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Functional analysis of Suppressor of IKKɛ (SIKE) in migration and phagocytosis
Author(s) -
Fahey Sean M,
Slykas Frank A,
DiChristofano Nick D,
Dawood Mariam K,
Bell Jessica K
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1075.6
Subject(s) - microbiology and biotechnology , cytoskeleton , biology , cell , genetics
Innate immunity is a critical, non‐specific early defense system that allows the body to respond to pathogens prior to the recruitment of specific antibody defenses. This process involves the recruitment and subsequent migration of nearby immune cells to the site of infection, allowing the pathogen to be consumed via phagocytosis to prevent widespread infection. Migration and phagocytosis require the rapid disassembly, rearrangement, and assembly of a network of structural proteins known as the cytoskeleton. The link between cytoskeletal rearrangements in these processes and detection of pathogen, like viral patterns by Toll‐like receptor 3, are not well defined. TLR3 initiates proinflammatory and type I interferon responses via the TANK binding kinase 1 (TBK1) pathway. Suppressor of IKKe (SIKE) was originally proposed as a suppressor of IKKe and TBK1, playing an important inhibitory role in interferon β production. However, more recent studies have indicated SIKE acts instead as a preferred substrate of TBK1. Mass spectrometry and confocal microscopy further indicate that SIKE interacts with the cytoskeletal proteins actin, tubulin, and a‐actinin. This revised understanding of SIKE suggests that SIKE acts as a bridge between the viral stimulation of TBK1 activity and the resulting cytoskeletal rearrangements. To examine the role of SIKE in cytoskeletal rearrangement, SIKE function in cellular migration and phagocytosis were assessed. A CRISPR/Cas 9 SIKE knockout was developed in the KMB7‐derived HAP1 cells. The HAP1 SIKE −/− showed no detectable SIKE expression compared to the parental HAP1 cells. To determine the role of SIKE in migration, parental and HAP1 SIKE −/− were used in cell migration assays +/− dsRNA to simulate viral infection conditions. A lentiviral‐mediated shRNA SIKE knockdown was completed in the mouse macrophage RAW264.7 cell line. Using SL1344 Salmonella typhimurium strain, gentamicin protection assays were completed to determine SIKE's role in phagocytosis. Introduction of SL1344 Salmonella typhimurium at MOI ≤ 100 into RAW 264.7 showed successful infection of cells by confocal microscopy and gentamicin protection assay. Support or Funding Information This work was supported by NIH grant R21AI107447 and University of San Diego SURE program.