Non-canonical STAT3 Serine phosphorylation is required for TLR-induced metabolic reprogramming.

Abstract

Macrophages are an essential component of the innate immunity which sense danger or pathogen signals and initiate inflammatory responses. Inflammatory macrophages require higher energy output to mediate this pro-inflammatory state. Toll-Like Receptors (TLRs) have evolved to function as sensory receptors that detect danger and pathogen signals, subsequently transmitting signals that trigger an inflammatory response. In macrophages, several studies have identified that aerobic glycolysis increases post-TLR signalling similar to ‘Warburg effect’ in cancer cells. However, the exact mechanism of how TLR signalling induces metabolic reprogramming remains unclear. This led to my hypothesis that non-canonical STAT3 activation is a critical mediator of TLR-induced mitochondrial programming and inflammation. In this study, I have identified for the first time that following TLR4 stimulation, the downstream mediators TRAF6 and TBK1 rapidly interacted with STAT3. Inhibition of TBK1 kinase function reduced serine phosphorylation of STAT3 (S727), which is required for its non-canonical mitochondrial function. This identified TBK1 as a novel serine kinase activator of STAT3 and demonstrating direct interaction between the TLR/JAK-STAT pathway. Subsequently, a pool of STAT3 migrated to the mitochondria resulting in metabolic reprogramming. By utilising a genetically engineered mouse model that cannot undergo STAT3 Ser727 phosphorylation, I found that STAT3 Ser727 phosphorylation is crucial for LPS-induced glycolysis, the production of the key immune regulatory metabolite succinate, and the generation of inflammatory cytokines in a model of LPS-induced inflammation. These studies identify for the first time a TRAF6/TBK1/STAT3 signalling nexus that is a central signalling intermediary for TLR4-induced glycolysis, macrophage mitochondrial reprogramming and inflammation, providing a mechanism for previous TLR immunometabolism studies. In an effort to address how STAT3 may regulate mitochondrial glycolysis, it has been observed that in cancer cell lines, the mitochondrial pool of STAT3 is described to bind to two proteins Leucine Rich Pentatricopeptide repeat containing (LRPPRC) protein, and Steroid receptor RNA activator Stem-Loop Interacting RNA Binding Protein (SLIRP), causing stabilised mitochondrial RNA and increased electron transport chain (ETC) complexes such as complex I and IV. I subsequently found that TLR4 stimulated macrophages induced a pool of STAT3 to translocate to the mitochondria, directly interacting with both proteins, which was dependent on TBK1 kinase activity. I subsequently identified that, similar to that identified in cancer cells, mitochondrial-localised STAT3 in complex with LRPPRC and SLRIP may lead to stabilised mitochondrial-associated genes of complex IV (COX1 and COX3) and complex I (ND4L and ND6). Conversely, inhibition of TBK1, or serine inactive STAT3S727A (STAT3 SA) macrophages displayed reduced RNA stability of ETC complex’s I and complex IV, further enhancing the critical role of this novel signalling nexus in mediating mitochondrial function. Critically from a potential therapeutic focus, small molecule targeting of TBK1 or using STAT3 SA peritoneal macrophages display reduced inflammatory, but increased anti-inflammatory cytokine production, following LPS challenge, suggesting that targeting this nexus could reduce the inflammatory burden associated with disease. Overall, these studies demonstrate the first characterisation of the molecular mechanism of STAT3 mediated glycolysis following mitochondrial localisation. It indicates that STAT3 interaction with LRPPRC and SLRIP may lead to the stabilisation of mitochondrial RNA, and extension of the ETC complex, regulating ‘late’ (ie. >12h) macrophage inflammation by regulating mitochondrial function. Equally, ACLY is a cytosolic enzyme which converts citrate to acetyl-CoA. Recently, it was found that post-LPS stimulation, ACLY underwent serine phosphorylation (ACLY Ser455) and induced histone acetylation which mediates the early innate immune inflammatory response (ie. gene regulation within the first 4 h post-TLR challenge). However, the mechanism of how ACLY is recruited to the TLR pathway remains poorly understood. Given STAT3 interacts with the serine kinase TBK1, I examined if this signalling nexus may play a role in mediating this early inflammatory gene regulation event. Subsequently, I found that TBK1, STAT3, and ACLY interacted rapidly following LPS stimulation, which was dependent on TBK1 kinase function. Importantly, specific inhibition of TBK1 led to reduced STAT3-TBK1-ACLY interaction which in turn reduced expression of a specific subset of ‘early’ pro-inflammatory genes, while increased anti-inflammatory genes. Uniquely, these studies identify the role of TBK1 and to a lesser extent, STAT, play in mediating activation of ACLY following LPS stimulation, linking this activation to early innate immune reprogramming via histone acetylation. Critically, it also further identifies potential therapeutic strategies to inhibit inflammation through identifying and targeting these novel signalling pathways and mediators such as TBK1. Together, these studies provide preliminary insights into TBK1-induced phosphorylation of ACLY via interaction with STAT3, providing the signalling ‘bridge’ for ACLY recruitment and post-translational modification by the TLR pathway to regulate the ‘early’ inflammatory. This thesis provides novel insights into cross-talk between the TLR and JAK/STAT pathway via non-canonical activation of STAT3. TBK1 is identified as a key mediator of TLR-induced immunometabolism in macrophages, with TRAF6 and STAT3 driving early pro- and anti-inflammatory gene regulation via ACLY and late regulation via mitochondrial reprogramming and stabilisation of mitochondrial ETC genomic DNA. Critically, it also highlights and characterises a previously unrecognised critical role for STAT3, and the signalling nexus of TRAF6 and TBK1, sequentially regulating both the ‘early’ and ‘late’ gene transcription pathways through post-translational cytosolic/mitochondrial localisation. Importantly, targeting TBK1 disrupts this network, offering potential therapeutic strategies for managing inflammation by modulating these pathways.