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Interferons and Lupus: Strategies for Blocking a Defender Gone Rogue

Interferons and Lupus: Strategies for Blocking a Defender Gone Rogue. Jerome A. Langer UMDNJ-Robert Wood Johnson Medical School Dept. of Pharmacology Piscataway , NJ. Molecular View of Human Anatomy: Immune System RU: 01:090:275:01; 04.29.13. Intro to the IFN system

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Interferons and Lupus: Strategies for Blocking a Defender Gone Rogue

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  1. Interferons and Lupus: Strategies for Blocking a Defender Gone Rogue Jerome A. Langer UMDNJ-Robert Wood Johnson Medical School Dept. of Pharmacology Piscataway, NJ Molecular View of Human Anatomy: Immune System RU: 01:090:275:01; 04.29.13

  2. Intro to the IFN system • What is Lupus (SLE)? • Evidence that Type I IFN expression is related to the pathogenesis of SLE • Strategies for blocking Type I IFN action • Development of a Type I IFN antagonist

  3. 1957 Peak production of baby-boomer babies Brooklyn Dodgers play last game at Ebbets Field Eisenhower enforces desegregation in Little Rock with US Army Congress passes the Civil Rights Act of 1957 Sputnik launched by Soviet Union 6. Interferon discovered!

  4. Viral interference - 1957 • Phenomenon: 1 virus interferes with another • Options: • Virus 1 produces product incompatible with Virus 2 • Virus 1 affects metabolism of cell (e.g., metabolite depletion) • Virus 1 stimulates cells to produce a substance that inhibits Virus 2 • (N.B. Experimentally, V1 can be an inactive virus)

  5. Cells (chick membranes) 37 °C, 24 h. Medium Cells (chick membranes) Wash Cells (new) 37 °C, 48 h. 37 °C, 48 h. Discovery of Interferon Reduced Virus No Virus Yield Isaacs A, Lindenmann J (September 1957). "Virus interference. I. The interferon". Proc. R. Soc. Lond., B, Biol. Sci. 147 (927): 258–67.

  6. Experimental infection of humans with wild-type influenza virus

  7. Interferon Families

  8. Type I Interferons • 13 IFN-αs. IFN-β, • IFN-ε, IFN-ω, IFN-κ • (variable functions) • Gene cluster on Chr. 9 • Generally co-expressed • (α’s) • Receptor: IFNAR • = IFNAR-1+ IFNAR-2

  9. To Arms! The viruses are coming!

  10. Cytokines – General Properties • Proteins • Synthesized and secreted in response to external stimuli • Often transient (controlled by transcription and post-translational mechanisms): turn on, turn off (why?) • Pleiotropic (1 cytokine → different effects on many cell types) • Redundant (different cytokines can have same or overlapping effects) • Often function in a concentration range of 10-9 to 10-12 molar (nanomolar to picomolar) • Autocrine, paracrine, endocrine actions

  11. Activities of Type I IFNs • Antiviral • Antiproliferative • Antitumor • Immunomodulatory Functions: NK Activity; T-cells; Macrophages; Induces MHC Class I expression; B-cell class-switching; T-cell survival; dendritic cell maturation

  12. IFN Membrane IFNAR Jak-STAT Signal Pathway Nucleus Signal mRNAs proteins ISRE ISG Cellular Effects

  13. 9 IFN 8 IFN 5 IFNAR 1 2 4 3 7 10 6 IFNAR “Target Cell” IFN Summary of Interferon Action 8. Binding to “Target Cell” 9. Signaling to nucleus 10. New protein synthesis 11. Cell-type specific changes: Antiviral state Change in cellular activation Etc. • Viral entry • Recognition by cytoplasmic “sensors” • Transmission of signal to nucleus: • Transcriptional activation of IFN genes • Secretion of Type I IFNs (e.g., α, β) • Feedback (autocrine action) • Additional regulation: antiviral state

  14. Cell Cell Cell Cell Cell IFN Apoptosis Direct Antiviral Effect Interactions with Innate and Adaptive Immunity Mechanisms of Type I IFN-based Defenses - MHC induction - NK cell activation - DC maturation - Th1 biasing - B-cell class switching

