Functional and Phenotypic Differences between Breast Milk CD14+ Cells Compared with Peripheral Blood CD14+ Populations Stephanie M. Dorosko, DVM, PhD – Geisel School of Medicine at Dartmouth, Lebanon, NH USA Introduction Results 1) Phenotypically, between forty and seventy percent of flow cytometry events from breast milk cell pellets were positive for CD14. 3) Functionally, breast milk CD14+ cells produced significantly lower concentrations of several pro-and anti-inflammatory cytokines compared to CD14+ cells derived from peripheral blood. Moreover, cytokine/chemokine release by breast milk CD14+ cells did not reach peripheral blood levels after 24 hrs exposure to LPS. Breast milk is considered to be protective to the infant gastrointestinal (GI) system as well as nourishing. However, it contains a population of cells expressing the surface marker CD14, a well-recognized receptor for bacterial lipopolysaccharide (LPS).Taking into consideration the facts that: 1) The binding of LPS to CD14 leads to initiation of inflammatory pathways and products, while 2) Breast milk contains an abundant population of CD14+ cells, yet is considered to serve an anti-inflammatory function to protect the infant intestine, we hypothesize that breast milk CD14+ cells differ from peripheral blood CD14+ cells in several important aspects. Methods Subjects: Milk from consenting lactating women between 1 week and 4 months postpartum was collected less than 4 hours post-expression, and the cell population was either prepared for flow cytometry or enriched for CD14+ cells via negative or positive selection using antibody-coated immunomagnetic nanoparticles. Laboratory: Phenotyping was conducted using primary stained antibodies and isotype controls. Functional assays conducted on the CD14+ -enriched populations included: 24hr cell culture with and without LPS exposure followed by cell supernatant cytokine/chemokine analysis by luminex and ELISA, and microarray analysis of mRNA extracted from breast milk CD14+ cells cultured under various conditions, including LPS exposure or co-culture with human intestinal Caco-2 cells cultured on transwell membranes. CD14+ cells derived from peripheral blood were used as controls. In additional, quantitative PCR (qPCR) was conducted on mRNA derived from CD14+ cells positively enriched from breast milk cells and control PBMC. This qPCR was performed after finding a number of genes showing statistically significant differences in expression between breast milk CD14+ cells and control CD14+ cells, as measured by microarray and analyzed using bioinformatics and DAVID analysis software and database. 2) Interestingly, CD14+ cells derived from breast milk were negative for the universal human hematopoetic surface marker CD45, compared to nearly one hundred percent CD45 staining of all peripheral blood cells (as noted in the table above). Additional flow cytometry staining for CD45 using multiple stains confirmed this finding. Messenger RNA was detected at low levels in CD14+ breast milk cells using real time qPCR, corroborating with the flow cytometry results. 4) Bioinformatics analysis of CD14+ enriched cells co-cultured with intestinal Caco-2 cells for 45 minutes demonstrated significant genetic upregulation of caseins α s1, κ, and β, metallothioneins (MT) 1 and 2, metal chelation factors, multiple transcription factors, and host-defense function genes, including pIgR, MHC I and MHC II. Analysis from 90-minute co-cultures demonstrated additional significant gene upregulation, including multiple chemokines responsible for recruiting T cells, neutrophils, and monocytes. 5) Further bioinformatics analysis comparing CD14+ cells positively enriched from both breast milk cell and control PBMC demonstrated statistically significant differences in expression of genes including caseins α s1, κ, and β, xanthine dehydrogenase (XDH), and lactotransferrin (LTF). Due to the unexpected nature of these results, qPCR was conducted to confirm these findings. Conclusions Our experimental efforts thus far have yielded results which suggest that breast milk CD14+ cells differ greatly in phenotype and function from peripheral blood monocytes. Elucidating the means by which these cells influence development of the infant GI immune system may shed light on new approaches for 1)optimizing vaccination strategies for infants, 2) treating necrotizing enterocolitis, and 3)understanding the pathogenesis of cell-associated diseases transmitted by breast milk, including human immunodeficiency virus (HIV)-1. Acknowledgements: Thank you to consenting milk donors; Suzanne Greeley for study subject recruitment; Ruth Connor, Emmanuel Balandya, and Gary Ward for mentorship and technical advice; Carol Ringelberg for bioinformatics analysis and advice; GSMD Immune Monitoring Laboratory and Genomics Core Facility for technical assistance. The author has no conflicts of interest to report, financial or otherwise.   Email contact: Stephanie.M.Dorosko@Dartmouth.edu or sdorosko@hotmail.com