The Ying and Yang of IFN-γ in Autoimmunity

Transcription

1 The Ying and Yang of IFN-γ in Autoimmunity Chander Raman, Ph.D. Early Late

2 Autoimmune neuroinflammation Experimental autoimmune encephalomyelitis (EAE) Rheumatoid arthritis (RA)

3 Autoimmune neuroinflammation Experimental autoimmune encephalomyelitis (EAE) Rheumatoid arthritis (RA)

4 Acknowledgements - Raman Lab Kevin Cashman Donald McGuire Amber Buel Christine Sestero Robert Axtell Rodrigo Naves Simer Preet Singh Funding NMSS, NIH-NIAID, NIH-NINDS UAB Collaborators Patrizia De Sarno Robin Lorenz John Mountz Hui-Chen Hsu Etty (Tika) Benveniste Stanford University Lawrence Steinman

5 Integrated signals from IFN-γ and type I IFN (IFN-α/IFNβ) in autoimmune neuroinflammation Naves et al, J. Immunol. 9:967, 03 Unpublished data Autoimmune pathogenesis IFN-γ IFN-α/IFN-β

6 Interferon Receptor Signaling MacMicking, J. Nat. Rev. Immunol. :367-38, 0

7 Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE) Multiple Sclerosis Autoimmune Disease characterized by: EAE Neurodegeneration Inflammation of central nervous system Effector cells are T-cells, dendritic cells, microglia, astrocytes, macrophages and others Induced mouse model of MS Disease develops after immunization with myelin-derived protein or peptides or transfer of encephalitogenic CD T cells. Shares many of the pathologies with human MS Disease initiated by autoreactive CD T-cells

8 Clinical Score Myelin Oligodendrocyte (MOG) Induced EAE 6 5 MOG Pertussis Toxin 3 Inductive Phase Effector Phase Days Post-Immunization 30

9 X X Mice Lines WT C57Bl/6 mouse Ifnαr-/- mouse - Abrogation of signaling from type IFNs Ifnγr-/- mouse Abrogation of signaling from type IFNs Ifnα/γr-/- mouse Abrogation of signaling from type and type interferons Plantanias, Nature Reviews Immunology, 5:375

10 Delayed onset but more severe EAE in IFNGR deficient mice Clinical Score * WT Ifngr -/ Days post-immunization * p<0.0

11 IFNAR-deficiency does not alter development and progression of EAE Clinical Score WT Ifnar -/ Days post-immunization

12 Removal of type IFN-signals attenuates severity in IFN-γ receptor deficient mice 6 Clinical Score 5 3 * WT Ifngr -/- Ifnar -/- Ifngr -/- * p< Days post-immunization

13 Inflammatory cell infiltration/activation in brain stem of IFN-γ receptor deficient mice UNIMM H&E LFB MPO GSI-B CD WT Ifnar -/- CD + T cells Ifngr -/- Ifnar -/- Ifngr -/-

14 Steinman, Nature Immunology : - (00) Th and Th7 encephalitogenic CD T cells contribute to MS and EAE. Question What is the T-cell intrinsic role of interferon signaling in EAE induced by Th or Th7 cells

15 Experimental design: EAE passive transfer Th Immunize WT or IFN-receptor deficient mice (50 g MOG 35-55, 500 g MT) Harvest spleen/ln (Day ) Th7 Restimulate with MOG under polarizing conditions i.v. transfer to naïve WT mice and monitor EAE 6 x 0 6 cells

16 T H Transfer WT IFNR-/- T H-cells WT mice

17 Clinical Score IFN-g WT IFNR-/- T H-cells WT mice T H Days Post Transfer Donor population WT IL-7

18 Clinical Score IFN-g IFN-γ signaling is necessary for persistence of Th EAE T H 5 3 WT IFNR-/- T H-cells Days Post Transfer WT mice Donor population WT Ifngr -/- IL-7

19 Clinical Score IFN-γ IFNAR signaling is necessary to maintain severity of Th EAE T H 5 3 WT IFNR-/- T H-cells Days Post Transfer WT mice Donor population WT Ifnar -/- IL-7

20 IFNAR signals do not compensate for requirement of IFN-γ signals in Th EAET H IFN-g WT IFNR-/- T H-cells WT mice 5 Donor population Clinical Score 3 WT Ifnar -/- Ifngr -/- Ifnar -/- Ifngr -/ Days Post Transfer IL-7

