NFkB activation and cytokine output in LPS‐treated RAW 264.7 macrophages
Authors: Reynoso Marinaliz , Geddis Alyssa , Mitrophanov Alexander , Matheny Ronald W , Hobbs Stuart 
Published: November 2023

During injury and infection of tissue, inflammation is a response by macrophages and other cell types to effect healing and repair; however, when inflammation becomes dysregulated, it causes a variety of pathologies. In macrophages, there is a lack of detailed information on the cell signaling kinetics that regulate the response of pro‐inflammatory mediator TNFα to lipopolysaccharide (LPS), which could guide the use of anti‐inflammatory drugs without affecting the normal reparative process. To address this gap, we used cultured RAW 264.7 (RAW) macrophage cells and set out to define intracellular signaling kinetics, mRNA expression, and cytokine production in cells treated with 10 ng/mL LPS over a 24‐hr period and in parallel vehicle‐treated cells All results except protein secretion are expressed relative to 15‐min vehicle control (P <0.05). We first measured the expression level of IκBα since it acts as a negative feedback to inhibit transcription factor NFκB activity by sequestering it in the cytoplasm. LPS stimulates IκBα degradation, releasing NFκB that translocates to the nucleus and regulates IκBα and TNFα expression. In cytoplasmic lysates, the abundance of IκBα was reduced by 78% 45‐min following LPS treatment, but began to increase at 10‐hr, reaching a maximum at 16 hr (2.4‐fold) and decreasing at 24‐hr. Transcription factor p65 NFκB governs cytokine expression, so we also measured its activation by assessing Serine 536 phosphorylation, nuclear translocation, and binding to DNA consensus sequences. NFκB phosphorylation in cytoplasmic lysates was significantly increased 45‐min following LPS treatment, was maximal at 2‐hr (3.8‐fold), and decreased to basal levels by 6‐hr. The abundance of NFκB in nuclear lysates was elevated 30‐min following LPS treatment, was maximal by 45‐min (2.6‐fold), and returned to basal levels by 24‐hr. Binding of nuclear NFκB to consensus oligonucleotide sequences in nuclear lysates followed a similar pattern to that observed for p‐NFκB. Finally, we measured expression of LPS‐stimulated inflammatory mediator TNFα and its negative feedback regulator IL‐10. Following LPS treatment, TNFα mRNA expression began at 1‐hr, was maximal at 6‐hr (18‐fold), and decreased starting at 10‐hr but remained significantly elevated up to 24‐hr. TNFα protein secretion in conditioned growth medium began at 4‐hr and accumulated to a maximal concentration by 16 hr (9.8 ng/mL). IL‐10 mRNA expression was induced by LPS at 10‐hr, was maximal at 16 hr (530‐fold), and remained elevated at 24 hr. IL‐10 protein secretion was induced at 16‐hr and continued to accumulate to a maximal concentration at 24‐hr (620 pg/mL). These data provide a foundation that could aid in identifying pharmacological targets that control the inflammatory process in tissues.

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