  15. Lupus (Systemic lupus erythematosus) • Autoimmune disease • Chronic inflammation • Multiple organ involvement • Heterogeneous presentation • Prevalence (est.: 40-120/100,000; or higher) • 120,000-360,000 in US (Others: >1 million) • 90% female; more common (3X) in AA than in Caucasian population; also more common in women of Latino, Asian, Native-American descent

  16. Diagnosis: The Issue(s) • There is no single diagnostic test for lupus • Lupus is a multi-system disease • Lupus is “a Great Imitator” • Slow development of disease symptoms • Variable presentation

  17. Diagnosis • Levels of Diagnosis • Obvious symptoms (rash, joint pain, kidney disease, low blood counts) • Problem: not specific for SLE • Addition: antibodies in the blood • Anti-nuclear antibodies (ANA)* • Anti-DNA antibodies • Meet 4 of 11 criteria (not equal)

  18. Type I IFNs and SLE • IFNs in patient serum (IFN-stimulated gene “signature” in patient WBCs) • Long-term treatment of patients (e.g., HBV, HCV, cancer) with IFN can produce SLE-like symptoms • (reversible upon removal of IFN!) • IFN system components implicated in GWAS • Type I IFN receptor inactivation (KO) in 2 murine autoimmune models decreases symptoms, increases life-span. • Development of plausible model relating IFN and SLE Interferon is involved in the pathogenesis of lupus.

  19. Type I IFN is in the Serum of Lupus Patients • 35-50% of SLE patients have IFN (protein/activity) in serum • Correlates with disease severity (generally) • in different patients • in serial samples of individual patients. • IFN is Type I by specific antibody neutralization • Issue: majority of IFN is also labile at pH 2 (“acid-labile Type I IFN”) • Q (1981): Why only 35-50% of patients? • Refs: • OT Preble et al. 1982. Science 216: 429-31. • O. Strannegard et al. 1982. Clin. exp. Immunol. 50, 246-252. • SR Ytterberg and TJ Schnitzer. 1982. Arth. Rheum. 25 (4), 401-406. • SV Skurkevich and EI Eremkina 1975. Annal Allergy 35, 357-360; • Early: JJ Hooks et al. 1979. NEJM 301 (1), 5-8; JJ Hooks et al. 1982. Arth. Rheum. 25 (4), 396-400)

  20. DNA Microarrays (“gene chips”) Expt: Extract mRNA from cells of control and SLE patients. Hybridize to slides with DNA representing large number of genes (e.g. 25,000). Detect mRNA levels by fluorescence. Compare control, SLE and any others. Represent graphically as an array. Try to make sense of it. No Change Increase Decrease

  21. SLE PBMCs show evidence of IFNMicroarray data: “heat map” Figure 1. SLE signature. Gene expression data by blood leukocytes of 9 healthy children, 30 with SLE, and 12 with juvenile chronic arthritis including 3 systemic arthritis. The SLE patients have been ranked according to their SLEDAI at time of blood draw. Each row represents a separate gene; each column a separate patient. 374 transcript sequences have been selected which were differentially expressed in SLE by comparison to healthy patients. The normalized expression index for each transcript sequence (rows) in each sample (columns) is indicated by a color code. Red, yellow, and blue indicate that expression of the gene is greater than, equal to or less than the mean expression across 9 healthy controls. (L. Bennett et al. J Exp Med. 2003. 197(6):711-23) (similar data from labs of V. Pascual and J. Banchereau; MK Crow; T. Behrens) Down-regulated Up-regulated

  22. Treatment with IFN can produce SLE-like symptoms • Autoimmunity after IFN-alpha therapy for mid-gut tumors (25/135): • Thyroid disease (18), SLE (1), pernicious anemia (4), vasculitis (2) time to onset: 3-17 mo. (LE Ronnblum et al. 1991. Annals Int. Med. 115: 178-183) • Treatment for neuroendocrine tumors (17/214) • Development of anti dsDNA Abs (17/214): polymyositis (1), hypothyroidism (3), (anti-DNA resolved (6) (K-M Kalkner et al. 1998. QJMed 91, 393-399) • Treatment for HCV • (LE Wilson et al. Sem. Arth. Rheum. 32 (3): 163-173) • Development of Raynaud’s Syndrome • (D. Schapira et al. 2002. Sem. Arth. Rheum. 32 (3): 157-162.

