Regulations of IRF5 activity in the TLR7 and type I IFN pathways
IRF5 is a member of the Interferon Regulatory Factor (IRF) family of transcription factors activated downstream of the Toll-Like receptors (TLRs). Following activation, IRF5 translocates into the nucleus where it regulates transcription of proinflammatory cytokines and type I IFN. Polymorphisms in IRF5 have been shown to be associated with the autoimmune disease Systemic Lupus Erythematosus (SLE) and other autoimmune conditions, suggesting a central role for IRF5 in the regulation of the immune response. Four different IRF5 isoforms are generated by alternative splicing and by the presence or absence of a 30 nucleotide insertion in IRF5 exon 6 tha t is included in the risk haplotype for SLE. Since the polymorphic region disturbs a PEST domain, we hypothesized that the isoforms bearing the insertion could have increased stability, thus explaining the association of IRF5 with SLE.
IRF family members IRF3, IRF7 and IRF8 have been shown to be targeted for proteasomal-mediated degradation by the E3 ubiquitin ligase TRIM21 following activation. In addition, two separate studies have shown that IRF5 and TRIM21 interact. In this study we therefore investigated whether IRF5 is subjected to regulation by TRIM21 and whether dysregulation of this mechanism could explain the association of IRF5 with SLE. We have thus been able to show that IRF5 is degraded following TLR7 activation and that TRIM21 is involved in this process, while confocal analysis has shown colocalization of IRF5 isoforms and TRIM21 in cytoplasmic vesicular and filamentous structures. Comparison of the individual IRF5 variants has shown that isoforms generated by alternative splicing are resistant to TRIM21-mediated degradation following TLR7 stimulation, while the presence of the 30 nucleotide insertion does not influence stability. Similarly, alternately spliced (thus more stable) isoforms present a lower degree of colocalization in TRIM21- containing vesicles following TLR7 stimulation, suggesting a possible role for these structures in protein degradation. Interestingly however, we have been able to show a possible role for TRIM21 in inhibition of TLR7-mediated nuclear translocation of these alternatively spliced isoforms, thus providing an additional mechanism for negative regulation of IRF5-mediated signaling in addition to regulation of stability. Taken together, these results indicate that alteration of splicing mechanisms in SLE, which would result in expression of IRF5 isoforms with increased stability, coupled to impaired TRIM21 function may account fo r increased activity of IRF5 in SLE patients, resulting in increased expression of proinflammatory cytokines and type I IFN characteristic of the disease.
We next focused on investigating novel mechanisms of regulation of IRF5 transcriptional activity and we have been able to show that IRF5 is phosphorylated on tyrosine residues following TLR7 and IFNa stimulation. In the TLR7 pathway we could identify Src as the effector tyrosine kinase involved in IRF5 phosphorylation. In the type I IFN pathway we investigated in detail the possible role fo r the IFNARassociated kinase TYK2, since polymorphisms in TYK2 have been shown to be associated with SLE and to have additive effects with polymorphisms in IRF5. We have thus been able to show that IRF5 is a target of TYK2 and that TYK2-mediated phosphorylation of IRF5 can influence its transcriptional activity with opposite effects on different promoters.
In conclusion, we have shown in this work novel mechanisms for regulation of IRF5 activity in the TLR7 pathway. In particular, by showing that IRF5 is negatively regulated by TRIM21, and since TRIM21 activity is dysregulated in SLE, we have provided a functional, albeit indirect, link between IRF5 and lupus. We have also provided the first evidence of a possible molecular link between IRF5 and TYK2, thus far only suggested by genetic interaction, and demonstrated a possible role fo r IRF5 in the type I IFN response. Given the pathogenic role of type I IFN in SLE, the evidence of IRF5 activation in this pathway may therefore provide additional insight on the role of IRF5 in lupus.