Alternative splicing takes control of cytokine signaling

Thesis (Ph.D.)--University of Washington, 2016-08

Immune responses must be tightly controlled for dose, location, strength and duration using genetic, epigenetic or biochemical regulation. Among these, the generation of alternatively-spliced transcripts is an efficient and dynamic way to increase transcriptional and proteomic diversity. Specifically, this thesis explains how splice variation dictates the biological functions of interleukin-22 (IL-22) binding protein (IL-22BP) and interferon lambda 4 (IFNλ4), two proteins that participate in key cytokine responses to infection and inflammation. IL-22BP is a soluble receptor for IL-22 that is expressed as three isoforms in humans, IL-22BPi1, IL-22BPi2 and IL-22BPi3. The murine homolog of IL-22BPi2 has been characterized as an antagonist of IL-22 while the physiological relevance of IL-22BPi1 and IL-22BPi3 are unknown. Here, we present findings demonstrating that alternative splicing tailors IL-22BP activity to specific spatiotemporal conditions. Inclusion of a unique third exon renders IL-22BPi1 inactive by preventing its secretion, while IL-22BPi3 is a weaker antagonist than IL-22BPi2 due to exclusion of exons 5 and 6. While IL-22BPi3 is the most dominant isoform under homeostatic conditions, stimulation by Toll-like receptor 2 (TLR2) or retinoic acid induces only IL-22BPi2. Response to environmental cues therefore generates a gradient of IL-22BPi2 and IL-22BPi3 as a rheostat for IL-22 activity. In the case of IFNλ4, alternative splicing suppresses protein expression and secretion to prevent its antiviral activity. Unlike the tightly linked IFNL3, IFNL4 is genetically associated with hepatitis C virus persistence. Thus, we examined whether any of its three natural protein-coding isoforms IFNλ4p107, IFNλ4p131 or IFNλ4p179 may account for unexpected pro-viral activity. Using overexpression systems and recombinant proteins, we found that only full-length IFNλ4p179 is bioactive and, like IFNλ3, exhibits antiviral activity without blocking other IFN signaling. Even so, little of this active cytokine is made as alternative splicing favors the expression of the inactive isoforms or unproductive transcripts instead . Thus, alternative splicing is a major gene regulatory mechanism that limits IFNλ4 bioactivity during infection, causing the genetically linked IFNλ3 to be the dominant antiviral effector instead. Overall, our data show that alternative splicing is an important response to pathogen sensing and infection that efficiently fine-tunes or controls expression of immune receptors and effectors. In these ways, it dictates the outcome of many cytokine signaling pathways.