Intestinal neutrophil extracellular traps promote gut barrier damage exacerbating endotoxaemia, systemic inflammation and progression of diabetic retinopathy in type 2 diabetes
Authors: Floyd Jason L. , Prasad Ram , Dupont Mariana D. , Adu-Rutledge Yvonne , Anshumali Shambhavi , Paul Sarbodeep , Li Calzi Sergio , Qi Xiaoping , Malepati Akanksha , Johnson Emory , Jumbo-Lucioni Patricia , Crosson Jason N. , Mason John O. , Boulton Michael E. , Welner Robert S. , Grant Maria B. 
Published: January 2025

Abstract

Aims/hypothesis
Within the small intestine, neutrophils play an integral role in preventing bacterial infection. Upon interaction with bacteria or bacteria-derived antigens, neutrophils initiate a multi-staged response of which the terminal stage is NETosis, formation of protease-decorated nuclear DNA into extracellular traps. NETosis has a great propensity to elicit ocular damage and has been associated with diabetic retinopathy and diabetic macular oedema (DME) progression. Here, we interrogate the relationship between gut barrier dysfunction, endotoxaemia and systemic and intestinal neutrophilia in diabetic retinopathy.

Methods
In a cohort of individuals with type 2 diabetes (n=58) with varying severity of diabetic retinopathy and DME, we characterised the abundance of circulating neutrophils by flow cytometry and markers of gut permeability and endotoxaemia by plasma ELISA. In a mouse model of type 2 diabetes, we examined the effects of diabetes on abundance and function of intestinal, blood and bone marrow neutrophils, gut barrier integrity, endotoxaemia and diabetic retinopathy severity. Pharmacological inhibition of NETosis was achieved by i.p. injection of the peptidyl arginine deiminase 4 inhibitor (PAD4i) GSK484 daily for 4 weeks between 6 and 7 months of type 2 diabetes.

Results
In human participants, neutrophilia was unique to individuals with type 2 diabetes with diabetic retinopathy and DME and was accompanied by heightened circulating markers of gut permeability. At late-stage diabetes, neutrophilia and gut barrier dysfunction were seen in db/db mice. The db/db mice exhibited an increase in stem-like pre-neutrophils in the intestine and bone marrow and a decrease in haematopoietic vascular reparative cells. In the db/db mouse intestine, enhanced loss of gut barrier integrity was associated with elevated intestinal NETosis. Inhibition of NETosis by the PAD4i GSK484 resulted in decreased abundance of premature neutrophils in the intestine and blood and resulted in neutrophil retention in the bone marrow compared with vehicle-treated db/db mice. Additionally, the PAD4i decreased senescence within the gut epithelium and yielded a slowing of diabetic retinopathy progression.

Conclusions/interpretation
Severity of diabetic retinopathy and DME were associated with peripheral neutrophilia, gut barrier dysfunction and endotoxaemia in the human participants. db/db mice exhibited intestinal neutrophilia, specifically stem-like pre-neutrophils, which was associated with elevated NETosis and decreased levels of vascular reparative cells. Chronic inhibition of NETosis in db/db mice reduced intestinal senescence and NETs in the retina. These changes were associated with reduced endotoxaemia and an anti-inflammatory bone marrow milieu with retention of pre-neutrophils in the bone marrow and increased gut infiltration of myeloid angiogenic cells. Collectively, PAD-4i treatment decreased gut barrier dysfunction, restoring physiological haematopoiesis and levels of haematopoietic vascular reparative cells.

Graphical Abstract