Protein tyrosine phosphatases as rheostats of Jak-STAT cytokine signals and determinants of disease heterogeneity.

Inflammation is the body's response to infection or injury and contributes to the healing process. In immune-mediated diseases such as rheumatoid arthritis, appropriate control of inflammation is lost, promoting joint disease. Patients with these diseases often display differences in the underlining pathology, affecting the rate of disease onset, the severity, and response to therapy. Studies outlined here will define the inflammatory mechanisms driving these differences in disease. Adopting newly developed models of arthritis that display disease hallmarks comparable with three forms of joint inflammation in rheumatoid arthritis, we will identify the decision-making process within the inflammatory cascade responsible for steering the course of pathology. The study is therefore designed to generate new hypotheses relevant to the diagnosis and treatment of this debilitating condition.

What is the background to the problem?
Cytokines are proteins that regulate inflammation and are considered major drug targets for the treatment of rheumatoid arthritis. These inhibitors target adverse cytokine activities driving joint inflammation and disease progression. However, not all patients respond to these drugs, suggesting that multiple, often independent, mechanisms contribute to joint disease. We propose that cytokines steer the type of inflammatory joint disease seen in rheumatoid patients. Increased understanding of the inflammatory processes responsible for this level of diversity will improve patient diagnosis and clinical decisions on the best course of patient therapy.

What are the research questions?
We previously identified a regulatory mechanism that alters the way cytokine cues are sensed and interpreted by cells within the inflamed joint. We propose that this mechanism affects the pattern and severity of the joint disease. The project is, therefore, designed to:

1. What are the inflammatory mechanisms that drive chronic joint disease?
2. How do the activities of particular cytokines affect the type of joint inflammation observed?
3. How are cytokine signals controlled to determine the course of the disease and joint pathology?
4. Can insights from mouse models of disease increase understanding of how different forms of joint inflammation arise in humans?

What is the experimental plan?
Studies will establish how cytokines promote the various forms of joint inflammation observed in patients. Here, differences in inflammation refer to the composition and specific organization of infiltrating immune and structural cells within the diseased tissue. Examining the response of these cells to cytokines, experiments will use cell-based systems and mouse models of arthritis to identify differences in gene expression, the mechanisms that affect these gene changes and the impact of these events on joint inflammation. Testing the relevance of any discoveries to human disease, we will use ultrasound-guided tissue biopsies from rheumatoid arthritis patients to generate new hypotheses affecting the clinical management of rheumatoid arthritis.

Why is this particular study important?
Biological drugs and small molecule inhibitors that block cytokines have revolutionised the treatment of immune-mediated diseases such as rheumatoid arthritis. However, patients often show inadequate responses to treatment and frequently display varying efficacies to certain classes of therapy. This lack of therapeutic response may reflect the clinical characterisation of a spectrum of disease subtypes affecting arthritis patients. Enhancing our understanding of arthritis pathology, we will identify the cellular and molecular processes of disease heterogeneity in rheumatoid arthritis. New insights captured through this study will generate new hypotheses on the development of arthritis and open new opportunities for future clinical investigation.

1. Define the regulation of STAT transcription factor by protein tyrosine phosphatases (PTP)
We will analyse RNA-seq and ATAC-seq data from IL-6, and IL-27, stimulated naïve and effector memory CD4+ T cells from Lck-Cre:Ptpn2fl:fl and Ptpn22-/- mice (and littermate controls). ChIP-seq of STAT1, STAT3 and P300 will identify the regulation of STAT transcription factors by PTP. Archived repository datasets will enhance the bioinformatic analysis. We will also consider the relevance of this mechanism to stromal cells from the joint using synovial fibroblasts cultured from mice with antigen-induced arthritis (AIA).

2. Establish the regulatory properties of PTP activity in synovitis
Histology assessments of joint pathology and bioinformatics of functional genomics will compare the induction of synovitis in Lck-Cre:Ptpn2fl:fl and Ptpn22-/- mice with corresponding data (already generated) from wild-type, Il6ra-/- and Il27ra-/- mice with AIA. Bioinformatics will establish how STAT transcription factors engage the genome during disease and reveal the action of PTP on Jak-STAT signalling. Data will identify common pathways driving synovitis and disease heterogeneity. As evidenced by our publications and pilot data, we will validate our mouse findings using clinical data from open access repositories (e.g., GEO, the accelerated medicine partnership) and our collaborators. This approach will also identify criteria for the design of future clinical studies.

3. Track the transcriptional profile of cells shaping synovial histopathology
Single-cell sequencing (scRNA-seq, scATAC-seq) of synovial tissue from mice with AIA will map gene signatures, epigenetic determinants (e.g., transcription factor binding sites) and cell types affecting disease onset. Bioinformatics will provide insights into alternate cellular dynamics, cell lineages and functions responsible for disease heterogeneity in synovitis.