Hypothalamic arcuate microglia in sepsis: AgRP circuit engagement and ARHGAP24-dependent remodeling.

BACKGROUND

Sepsis remains a leading cause of death in intensive care, and effective therapies that restore host adaptation are limited. The hypothalamus is a central hub that coordinates endocrine and immune homeostasis, yet how defined hypothalamic microcircuits are engaged in sepsis induced neuroimmune dysregulation is poorly understood. Within the hypothalamus, the arcuate nucleus lies adjacent to the fenestrated median eminence, placing microglia close to circulating inflammatory cues, but how these cells respond during systemic inflammation remains unclear.

METHODS

Polymicrobial sepsis was induced in mice by cecal ligation and puncture (CLP). Arcuate microglial responses were evaluated by immunofluorescence, confocal and electron microscopy, acute slice live imaging, and transcriptomic profiling of the mediobasal hypothalamus using Smart-seq2 of sorted microglia and 10× single-nucleus RNA sequencing (snRNA-seq) with bioinformatic analyses. Microglia were depleted using a Tmem119 driven diphtheria toxin strategy. AgRP neuronal activity was assessed by c-Fos staining, whole-cell patch clamp recordings, and synaptic analysis. AgRP neurons were manipulated by chemogenetics. Sepsis outcomes were assessed by survival, clinical scores, systemic cytokines, endocrine hormones, and open field behavior. ARHGAP24 function was tested by lentiviral knockdown in LPS stimulated BV2 microglia, and Rac1 and Cdc42 signaling was pharmacologically modulated in vitro and via third ventricle delivery in vivo.

RESULTS

CLP rapidly activated arcuate microglia and this activation persisted, accompanied by cytoskeletal remodeling and increased motility. AgRP neurons underwent sustained hyperexcitability after CLP, showing increased firing at both 1 day and 7 days with stage specific electrophysiological remodeling. Microglial depletion reduced AgRP activation, worsened sepsis severity, blunted corticosterone release, impaired inflammatory resolution, and compromised open field performance. Chemogenetic manipulation indicated that AgRP activity tracked sepsis outcomes independent of feeding behavior. snRNA-seq of the mediobasal hypothalamus revealed enhanced microglia-AgRP communication after CLP and identified Arhgap24 as a microglial state associated regulator induced along the activation trajectory. Arhgap24 knockdown enhanced microglial protrusive remodeling and amplified LPS induced cytokine responses. Pharmacological inhibition of Rac1 and Cdc42 using AZA1 restrained microglial remodeling and improved sepsis outcomes, whereas pathway activation using CN02 exacerbated these responses.

CONCLUSION

Arcuate microglia act as central neuroimmune sensors that couple systemic inflammation to sustained functional remodeling of AgRP neurons, thereby linking sepsis to endocrine and behavioral adaptation. ARHGAP24 restrains Rac1/Cdc42 dependent cytoskeletal remodeling in microglia, maintains an adaptive microglial state, and shapes sepsis outcomes.