Interleukin 6 (IL6) transsignaling is a non-canonical IL6 signaling paradigm that is a biological modifier of multiple inflammatory diseases. IL6 transsignaling is driven by localized or systemically elevated levels of soluble IL6 receptors (sIL6R) that are generated when the membrane bound IL6 receptor is excised or ?shed? from the extracellular cell surface. In cells that do not express IL6 receptor, this extracellular sIL6R can bind IL6, form an active complex with ubiquitously expressed membrane-bound glycoprotein 130 (gp130), and then initiate dysfunctional activation of the IL6 signal transduction pathway. In humans, IL6 receptor shedding is greatly enhanced by the inheritance of the IL6R Ala358 isoform (rs2228145; Asp358Ala), and this coding change has been genetically associated with multiple diseases, some of which are targets of anti-IL6R therapies. IL6 transsignaling has become a research focus in multiple disciplines at Wake Forest School of Medicine, however no IL6 transsignaling animal models have been established to complement the in vitro cell and clinical studies being performed at Wake Forest. Within the parameters of the R03 mechanism to ?develop new research technology?, we are proposing to construct a mouse model of IL6 transsignaling that will benefit multiple research efforts at Wake Forest. The goals of this proposal are: 1. To generate a CRISPR/Cas9 knock-in C57BL/6 mouse model of IL6 receptor shedding by incorporating termination codon at the Glu357 codon in the mouse il6ra gene. Termination of the IL6 receptor peptide at this amino acid will remove the transmembrane binding domain and will allow the IL6 receptor to be excreted into the extracellular compartment, thus increasing the extracellular levels of sIL6R. The increase in sIL6R will be measured in serum, cerebrospinal fluid (CSF), and lung brochoalveolar lavage (BAL) fluid in this C57BL/6 Il6raE357Ter mouse model and compared the sIL6R levels in wild type C57BL/6 . 2. Assess the ability of increased soluble IL6 receptor levels in C57BL/6 Il6raE357Ter mice to induce IL6 transsignaling by measuring the increase in Stat3 and Socs3 expression in tissues that highly express IL6 receptor (ex. liver, lung, peripheral blood cells) and in tissues that express low levels of IL6 receptor (ex. prostate, brain, kidney). This IL6 transsignaling model will be innovative in that it will very closely resemble the IL6 transsignaling state induce by the IL6R Asp358 in humans and can be crossbred with other mouse models of disease to determine the modifying effects of IL6 transsignaling in those diseases and determine the efficacy of anti-IL6R therapies.
Interleukin 6 (IL6) transsignaling is a non-canonical IL6 signaling paradigm that appears to be a biological modifier of multiple inflammatory diseases. In humans, IL6 transsignaling is driven by the inheritance of the common human IL6R Ala358 mutation and is considered a disease modifying mutation. In this proposal, we will be constructing a mouse model of IL6 transsignaling using CRISPR/Cas9 methods to knock-in a mutation into the mouse Il6ra gene. This mutation will mimic the function of the human IL6R Ala358 mutation, thus increasing the levels of IL6 transsignaling. This mouse model will be shared across several research areas at Wake Forest to study the effects of IL6 transsignaling on disease initiation, progression, and severity. This model will also be shared with any other NIH funded investigator interested in how IL6 transsignaling modifies other diseases.