21 T Cell intrinsic IFNGR signals are necessary for persistence of encephalitogenic Th cells CD5. WT IFNR-/- T H-cells CD5. WT mice Clinical Score Days Post Transfer WT Ifngr -/- CD + T-Cells x CD5. + (recipient) WT Ifngr-/- WT Ifngr-/- WT Ifngr-/- CD5. + (donor) WT Ifngr-/- d d5 d d5

22 T H 7 Transfer WT IFNR-/- T H7-cells WT mice

23 IFN-g T H 7 Clinical Score 5 3 WT IFNR-/- T H7-cells WT mice Donor population WT IL Days Post Transfer

24 Clinical Score IFN-g IFN-g 5 3 Type I and type II IFN signaling in T-cells suppress Th7 EAE WT IFNR-/- T H7-cells Days Post Transfer WT WT mice Donor population Ifnar -/- Ifngr -/- Ifnar -/- Ifngr -/- T H 7 IL-7

25 IFN-g MODEL 5 WT IFNR-/- T H-cells WT mice Clinical Score 3 Donor population Ifngr -/- Ifnar -/- Ifngr -/ Days Post Transfer Immature DC Naïve macrophage & microglia Mature DC Activated macrophage & microglia IL-7 Apoptosis IFN-γ elevated IFN-γ NO superoxides

26 Hypothesis: The ability of recipient macrophages/microglia to respond to IFN-γ is necessary to attenuate Th EAE

27 Immunize WT mice Harvest spleen/ln (Day ) T H Restimulate with MOG under polarizing conditions i.v. transfer T H to naïve WT or IFN-γR-/- mice and monitor EAE Recipients cannot respond to IFN-γ

28 IFN-γ signaling in recipient is necessary to control Th EAE C lin ic a l S c o re WT T H -cells WT Ifngr -/- mice Recipient Mouse W T Ifn g r -/ T im e a fte r tr a n s fe r (d )

29 Does IFN-γ have a dual role in EAE? Clinical Score Days post-immunization WT Ifngr -/- Experiment: - Immunize WT or IFNAR-/- mice. - Treat with rifn-γ - day -0 (induction phase) - day 0-0 (effector phase) - Follow disease course

30 Outcome of interferon-γ treatment is dependent of stage of disease Clinical Score IFN-g PBS rifn-g * Days post-immunization

31 Clinical Score Outcome of interferon-γ treatment is dependent of stage of disease IFN-g * Clinical Score IFN-g Days post-immunization PBS mrifn-g g mrifn-g 00 ng

32 Does type I IFN receptor modulate therapeutic effectiveness of IFN-γ? Experiment: Test if IFNγ can inhibit EAE in IFNAR-/- mice

33 5 WT/PBS WT/IFN-g Clinical Score IFN-g Time after induction (d)

34 5 WT/PBS WT/IFN-g Ifnar -/- /PBS Clinical Score IFN-g Time after induction (d)

35 IFN-γ requires type I-IFN signaling to suppress EAE 5 WT/PBS WT/IFN-g Ifnar -/- /PBS Ifnar -/- /IFN-g Clinical Score IFN-g Time after induction (d)

36 STAT STAT IFNAR IFNAR Tyk JAK STAT3 STAT P IRF9 P STAT5 STAT6 Test if therapeutic effectiveness of IFN-γ is dependent on STAT

37 IFN-γ mediated suppression is STAT- dependent Clinical Score 6 5 WT/PBS WT/IFN-g Stat -/- /PBS IFN-g Time after induction (d)

38 IFN-γ mediated suppression is STAT- dependent Clinical Score WT/PBS WT/IFN-g Stat -/- /PBS Stat -/- /IFN-g IFN-g Time after induction (d)

39 Steinman, Nature Immunology : - (00) Does therapeutic effectiveness of IFN-γ segregate on a Th - Th7 axis? Encephalitogenic T H cells T H 7 cells PBS or IFN-γ Naïve mice

40 IFN-γ suppresses Th EAE but not Th7 EAE Clinical Score 6 WT/PBS WT/IFN-g 5 3 Th IFN-g Time after transfer (d) Clinical Score 6 WT/PBS WT/IFN-g 5 3 Th IFN-g Time after transfer (d)

