TRAIL, Death Receptors, TRA-8, and Apoptosis

Transcription

1 TRAIL, Death Receptors, TRA-8, and Apoptosis DR5 is a pro-apoptotic molecule, which can induce cell death in a variety of cells by engaging the it ligand, TRAIL or anti-dr5 antibody. TRA-8 is a monoclonal anti-human DR5 antibody, which induces apoptosis upon binding to its receptor, DR5. TRA-8/TRAIL Decoy DR5 TRAIL - R1 R2 R3 R4 Osteoprotegerin Cell Res Oct;14(5): Apoptosis

2 Anti-human DR5 Antibody Triggers Apoptosis in RA Synovial Fibroblasts TRA-8 can induce apoptosis in human RA synovial fibroblasts. Rheumatoid Arthritis Synovial Fibroblasts Isotype control TRA-8 Advantages of TRA-8 compared to TRAIL: Specificity to DR5; Long half-life; No hepatocellular toxicity. Can TRA-8 induce apoptosis of pathogenic macrophages and T cells in RA?

3 Strategies Used to Determine the Arthritis Therapeutic Effects of TRA-8 in Mice 1. Creation of a mouse model with a humanized DR5 reactive with TRA Control of expression through a Cre/loxp system, using both general (Ubiquitous C) and macrophage specific (Lysozyme M) expression Cre mice. 3. Murine models of inflammatory arthritis (Collagen IIinduced arthritis). 3

4 Generation of human/mouse Chimeric DR5 Transgenic Mice Human, mouse and chimeric DR5 Human DR5 Mouse DR5 Chimeric DR5 Not recognized by anti-hu DR5 Recognized by anti-hu DR5 Doesn t trigger apoptosis in mouse cells Trigger apoptosis in mouse cells Generation of hu/mo chimeric DR5 Tg mice c-flip L Mouse DR5 Promoter cflip or ciap2 ciap2 Floxed STOP Chimeric DR5

5 Generation of human/mouse Chimeric DR5 Transgenic Mice Mo 3kb promoter x Floxed STOP hu/mo DR5 Ubc.Cre Ubc/Cre x Floxed STOP hu/mo DR5 LysM/Cre LysM.Cre 5

6 Tg DR5 Expression in Draining LN of Ubc.Cre DR5 Mice (2-mo post CII) B6 Ubc.Cre DR5 Ubc.Cre Count CD4 + CD8 + CD19 + CD11b + CD11b + CD11b - Gr1 + Ly6C + Ly6C + Hu/mo DR5 CD4 + B6 CD8 + Ubc.Cre CD19 + DR5 Tg + Ubc.Cre CD11b + CD11cGr1 Gr1 + CD11b + Ly6C + Ly6C+CD11b+ CD11b + CD11b - Ly6C-CD11b hu/mo DR5 + (%) Induction of CIA. CIA was induced using DR5 Tg mice of C57BL/6 background that were 8- to 16- weeks old. Mice were immunized by intradermal administration of chicken Type II collagen in complete CFA, followed by injection of chicken CII in IFA on day 30 after the primary injection. To ensure a higher incidence of CII arthritis, an adenovirus expressing mouse IL-17 (2x10 9 pfu/mouse) was administered IV to all mice 2 days prior to the primary immunization with CII. TRA-8 treatment of CIA mice. TRA-8 (dissolved in PBS, 0.2 mg per mouse) or IgG1 isotype control was administered i.v. or i.p. twice/week starting on day 0 (early treatment) or on day 30 (late treatment) until mice were sacrificed. 6

7 TRA-8 Suppresses the Development of CIA in Ubc.Cre DR5 Mice Arthritis Scores DR5 Tg + UbcCre Isotype DR5 Tg + UbcCre Early TRA-8 (from day 0) DR5 Tg + UbcCre Late TRA-8 (from day 30) ** ccii Early TRA-8 Late TRA-8 ccii DR5Tg - DR5Tg + H&E Mac-3 H&E Mac-3 Day 60 post 1 ccii 7

8 TRA-8 Treatment Depletes Macrophages Question: What is the TRA-8 effect if TgDR5 expression is restricted in macrophages? 8

9 M1/M2 Imbalance in Rheumatoid Arthritis - Can It Be Corrected by TRA-8? Current therapeutic targets related to macrophages Inflammatory Anti-GM-CSF Anti-TNF-α GM-CSF LPS IFN-γ TNF-α Imatinib mesylate Tofacitinib Clodoronate Liposomes Anti-Inflammatory M-CSF IL-4 IL-13 IL-10 Anti-M-CSF Blocker TRA-8 Ligand or agonistic antibody TNF-α CD80,86 IL-1, 6, 12, 23 IFN-I RNI ROI CXCL9,10,11 CCL2,3,4,5,15,20 inos DR5 Tyrosine kinase JAK Tyrosine phosphatase Fas TLR2,4 IRF5 RANKL Denosumab RANK IRF4 Osteoclast IL-4,10,IL-1Ra, CD163, MR, GR CD200R TGF-β Ym1/2 Fizz1 CCL1,16,17,18,22 Arg1 IL-4R IL-10R CD200-Fc Adapted from J. Li, et al 2012

10 Generation of Chimeric DR5 Transgenic LysM.Cre Mice Mo 3kb promoter x Floxed STOP hu/mo DR5 Ubc.Cre Ubc/Cre x Floxed STOP hu/mo DR5 LysM/Cre LysM.Cre

