Negative regulation of the JAK/STAT signalling pathway in inflammatory arthritis

Background ¾ Rheumatoid arthritis (RA) is one of the most common forms of autoimmune disease, affecting about 1% of the population and causing chronic inflammation that primarily affects the joints. Cytokines that signal via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway are major drivers of synovitis in patients with RA and contribute to the rapid progression of the disease. Biological agents targeting the cytokine interleukin (IL)-6, its receptor system or downstream pathway have revolutionised the treatment of immune-mediated diseases and can induce drug-free remission. However, RA is a highly heterogeneous and complex disease and consequently, around 40% of patients do not respond to current frontline biologics. This has raised the need for alternative therapeutic strategies or precision medicine approaches that improve treatment decisions for RA patients. My thesis aimed to investigate the action of novel therapeutic modalities that target the IL-6 receptor system or downstream signalling cassette to improve understanding of the underlying inflammatory processes that drive synovitis. Results ¾ Exploiting three novel classes of IL-6/STAT3 inhibitors, I have interrogated their mode of action in in vitro model systems and animal models of synovitis. (1) Using histopathology and RNA-sequencing of the inflamed synovium, I demonstrated that an anti-cancer therapy (CpG-Stat3siRNA) improved arthritis outcome, altered the balance of STAT1/STAT3 signalling and reduced the incidence of ectopic lymphoid-like structures in synovitis. (2) I also used a virus-derived SOCS3 modulator peptide that suppresses STAT3 activity through the induction of SOCS3. This agent was shown to block pathogenic Thelper (Th)17 cell differentiation in vitro and reduced disease pathology in mice with antigen-induced arthritis. (3) Blocking of IL-6 trans-signalling pathway with engineered inhibitors (cs-130Fc) based on regulatory domains of the sgp130 receptor proved efficacy over other related therapy variants (e.g., olamkicept) in inhibiting STAT3-driven Th17 cell expansion in vitro. In a final approach, I also examined the biology of genes that are suppressed by IL-6 and IL-27 in CD4+ T-cells. These factors might have immune-protective function in synovitis, and initial studies are presented on the identification of CRTAM as one such factor and its inflammatory regulation in mouse synovitis. Conclusions ¾ These studies showcase how JAK/STAT signalling through the IL-6 receptor cassette may be controlled at multiple levels and further demonstrate how investigations into their mode of action help to unearth new understanding of IL-6 biology in RA

IL-6 trans-signaling links inflammation with angiogenesis in the peritoneal membrane

Vascular endothelial growth factor (VEGF) is implicated in the peritoneal membrane remodeling that limits ultrafiltration in patients on peritoneal dialysis (PD). Although the exact mechanism of VEGF induction in PD is unclear, VEGF concentrations in drained dialysate correlate with IL-6 levels, suggesting a link between these cytokines. Human peritoneal mesothelial cells (HPMCs), the main source of IL-6 and VEGF in the peritoneum, do not bear the cognate IL-6 receptor and are thus unable to respond to classic IL-6 receptor signaling. Here, we investigated whether VEGF release by HPMCs is controlled by IL-6 in combination with its soluble receptor (IL-6 trans–signaling). Although treatment with either IL-6 or soluble IL-6 receptor (sIL-6R) alone had no effect on VEGF production, stimulation of HPMCs with IL-6 in combination with sIL-6R promoted VEGF expression and secretion through a transcriptional mechanism involving STAT3 and SP4. Conditioned medium from HPMCs cultured with IL-6 and sIL-6R promoted angiogenic endothelial tube formation, which could be blocked by silencing SP4. In vivo, induction of peritoneal inflammation in wild-type and IL-6–deficient mice showed IL-6 involvement in the control of Sp4 and Vegf expression and new vessel formation, confirming the role of IL-6 trans–signaling in these processes. Taken together, these findings identify a novel mechanism linking IL-6 trans–signaling and angiogenesis in the peritoneal membrane

Selective inhibition of IL-6 trans-signaling by a miniaturized, optimized chimeric soluble gp130 inhibits TH17 cell expansion

The cytokine interleukin-6 (IL-6) signals through three mechanisms called classic signaling, trans-signaling, and trans-presentation. IL-6 trans-signaling is distinctly mediated through a soluble form of its transmembrane receptor IL-6R (sIL-6R) and the coreceptor gp130 and is implicated in multiple autoimmune diseases. Although a soluble form of gp130 (sgp130) inhibits only IL-6 trans-signaling, it also blocks an analogous trans-signaling mechanism of IL-11 and its soluble receptor sIL-11R. Here, we report miniaturized chimeric soluble gp130 variants that efficiently trap IL-6:sIL-6R but not IL-11:sIL-11R complexes. We designed a novel IL-6 trans-signaling trap by fusing a miniaturized sgp130 variant to an IL-6:sIL-6R complex–binding nanobody and the Fc portion of immunoglobulin G (IgG). This trap, called cs-130Fc, exhibited improved inhibition of as well as increased selectivity for IL-6 trans-signaling compared to the conventional fusion protein sgp130Fc. We introduced affinity-enhancing mutations in cs-130Fc and sgp130Fc that further improved selectivity toward IL-6 trans-signaling. Moreover, cs-130Fc efficiently inhibited the expansion of T helper 17 (TH17) cells in cultures of mouse CD4+ T cells treated with IL-6:sIL-6R. Thus, these variants may provide or lead to the development of more precisely targeted therapeutics for inflammatory disorders associated with IL-6 trans-signaling

Activation of naïve CD4+ T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4+ T cells

The cytokine IL-6 controls the survival, proliferation and effector characteristics of lymphocytes through activation of the transcription factors STAT1 and STAT3. While STAT3 activity is an ever-present feature of IL-6 signaling in CD4+ T cells, prior activation via the T cell antigen receptor limits IL-6’s control of STAT1 in effector and memory populations. Here we found that phosphorylation of STAT1 in response to IL-6 was regulated by the tyrosine phosphatases PTPN2 and PTPN22 expressed in response to the activation of naïve CD4+ T cells. Transcriptomics and chromatin immunoprecipitation–sequencing (ChIP-seq) of IL-6 responses in naïve and effector memory CD4+ T cells showed how the suppression of STAT1 activation shaped the functional identity and effector characteristics of memory CD4+ T cells. Thus, tyrosine phosphatases induced by the activation of naïve T cells determine the way activated or memory CD4+ T cells sense and interpret cytokine signals