  23. Type I IFN involvement in autoimmunity:Evidence from murine models • Murine strains naturally developing autoimmunity: • NZB x IFNAR-1(-/-) • (ML Santiago-Raber et al. 2003 J. Exp.Med. 197, 777-788) • C57Bl/6-lpr xIFNAR-1(-/-) • (D. Braun et al. 2003. J. Autoimmun. 20, 15-25) • Type I IFN receptor inactivation (KO) in 2 murine autoimmune models decreases symptoms, increases life-span

  24. Development of plausible model relating IFN and SLE • Type I IFN is produced by dendritic cells (NIPC or pDC) • Serum of SLE patients contains non-viral IFN inducers • Complex of dsDNA + anti-dsDNA; others (L. Ronnblom, G. Alm) • Type I IFN can cause maturation of dendritic cells • Dendritic cells are primary antigen-presenting cell • Type I IFNs cause other consistent effects on immune system.

  25. Type I IFN and Lupus: A Model Crow, MK. Arthritis Rheum. 2003. 48(9):2396-401.

  26. Diseases linked (?) to Type I IFN • SLE • Sjögren’sSyndrome • Dermatomyositis • Type I Diabetes (?) • Aicardi-Goutières syndrome PG Bronson, C Chaivorapol, W Ortmann, TW Behrens, RR Graham. 2012. The genetics of type I interferon in systemic lupus erythematosus. Current Opin. Immunol. 24 (5) 530–537.

  27. Goal To block interferon action and test the effect of such blockage on autoimmune disease (in mice or other models) and SLE (in humans)

  28. IFN-producing Cell (IPC) 1 2 Signal Transduction Blocking Type I IFN action:The Interferon Life-Cycle Stimulus IFN IFNAR

  29. Roles of IFNAR Subunits B A B B B B A A B B A 2 Signal Transduction Human IFNAR-1 and IFNAR-2 • Both bind directly to Type I IFNs. • HuIFNAR-1- low affinity • HuIFNAR-2- moderate-high affinity (nM) • - The complex has higher affinity than IFNAR-2 alone. • - Both subunits are important

  30. IFNAR-2 Binding Site on IFN-α2 J Biol Chem. 2000 Dec 22;275(51):40425-33. New structural and functional aspects of the type I interferon-receptor interaction revealed by comprehensive mutational analysis of the binding interface. Piehler J, Roisman LC, Schreiber G. Weizmann Institute of Science,Rehovot, Israel.

  31. IFN (native) Agonist IFN Antagonist IFN IFN 2 1 X 2 X 2 1 2 IFNAR-1 IFNAR-1 IFNAR-2 IFNAR-2 No Signal Transduction Signal Transduction Designing an Interferon Antagonist If “X” marks the spot, where, exactly, is “X?

  32. ? IFNAR-2 IFNAR-1 Can we change an IFN into an IFN antagonist? • Mutate residues of HuIFN-a2 • Test in vitro for: • Lack of biological activity • Binding to cells, IFNAR-2 • Ability to block IFN binding & activity • Test in vivo

  33. Potential IFNAR-1 Binding Site on IFN-α2 Fig. 2. Model of IFN-α viewed from the Helix B/C face (putative IFNAR-1 site). Surface residues of Helices B and C (space-filling) are shown against the rest of the molecule (stick representation; background). Other helices are indicated by ribbons. Central aromatic surface residues are in yellow (see text). (from grant application to the Alliance for Lupus Research, 2004)

  34. WHOOPS!!! Roisman, LC, JaitinDA, Baker, DC, Schreiber, G. 2005. Mutational Analysis of the IFNAR1 Binding Site on IFNα2 Reveals the Architecture of a Weak Ligand-Receptor Binding-site. J. Mol. Biol. 353 (2) 271 – 281. Figure 5. The functional epitope for binding IFNAR1 on IFNα2. Residues that upon binding increase or decrease the binding affinity by >2-fold are colored red or blue, respectively. Residues previously found to bind IFNAR2 37 are colored green. Residues with no significant effect on binding are depicted in brown. Helices H2, H3 and H4 stand for helix B to D, respectively.

