LXB₄ Treatment Reduces Astrocyte Reactivity in Response to Cytokines and Ocular Hypertension

Abstract

Purpose : We recently proposed that therapeutic administration of endogenous lipoxin B₄ (LXB₄) provides marked neuroprotection by reducing RGC loss and preventing Müller glia cell reactivity in experimental glaucoma. We investigated whether LXB₄ treatment reduces astrocyte reactivity in response to models of cytokine-induced stress, and both acute and chronic ocular hypertension (OHT).

Methods : Primary human brain astrocytes (HBA) were treated with IL-1α, TNF- α, and C1q cytokines for 24 hrs to induce astrocyte reactivity. HBA were treated with 1μM of LXB₄ for 20 mins prior to cytokine treatment. After 24 hrs, cells were collected for qPCR, immunofluorescence, and LC/MS/MS-based lipidomic analysis. The protective activity of LXB₄ was investigated in vivo using the mouse Silicone-oil model of acute and chronic OHT (n=10). Astrocyte reactivity in retinas and optic nerves (ON) were analyzed by RNA-seq and qPCR.

Results : HBA qPCR data showed significant upregulation of reactive astrocyte markers including Lcn2, Serping1, and C3 following cytokine treatment (p<0.05, n=4) compared to controls. LXB₄ treatment downregulated astrocyte reactivity markers. Immunofluorescence data was consistent with the qPCR findings. Reactive astrocytes also showed increased 5-Lipoxygenase (5-LOX) expression compared to controls. LXB₄ treatment reduced 5-LOX in reactive astrocytes. Lipidomic analysis of HBA culture media established the formation of lipoxin pathway intermediates (5-HETE, 12-HETE, 15-HETE), PUFAs, and lipoxins (LXA₄ and LXB₄). More importantly, reactive astrocytes stopped generating LXA₄, but LXB₄ treatment restored LXA₄ formation. RNA-seq analysis performed in the retina and ON revealed significant upregulation of astrocyte markers in response to acute OHT at two weeks (p<0.05, n=3). Expression of Gfap, Lcn2, Steap4, Serping1, Gbp2, and Fbln5 were upregulated in chronic mild OHT at 2, 4, and 8 weeks but significantly downregulated with LXB₄ treatment in the retina (p<0.05, n=4).

Conclusions : We demonstrate that the LXB₄ downregulates the reactivity of astrocytes in response to cytokine-induced activation and chronic OHT, identifying a novel mechanism. Moreover, the expression of the lipoxin pathway in the human brain astrocytes, along with the mouse retina and optic nerve, indicates that this neuroprotective pathway has the potential to influence neurodegenerative disease, including glaucoma.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.