11 TRA-8 Treatment Eliminates Inflammatory Macrophages in DR5 Transgenic LysM.Cre CIA Mice

12 Apoptosis Induced by TRA-8 in Joints of DR5 Transgenic LysM.Cre Mice with CIA Caspase Activity Before TRA-8 treatment DR5 Tg - DR5 Tg + After TRA-8 treatment DR5 Tg - DR5 Tg + AB50-Cy5 Intensity/Joint T g h u D R 5 - T g h u D R 5 + T g h u D R 5 - T g h u D R 5 + DR5 Tg - DR5 Tg + DR5 Tg - DR5 Tg + Before TRA-8 B e f o re T R A -8 A f te r T R A -8 ** After TRA-8 Measurement method: Mice were induced to develop CIA. At 8 week after the primary ccii injection, Mice were then treated with TRA-8 (0.2 mg on day 0 and day 3) and apoptosis imaging using AB50-Cy5 was performed on the same mice on day 6 after initiating TRA-8 treatment. 12

13 Apoptosis Induced by TRA-8 in Joints of DR5 Transgenic LysM.Cre Mice with CIA DR5 Tg - E H&E TUNEL Mac-3 H E H E H H H M H DR5 Tg + E H E H E TU M H H H H TUENL + cells in the sub synovial lining layer(%) ** ** DR5 Tg - DR5 Tg + Tg hudr5- Tg hudr5+ ** Total Macrophages Fibroblasts Total Mφ Fibroblasts Measurement method: Mice were induced to develop CIA. At 8 week after the primary ccii injection, Mice were then treated with TRA-8 (0.2 mg on day 0 and day 3) and were sacrificed for in situ staining of joint apoptosis (TUNEL) and joint macrophage (Mac-3) infiltration. 13

14 TRA-8 Treatment Ameliorates the Severity of CIA in DR5 Transgenic LysM.Cre Mice Arthritis score DR5 Tg + Isotype DR5 Tg + TRA-8 DR5 Tg+ Isotype DR5 Tg+ TRA ** Arthritis score DR5 Tg - Isotype DR5 Tg - TRA-8 DR5 Tg- Isotype DR5 Tg- TRA ccii TRA-8 ccii ccii ccii DR5 Tg + TRA-8 Treatment H&E Mac-3 TRAP DR5 Tg TRA-8 Treatment H&E Mac-3 TRAP 20 X 10 X H E M H E E H TR 14

15 Immuno-modulatory Effects of TRA-8 in DR5 Transgenic LysM.Cre Mice with CIA 15

16 Summary I 1. In human/mouse DR5 Tg Ubc.Cre mice with CIA, Tg DR5 is expressed on CD11b + macrophages and CD4 + T cells; In DR5 Tg LysM.Cre mice, Tg DR5 is restrictively expressed on macrophages; 2. TRA-8 (anti-human DR5) treatment results in depletion of pathogenic macrophages and Th17 cells, increased Treg cells in draining LNs of DR5 Tg CIA mice; 3. TRA-8 is potential novel biologic agent for rheumatoid arthritis. QUESTION: What is the TRA-8 effect in a systemic autoimmune disease model? 16

17 Chimeric DR5 Transgenic x Viable Motheaten Mice (me v /me v ) H.E. Joint Mac-3 Joint Lung Prominent disorders of SHP-1 (PTPN6) deficient mice: 1. Myeloid hyper-proliferation and inappropriate activation. 2. Rapidly progressive patchy dermatitis, limb necrosis, recurrent infections, and premature death consequent to hemorrhagic pneumonitis.

18 DR5 expression and TRA-8 Induced Depletion of Inflammatory Macrophages in DR5 Tg me v /me v Mice

19 DR5 expression and TRA-8 Induced Depletion of Pathogenic CD4 T Cells in DR5 Tg me v /me v Mice

20 TRA-8 Treatment Reduces Tissue Inflammation and Increases Lifespan of DR5 Tg mev/mev Mice

21 TRA-8 Eliminates Inflammatory M1 Macrophages and CD4 T Cells in Human RA Patients with Low PTPN6 M1 signature genes Th17 signature genes

22 Summary II Summary 1. DR5 expression is upregulated in inflammatory macrophages and pathogenic CD4 T cells in DR5 Tg me v /me v Mice; 2. TRA-8 selectively eliminates IRF5 + IL-23 + pathogenic macrophages and IL-17 + GM-CSF + CD4 T cells in both human and mouse autoimmune conditions, resulting in immune homeostasis and resolution of inflammation. 3. Anti-human DR5 (TRA-8) can deplete inflammatory cell populations that result from a hyperactive GM-CSF/IRF5 AXIS, and it is a novel biologic agent for RA and other autoimmune diseases. DR5+ IRF-5+ M1 Mφ Naïve CD4 IL-23+ IL-23R+ IL-6, IL-23 GM-CSFR+ RA inflammation TRA-8 IL-10+ positive TGF + β feedback loop M-CSFR+ IRF-4+ M2 Mφ Monocytes GM-CSF Th1/17 TRA-8 Treg IL-10+ Foxp3+ DR5+ GM-CSF+ IL-17+ [IFNγ+] IL-23R+ RORγt+

23 Acknowledgement Division of clinical rheumatology, UAB Dr. John D. Mountz and lab members Dr. Hui-Chen Hsu and lab members Dr. Robert P. Kimberly Dr. S. Louis Bridges Dr. David M. Spalding Dr. W. Winn Chatham Dr. Laura Hughes Thank you for your support