Particles in Raw Sheep Milk Can Modulate the Inflammatory Response in THP-1, a Human Monocyte Cell Line, In Vitro

Abstract

Background: The UK dairy sheep industry is relatively small but growing, particularly for cheese and yogurt products. Anecdotally, sheep milk (SM) may be better tolerated by humans than cows’ milk and could have environmental as well as health benefits. All milk contains sub-micron particles called extracellular vesicles (EVs) which are mainly derived from the mammary epithelium. Physiologically, milk-derived EVs are thought to aid in the development of infant immunity and the microbiome, but may also have health benefits to adult humans. The purpose of this study was to determine whether EVs could be isolated from raw sheep milk and whether they have any effect on inflammatory responses in THP-1, a human monocyte cell line, in vitro. Methods: Using sequential ultracentrifugation, vesicles of <1 µm (LEV) followed by <200 nm (sEVs) were isolated from six individual sheep during mid-lactation. RNA was extracted and microRNA analyzed by RTqPCR for sequences previously identified in cows’ milk. Human THP-1 monocytes were differentiated into macrophages and incubated with SM-derived LEVs and sEVs in the presence of pro-inflammatory LPS to measure the effects on the secretion of the chemokine CCL-2 or in the presence of DMNQ and fluorescent dihydrorhodamine-1,2,3 to measure reactive oxygen species. Results: LEVs induced an increase in ROS in both monocytes and macrophages, whilst sEVs decreased DMNQ-mediated ROS in macrophages but not monocytes. Interestingly, the LEVs did not induce CCL2 release; however, they increased LPS-induced CCL2 secretion in monocytes but not macrophages. miR26a, miR92a, miR125b, miR155 and miR223 were identified in both sEVs and LEVs by RT-qPCR and could be responsible for the modulation of ROS and CCL2 expression. Conclusions: These findings suggest that like cows’ milk, sheep milk contains EVs, and they can influence human monocyte/macrophage responses, and so is worthy of further investigation for its potential human- and non-human-animal health benefits.

Contribution of Extracellular Particles Isolated from Morus sp. (Mulberry) Fruit to Their Reported Protective Health Benefits: An In Vitro Study

“…DM and WM were photographically assessed, and pH measured, to confirm that they were of different species ( Supplemental data ). Following the modification of our laboratory’s well-established ultracentrifugation protocol [ 25 ], we isolated EVs from both WM and DM. As shown in Figure 1 , we were able to separate two populations: a population of LEVs which could be identified by the binding of annexin V to the surface and detection via flow cytometry ( Figure 1 C,F) and a population of sEVs which had a typical peak between 136 nm (WM sEVs) and 149 nm (DM sEVs; Figure 1 D,G) per nanotracker analysis (NTA).…”

“…As previously described in our laboratory [ 25 , 39 ], THP-1 monocytes were pelleted, washed, counted, and then incubated with 5 μM of dihydrorhodamine-1,2,3 (DHR) for 10 min in the dark. They were then seeded onto 96-well plates at 10,000 cells per well.…”