Exosomes and their genetic contents can regulate a variety of cellular pathways through regulation of the expression of multiple target genes in recipient cells 
. In this regard, exosomes have been suggested to function as immune-response modifiers because these vesicles are secreted by many types of tumors cells. Exosomes were previously found to be secreted in saliva 
; although, no physiological function was assigned. Exosomes are released into the saliva via either ductal or acinar cells 
. Essentially salivary glands have been implicated in a constitutive-like secretory pathway involved in secretion of exosomal-like vesicles. These secretory vesicles are derived directly from the trans-Golgi or involve elements of the endosomal-lysosomal trafficking pathway 
. In this study, we isolated saliva exosomes and showed that these vesicles were, in fact, physiologically active. Consistent with previous EM images of exosomes in body fluids 
, ultrastructural examination of saliva exosomes revealed small vesicles with diameters <100 nm and a unique “cup-like” shape, which are both characteristic features of exosomes. AFM also revealed the ultrastructural features and distribution of the exosomes.
In addition, microarray analysis indicated the presence of mRNA inside the exosomes, and these nucleic acids were protected against ribonucleases in saliva. Furthermore, the exosomal RNA analysis of Valadi et al. 
demonstrated that virtually no ribosomal RNA was present and that most of the RNA molecules were <200 nucleotides in length. Moreover, saliva exosome RNA exhibited characteristic features similar to mast cell-derived exosomal RNA. Finally, RNA present in exosomes was functional as modulation of gene expression was observed in keratinocytes incubated with the exosomes. This finding was in accord with recent reports that exosomes can transfer mRNA horizontally to neighboring cells 
. The notion that exosome RNA is delivered to other cells provides added functional significance to salivary exosomes.
The functions of exosomes should be reflected by their proteins and mRNA molecules, which originate from endocytic release. Because exosomes are formed as MVBs, these particles likely contain factors required for MVB formation and protein sorting. Analysis of exosomes derived from human mast cells, dendritic cells, and epithelial cells as well as other cell types revealed the presence of common and cell type-specific proteins and mRNA. For example, the aquaporin family of proteins was especially enriched in exosomes derived from body fluids such as urine and amniotic fluid 
. Human saliva and saliva exosomal proteins have been identified and cataloged in detail 
including aquaporins, cytoskeleton proteins, and membrane proteins, which have also been found in exosomes from other cell types. Importantly, sorting of disease-specific proteins into exosomes is quite useful for diagnostic applications 
. The molecular factors and mechanisms behind this cell-specific sorting process in exosomes are still unknown, and such an analysis may help their translational utility.
Proteomics analysis of saliva ductal fluids revealed 1,166 proteins, including various membrane-bound proteins 
. Interestingly, the aquaporin protein family, which is involved in water flow through membranes, was identified in saliva. Aquaporins have been identified on both the apical and basolateral membranes of secretory acinar cells of salivary glands 
. Surprisingly, AQP1 and AQP2 proteins identified in urine exosomes via secretion through renal ductal cells were implicated in pathophysiological processes in urinary epithelial cells 
. Additionally, decreases in aquaporin expression are linked to various kidney and pancreas diseases, while reduced aquaporin expression in salivary glands is linked to Sjögren's syndrome 
. Furthermore, the annexin family of proteins bind to intracellular membranes and is involved in intracellular membrane fusion 
. Association of annexins with exosomes may result from the presence of phosphatidylserine in these vesicles 
. Interestingly, Annexins and Alix proteins are reportedly present in saliva exosomes 
. Differential expression of annexin A1, annexin A2, moesin, and OS-9 proteins indicated the influence of saliva exosomes in oral keratinocytes. Interestingly, presence of annexin A1 mRNA in saliva exosomes may translate protein in the recipient cell gene expression. In addition, moesin, which is an actin-binding protein of the ERM family in exosomes, has been demonstrated to play a role in de novo
actin assembly on phagosomal membranes 
. Further, moesin has been reported to be present in B cell-derived exosomes 
and breast milk 
Clearly, exosome-like microvesicles are present in body fluids such as saliva, blood, amniotic fluid, and pleural effusions under both healthy and disease conditions; however, the origin of these exosomes and their intended destination for stimulation of distal cells remains unclear. Here, we demonstrated that saliva exosomes can be taken up by oral keratinocytes. Interestingly, our observation establishes another dimension of cell-cell communication of body fluid exosomes. Notably, keratinocytes are able to secrete exosomes and externalize stratifin protein, which is a potent stimulant of metalloproteinases in fibroblasts 
. Arguably, both keratinocytes and saliva exosomes engage in cell-cell communication, and the possibility exists that part of the saliva exosomes originates from oral keratinocytes. Whether these interactions are involved in a novel mechanism of cell-cell communication is an intriguing, yet unanswered, question. Our studies do not directly identify the functional consequence of mRNA release via exosomes; however, saliva exosomes carrying mRNA transcripts of these specific altered proteins suggest that these RNAs could possibly be translated into proteins at their new location. Also, recent studies suggest an important role for exosomes in the modulation of host gene expression levels. Interestingly, exosomes purified from mast cells 
and neuronal cells 
are enriched in mRNA molecules that stimulate and alter gene expression of recipient cells. These data have led to the suggestion that secreted exosomes expressing relevant mRNAs may play a role in the generation of new genes and modulate gene expression of recipient cells. Indeed, annexin and moesin are overexpressed in keratinocytes following incubation with saliva exosomes. We cannot rule out the presence of lipids and proteins in saliva exosomes that also can trigger gene expression at their new recipient cells. We have observed several ceramide lipid species in saliva exosomes that could potentially have impact on oral keratinocytes (unpublished observation). Finally, the source of these exosomes in saliva, however, is probably heterogeneous, and formal demonstration that salivary glands secrete exosomes in vivo
awaits further analyses.
In summary, saliva exosomes may regulate cell-cell environment by altering their gene expression. This study extends our knowledge about human saliva exosomes. In addition to genetic regulation, as mentioned above, saliva exosomes are involved in protecting nucleic acids against nucleases in the oral cavity. Thus, saliva exosomes, like other types of exosomes, clearly have multiple functions. We expect that more saliva exosome targets will be identified in the near future using the same proteomic approach for various systemic diseases. These discoveries will allow us to better understand the molecular basis of oral diseases. The studies of Valadi 
, Skog 
, and Ratajczak 
as well as the present study open up a new research perspective on the use of exosomal transfer of mRNA to target another cell type. In particular, the results of the present study indicate that exosomes derived from human saliva activate or modulate gene expression in oral keratinocytes.