2.3. (b) Rv2615c leads to up-regulation of TLR4 and HLA-DR expression
Expression profile of TLR2 and TLR4 was estimated by flow cytometry in response to Rv2615c-stimulation in THP-1 macrophages. Both Rv2615c and LPS-a TLR4 agonist resulted in significant 1.27-fold increase in TLR4 percent positive cells at 24h than unstimulated control. There was no up-regulation of TLR4 expression observed for cells stimulated with cell wall fraction of Mtb. When THP-1 cells were blocked with Anti-TLR4 antibody prior to stimulation with Rv2615c or controls; the percentage of TLR4 positive cells significantly decreased by 1.4-fold implicating that the function of Rv2615c is TLR4 mediated. Further, there was no up-regulation in TLR2 expression in response to both LPS and Rv2615c-stimulation at all-time points. Cell wall fraction which was included as positive control for TLR2 expression showed a significant 1.2-fold increase in TLR2 expression at all-time points (Fig 3b).
For determining the effect of Rv2615c-stimulation on TLR-associated antigen presenting molecule HLA-DR, we performed flow cytometry analysis to study the expression of HLA-DR. Rv2615c showed significant increase in percentage of HLA-DR positive cells at 24h and 48h. At 24h, there was significant 2-fold increase in percentage of HLA DR positive cells induced by Rv2615c in comparison to unstimulated cells (Fig 3b).
In comparison to unstimulated cells, LPS and CWF significantly (~1.5-fold ) raised the levels of HLA-DR positive cells. We examined the HLA-DR levels in cells inhibited with anti-TLR4 antibody prior to stimulation to determine the TLR4-dependent up-regulation of Rv2615c-induced HLA-DR expression. In comparison to unstimulated cells, we observed a 2.8-fold decrease in HLA-DR expression in control (LPS and CWF) and Rv2615c-stimulated THP-1 macrophages (Fig 3c and 3d).