Materials and MethodsIsolation of peripheral blood mononuclear cells and platelets Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtainedmfrom the Sanquin blood bank Nijmegen while platelets were isolated from citrateanticoagulated venous blood collected from healthy adult volunteers as described 91 previously (21). Briefly, PBMCs were isolated by density gradient centrifugation on FicollHypaque (Pharmacia Biotech, Uppsala, Sweden). PBMCs were washed fivetimes with normal saline then resuspended in RPMI-1640 Dutch Modification culture medium(ICNBiomedicals, Costa Mesa,CA) and adjusted to 5 × 1092 cells/mL.

For platelet isolation, 3.2% sodium-citrate-anticoagulated whole blood (Becton Dickinson,Franklin Lakes, NJ) was centrifuged for 15 min at 156 g without brake to obtain platelet-richplasma (PRP). PRP was further centrifuged at 330 g for 15 min to obtain platelet pellets andresuspended in HEPES tyrode buffer.

ResultsWe first investigated whether platelets modulated RSV infection of monocytes. PBMCswere infected with RSV at MOI of 10 in the presence and absence of platelets. Addition ofplatelets significantly decreased RSV infection of monocytes after 48 h of incubation (Fig.1A and Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/vim). CD16 expression on monocytes, a marker for monocyte activation, also tendedto be lower when PBMCs were incubated with RSV in the presence of platelets althoughthe effect was not significant (Fig. 1B, C and Supplementary Fig. S2).

ConclusionCollectively, our current study shows that platelets play an important role in host innatedefenses against RSV by reducing RSV infection of monocytes and monocyte activationin a type I IFN dependent manner. Platelets can also internalize viral particles and maytherefore be important in preventing RSV dissemination and disease exacerbation.