  35. Known residues of binding site 1A for IFNAR-1 do not play a critical role for all IFN-αs, nor is all biological activity lost in A4 (=NLYY) mutant. → There are other residues or another IFNAR-1 binding site on IFN-α2/α1 (and possibly all Type I IFNs?): Examine Helix D.

  36. Binding Site 1A for IFNAR-1 • Roisman, LC, Jaitin DA, Baker, DC, Schreiber, G. 2005. Mutational Analysis of the IFNAR1 Binding Site on IFNα2 Reveals the Architecture of a Weak Ligand-Receptor Binding-site. J. Mol. Biol. 353 (2) 271 – 281.

  37. Helix D mutants show strongly reduced antiviral activity M. Pan et al. 2008. Mutation of the IFNAR-1 receptor binding site of human IFN-α2 generates Type I IFN competitive antagonists. Biochem. 47, 12018-27

  38. Sites 1A and 1B (novel!) for IFNAR-1

  39. IFN-α2

  40. A novel second site on IFN-αs ("Site 1B") is critical for the binding of both IFN-α2 and the chimeric IFN-α2/α1. Since Site 1B mutants have no detectable biological activity, do they also act as antagonists?

  41. Antagonism of IFN-2: Antiviral Assay R120E R120E-α8 tail • L117A/R120A/K121A

  42. IFN X 2 1 Summary of Antagonists Several variants - Site 1B (R120E) - Site 1A+1B (NLYY/R120A) Criteria - Lack detectable biological activity - Antiviral, antiproliferation, other - Bind normally to IFNAR-2 - Lack detectable binding to IFNAR-1 - Block biological activity of native IFNs (tissue culture data)

  43. Experimental Design: Type I IFNR Antagonist in Acute SIV Infection 4 24 0 Assess effects of type I IFNR antagonist in PB, BAL, gut, LN: • Interferon stimulated genes (ISG) • CD4 T cell frequency • CD4 and CD8 T cell activation: HLA-DR, CCR5, Ki67 • Interferon stimulated genes (ISG) SIV RNA levels • Overall morbidity and mortality Week SIV Mac251 Follow-up 6 RM IFN receptor antagonist 1mg daily 6 RM Placebo Follow-up Sandler, NG, Zhu, RT, Estes, JD, Bortiz, E, Lifson, JD, Levin, D, Langer, JA,Schreiber, G, Rao, SS and Douek, DC. 2013. Blockade of Type I Interferon During Acute SIV Infection Results in Accelerated Progression to AIDS and Death. 20th Conference on Retroviruses and Opportunistic Infections (CROI2013), Atlanta, GA. March 3-6, 2013.

  44. Effect of IFNRa on Plasma Virus Load IFNRa Placebo * * * * P < 0.05 SIV RNA levels become significantly higher in the IFNR antagonist-treated animals by 6 months of SIV infection

  45. Clinical Outcome 100 IFNRa (n = 6) Placebo (n = 6) P = 0.01 50 % Survival 0 0 2 4 6 8 10 12 Months after infection At month 6 two IFNRa-treated animals died of AIDS At months 7-8 the remaining IFNRa-treated animals were euthanized for humanitarian reasons

  46. IFN/IFNAR Complex

  47. Type I IFN Ternary Complex IFN-a2 IFNAR1 IFNAR1

  48. Summary of Progress • We have mapped a new region (Site 1B) on IFN- which is important for IFNAR-1 binding. • We have identified IFN-2 variants that act as potent Type I IFN antagonists in vitro • Current work: Proof-of-principle application to rhesus macaques chronically infected with SIV (collaboration with Danny Douek (NIH) and Gideon Schreiber (Weizmann Inst) • Future work: Improve performance: • Increased testing of several current variants • Higher affinity for IFNAR2 • Increased serum half-life

  49. IFN-producing Cell (IPC) 1 2 Signal Transduction Blocking Type I IFN action:The Interferon Life-Cycle Stimulus IFN • Phase I/II Clinical Trial • Anti-IFN-a: • Sifalimumab: MedImmune • Rontalizumab: Genentech IFNAR

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