2.3 (d) Rv2615c leads to up-regulation of pro-inflammatory
cytokine TNF-α but down-regulation of IL-1β
A plethora of pro-inflammatory cytokines are secreted when an immune
response is triggered. Additionally, pro-inflammatory immune response
activation is also involved in various cell death mechanisms that take
place at the host-pathogen interface. TLR4-NF-ƙB signaling is a widely
studied cascade known to activate key pro-inflammatory cytokines such as
TNF-α. We found significantly up-regulated levels of TNF-α at all the
time points in response to LPS and Rv2615c protein stimulated THP-1
macrophages as compared to unstimulated cells (Fig 4a). Both Rv2615c and
LPS showed approximately ~ 3 to 4-fold increase in TNF-α
expression till 48h of stimulation. Another pro-inflammatory cytokine,
IL-1β, is also reported to be released as a result of TLR4-NF-ƙB
signalling. IL-1 β is a modulator of extrinsic apoptosis via Caspase 8
activation and inflammasome activation. Interestingly, IL-1β levels in
Rv2615c-stimulated macrophages were either equivalent to or lower than
in unstimulated cells at all time periods. (Fig 4b). IL-1β expression in
response to LPS stimulation was found to be significantly 2 to 3-fold
up-regulated than unstimulated cells till 24h.
To further ensure that the release of TNF-α in response to Rv2615c
protein stimulation was mediated by TLR4; we evaluated the expression of
TNF-α after blocking the expression of TLR4. There was a 2-fold decrease
in TNF-α release in response to Rv2615c and control stimulated THP-1
macrophages when blocked with Anti-TLR4 antibody prior to stimulation
(Fig 4c).