Presentation: Toll-Like Receptor-Inducible Upregulation of Alternative IRF5 mRNA Isoforms Correlates with Differential Tissue Targeting in Human Anti-RNP Autoimmunity (2008)

1466 Toll-Like Receptor-Inducible Upregulation of Alternative IRF5 mRNA Isoforms Correlates with Differential Tissue Targeting in Human Anti-RNP Autoimmunity

Purpose: This study assessed whether TLR3 and TLR7 activation induce different patterns of IRF5 expression, and whether this correlates with the presence or absence of clinical lung disease.
Methods: 293 cells expressing either TLR3 or TLR7 were stimulated with TLR3 and/or TLR7-specific agonists, and lysed for real time PCR and northern blots. PBMC from 25 RNP+ patients characterized for the presence of lung disease (pHTN, pulm fibrosis, or %pred DLCO < 60) were used for genotyping and real time PCR for IRF5 mRNA in IRB-approved protocols. Real time PCR used 18S ribosomal RNA as calibrator and RNA from unmanipulated 293 cells or from normal human PBMC as controls. IRF5 primers were specific for the conserved Exon 3 region, or for Exon 9 just distal to the AU-Rich Element (ARE) in the 3' UTR. For Northern blot analysis, samples were hybridized with antisense probe specific to the exon 3 region of IRF5. Genotyping of the lupus-linked IRF5 Exon 9 ARE region was determined by BsmI RFLP analysis by introducing an allele-specific restriction site with a mismatch 3’ PCR primer.
Results: The 293 cell genotype showed heterozygosity for the IRF5 ARE SNP. TLR7 stimulation induced upregulation of lower molecular weight IRF5 mRNA by northern blot that contained Exon 3 but not Exon 9 by real time PCR. In contrast, TLR3 stimulation induced upregulation of a higher molecular weight IRF5 mRNA by northern blot that contained both Exon 3 and Exon 9 by real time PCR. IRF5 mRNA was upregulated in patients with anti-RNP autoimmunity compared to normal controls. All 6/6 patients with lung disease in our cohort upregulated the full-length IRF5 mRNA, while 12/19 patients without lung disease upregulated Exon 3 of IRF5 mRNA but not Exon 9 (Fisher’s Exact p = 0.01). Genotyping showed that the IRF5 ARE SNP allele frequencies were the same for patients upregulating the truncated form and the full-length form of IRF5 mRNA in this cohort.
Conclusions: TLR3 and TLR7 activation upregulate distinguishable forms of IRF5 mRNA. A truncated form of IRF5 mRNA previously linked to lupus risk is upregulated by TLR7. A full-length form of IRF5 mRNA is upregulated by TLR3. Increased expression of full-length IRF5 mRNA is associated with clinical lung disease in RNP+ autoimmune disease patients. These studies identify changes in ARE fate (on IRF5 and potentially other genes) as a novel site of differential action of TLR3 versus TLR7 that may account for differences in TLR-induced tissue targeting.

 M. Carpintero, None; I. Fernandez, None; R.W. Hoffman, None; E.L. Greidinger, None.