41 Summary - Integrated regulation of EAE pathogenesis by IFN-γ and type I IFN signals IFN-γ signaling in T-cells is necessary to sustain Th EAE but not Th7 EAE IFN-γ and type I IFNs independently regulate severity of Th7 pathogenesis. Janus-like activity of IFN-γ treatment in EAE: exacerbates disease during induction and suppresses in active disease exacerbates Th7 disease, attenuates Th disease

42 Autoimmune neuroinflammation Experimental autoimmune encephalomyelitis (EAE) Rheumatoid arthritis (RA)

43 Acknowledgments Chander Raman S. Louis Bridges Molly Boland Yanna Ding Surabhi Vinod Qi Tang Keith Wanzeck Stephanie Ledbetter Dongmei Sun Rajeshwari Chellappan Clark Ren Setphanie Boas UAB Collaborators Patrizia De Sarno Maria Danila John D. Mountz Hui-Chen Hsu Richard Reynolds Xiangqin Cui William Meador Khurram Bashir John Rinker UAB Comprehensive Flow Cytometry Core

44 IFNGR Expression in PBMCs is Associated with Radiographic Severity of Rheumatoid Arthritis in African- Americans 583 African-American RA 583 African-American RA 583 African-American RA n=7 n=00 n=99 n=38 n=88 n=58 n=7 n=76 n=37 n=3 n=8 n=55 n=7 n=6 n=60 n=3 n=86 n=59.tang Q et al. Arthritis Rheumatol. 67:65-70, 05

45 Elevated Expression of IFNGR Associates with RA Disease in African-Americans.Tang Q et al. Arthritis Rheumatol. 67:65-70, 05

46 O'Shea JJ et al. N Engl J Med 03;368:6-70.

47 HC RA HC RA HC RA HC RA HC RA HC RA py70 STAT Log fold control MFI Enhanced IFN-γ induced STAT activation in RA vs HC in naïve and CM CD T cells 6 8 Naïve CD5RA +, CCR7 + * 6 8 Central memory CD5RA -, CCR7 + * 6 8 Effector memory CD5RA -, CCR Basal IFN-γ Basal IFN-γ Basal IFN-γ Data have not been stratified on disease activity. *p<0.05.

48 HC py70 STAT Log fold control MFI RA HC RA HC RA HC RA Enhanced IFN-γ induced STAT activation in RA vs HC in Tfh and Treg cells Tfh PD- + CXCR5+ Treg CD5 hi 3 6 * 3 6 * Basal IFN-γ Basal IFN-γ *p<0.05.

49 The activation of STAT by IFN-γ in all CD8 T cell populations was similar in healthy controls and RA. Due to the small sample number (37 RA), the data have not yet been stratified on disease severity. Effector CD populations - Th and Th7 will be examined

50 IL- - Regulation of pathogenesis Autoimmunity/RA Tfh Treg Th IL- Th7

51 O'Shea JJ et al. N Engl J Med 03;368:6-70.

52 py69 STAT5 Log fold control MFI HC RA HC RA HC RA HC RA HC RA HC RA In RA enhanced IL- induced STAT5 activation observed only in EM CD T cells Naïve CD5RA +, CCR Central memory CD5RA -, CCR Effector memory CD5RA -, CCR7 - * * Basal IL- Basal IL- Basal IL- *p<0.05.

53 From Furqan et al, Biomarker Research DOI:0.86/ , Jan 6 03

54 IL- - Regulation of pathogenesis Autoimmunity/RA Tfh Treg Th IL- Th7 STAT5 phosphatase?

55 py69 STAT5 Log fold control MFI py69 STAT5 Log fold control MFI In RA inhibition of phosphatase by phenylarsine oxide (PAO) enhances IL- induced STAT5 activation Naïve Central Memory Effector Memory PAO PAO IL- Stimulated Treg (CD5 hi ) Tfh * IL- Stimulated * 6 ** HC RA

56 PAO treatment has no effect on IFN-γ induced STAT activation

57 IL- - Regulation of pathogenesis Autoimmunity/RA Tfh Treg Th IL- Th7 STAT5 phosphatase?

58 Conclusions In African American population Ifngr expression associates with RA and Igngr associates with enhanced radiographic severity. IFNg-induced STAT activation is greater in RA naïve, central memory CD T cells, Treg and Tfh cells than healthy controls. IL- induced activation of STAT5 is similar between RA and HC in all CD and CD8 T cell populations with the exception of effector memory CD T cells. Treatment with phenylarsine oxide (PAO) enhances STAT5 activation in RA. PAO treatment had no effect on IFN-γ induced STAT activation.