Translatome analysis reveals microglia and astrocytes to be distinct regulators of inflammation in the hyperacute and acute phases after stroke

Abstract

Neuroinflammation is a hallmark of ischemic stroke, which is a leading cause of death and long-term disability. Understanding the exact cellular signaling pathways that initiate and propagate neuroinflammation after stroke will be critical for developing immunomodulatory stroke therapies. In particular, the precise mechanisms of inflammatory signaling in the clinically relevant hyperacute period hours after stroke have not been elucidated. We used the RiboTag technique to obtain astrocyte and microglia-derived mRNA transcripts in a hyperacute (4 hours) and acute (3 days) period after stroke, as these two cell types are key modulators of acute neuroinflammation. Microglia initiated a rapid response to stroke at 4 hours by adopting an inflammatory profile associated with the recruitment of immune cells. The hyperacute astrocyte profile was marked by stress response genes and transcription factors, such as Fos and Jun, involved in pro-inflammatory pathways such as TNF-α. By 3 days, microglia shift to a proliferative state and astrocytes strengthen their inflammatory response. The astrocyte pro-inflammatory response at 3 days may be driven by the upregulation of the transcription factors C/EBPβ, Spi1, and Rel, which comprise 25% of upregulated transcription factor-target interactions. Surprisingly, few sex differences across all groups were observed. Expression and log2fold data for all sequenced genes are available on a user-friendly website for researchers to examine gene changes and generate hypotheses for stroke targets. Taken together our data comprehensively describe the astrocyte and microglia-specific translatome response in the hyperacute and acute period after stroke and identify pathways critical for initiating neuroinflammation.

A role for astrocytic insulin-like growth factor I receptors in the response to ischemic insult

“…52 Recent data reinforce the need to separately analyze a possible differential role of IGF-IR in astrocytes and microglia in the response to stroke. 53 GFAP-IGF-IR KO mice showed cellular and molecular changes after ischemia compatible with aggravated responses to damage. Firstly, there was increased expression of a total of 20 genes involved in inflammatory, cell-adhesion and angiogenic pathways, which likely reflects potentiation of these putatively protective mechanisms.…”

Role of microglia in stroke

“…It is estimated that microglia start to produce pro‐inflammatory cytokines TNF‐α and IL‐1β at the peri‐infarct regions within the first hours after stroke onset (Lambertsen et al, 2012). Among other inflammatory mediators, strong upregulation of pro‐inflammatory CC motif chemokine ligand 3 ( Ccl3 ) and Ccl4 mRNA was detected 4 h after induction of permanent ischemia in mice (Hernandez et al, 2023). Both chemokines signal through CCR5 receptor, which is involved in immune cell recruitment and mediates complex effects in brain ischemia (Jing et al, 2023).…”

“…Both chemokines signal through CCR5 receptor, which is involved in immune cell recruitment and mediates complex effects in brain ischemia (Jing et al, 2023). Notably, brain injury downregulates the expression of typical homeostatic genes, including Tmem119, P2ry12, Sall1 , and Gpr34 (Hernandez et al, 2023; Mercurio et al, 2022; Paolicelli et al, 2022). However, antibodies against TMEM119 or P2Y12 can still help to differentiate microglia from infiltrating macrophages by immunofluorescence (e.g., Otxoa‐de‐Amezaga et al, 2019).…”

C/EBPβ: A transcription factor associated with the irreversible progression of Alzheimer's disease

“…The activation of astrocytes is influenced by various stimuli, including lipopolysaccharide (LPS), interleukin‐1β (IL‐1β), and tumor necrosis factor‐alpha (TNF‐α), which induce the expression of C/EBPβ and C/EBPδ genes in primary astrocytes, thereby promoting astrocyte activation 40,41 . Notably, LPS concentration dependently upregulates the expression of IL‐1β and TNF‐α in astrocytes 42 .…”