title: Gatekeepers of cytokine receptor signalling in the pathogenesis of systemic lupus erythematosus
Creators: name: Zhang, Yaoyuan
Publisher: name: The Australian National University
Year: 2022

description: Systemic lupus erythematosus (SLE) is a multifactorial systemic autoimmune disease with complex aetiology including genetic variations, environmental triggers, and cytokine dysregulation. Cytokines are responsible for intercellular communication primarily transduced by signalling cascades named JAK-STAT signalling pathways. To date, many cytokines and their corresponding JAK-STAT signalling pathways have been implicated in autoimmune diseases. Our group previously identified that SLE-patients had increased frequencies of rare variants in two genes of the JAK-STAT signalling pathways, SH2B3 and TYK2, compared to those in healthy controls. We hypothesised that SLE-patient-specific variants in these two genes may increase the risk of autoimmunity in carriers. SH2B3 encodes a negative regulator of JAK-STAT signalling. Functional assays revealed that four out of five rare SLE-patient-specific SH2B3 variants encode proteins with impaired ability to suppress IFNGR signalling, suggesting loss-of-function (LOF) or hypomorphic alleles. We generated orthologous mouse models of two variants; the E400K (mouse E372K; cav allele) and the R566Q (mouse R530Q; ldf allele). Immunophenotyping analysis demonstrated that both alleles exhibited increased leukocytes, particularly lymphocytes and B cell phenotypes that were previously described for Sh2b3-deficient mice. However, phenotypes were weaker than in Sh2b3-deficient mice, suggesting a hypomorphic allele rather than a complete LOF. The most consistent and interesting phenotypes were observed in B cells, especially the increase in immature and transitional 1 (T1) B cells, which can harbour cells with considerable levels of autoreactivity that can contribute to autoantibody production if tolerance is breached. An increase in the equivalent cell type, transitional B cells, was also observed in human SLE patients with rare SH2B3 variants. Although Sh2b3cav and Sh2b3ldf mice failed to develop clear evidence of spontaneous autoimmunity, by utilising the SWHEL-mHEL3x BM chimera model, we found that Sh2b3cav autoreactive B cells can breach B cell tolerance, and that this predisposed them to autoimmunity given environmental triggers. Indeed, induction of lupus-like systemic autoimmune disease with pristane resulted in higher levels of anti-DNA autoantibodies in mice heterozygous or homozygous for the cav allele. In addition, when crossed to the 564Igi strain, mice carrying the cav allele displayed increased frequencies of autoreactive B cells with activated phenotypes. In contrast to SH2B3, TYK2 is a mediator of JAK-STAT signalling including type-1 interferon (T1-IFN). Thus, given the constitutively high levels of T1-IFN observed in most of SLE patients, patient-associated TYK2 variants were predicted to increase protein activity downstream of the T1-IFN receptors (IFNAR). Consistently, our preliminary functional assays revealed that many SLE-associated TYK2 variants displayed gain-of-function properties including increase IRSE activity downstream of IFNAR, increased STAT1 and STAT3 phosphorylation was also observed in cells overexpressing patient-specific variants in the pseudokinase domain but not in those overexpressing FERM/SH2 domain variants. In summary, this study has demonstrated that rare SLE patient-specific variants in SH2B3 and TYK2 can impair their functions as gatekeepers of cytokine signalling to potentially contribute to an increased risk to autoimmune disease pathogenesis. Although preliminary, our results provide a theoretical basis for the potential use of JAK inhibitors in treating SLE patients with mutations in these two genes that could be tested in the orthologous mouse models.