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).