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).