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Regulation of alternative pre-mRNA splicing in the TLR pathway to limit inflammation.

Inflammation study diagramOne negative feedback loop that limits persistent TLR-induced inflammation is the production of negatively acting proteins specified by alternative mRNA splice forms of TLR pathway components. Production of these negative regulators (including a negatively acting isoform of the TLR adaptor MyD88) is induced by TLR stimulation, and thus production of these alternative splice forms constitutes a negative feedback loop to limit inflammation (see Figure).

Our studies suggest that genes in the TLR pathway are poised to undergo alternative splicing to terminate persistent inflammation. Thus, a significant focus in our laboratory is on investigating the mechanisms that mediate this important negative feedback loop. Using genetic, genomic, biochemical, and computational approaches, we have identified components of the TLR signaling pathway and components of the core pre-mRNA splicing machinery that mediate lipopolysaccharide (LPS)-induced alternative splicing in the TLR signaling pathway. These mechanistic studies are informing on a key fundamental biological question, how signal-induced alternative splicing is regulated.

We also are examining the effects of this alternative splicing in two models of diseases with an inflammatory component, acute respiratory distress syndrome (ARDS) and acute myeloid leukemia (AML). In our lung studies, we are using mouse models to determine the effect of alternative splicing on inflammatory lung disease. We also are testing if splicing in the TLR pathway is perturbed in patients with ARDS. For our leukemia studies, we are examining the effect of spliceosome mutations on inflammation and cancer pathogenesis. One factor known to influence the pathogenesis of leukemia is altered inflammatory signaling. A second factor implicated in leukemia pathogenesis is altered splicing; spliceosome genes are recurrently mutated at a high frequency in leukemia. Our discovery that these splicing factors regulate inflammation unifies the disparate observations on the roles of inflammation and mRNA splicing in the development of leukemia, suggesting that spliceosome mutations could enhance inflammation to affect disease.

Spatiotemporal regulation of TLR signaling by a novel RAB-GAP

Using mouse and macrophage models, we have identified Tbc1d23, a candidate RAB-GAP, as an inhibitor of the maintenance but not the initiation of TLR signaling. RABs are members of the RAS superfamily of small GTPases that mediate vesicle trafficking within the cell. RAB-GAPs inhibit their cognate RABs by facilitating the hydrolysis of the GTP on the RAB. Our studies demonstrate that Tbc1d23 likely acts by regulating a RAB and thus controls cellular trafficking to inhibit TLR signaling. We are currently investigating what RAB is regulated by Tbc1d23, what vesicles/proteins are trafficked by Tbc1d23, and how Tbc1d23 impacts the TLR signaling pathway. We also have identified other proteins that bind to Tbc1d23 and mediate its effects. Thus, we have discovered a new signaling pathway that controls the maintenance of TLR signaling several hours after challenge; this pathway regulates the canonical TLR pathway. Our expectation is that targeting this pathway pharmacologically will be a useful approach to prevent chronic inflammation and chronic inflammatory disease without abolishing the essential initial anti-pathogen response.

Inflammation diagram