Modulating the Inflammatory Response through Molecular Targeting
Authors: Kolb Alexander Louis , Reynoso Mainaliz , Geddis Alyssa , Matheny Ronald 
Published: April 2020

Inflammation is a highly regulated process, however due to the myriad of signaling pathways involved, can be extremely difficult to manipulate to study specific physiological processes. In this research, we focused on the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB) inflammatory signaling pathway due to its role in both pro‐inflammatory and anti‐inflammatory signaling. Activation of NFκB signaling induces the expression of tumor necrosis factor alpha (TNFα), a pro inflammatory signaling molecule. It also induces an anti‐inflammatory state through by expressing interleukin 10 (IL‐10). Given this, we investigated NFκB signaling kinetics in RAW 264.7 macrophage cells treated with lipopolysaccharide (LPS) for 15 minutes to 72 hours. Changes in inflammatory cytokines were measured by ELISA; statistical values were generated by normalizing LPS treated cells to DMSO (vehicle)‐treated cells. Stimulation of Raw 264.7 macrophages with LPS induced an immune response demonstrated by a 4‐fold increase in nuclear NFkB phosphorylation (p‐NFκB p65) within 2 hours of LPS treatment (p <0.001). Analysis of anti‐inflammatory signaling demonstrated a 500‐fold increase in IL‐10 transcript levels at 16 hours (p<0.001), and a 600‐fold increase in IL‐10 protein levels (p<0.001) 24‐hours following LPS treatment. This suggests that LPS stimulation induced a rapid inflammatory response, possibly mediated by p‐NFκB p65. Using these observations we proceeded to genetically manipulate the NFκB signaling pathway using bone marrow derived macrophages (BMDMs) with conditional knockouts of two adapter proteins in the NFκB signaling pathway, myeloid differentiation primary response 88 (“MyD88‐KO”) and TIR‐domain‐containing adapter‐inducing interferon‐β (“TRIF‐KO”). To define the role of each adapter protein, cells were treated with LPS and the secretion of TNFα and IL‐10, and analyzed at 6, 24, and 48 hours. Analysis of pro‐inflammatory signaling indicated BMDMs lacking MyD88 had a 97% reduction in TNFα (p<0.01), whereas cells lacking TRIF showed no change in TNFα levels following LPS treatment. Analysis of anti‐inflammatory signaling indicated cells lacking MyD88 had a 100% reduction in IL‐10 at 24 and 48 hours (p<0.001: p<0.001), and cells lacking TRIF had a 76% and 83% reduction in IL‐10 at 24 and 48 hours(p<0.001: (p<0.001). To determine if Myd88 was responsible for maintaining TNFα levels in TRIF‐KO cells we utilized CRISPR to knockout MyD88. Cells were treated with LPS and media was collected at 6, 24, and 48 hours post LPS treatment. Our data demonstrated the addition of CRISPR had no significant effect on TNFα secretion, but leads to a 72% reduction in IL‐10 levels at 24 post LPS treatment. (p<0.01). Overall our data details temporal signaling kinetics of the NFκB signaling pathway over the first 72 hours and that it is possible to genetically manipulate NFκB signaling pathway. The ability to genetically manipulate inflammatory signaling aids in the development of enhanced therapeutics for the treatment of inflammatory diseases and musculoskeletal trauma.

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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.