Research Article: Neutrophil–microglia communication through the C3–C3aR1 axis contributes to secondary neuroinflammation after traumatic brain injury

Abstract:
Traumatic brain injury (TBI) triggers a rapidly evolving neuroinflammatory response that contributes to secondary brain injury. Although infiltrating leukocytes and resident microglia are both prominent components of this response, the cellular signals that connect peripheral immune-cell recruitment to microglial state remodeling remain incompletely defined. We integrated and reanalyzed publicly available single-cell RNA-sequencing datasets from mouse TBI models to define temporal changes in brain-cell composition and transcriptional states. CellChat and NicheNet were used to prioritize candidate neutrophil-to-microglia signaling pathways. The predicted cellular distribution of C3 and C3aR1 was evaluated by immunofluorescence and flow cytometry. The effects of JR14a treatment on microglial inflammatory responses, blood–brain barrier (BBB) integrity, and neurological outcomes were examined in a mouse TBI model. Single-Cell rEgulatory Network Inference and Clustering (SCENIC), CellOracle, a gene regulatory network inference and in silico transcription-factor perturbation framework, and scTenifoldKnk, a single-cell gene regulatory network-based virtual knockout framework, were used for gene regulatory network inference and in silico transcription-factor perturbation analyses in C3ar1 -high microglia. Analysis of 254,512 cells revealed a transient increase in infiltrating neutrophils during the acute phase of TBI and the emergence of inflammatory microglial states. CellChat and NicheNet independently prioritized C3-associated signaling between neutrophils and Microglia4. C3 expression was enriched in infiltrating neutrophils, whereas C3ar1 was preferentially expressed by inflammatory microglia. Flow cytometry showed that C3aR1-positive microglia expressed higher levels of IL-1? and TNF-? than C3aR1-negative microglia. JR14a treatment reduced microglial inflammatory cytokine production, preserved Claudin-5 expression, limited Evans blue extravasation, and improved neurological and cognitive outcomes after TBI. Exploratory regulatory analyses further prioritized Ets2, Fosl2, and Irf7 as candidate regulators of the C3ar1 -high microglial program. These findings implicate a neutrophil-associated C3/C3a–C3aR1 communication pathway in inflammatory microglial remodeling after TBI and support further investigation of C3aR1-associated signaling as a therapeutic target for secondary neuroinflammation.

Introduction:
Traumatic brain injury (TBI) triggers a rapidly evolving neuroinflammatory response that contributes to secondary brain injury. Although infiltrating leukocytes and resident microglia are both prominent components of this response, the cellular signals that connect peripheral immune-cell recruitment to microglial state remodeling remain incompletely defined.

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