Using Phosphoflow to Dissect Alterations in Cytokine- Induced Activation of Jak/STAT Pathway in Rheumatoid Arthritis
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1 Using Phosphoflow to Dissect Alterations in Cytokine- Induced Activation of Jak/STAT Pathway in Rheumatoid Arthritis Molly Boland, Pathobiology and Molecular Medicine Mentors: Chander Raman, PhD S. Louis Bridges, MD, PhD
2 The Jak/STAT Pathway
3 Expression of Interferon Receptors indicates presence and severity of RA Rheumatoid Arthritis Increased IFNGR1 expression in RA patients compared to controls Increasing IFNGR2 expression indicates radiographic damage No change in serum levels of IFN-γ indicating that the limiting factor is the expression of receptors Tang Q, et al., 2015
4 is an important regulator of disease Nuetralization of in mouse model inhibits the proliferation of CD25+CD4+ regulatory T cells, causing autoimmunity. 1 T reg proliferation driven by suppresses differentiation of Th17 cells. Suppression of Tfh populations cause a decrease in germinal center formation JEM, 2005, Setoguchi 2. Immunity, 2012, Ballesteros-Tato
5 Advantages of Multi-parameter Phospho Flow Cytometry Evaluate activation-dependent post-translational changes in 3 or more STATs and/or other signaling molecules simultaneously Ability to evaluate changes in expression/activation in subpopulations within a mixed population. This is not feasible by Western blot due to cell number limitations. Ability to examine multiple patients/controls at the same time. Reproducible, sensitive and with high specificity.
6 SSC CD45RA SSC FSC-H SSC Gating Strategy Lymphocytes Single Cells Live Cells FSC FSC-A Live/Dead CD8 CD4 CD25 CCR7
7 CD45RA SSC Phosphoflow Analysis CCR7 Central Memory CD4 CD25 Treg IFN-ɣ IFN-ɣ p-stat1 p-stat5 p-stat1 p-stat5
8 RADAR 350 patients currently enrolled ~ 93% of patients have agreed to routine venipuncture for obtaining blood for IRB-approved studies. Database includes the following patient information Current medications Number of tender or swollen joints from 28 evaluated joints (CDAI, DAS28 score) Clinical evaluation by doctor or nurse practitioner Saved for multiplex cytokine analysis Lymphocyte isolation Frozen at -80 in order to normalize results
9 Fold change of isotype Activation of STAT1 by IFN-γ 20 RA HC Gout 15 Central memory Naïve CD Treg
10 Fold change of isotype Activation of STAT5 by 60 RA HC Gout Central memory Naïve CD Treg
11 Role of Phosphatases Phosphatases (PTPs) are critical regulators of many signaling pathways. Many PTPs may be associated with autoimmunity A single nucleotide polymorphism in the Ptpn22 gene is strongly associated with RA and other autoimmune diseases. 1 PTPN22 is a direct target of FoxP3 and could be pathogenic if mutant form is overactive in Tregs Seminars in Immunology, 2006, Bottini 2. Nature, 2007, Marson
12 Fold change of isotype Phosphatase inhibitor increases stimulated p-stat5 RA HC Gout 60 + PAO + PAO + PAO Central memory PAO + PAO + PAO 40 Treg 20 0 PAO = phosphatase inhibitor
13 Fold difference of isotype Total STAT5 decreased with phosphatase inhibitor 25 Total STAT5 + PAO PAO = phosphatase inhibitor
14 Summary is a known regulator of autoimmune disease There is phosphatase activity specific to RA and p-stat5 Attenuation of activation of STAT5 could be pathogenic in RA
15 Do circulating levels of cytokines contribute to differences in cytokine-induced activation of the Jak/STAT pathway?
16 LEGENDplex: using flow cytometry for multiplex cytokine analysis Ability to evaluate up to 13 cytokines simultaneously In-plate assay allows for up to 80 samples to be run at one time Customizable panels for mouse and human
17 APC SSC LEGENDplex assay Beads A Beads B Beads A Beads B IL-10 IL- IL-6 9 IL-13 IL-5 2 IL-4 IL-17A IFN-γ 1 IL-17F TNFα FSC PE Beads A Beads B APC APC
18 Plasma cytokine levels are not significantly different in RA vs Controls Potential biomarkers for treatment may include the cellular response to cytokines in different sub-populations of T cells RA patients HC Gout
19 Acknowledgements Raman Lab Surabhi Vinod Clark Ren Yanna Ding, PhD L. Ashley Haynes Christine Sestero, PhD Bridges Lab/Group Keith Wanzeck Krishnan Raman, PhD Vincent Laufer Stephanie Ledbetter UAB collaborators John Mountz, MD, PhD Hui-Chen Hsu, PhD Patrizia De Sarno, PhD Comprehensive Flow Cytometry Core (CFCC) Enid Keyser NIH P30 AR NIH P30 AI27667 Funding from the NIH
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PRINCIPAL INVESTIGATOR: Stanley Louis Bridges, Jr., MD, PhD. CONTRACTING ORGANIZATION: University of Alabama at Birmingham Birmingham, AL
AWARD NUMBER: W81XWH-16-1-0537 TITLE: The Role of the Interferon-Gamma-Jak/STAT Pathway in Rheumatoid Arthritis PRINCIPAL INVESTIGATOR: Stanley Louis Bridges, Jr., MD, PhD CONTRACTING ORGANIZATION: University
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