Background/Purpose: Microarray expression analysis of blood taken from patients with Systemic Lupus Erythematosus (SLE) has revealed two characteristic signatures: one that reflects exposure to type 1 interferon (IFN) and the other reflective of immature circulating granulocytes.  Whereas the IFN signature is thought to arise predominantly from the activation of TLR7 in plasmacytoid dendritic cells (pDC) by RNA containing nucleoprotein immune complexes (ICs), the generation of the granulocyte signature is uncertain. Mice that overexpress TLR7 (TLR7 transgenic (Tg) mice) not only develop a lupus-like syndrome, but also a myeloid expansion in the spleen.

Goals: A) To determine the relationship between the myeloid expansion and the pathology in TLR7 Tg mice. B) To determine whether the myeloid expansion and immunopathology is driven by the TLR7 ligand, RNA, and whether it is also IFN dependent.  

Methods: Granulocytes (CD11bhigh Ly6Ghigh Ly6Chigh) and inflammatory monocytes (CD11bhigh Ly6Chigh Ly6Gnegative) were flow sorted from the spleen. Microarray analysis on RNA isolated from whole spleen was performed using Illumina’s MouseWG-6 v2.0 Expression BeadChips. Chips were scanned on an Illumina HiScanSQ  System and >2.0-fold changes in gene expression by TLR7 Tg compared with B6 were analyzed by Ingenuity software. To create TLR7 mice that either overexpressed RNase A or were deficient in the type 1 interferon receptor (IFNAR), TLR7 mice were crossed to RNase Tg or IFNAR deficient mice respectively. Parenchymal organs were examined by light microscopy, Mac2 staining and by other methods as appropriate.    

Results:   Transcript profiling of TLR7 Tg and B6 spleens (n=10 in each group) revealed a striking granulocyte signature in the TLR7 Tg spleen (e.g. proteinase 3, myeloperoxidase and elastase were expressed at 16 x wild type). Surprisingly, Q-PCR of flow sorted subpopulations revealed that it was the inflammatory monocytes rather than the granulocytes that expressed high levels of these genes. These myeloid populations from TLR7 Tg mice expressed 5-10-fold more TLR7 mRNA compared to B6 mice and the inflammatory monocytes from TLR7 Tg mice  expressed more inflammatory cytokines in response to the TLR7 agonist gardiquimod. Pathologic studies of TLR7 Tg mice revealed only mild kidney disease whereas a striking inflammatory infiltrate predominantly of myeloid cells was observed in the liver. This infiltrate was associated with hepatic necrosis. The inflammatory infiltrate was markedly reduced in double mutant TLR7 Tg mice that either co-expressed RNase or that were deficient in IFNAR expression.

Conclusion: Our results indicate that TLR7 Tg mice express a granulocyte signature similar to that observed in humans with SLE. This signature was a consequence of high expression of neutrophil genes in inflammatory monocytes which is likely explained by increased numbers of immature cells released by the bone marrow suggestive of altered myelopoiesis. Attenuation of disease in the double mutant mice indicate that myelopoiesis and /or activation of myeloid cells is caused by exposure to RNA in an IFN dependent process. These results have important implications for therapy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-granulocyte-signature-and-organ-inflammation-in-tlr7-responsive-mice-is-rna-and-type-1-interferon-dependent/