Figure 4. Live cell imaging of opioid-induced TLR4 signaling.

Opioid interactions with TLR4 can be imaged in real time using a RAW264.7 mouse macrophage cell line that stably expresses green fluorescent protein labeled Akt1 (GFP-Akt1), as Akt is a component of one of the 3 parallel signaling pathways activated in response to TLR4 agonists. (A) Under basal conditions, Akt1 is diffusely distributed in the cytosol. (B) On activation, Akt1 rapidly moves to the membrane site where an Akt1 activating event is occurring. Membrane ruffling is also reflective of activation of the PI3K/Akt pathway downstream of TLR4. (C) Mobilization of GFP-Akt1 from the cytosol is easily quantified. At the first (downward) arrow, a TLR4 antagonist [Gray Triangles; either the competitive TLR4 antagonist LPS-RS (an LPS variant expressed by the bacterium Rhodobacter sphaeroides which helps the pathogen evade the host’s immune system by blocking TLR4), (+)-naloxone, or (-)-naloxone] or vehicle [Black Squares] is added to the live cell cultures. Cytoplasmic Akt1 fluorescence remains unchanged until TLR4 agonists (the classic TLR4 agonist LPS, (+)-morphine, or (-)-morphine) are added at the second (upward) arrow. Cells that are exposed only to TLR4 agonists (Black Squares) show a rapid loss of fluorescence from the cytosol as GFP-Akt1 mobilizes to the cell membrane. In the presence of TLR4 antagonists (Grey Triangles), TLR4 agonists now fail to induce GFP-Akt1 activation, so cytosolic fluorescence remains stable. As a positive control that these antagonist-blocked cells are in fact responsive, complement 5a (C5a) is then added at the third arrow (White Triangles) as a positive control since C5a activates PI3K/Akt1 but via a pathway independent of TLR4. Panel 3 is modified from Hutchinson et al.⁴⁵.