Establishing tools to study the involvement of Keap1 in inflammatory signaling upon infection with M. avium
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Mycobacterium avium is a non-tuberculous mycobacteria that causes opportunistic infections in immunocompromised individuals such as AIDS patients. The pathogen is receiving more attention due to increasing resistance towards several antibiotics, and a high degree of genetic variability within the strain. The treatment towards M. avium is lengthy, highlighting the importance of elucidating the molecular mechanisms of infection in the host cells in search for novel drug targets. M. avium infects macrophages, where they reside and avoid degradation by blocking maturation of the phagosomes and fusion with lysosomes like the more virulent M. tuberculosis, the causative agent of Tuberculosis. Reactive oxygen species released due to activation of Pattern recognition receptors are thought to be important for killing intracellular mycobacteria, but excessive amounts could be damaging to the cells. Keap1 is a sensor for ROS and a substrate adaptor for the Cullin3- based E3 ubiquitin ligase, shown to regulate IKKβ and NF-kB signaling among other important targets. Our group has shown that Keap1 is recruited to mycobacterial phagosomes and regulates inflammatory signaling in human primary macrophages. The study further raised the evidence for a direct or indirect regulation of TBK1, involved in the IRF-pathways and production of type 1 Interferons. In addition it suggests the role of Keap1’s contribution to increased survival of M. avium intracellulary. The limitations with the use of primary macrophages due to donor variations and difficulties in modulating protein levels, further addressed the need for a macrophage-like model cell-line to further detail the mechanism of Keap1’s regulation of IKKβ and TBK1 using transfection or transduction of tagged full-length Keap1 and deletion constructs of Keap1. In this project we examined the U373-CD14 and THP1 cell-lines for response towards M. avium, both phagocytic clearance and inflammatory cytokine responses. As the U373-CD14 cell-line did not show a high up-regulation of the cytokines of interest, the cell-line was transiently transfected with the endogenously absent TLR2 and TLR8, and an increased response was observed towards infection, especially for TLR2 transfected cells. But the transient transfection displayed low efficiency in the cells, and it was decided to modify the cell-line with lentiviral transduction and gateway cloning with TLR2 and TLR8 in separate cell-lines. The U373-CD14 TLR2 cell-line responded to M. avium with a significant high up-regulation of TNF-α and IL-8, and a significant low up-regulation of IFNβ, which highlighted the importance of the receptor for M. avium induced inflammation. The TLR8- expressing cell-line did not display an efficient up-regulation of screened cytokines towards M. avium, and this could either be explained by the short infection time or the incompetence of the TLR8 receptor in infection because of absent components in the pathway. This project has provided new tools to study the regulation of IKKβ and TBK1 by Keap1 upon M. avium infection. In addition it has raised new questions regarding the TLR8 receptor’s role in mycobacteria infection, and highlighted the importance of TLR2 for an efficient immune response. It further remains to investigate whether the knockdown of Keap1 leads to an up-regulation of the cytokines of interest, and to reveal the regulation mechanisms of the kinases upon infection.