs, in mice with chronic stress. C57BL/6 mice were exposed to chronic unpredictable mild stress (CUMS) and corticosterone drinking, respectively, for 4 weeks. Negative emotional behaviors were comprehensively investigated. Microglial morphology, oxidative stress, and inflammation, as well as C-type lectin receptor 2D (Clec2d) and Toll-like receptor 9 (TLR9) expression in medial prefrontal cortex (mPFC) were assessed with flow cytometer and cell sorting. Specifically, microglial pro-inflammatory activation and inflammation were further investigated with stereotactic injection of recombinant nucleosomes and histones in mPFC and further evaluated with AAV-Clec2d knocking-down, DNase I, and activated protein C (APC) pretreatment. Moreover, the rescue effect by AAV-Clec2d knocking-down was observed in mice with chronic stress. Mice with chronic stress were presented as obviously depressive- and anxiety-like behaviors and accompanied with significant microglial oxidative stress and inflammation, indicating by reactive oxygen species (ROS) production, primed nuclear factor-κB (NF-κB) signaling pathway, activated NACHT, LRR, and PYD domain–containing protein 3 (NLRP3) inflammasome, and upregulated Clec2d and TLR9 in mPFC, together with histones dictation in cerebrospinal fluid and extracellular trap formation. Stereotactic injection of nucleosomes was contributed to promote microglial inflammation rather than histones in mPFC, indicating that the pro-inflammatory role was derived from extracellular histones-bound DNA but not freely histones. AAV-Clec2d knocking-down, DNase I, and APC were all effective to inhibit nucleosome-induced microglial oxidative stress and inflammation. Moreover, AAV-Clec2d knocking-down in mice with chronic stress exhibited reduced microglial inflammation and improved negative emotional behaviors. Our findings reveal a novel mechanism of DAMP-associated inflammation that extracellular nucleosomes accelerate microglial inflammation via Clec2d and TLR9, and then contribute to chronic stress-induced emotional disorders.

Microglial Responses to Stress-Induced Depression: Causes and Consequences

“…Chronic stress can trigger not only previously well-recognized DAMPs but also extracellular nucleosomes and histones. Increased histones and nucleosomes were found in the cerebrospinal fluid of the CUMS mice, which positively correlated with IL-1β levels in PFC [44]. Higher levels of nucleosomes promoted microglial inflammatory signaling in a C-type lectin receptor 2D (Clec2d)-dependent manner, increasing oxidative stress and IL-1β secretion [44].…”

“…Increased histones and nucleosomes were found in the cerebrospinal fluid of the CUMS mice, which positively correlated with IL-1β levels in PFC [44]. Higher levels of nucleosomes promoted microglial inflammatory signaling in a C-type lectin receptor 2D (Clec2d)-dependent manner, increasing oxidative stress and IL-1β secretion [44]. Knockdown of Clec2d in PFC reduced microglial inflammatory activation and depressive-like behavior in CUMS mice.…”

Chronic Stress-Induced Neuroinflammation: Relevance of Rodent Models to Human Disease

“…A few KD studies have revealed the similar involvement of additional TLRs in chronic stress resilience; KD of cyclic adenosine monophosphate response element-binding protein (CREB) in the HPC of UCMS mice increased resilience to stress and was associated with the downregulation of TLR1 and TLR6 [10]. Increased TLR9, and increased Clec2d, a receptor involved in sensing cellular stress, were observed in the medial prefrontal cortex (mPFC) of UCMS mice, while the KD of the latter inhibited microglial inflammation and reduced stress-induced negative emotional behaviors [88]. This indicates that other TLRs should be investigated in chronic stress and that the TLR-NF-κB pathway is one of the main mechanisms contributing to inflammation and chronic stress.…”

C-type lectin receptor 2d forms homodimers and heterodimers with TLR2 to negatively regulate IRF5-mediated antifungal immunity

“…Lai et al expanded the role of CLEC2D serving as a sensor for cell death to detect histones released from necrotic cells 21 . CLEC2D has been found to carry histone-bound DNA complexes into endosomes, stimulating TLR9-mediated pro-inflammatory responses in macrophages and microglia 21 , 22 . Our recent findings demonstrate that CLEC2D recognizes glucuronoxylomannan from Cryptococcus neoformans to promote the p38-mediated production of arginase-1, leading to the induction of immunosuppressive activity of neutrophilic myeloid-derived suppressor cells (MDSCs), and subsequently inhibiting T cell-mediated anti-fungal responses 23 .…”