Keap1 in the inflammatory response and autophagy regulation in mycobacterial infections and bacterial sepsis

Emerging and rising trends in antibiotic resistance has urged the need for novel drug targets to bacterial infections. Identification of drug targets can be approached both from exploring molecular and genetic mechanisms of host defense to infection, and characterizing evasion mechanisms employed by the bacterium to avoid killing within the host. We hypothesise that Keap1 plays a central role in mycobacterial infections and bacterial sepsis, and propose to characterize in detail how Keap1 and related molecules may regulate the 3 pathways of phagocytosis, inflammatory signaling and autophagy. Based on our preliminary results that Keap1 regulates inflammation /NF-kappaB-driven cytokines, we hypothesize that Keap1 genetic variations maybe associated with the development and clinical manifestations of bacterial sepsis. Thus we propose to investigate if Keap1 gene polymorphisms may be associated with the development and progression of bacterial sepsis. SP1: Keap1 in regulation of mycobacteria-induced innate immune responses. We have shown that Keap1 negatively regulates inflammatory cytokines and type I IFNs induced by M. avium, and we have observed that Keap1 may also regulate the IKK complex and TBK-1 protein levels (published). To further investigate the mechanism of how Keap1 controls inflammation, we have been looking at other interaction partners that might be relevant apart from the ones we have published. We have been focusing on the interaction between Keap1 and a mitochondrial protein called PGAM5 which plays a role in inflammasome activation and cell death. These processes are both important in disease progression. Tagged Keap1 constructs have been obtained from our collaborators and we are currently subcloning these constructs into lentiviral vectors for transfection as well as PGAM5 and inflammasome components like NLRP3 (Ongoing). PGAM5 has also been implicated in mitophagy, an autophagic process that specifically clears mitochondria. We have observed that Keap1 is recruited to M. avium containing phagosomes, to a lesser degree accompanied with LC3. Our preliminary results from siRNA knockdown and imaging indicate that this recruitment may be independent of p62 contrary to what we hypothesised. PGAM5 might be the connection here and we are currently developing the tools needed to investigate this. SP2: Keap1 and LC3 in regulating mycobacterium-specific T-cell responses (Not yet commenced). SP3: Keap1 gene polymorphisms may be associated with the development and progression of bacterial sepsis- (We ran a GWAS analysis of some HUNT 2 data on bacterial sepsis with promising results earlier reported. We are establishing collaboration with a research group in the US that has Keap1 knockout mice that will be useful in staging a sepsis model in mice to further investigate this). Finally, in order to study these mechanisms, a good understanding of current literature is important. We thus accepted an invitation to write a review that was published in November, 2016 on the various mycobacterial strategies employed to evade the immune system and particularly macrophage killing (Awuh, J.A. & Flo, T.H. Cell. Mol. Life Sci. (2016). doi:10.1007/s00018-016-2422-8).

