Figure 4.

RANK-L is secreted by HIV-1-infected MF and promotes 3D migration of OC precursors. (A) MF were infected with HIV-1 (HIV-MF, NLAD8 strain) for 7 days, and the level of released RANK-L was measured by ELISA in the supernatant and compared to uninfected MF (NI-MF) from the same donor. n = 5. (B) Monocytes were differentiated for 3 days in presence of sub-optimal concentrations of RANK-L and then exposed to the supernatant of infected (CmHIV) or uninfected (CmNI) MF for additional 10 days. Cells were then fixed and the fusion index was quantified by IF. Bars represent median, n = 6 donors. (C, D) CmHIV or CmNI pre-treated or not with OPG (100ng/mL) (C), or different concentrations of recombinant RANK-L (D) was added at the bottom of 3D Matrigel matrices. OC precursors were then seeded at the top of the matrices and allowed to migrate. Left: 3D representation of the positions of OC precursors that have migrated (blue dots) or not (red dots) in a representative migration assay. Ticks represent depth in microns. Right: quantification of the percentage of cells inside the matrix. (C) n = 4 donors; (D) n = 7 donors, performed in triplicates. Histograms represent median and error bars are interquartile range. * p ≤ 0.05; ** p ≤ 0.001, ns: not significantly different.