MYD88 Expression Is Required for Time Phase‐Specific LPS‐Stimulated TNFα and IL‐10 Expression in RAW 264.7 Macrophages
Authors: Reynoso Marinaliz , Matheny Ronald W. , Mitrophanov Alexander Y. , Hobbs Stuart 
Published: June 2021

Macrophages are a critical component of the inflammatory response to effect healing and repair during injury and infection. Abnormal responses in TNFα and IL‐10 production by macrophages are thought to play a key role in a number of inflammatory diseases. Currently, there is incomplete knowledge of how and to what extent the TLR4 signaling adapter MYD88 contributes to the kinetic responses of these cytokines stimulated by inflammatory inducers such as lipopolysaccharide (LPS). To clarify this, we investigated the role of MYD88 expression in the kinetic profile of TNFα at 6, 16, and 24 hours; and IL‐10 expression at 24, 48, and 72 hours induced by 10 ng/mL LPS. We transfected cells with control small interfering RNA (siRNA) or MYD88 siRNA to test the effect of MYD88 protein expression knock‐down. In control siRNA‐transfected cells, LPS induced TNFα mRNA expression (5.7‐fold compared to Mock‐PBS‐treatment) at 6 hours, which peaked (7.9‐fold) at 16 hours, and decreased (5.8‐fold) at 24 hours. The secreted TNFα protein concentration increased (14.1 pg/mL*ug total protein) at 16 hours, with no additional increase at 24 hours, that then increased (20.1 pg/mL*ug total protein) 48 hours after LPS treatment. Despite a decrease in MYD88 expression with MYD88 siRNA transfection, TNFα mRNA expression was unaffected at 6 and 24 hours after LPS stimulation, but was decreased 32% at 16 hours after LPS stimulation (P<0.05). Knock‐down of MYD88 protein expression decreased secreted TNFα protein expression by 53% at 16 hours after LPS treatment, but did not significantly affect expression at 24 or 48 hours. In control siRNA‐transfected cells, LPS induced IL‐10 secreted protein expression (0.107 pg/mL*ug total protein) at 24 hours that continued to increase (0.251 pg/mL*ug total protein) at 48 hours and increased further (0.523 pg/mL*ug total protein) at 72 hours. MYD88 siRNA transfection reduced secreted IL‐10 protein expression by 73% at 48 hours after LPS treatment, but did not affect expression at 24 or 72 hours. These data suggest that signaling coupled to MYD88 regulates TNFα mRNA and IL‐10 protein expression mid‐course, but not early and late, and TNFα secreted protein early in the LPS response. It is also possible that the early and late inductions of these cytokines are less sensitive to alterations in signaling resulting from decreased expression levels of MYD88. The simultaneous effect on TNFα mRNA expression and secreted TNF protein expression at 16 hours upon MYD88 knock‐down suggests that MYD88 may have a role in TNF mRNA transcription/stability, translation, and/or processing of immature TNF by matrix metalloproteases. Overall, these data indicate that MYD88 is a possible target for manipulating the inflammatory process in injury and infection.The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This abstract has been approved for public release with unlimited distribution.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.