Background/Purpose:

The genetic analysis of the lupus prone NZM2410 mouse has identified a suppressor locus, Sle2c2, which confers resistance to both spontaneous and chronic graft-vs.-host disease (cGVHD) induced lupus. We hypothesized that a non-synonymous polymorphism in the granulocyte colony stimulating factor (G-CSF) receptor 3 (Csf3r) gene in Sle2c2 is responsible for providing protection. G-CSF acts as a pleiotropic growth factor and mediates its biological functions including granulopoiesis by binding specifically to G-CSF-R. Exogenous G-CSF restored cGVHD susceptibility in Sle2c2, suggesting that the Csf3r rs13377964 coding SNP results in a loss-of-function in signaling modulating the expression of targets in myeloid cells expressing G-CSF-R, including neutrophils and dendritic cells, and thus mediating lupus resistance.

Methods:

We investigated the functional differences between the wild type B6 and congenic Sle2c2 alleles of Csf3r transduced in the murine Csf3r-negative myeloblast- like 32d.cl3 cell line with a lentiviral vector. G-CSF-R signaling was measured using phospho-flow staining and gene expression assays in transduced 32d.cl3 cells stimulated with mouse G-CSF. Furthermore, the assays were repeated using primary bone-marrow derived dendritic cells, splenocytes and peripheral blood leukocytes of B6 and congenic B6.Sle2c2mice. In addition, the progression and activation of G-CSF-R expressing myeloid cells throughout the entire course of cGVHD was compared between B6 and congenic mice by flow cytometry.

Results:

32d.cl3 cells expressing the Sle2c2 allele of G-CSF-R showed a reduced expression of Jak and Src targets in response to G-CSF with both phospho-flow and gene expression assays. The Sle2c2 allele of G-CSF-R induced a reduced phosphorylation of STAT3, ERK and p38 and lowered the expression of genes such as SOCS3, cFOS, Fcgr3, IRF1 compared to B6 allele. The effects of phospho signaling associated with Jak-STAT and ERK pathways were recapitulated with ex-vivo experiments using primary BM derived DCs as well as total splenic and blood neutrophils. Differences in frequency of G-CSF-R positive neutrophils and splenic dendritic cell subsets as well as levels of G-CSF-R expression during the course of cGVHD further suggest the functional relevance of the G-CSF-R SNP in lupus.

Conclusion: Our results show that the coding rs13377964 SNP affects G-CSFR signaling, supporting the hypothesis that it could confer resistance to lupus. The defective signaling observed downstream of G-CSF-R primarily within the Jak and Src pathways in both primary cells and cell lines transduced with Csf3r alleles suggest the functional relevance of the polymorphism. Both Jak and Src kinase pathways are involved in differentiation, proliferation and survival of myeloid progenitor cells and activation of neutrophils. Future experiments aimed at elucidating the differences in functions of these pathways using cell lines and myeloid cells such as neutrophils and dendritic cells will enhance our understanding of the GCSFR axis in lupus pathogenesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/characterizing-the-effects-of-the-g-csf-r-coding-rs13377964-snp-located-within-murine-lupus-susceptibility-locus-sle2c2/