Role of TIM-4 in exosome-dependent entry of HIV-1 into human immune cells

Abstract

Exosomes, 30–200 nm nanostructures secreted from donor cells and internalized by recipient cells, can play an important role in the cellular entry of some viruses. These microvesicles are actively secreted into various body fluids, including blood, urine, saliva, cerebrospinal fluid, and breast milk. We successfully isolated exosomes from human breast milk and plasma. The size and concentration of purified exosomes were measured by nanoparticle tracking, while Western blotting confirmed the presence of the exosomal-associated proteins CD9 and CD63, clathrin, and T cell immunoglobulin and mucin proteins (TIMs). Through viral infection assays, we determined that HIV-1 utilizes an exosome-dependent mechanism for entry into human immune cells. The virus contains high amounts of phosphatidylserine (PtdSer) and may bind PtdSer receptors, such as TIMs. This mechanism is supported by our findings that exosomes from multiple sources increased HIV-1 entry into T cells and macrophages, and viral entry was potently blocked with anti-TIM-4 antibodies.

Biological Function of Exosomes as Diagnostic Markers and Therapeutic Delivery Vehicles in Carcinogenesis and Infectious Diseases

“…There are several common techniques that are used to determine the quantity, morphology, and size of exosomes following purification. Exosomes can be characterized using the following techniques: flow cytometry [116], electron microscopy (EM) [117], NanoSight tracking analysis (NTA) [92,117,118], Raman spectroscopy (RS) [119], Western blot (WB) [42,92,120], and/or ExoCarta database [117,121].…”

“…NTA measures the concentration and size distribution of exosomes [92,117,126,127]. An NTA device is composed of a laser light scattering microscope connected to a sensitive chargecoupled device camera, a complementary metal-oxide-semiconductor camera, a hydraulic pump, a measuring chamber, and an analytical software [117,121].…”

“…First, cell culture is subjected to a low speed centrifugation using a Sorvall RT600 centrifuge with a swinging bucket rotor (Thermo Fisher Scientific). This is applied to remove cells and apoptotic debris [92,93]. Next, a higher speed is used to administer and eliminate larger vesicles, whereas,…”

“…Nanomedicines the remaining media is re-suspended in phosphate buffered saline. Lastly, a high speed of centrifugation using a SW41T1 swinging rotor in a Beckman Coulter (Brea, CA, USA) (Optima L-70 K ultracentrifuge) is performed to precipitate exosomes; and the exosome pellet is stored at −80°C until further use [92].…”

“…Filtration is a size-based technique that is often used in combination with ultracentrifugation, as depicted in Figure 3, for the isolation of exosomes in in vitro studies [92][93][94]. Depending on the size of vesicles, filtration is applied to separate exosomes from proteins and other particles [79].…”