Characterization of exosomes in patients with ocular toxoplasmosis

Abstract

Purpose : Ocular toxoplasmosis (OT) is caused by the intracellular protozoan Toxoplasma gondii and is the main cause of posterior uveitis worldwide. Extracellular vesicles (EVs) are related to the dissemination of the pathogen and to the regulation of the host immune system in infectious diseases. However, the role of EVs in ocular toxoplasmosis remains unclear. The aim of this study is to identify and to characterize EV markers isolated from aqueous humor (AH) and plasma from patients with ocular toxoplasmosis (OT) and cataract (control group).

Methods : AH and plasma were collected from six patients with active OT and six cataract patients at the Department of Ophthalmology, UNIFESP / EPM, Brazil. All patients were clinically evaluated. EVs were isolated using the exoRNeasy Serum / Plasma Maxi Kit membrane affinity column method (Qiagen, Valencia, CA, USA), quantified and characterized for size and distribution using Nanosight. For validation of EVs, western blotting was performed for CD63 and TSG101 markers. Mass spectrometry was then performed to characterize the proteomic profile of EVs from all samples.

Results : The presence of EVs in plasma and aqueous humor samples from OT patients (n = 6) and cataract patients (control group, n = 6) was confirmed. Nanosight data showed a mean concentration of EVs in AH was significantly lower in OT (ranged from 1.0 × 10¹¹ to 8.6 × 10¹¹ particles/mL) compared to cataract (ranged from 2.9 × 10¹¹ to 1.3 × 10¹² particles/mL) (P = 0.03); However, in the plasma, all groups presented a similar concentration of EVs. Size distribution of EVs was the same in the AH and plasma of OT patients when compared to the cataract group. Plasma samples were all positive for TSG101 and CD63 markers, while aqueous humor samples were positive only for CD63 marker. Cataract patient samples were negative for both markers, CD63 and TSG101.

Conclusions : To the best of our knowledge, this is the first time that markers of EVs in AH and plasma of patients with ocular toxoplasmosis were identified and size and concentration were characterized. This may represent a highly specific marker for the diagnosis of atypical clinical presentations of ocular toxoplasmosis.

This is a 2020 ARVO Annual Meeting abstract.