siRNA knockdown of <i>Dusp3</i> and <i>Psme3</i> result in significant elevation of cytokine production, consistent with the pattern of bone marrow derived macrophages from CSS11 as compared with C57BL/6J (GM-CSF, IL-1β, IL-6 and TNF-α).

Abstract

<p>(<b>A</b>) Bone-marrow derived macrophages (BMDMs) from A/J and C57BL/6J have similar <i>S. aureus</i> phagocytosis ability. 2×10⁶ BMDMs were seeded to single wells in a 6-well plate the day before phagocytosis and incubated with hexidium iodide stained <i>S. aureus</i>. Phagocytic efficiency as determined by the mean fluorescence intensity (MFI) is not significantly different in BMDMs from C57BL/6J and CSS11 mice. Representative histogram of 3 separate experiments. (<b>B</b>) BMDMs from CSS11 mice produced significantly higher cytokine levels as compared to C57BL/6J. 4×10⁵ BMDMs from both C57BL/6J and CSS11 mice were seeded to single-wells of a 24-well plate the day before infection. Infection was simulated by adding <i>S. aureus</i> particles at 10 µg/ml. At 24 hours post-infection the supernatants were harvested and subjected to Luminex cytokine assaying. BMDMs from CSS11 mice significantly enhanced cytokine production, including GM-CSF, IL-1β, IL-6, and TNF-α. (<b>C</b>) Down-regulation of <i>Dusp3</i> and <i>Psme3</i> by siRNA led to up-regulation of cytokine production upon <i>S. aureus</i> challenge in RAW264.7 macrophages. RAW264.7 cells were transfected by either scramble or <i>Dusp3</i> or <i>Psme3</i> siRNA, and then infected with <i>S. aureus</i> particles at 10 µg/ml as before <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004149#ppat.1004149-Ahn1" target="_blank">[11]</a>. At 24 hours post-infection, the supernatants were harvested and subjected to Luminex-multiplex cytokine assaying. The down-regulation of <i>Dusp3</i> significantly enhanced cytokine production, including GM-CSF, IL-1β, IL-6, and TNF-α, as compared to scramble siRNA control. The down-regulation of <i>Psme3</i> also significantly elevated GM-CSF and IL-6 production. (<b>D</b>) Down-regulation of <i>Dusp3</i> or <i>Psme3</i> by siRNA led to up-regulation of cytokine production upon <i>S. aureus</i> challenge in BMDMs. BMDMs from C57BL/6J were transfected by either scrambled, <i>Dusp3</i> or <i>Psme3</i> siRNA, and then infected with <i>S. aureus</i> particles at 10 µg/ml. At 24 hours post-infection, the supernatants were harvested and subjected to cytokine analysis. The down-regulation of <i>Dusp3</i> significantly enhanced cytokine production, including IL-6 and TNF-α, as compared to scrambled siRNA control. The down-regulation of <i>Psme3</i> also significantly elevated TNF-α production.</p