These findings led to experiments designed to assess infection of human skin in a controlled study of live spirochetes infecting full thickness human skin explants (keratomes). Blinded analysis of low power fields Erlotinib assessed the number of CD1 expressing cells within the dermis and epidermis. There were no significant changes in the number, apparent brightness or size of CD1a expressing Langerhans cells (LCs) in the epidermis, when comparing infected or sham-treated

keratomes (Fig. 1B and C). The number of CD1a expressing cells in the dermis (4.1% of all cells) increased slightly after infection (6.1%) but did not reach statistical significance (p=0.34). However, the number of CD1b (p<0.0027) or CD1c (p<0.0086) expressing cells showed a significant increase after infection (Fig. 1C). Also, we observed marked increases in brightness of staining in each of three experiments. Although PS341 CD1d could be detected at very low levels in flow cytometry experiments

(Fig. 2), CD1d staining was not seen at levels higher that isotype-matched staining control samples (Fig. 1C). We conclude that evaluation of CD1a induction was limited by constitutively positive LCs, but increased CD1b and CD1c expression is induced during B. burgdorferi infection of human skin. To study the cellular mechanisms of CD1 induction by B. burgdorferi, we measured CD1 expression on human monocytes in culture. To determine whether the events seen ex vivo could be modeled in vitro, we first measured CD1 expression on monocytes after infection with live bacteria or by treatment of cells with lipids extracted from bacteria with chloroform and methanol. Fresh monocytes and control monocytes sham treated with medium for 3 days did not detectably express CD1a, CD1b or CD1c proteins at the surface, but CD1d was detected at low density on some cells (Fig. 2A and data not shown). Ex vivo infection with live spirochetes (data not shown) or cell wall lipids (Fig. 2A) increased cell surface expression of CD1a, CD1b and CD1c proteins to high levels. CD1a surface density increased

in a dose-dependent fashion (Fig. 2B). The resultant CD1a cell surface expression of was sufficient to activate a CD1a autoreactive T-cell line (Fig. 2C). The low levels of baseline expression of CD1d were unaltered or slightly decreased, so that they were undetectable (Fig. 2A). These results confirm that B. burgdorferi potently activates group 1 CD1 expression on monocyte-derived DCs in a model that mimics many aspects of the in vivo observations. In particular, these data show selective upregulation of group 1 CD1 proteins over 3 days. Activation of myeloid cells by B. burgdorferi lipoproteins is mediated through TLR-2 29. Also, a synthetic TLR-2 agonist triacyl-CSK4, which mimics the structure of the N-terminus of a borrelial lipoprotein, can induce CD1 expression 30.