Emerging and rising trends in antibiotic resistance has urged the need for novel drug targets to bacterial infections. Identification of drug targets can be approached both from exploring molecular and genetic mechanisms of host defense to infection, and characterizing evasion mechanisms employed by the bacterium to avoid killing within the host. We hypothesise that Keap1 plays a central role in mycobacterial infections and bacterial sepsis, and propose to characterize in detail how Keap1 and related molecules may regulate the 3 pathways of phagocytosis, inflammatory signaling and autophagy. Based on our preliminary results that Keap1 regulates inflammation /NF-kappaB-driven cytokines, we hypothesize that Keap1 genetic variations maybe associated with the development and clinical manifestations of bacterial sepsis. Thus we propose to investigate if Keap1 gene polymorphisms may be associated with the development and progression of bacterial sepsis. SP1: Keap1 in regulation of mycobacteria-induced innate immune responses. Cytokine responses: We have shown that Keap1 negatively regulates inflammatory cytokines and type I IFNs induced by M. avium, and we have observed that Keap1 may also regulate the IKK complex and TBK-1 protein levels. Using siRNA knockdown of Keap1-associated partners (p62, Nrf2, Cul3 and Rbx1) in primary human macrophages subsequently infected with M. avium we find that the effect of Keap1 on inflammation is independent of p62 and Nrf2 but may be associated to the E3 ligase complex that it is a part of, along with Cul3 and Rbx1. (This data is now published, Awuh JA, et al., 2015. Keap1 regulates inflammatory signaling in Mycobacterium avium-infected human macrophages. PNAS: 112(31):E4272-80.). To study the mechanism of how Keap1 controls inflammation, we need to find an appropriate cell line that can be infected, produce inflammatory cytokines and be transfected. We have thus screened various cell lines and currently selected 2 macrophage lines (THP1 and U373 cells) to stably overexpress Keap1 constructs by lentiviral transfection. (These cell lines have now been established – THP1-Keap1 Knockdown and U373 Keap1 Knockdown cells). Tagged Keap1 constructs have been obtained from our collaborators and we are currently subcloning these constructs into lentiviral vectors for transfection (Ongoing). Autophagy and LC3-associated phagocytosis of mycobacteria: Still developing the tools needed. We have observed that Keap1 is recruited to M. avium containing phagosomes, to a lesser degree accompanied with LC3. Our preliminary results from siRNA knockdown and imaging indicate that this recruitment may be independent of p62 contrary to what we hypothesised. SP2: Keap1 and LC3 in regulating mycobacterium-specific T-cell responses (Not yet commenced). SP3: Keap1 gene polymorphisms may be associated with the development and progression of bacterial sepsis- (Study started where we have run a GWAS analysis of some HUNT 2 data on bacterial sepsis. Preliminary results show some associations with susceptibility and mortality to bacterial sepsis of some SNPs that might be associated with the Keap1 E3 ligase complex. We are currently investigating the biological significance of this in the lab.)

Emerging and rising trends in antibiotic resistance has urged the need for novel drug targets to bacterial infections. Identification of drug targets can be approached both from exploring molecular and genetic mechanisms of host defense to infection, and characterizing evasion mechanisms employed by the bacterium to avoid killing within the host. We hypothesise that Keap1 plays a central role in mycobacterial infections and bacterial sepsis, and propose to characterize in detail how Keap1 and related molecules may regulate the 3 pathways of phagocytosis, inflammatory signaling and autophagy. Based on our preliminary results that Keap1 regulates inflammation /NF-kappaB-driven cytokines, we hypothesize that Keap1 genetic variations maybe associated with the development and clinical manifestations of bacterial sepsis. Thus we propose to investigate if Keap1 gene polymorphisms may be associated with the development and progression of bacterial sepsis. SP1: Keap1 in regulation of mycobacteria-induced innate immune responses. Cytokine responses: We have shown that Keap1 negatively regulates inflammatory cytokines and type I IFNs induced by M. avium, and we have observed that Keap1 may also regulate the IKK complex and TBK-1 protein levels (our unpublished data). Using siRNA knockdown of Keap1-associated partners (p62, Nrf2, Cul3 and Rbx1) in primary human macrophages subsequently infected with M. avium we find that the effect of Keap1 on inflammation is independent of p62 and Nrf2 but may be associated to the E3 ligase complex that it is a part of, along with Cul3 and Rbx1. To study the mechanism of how Keap1 controls inflammation, we need to find an appropriate cell line that can be infected, produce inflammatory cytokines and be transfected. We have thus screened various cell lines and currently selected 2 macrophage lines (THP1 and U373 cells) to stably overexpress Keap1 constructs by lentiviral transfection. Tagged Keap1 constructs have been obtained from our collaborators and we are currently subcloning these constructs into lentiviral vectors for transfection. Autophagy and LC3-associated phagocytosis of mycobacteria: Still developing the tools needed as above. We have observed that Keap1 is recruited to M. avium containing phagosomes, to a lesser degree accompanied with LC3. Our preliminary results from siRNA knockdown and imaging indicate that this recruitment may be independent of p62 contrary to what we hypothesised. SP2: Keap1 and LC3 in regulating mycobacterium-specific T-cell responses Not yet commenced. SP3: Keap1 gene polymorphisms may be associated with the development and progression of bacterial sepsis Not yet commenced